A new generation of microfluidic platforms based on smart and multifunctional materials

Universidade do Minho Escola de Engenharia Ricardo Jorge Brito Gonçalves Pereira A new generation of microfluidic platforms based on smart and multifunctional materials janeiro de 2023 UMinho | 2023 Ricardo Jorge Brito Gonçalves Pereira A new generation of microfluidic platforms based on smart and multifunctional materials A new generation of microfluidic platforms based on smart and multifunctional materials Ricardo Jorge Brito Gonçalves Pereira Tese de Doutoramento Engenharia de Materiais Trabalho efetuado sob a orientação de Doutora Vanessa Fernandes Cardoso Professor Senentxu Lanceros-Méndez janeiro de 2023 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial CC BY-NC https://creativecommons.org/licenses/by-nc/4.0/ iii ACKNOWLEDGEMENTS The completion of my dissertation was the product of extensive labor, hours of research, and reflection, all of which led to the achievement of this objective. However, in order to complete my project, I required the personal and professional accompaniment, assistance, and support of others. First, I want to thank the support of the Foundation for Science and Technology-FCT (grant SFRH/BD/140698/2018). I would like to thank my supervisors, Dr Vanessa Cardoso and Professor Senentxu Lanceros-Mendez, for accepting the guidance of this project. I also thank you both, for your scientific knowledge, as well as your support, effort, availability and friendliness during these years. A special thanks to my friends, André, Clarisse, Costa and Pedro, which were essential in the good and the bad times; they were always present to help me, even when the things appeared difficult, sometimes with real big gestures, sometimes with just a friendly word of encouragement, that made the difference. I also thank the ESM research group for their support and welcome, which has always allowed me to develop my work, in the best way. This work was partly made of trips and scientific stays, I would like to thank Professor Catherine Klapperich and her group at Boston University, for the great experience with them, it were unforgettable times, that I miss and cherish. I would also like to thank Professor Gerardo Hernandez, for the reception in the beautiful Heidelberg, at the Karlsruhe Institute of Technology, I learned a lot scientifically, I gained friends and experiences that I will keep forever. I would also like to thank David Durán and his supervisors at the Centro de Cirugía de Mínima Invasión Jesús Usón, in Caceres, for the fantastic work and experience that we have developed together, which has allowed me to greatly expand my knowledge. Finally, I would like to thank BCMaterials, in Bilbao, for the support in the various works carried out and for the always very kind reception in the various visits made throughout the PhD. I would like to leave a very special thanks to Paulina, we both started and finished this step, we went through its difficulties and ended up overcoming it, with total affection and unconditional support. To my family, thank you from the bottom of my heart! Mom you are the best and I will never be able to thank you, for everything you do for me. iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v Abstract A precise diagnostic is a key part of the clinical practice as it is essential to provide a correct clinical decision-making for both health professionals and policy makers in terms of public health. In this respect, the primary goal of this project is to develop new solutions that take advantage of the beneficial qualities of Portable Analytical Devices (PADs) and the microfluidic paper-based analytical devices (µPADS), while addressing their technological limitations such as the uncontrolled passive flow rate, the weak mechanical properties or the lack of simple functional reactions, that have a significant impact on their potential to achieve real-life applications and commercialization. To achieve such objective, different polymer-based substrates have been developed, characterized and tested for the printing of PADs and different fillers have been added to printable waxes aiming to induce multifunctionality on the PADs. In this context, poly(l-lactic acid) (PLLA), poly(vinylidene fluorideco -trifluorethylene) (P(VDF-TrFE)), poly(D,L-lacticco - glycolide) (PDLG), silk and silk fibroin, were processed in different morphologies. Each substrate revealed characteristics of great interest, such as the maximum capillary flow rate of PLLA oriented fiber substrates (70.2 ± 1.9 mm.min-1), the high mechanical strength of P(VDF-TrFE) substrates (ranging from 71.4 ± 2.9 to 163.4 ± 5.1 MPa), the rapid degradation rate of PDLG (90 % weight loss in six weeks), or the stability of silk-based substrates which are capable of maintaining high accuracy after three utilizations, using different analytes (albumin, uric acid and glucose), with an minimum R2 of 0.967. The next objective of this project was to increase the versatility of the wax printing technique in terms of functional materials. Three different types of hydrophobic wax-based inks have been developed: magnetic, dielectric and conductive. Magnetic waxes (with Fe3O4 nanoparticles) exhibited an actuation capability with a deformation of ≈12 mm when a magnetic field of 105 mT was applied, and an energy harvesting aptitude with an energy density of about 9.7 mW.cm−3. Active waxes with dielectric properties (with BaTiO3 nanoparticles), exhibited optimized dielectric properties up to ε= 18 at 10 kHz. Conductive waxes (with carbon nanotubes and graphene) exhibited high electrical conductivity values (up to ≈ 2.5×10-4 S.m-1), high gauge factor (up to 11), and a pressure sensitivity of (up to 0.17 MPa-1). In order to demonstrate the technological significance of the produced materials, two µPADS systems have been developed: i) a magnetic origami system completely printed on paper suitable for the detection of infectious diseases and magnetic biomarkers; and ii) a low-power thermal actuation system, based on the printing of conductive wax composites with integrated graphene nanoplatelets (GNP), that was able to work simultaneously as a barrier and as a heater. Thus the materials developed in this thesis hold great promise for the next generation of versatile, effective and accurate PADs and µPADS. Keywords: Microfluidic; Multifunctional waxes; Point-of-care; Polymer-based substrates vi Resumo Um diagnóstico preciso é uma parte essencial da prática clínica uma vez que é crucial para uma correta tomada de decisão quer por parte profissionais de saúde quer pelos decisores políticos em termos de políticos de saúde pública. O principal objetivo deste projeto foi o desenvolver novas soluções que aproveitassem, em simultâneo, as mais valias dos dispositivos analíticos portáteis (PADs) e dos dispositivos microfluídicos analíticos baseados em papel (µPADS), enquanto se procurava solucionar também as suas limitações tecnológicas como a sua taxa de fluxo passivo não controlada, as fracas propriedades mecânicas e a falta de reações funcionais, uma vez que todas estas limitações têm um impacto negativo na sua aplicabilidade/comercialização. Para alcançar este objetivo, foram desenvolvidos diferentes materiais de base polimérica e testados diversos reforços (promotores de multifuncionalidade). Nesse sentido foram usados o poli(L–acido lactico) (PLLA), o poli(fluoreto de vinilideno – trifluoretileno) (P(VDF-TrFE)), o poli(D,L-láctico-co-glicólido) (PDLG) e a fibroína de seda com diferentes morfologias. Cada substrato polimérico evidenciou características de elevado interesse, tais como a velocidade e fluxo capilar dos substratos de fibras orientadas de PLLA (70.2 ± 1.9 mm.min-1), a elevada resistência mecânica dos substratos de P(VDF-TRFE) quando molhados (de 71.4 ± 2.9 até 163.4 ± 5.1 MPa), a rápida taxa de degradação do substratos de PDLG, cerca de 90 % em seis semanas, e a elevada estabilidade da seda com a capacidade de manter uma elevada precisão após três utilizações, em diferentes reagentes (albumina, acido úrico e glucose), com um R2 de 0.967. Outro dos objetivos deste trabalho foi o de aumentar as soluções proporcionadas pela técnica de impressão a cera utilizada no fabrico dos µPADs, tendo sido desenvolvidas ceras com propriedades magnéticas, dielétricas e condutoras. As ceras magnéticas (com a adição de nanopartículas de Fe3O4) evidenciaram uma boa capacidade de atuação (12 mm quando aplicado um campo de 105 mT) e uma elevada capacidade de recolha energética (cerca de 9.7 mW.cm− 3); as ceras ativas dielétricas (com a adição de nanopartículas de BaTiO3) exibiram uma resposta dieléctrica optimizada (ε=18 a 10 kHz); e as ceras condutoras (com nanotubos de carbono e grafeno) mostraram uma elevada condutividade elétrica (até ≈2.5×10-4 S.m-1), um gauge factor (GF) de 11 e uma pressão de sensibilidade (PS) de 0.17 MPa-1. Por fim e para demonstrar a aplicabilidade dos materiais desenvolvidos, foram desenvolvidas dois µPADS: i) um origami magnético totalmente impresso em papel para deteção de doenças infeciosas e biomarcadores magnéticos; e ii) um sistema de aquecimento em papel, baseado na impressão de compósitos hidrofóbicos multifuncionais com nano-folhas de grafeno (GNP) integradas na matriz de cera. Os resultados deste projeto demonstram que os materiais desenvolvidos nesta tese poderão ser usados numa próxima geração de PADs e µPADs: versáteis, eficazes, e com elevada precisão. Palavras chave: Ceras multifuncionais; Microfluídica; Point-of-care ; Substratos de base polimérica vii Index 1.1. Introduction.......................................................................................................................... 2 1.2. Microfluidics ......................................................................................................................... 4 Diminutive reagent utilization ........................................................................................ 5 High surface to volume ratio ......................................................................................... 5 High-throughput applications......................................................................................... 5 Portable systems .......................................................................................................... 6 1.3. Conventional materials to fabricate microfluidic devices ........................................................ 6 Silicon and glass ........................................................................................................... 6 Polymers ...................................................................................................................... 7 Paper ........................................................................................................................... 8 Smart and bio-based polymers ...................................................................................... 9 1.4. Portable analytical devices applications .............................................................................. 11 1.5. Printing techniques applied on portable analytical devices and microfluidic paper analytical devices …………………………………………………………………………………………………………………………18 1.6. Objectives and methodology .............................................................................................. 22 1.7. Structure of the thesis ....................................................................................................... 23 1.8. References ........................................................................................................................ 24 Introduction ............................................................................................................... 37 Experimental procedures ........................................................................................... 39 Experimental Results and Discussion ......................................................................... 44 Conclusions ............................................................................................................... 55 xiv Figure 3.1.1. Graphical representation of the preparation procedure of the Fe3O4/wax cartridges and the posterior printing process. .............................................................................................................. 131 Figure 3.1.2. a) TEM image of the Fe3O4 nanoparticles. Insets: Representative X-ray diffraction pattern. b) Size distribution of the Fe3O4 nanoparticles obtained from TEM. ....................................................... 134 Figure 3.1.3. Surface (500x) SEM images of Fe3O4/wax composites with different wt.% of Fe3O4: a) 0; b) 5; and c) 10. Cross section (150x) SEM images of Fe3O4/wax composites with different wt.% of Fe3O4: d) 1; and e) 10. f) Cross section SEM image (20000x) of the Fe3O4/wax composite cartridges with 10 wt.% Fe3O4 content. ................................................................................................................................. 135 Figure 3.1.4. a) Room-temperature magnetization of the Fe3O4/wax cartridges. The inset reveals the roomtemperature magnetization of the neat Fe3O4 powder. b) Relation between Fe3O4 wt.% in the composite blocks: left, the Fe3O4 theoretical wt.% (in melt state) and, right, the corresponding magnetization saturation values (MagnetizationSAT) The inset shows a photograph of the Fe3O4/wax composite block with 10 wt.% of nanoparticle content. ..................................................................................................... 136 Figure 3.1.5. a) First DSC scans of Fe3O4/wax composites at heating and cooling rates of 5 °C.min-1 between 0 and 120 °C and; b) DSC second scans of Fe3O4/wax composites at heating and cooling rates of 5 °C.min-1 between 0 and 120 °C. Heat flow indicates exothermic phase transitions up. ............. 137 Figure 3.1.6. - Surface and cross-section SEM/EDX elemental mapping images of the magnetic paper samples. On the first column are placed the SEM images and in the other three columns are shown the different chemical elements (oxygen, iron and carbon). The lines correspond to the different paper samples. ........................................................................................................................................ 139 Figure 3.1.7. a) Characteristic room-temperature stress-strain curves on both paper and magnetic paper samples. b) Young’s Modulus values obtained from Figure 3.1.7a. .................................................. 140 Figure 3.1.8. Room-temperature magnetization of the Paper@Fe3O4/wax_10 samples with three printed widths (1, 2, 3). The inset shows the neat magnetization for each sample. b) Magnetic actuation ability of the Paper@Fe3O4/wax_10/3 sample. .......................................................................................... 141 Figure 3.1.9. a) Scheme of the displacement calculation on the magnetic paper. b) An interactive/smart book, one of the potential applications of the developed materials. .................................................. 141 Figure 3.2.1. Printing procedure of the proposed sensing/harvesting platform. ................................ 150 Figure 3.2.2. Working principle of the proposed sensing/harvesting platform. .................................. 152 Figure 3.2.3. Schematic representation of the electronic circuit that ensures IoT connectivity. .......... 153 Figure 3.2.4. a) Relation between the |d33| piezoelectric coefficient and the P(VDF-TrFE) weight content in the printed polymer prepared with different P(VDF-TrFE)/ DMF ratios. b) Room-temperature magnetic xv hysteresis cycles for the printed wax-CoFe2O4 compositions with different CoFe2O4 wt.% content. The inset reveals the relation between the CoFe2O4 wt.% in solution and the CFO wt.% in the corresponding printed layers. c) Contact angle at room-temperature and at 80 °C of different waxCoFe2O4 (80 wt.% of CoFe2O4) drops on printed P(VDF-TrFE) with silver electrodes. d) Young’s Modulus of the different samples. The inset shows a representative stress-strain curve for each sample. Representative e)-cross section and f)- surface SEM images of the P(VDF-TrFE)/silver//Wax-CFO/Resin 3-layered composite. .................... 154 Figure 3.2.5. Output voltage of a PVDF R1F sample as a function of: a) CFO wt.%; b) magnetoactive layer thickness; c) minimum distance to the magnet; d) frequency of the magnetic stimulus; e) cycling solicitation; and f) magnetic prints design. ....................................................................................... 156 Figure 3.2.6. a) Harvested power (Po) and magnetic field (Bm) as a function of the distance to the magnet without the mechanical deformation resulting from the impact between the magnet and the printed composite. b) Relation between the measured power, load resistance (R) and current (I) without the mechanical impact between the magnet and the printed composite. c) Harvested power (Po) as a function of the cycling solicitation (f=3.3 Hz). d) Relation between the measured power, load resistance (Rl) and current (I) with mechanical impact between the magnet and the printed composite. e) Data sent (through Bluetooth) by the microcontroller and received by a smartphone. .................................................... 158 Figure 3.3.1. Experimental procedure for the preparation of the wax and wax composites for the different barium titanate contents a). Moulded b) and screen-printed c) samples illustrating the materials processability. ................................................................................................................................ 168 Figure 3.3.2. SEM images of the wax (A and B) and wax composites with the lower and higher BT content, 10 (C and D) and 50 wt.% (E and F) respectively. The orange circles show the barium titanate agglomerates in the composite. The magnifications are 1 000× and 5 000× for both composites. ... 171 Figure 3.3.3. SEM and EDX analysis of the composites where are represented the barium (Ba) and titanium (Ti) contents of representative composites. ........................................................................ 172 Figure 3.3.4. Chemical characteristics and thermal properties of the wax and BT/W composites with 10 and 50 wt.% of BT. A and B) FTIR specta of the materials with representatinve abspoption bands and C and D) DSC analysis with melting temperature (Tm) of the materials. ................................................ 173 Figure 3.3.5. - Mechanical compression measurements of the wax and 20BT/W molded samples A-D) and bending evaluation E) of the screen-printed 20BT/W composite. ............................................... 175 Figure 3.3.6. - Dielectric response of wax and corresponding composites up to 50 wt.% BT as a function of frequency (A) and ceramic content at a frequency of 1 kHz (B). ................................................... 177 xvi Figure 3.3.7. Experimental results and theoretical models (Maxell-Garnet, Bruggeman, Tinga and Wiener models) for BT/wax composites up to 50 wt.% BT content with a filler diameter of 100 nm. The experimental results correspond to a frequency of 1 kHz. ................................................................ 179 Figure 3.3.8. A) 2D illustration of the electrical potential and B) the computed capacitance for the simulated different dielectric areas as a function of BT content. ....................................................... 181 Figure 3.4.1. Experimental procedure used to prepare the wax (black dye) rGO/W and CNT/W composites a) and the prepared samples for several contents of rGO b) and c) CNT. ......................................... 191 Figure 3.4.2. Illustration of the (i) screen-printing process of the wax composites (ii) on the flexible substrates (Kapton). The experimental process was carried out at 100 °C. ...................................... 192 Figure 3.4.3. Schematic representation of the electromechanical measurements of the functional G/W and CNT/W composites. a) Cubic samples (volume of 1 cm3) in compression mode up to a force of 50 N and b) 4-point-bending mode up to 5 mm deformation. The piezoresistive sensibility measured by the 4-point-bending method presents two contributions: the geometrical effect (1+2υ=1.74) and the intrinsic one that depends on the conductivity variation due to the fillers network reconfigurations [50]. ........ 194 Figure 3.4.4. Representative SEM images of pristine wax a) and composites with CNT contents of 0.25 wt.% b) and 2 wt.% c) and rGO contents of 2 wt.% (d and e) and 25 wt.% f). a) and d) correspond to SEM surface images and b), c), e) and f) represent cross-section images of the samples. The magnifications used were 1 000× and 15 000×. .................................................................................................... 195 Figure 3.4.5. a) FTIR spectra and b) DSC thermograms for pristine wax and composites with 0.5 and 4 wt.% of CNT and 10 and 25 wt.% of rGO. Characteristic absorption bands of wax in the FTIR spectra are highlighted. .................................................................................................................................... 196 Figure 3.4.6. Illustration of the force-deformation characteristic mechanical curves under cyclic loadingunloading for wax (a) and for the 4CNT/W composite (b). Measurements were performed at several compression forces up to 50 N. ...................................................................................................... 197 Figure 3.4.7. a) Characteristic I-V measurements of wax and the different wax composites with rGO (above) and CNT (below). b) Electrical conductivity of the wax composites reinforced with CNT and rGO up to 4 and 25 wt.%, filler content, respectively. Inset: determination of the percolation threshold. ... 198 Figure 3.4.8. Piezoresistive response of the molded CNT/W and rGO/W composites. Compression cycles (10 cycles) for different applied maximum forces up to 50 N for a) 05CNT/W and b) 25rGO/W composites, respectively. Linearity of the electromechanical responses for (c) 05CNT/W and pressure sensibility (d) of the composites as a function of applied force, determined by average og the 10 cycles. ...................................................................................................................................................... 200 xvii Figure 3.4.9. a) Piezoresistive bending response of the 25rGO/W samples for cycling deformations (10 cycles) from 0.1 to 5 mm and b) linear beahviour between strain (ε) and resistance changes (ΔR/R0). c) Cycling stability measurements for 1000 cycles at 5 mm of deformation (higlighted 25-30 cycles for the initial and the last cycles) and d) piezoresistive sensibility as a function of bending deformation up to 5 mm. ............................................................................................................................................... 202 Figure 3.4.10. - a) Illustration of the wax composite working in the bending mode. b) Composite reinforced with 25 wt.% of graphene, processed by screen-printing, connected to the electronic circuit (left) measuring the bending stimulus applied to the sample (right). ........................................................ 204 Figure 4.1.1. Proposed working principle of the printed magnetic origami portable analytical system applied to malaria: i) the infected mosquito bites a person; ii) a blood sample is collected from the person; iii) a few blood drops are placed on the testing rectangle and the printed magnets (black rectangles) concentrate the haemozoin crystals exhibiting a paramagnetic behaviour; iv) the detection of haemozoin crystals can be performed through four different techniques (Infrared spectroscopy; X-ray spectroscopy, Magnetometry, and Color evaluation); and v) the person who was bitten is free to go (in the case of a negative test) or referred for medical treatment (in the case of a positive test). ................................. 215 Figure 4.1.2. Experimental scheme (a-d) regarding the printing of the MOPAS. a) Mechanical mixing of the Wax and/or Wax-NdFeB inks; b) Molding process of the Wax and/or Wax-NdFeB cartridges; c) Printing of the MOPAS on a Whatman nº1 cellulose paper substrate using a Xerox ColorQube 8880 printer; d) A4 Whatman nº1 cellulose paper sheet with 23 printed MOPAS. MOPAS working principle (e-g): e) initial coniguration and dropping the Fe3O4@H2O suspension (50 μl) on the center of the white rectangle (10mm x 7mm); f) folded MOPAS configuration; and g) Fe3O4@H2O suspension flowing through the escaping channels (4 white lines: 26mm x 1mm) without the action of the printed wax/NdFeB magnets (10mm x 7mm). ............................................................................................................................................ 218 Figure 4.1.3. a) Shear stress vs. shear rate of the Wax and Wax/NdFeB inks at 120 C. b) Viscosity values of the Wax and Wax/NdFeB inks as a function of the shear rate at 120 C. c) Wax ink's storage (G') and loss (G'') modulus, and tg (δ) values as function of oscillation strain at 120 C. d) Wax/NdFeB ink's storage (G') and loss (G'') modulus, and tg (δ) values as function of oscillation strain at 120 C. Please note that during the printing procedure the cartridge is at 120 C. ................................................... 222 Figure 4.1.4. a) Proposed printed MOPAS: i) the black squares represent the printed Wax/NdFeB magnets, the yellow rectangles represent the wax layer printed on the paper substrates and adjacent to the Wax/NdFeB magnets, the green rectangle represents the hydrophobic wax layer printed on the paper substrates and adjacent to the testing zone, and the white cross represents the hydrophilic testing zone; xviii ii) the testing rectangle can be placed between the two printed magnets; being ready for the multi-tool diagnostics evaluation. b) Cross-section SEM image of the Wax/NdFeB-Wax interface. c) Surface SEM image of the hydrophilic (wax)-hydrophobic (paper) interface. .......................................................... 224 Figure 4.1.5. a) Stress-strain curves obtained at room-temperature (30 C) for the different samples. b) Young’s Modulus values obtained from (a). c) Force profile of the peeling test for the different samples as a function of time and pressure. d) Weight loss for the different samples as a function of the peeling force. ...................................................................................................................................................... 225 Figure 4.1.6. a) Room-temperature magnetization as a function of the applied DC magnetic field for the NdFeB powder, the wax/NdFeB printed magnet and, for the Fe3O4 powder. b) Color map obtained by EDS in the same sample and location of Figure 5b): Green: B; blue: Carbon; Yellow: Oxygen; Cyan: Nd; and Red: Iron. c) TEM image of Fe3O4 nanoparticles. The inset reveals the corresponding X-ray diffractions pattern. d) Size distribution of the Fe3O4 nanoparticles. .................................................................... 227 Figure 4.1.7. a) Color map obtained by EDS in the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: iv) 0.8 mg.ml-1; iii) 0.2 mg.ml-1; ii) 0.05 mg.ml-1; and i) 0.003125 mg.ml1. b) Relation between Fe wt.% in the testing zone rectangle (obtained from EDS) and the Fe3O4 concentration. c) Room-temperature magnetization as a function of the applied DC magnetic field for the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: 0.8 mg.ml-1; 0.2 mg.ml-1; 0.05 mg.ml-1; 0.0125 mg.ml-1; and 0.003125 mg.ml-1. d) Relation between the saturation magnetization (at 20 kOe) found on the testing zone rectangle and the Fe3O4 concentration. e) Infrared absorbance vs. wavenumber for the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: 0.8 mg.ml-1; 0.2 mg.ml-1; 0.05 mg.ml-1; 0.0125 mg.ml-1, and 0.003125 mg.ml-1. f) Relation between the 530 cm−1 FTIR band (reported as representing Fe-O vibrations in Fe3O4) absorbance and the Fe3O4 concentration on the drop added to the testing zone rectangle. g) Mobile phone photographs (iPhone 12) of the origami testing platform into which were added 50 μl of the mixture with Fe3O4 content of: v) 0.8 mg.ml-1; iv) 0.2 mg.ml-1; iii) 0.05 mg.ml-1; ii) 0.0125 mg.ml-1, and i) 0.003125 mg.ml-1. h) Relation between the mean grey value (obtained through image J software) and the Fe3O4 concentration of the drop added to the testing zone rectangle. ........................................................................................ 228 Figure 4.2.1. Representative illustration of the a) Whatman nº1 paper devices with arrays of 6 parallel channels and b) portable printed folded Whatman nº1 paper device, in which a conductive square is connected to a small battery. .......................................................................................................... 241 Figure 4.2.2. a) Variation of viscosity of the inks as a function of shear rate for solutions with different melted GNP/Wax concentrations. b) Viscosity at 2 s-1 shear rate for solutions with different melted xix GNP/Wax concentrations. c) Contact angle between different melted GNP/Wax concentration drops and paper at 30 C (left) and 80 C (right); d) corresponding representative photograph’s of the waxes in contact with paper. ......................................................................................................................... 242 Figure 4.2.3. Representative surface SEM images of a) Whatman nº1 paper; b) printed cured neat wax on paper; c) Printed cured 5 wt.% GNP/Wax on paper; d) printed cured 20 wt.% GNP/Wax on paper. e)- h) Respective representative cross-section images. .......................................................................... 243 Figure 4.2.4. a) Force profile of the peeling assays for the various wax printed samples as a function of time and compression force; b) respective weight loss after the peeling assays as function of the compression force; c) stress-strain curves for the various wax printed samples obtained at room temperature; d) respective Young’s modulus values obtained from a). ............................................. 244 Figure 4.2.5. a) Electrical conductivity values of the various wax based printed samples as a function of GNP concentration. b) Temperature at the top (T) and bottom (B) of the different GNP/Wax lines printed and cured on Whatman nº1 paper as function of the applied voltage. c) Temperature variation as a function of time for various applied voltages measured at the top of the printed and cured 20 wt.% GNP/Wax lines. d) Temperature measured at the top of the substrate in the dry and wet state as function of the applied voltage, 1 and 2 mm away from the printed and cured 20 wt.% GNP/Wax lines. ........ 246 Figure 4.2.6. Photographs of a) µPADs system with individual hydrophilic channels delineated by printed and cured hydrophobic neat wax and hydrophobic conductive 20 wt.% GNP/Wax lines; b) two thermochromic inks at room temperature (red activated at > 47 C and green activated at >28 C); Resulting colour changes when specific conductive lines are subject to an electrical voltage of c) 12 V and d) 14V indicated by the blue arrows. Corresponding thermal images in inset. .................................. 249 Figure 4.2.7. Photographs of a) portable printed folded Whatman nº1 paper devices, in which a 20 wt.% GNP/Wax square is connected to a battery (1.5, 9 or 12 V) placed inside the 3D printed support, in order to provide heat to the thermochromic ink contained in the circular testing area delimited by neat wax; b) thermochromic ink (activated at 28 °C) with conductive square subjected to a voltage of 1.5, 9 and 12 V and corresponding thermal images; c) thermochromic ink (activated at 47 °C) with conductive square subjected to a voltage of 1.5, 9 and 12 V and corresponding thermal images. ...................... 250 xx List of tables Table 1.1. Some relevant and representative works on the use of microfluidic technology in different areas. ............................................................................................................................................... 12 Table 1.2. Schematic overview of the most used printing techniques for PADs and µPADs fabrication. 19 Table 2.2.1. Comparative characteristics of commercial and P(VDF-TrFE) membranes as substrates for microfluidic applications……………………………………………………………………………………….……………..78 Table 2.4.1. Crystallinity degree of the Bombyx mori cocoons and electrospun SF samples. ............. 117 Table 3.1.1. Melting and crystallization temperatures, obtained from DSC, of the Fe3O4/wax composites. ...................................................................................................................................................... 137 Table 3.1.2. Latent heat of fusion and crystallization of the Fe3O4/wax composites. .......................... 138 Table 3.2.1. Power densities of energy harvesting technologies. ...................................................... 159 Table 3.3.1 – Summary of the theoretical models considered in this work, where ν and ε values represent the volume fraction and dielectric constant, respectively. The subscripts 1 and 2 represent the matrix and filler, respectively and d is the system dimensionality. ..................................................................... 178 Table 3.3.2. - A) Relative percentage deviation of the Maxwell-Garnett and Brugemann models in function of the volume fraction and B) the factors n and n’ for Tinga and Wiener models with the corresponding Rsquare obtained by computational iteration by fitting the experimental data. .................................... 179 Table 4.1.1. - Contact angle between Wax and Wax/NdFeB inks and the paper substrate at 30 C and 80 C, and between Fe3O4@H2O suspension and the 2 different substrates: paper and paper@wax. 220 xxi List of symbols a Distance between bending points A Electrode area Bm Magnetic field C Capacity d Thickness d33 Piezoelectric coefficient ε Electrical resistivity ε0 Vacuum permittivity ε’ Real part of dielectric constant ɛbreak Stain-to-Failure ɛexp Experimental data measured ɛmod Predicted dielectric constant ɛp Porosity ɛt Strain f Frequency Fβ β-phase content G' Storage modulus G'' Loss modulus GF Gauge factor HAC Amplitude of the alternating current magnetic field I Current Id Mechanical displacement It Sample thickness Kα Absorption coefficient of α-PVDF Kβ Absorption coefficient of β-PVDF L Distance between electrodes Msat Magnetic saturation pc Percolation threshold p Filler content xxii P Applied pressure P0 Harvested Power PS Compression applied r Surface electrical resistivity R Electrical resistance Rl Load resistance R0 Initial resistance s Universal critical exponent t Thickness of the piezoelectric layer tan δ Dielectric loss tg Storage modulus Tm Melting temperature V Voltage ω Angular frequency wt.% Weight percentage Xc Degree of crystallinity Y Young’s modulus Z Vertical displacement α33 Transverse magnetoelectric voltage coefficient ΔHf Melting enthalpy ΔR Electrical resistance variation ∆V Output voltage σ Electrical conductivity σs Stress σ0 Filler conductivity σbreak Ultimate tensile strength δr Relative percentage deviation xxiii List of abbreviations A ATR Attenuated total reflection ASSURED Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users B BM Basal medium BSA Bovine serum albumin BT Barium Titanate C CA Contact angle CFO CoFe2O4 D DAPI 4',6-diamidino-2-phenylindole DLS Dynamic light scattering DM Differentiation medium DMEM Dulbecco’s modified eagle’s medium DMF N, N-dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSC Differential scanning calorimetry DTG Differential thermogravimetric E ECM Extracellular matrix EDX Energy-dispersive X-ray spectroscopy F FBS Fetal bovine Serum FDA Food and Drug Administration FITC Phalloidin fluorescein isothiocyanate FTIR Fourier transformed infrared spectroscopy G Chapter 1 – Introduction 4 paper becomes essential, in order to combine proper absorption and flow rate with the ability to integrate materials that allow new responses, improved flow control and to allow analysis methods applicable in practical contexts and under a large variety of conditions [14]. This PhD thesis aims to address those issues by developing alternative materials with improved properties to both complement and replace paper. Natural and synthetic polymers featuring controlled physicochemical properties were developed to be used as novel microfluidic substrates with enhanced and even new and tailorable properties [15]. Concurrently, hydrophobic components are required to define the channels and confine the fluid flow within the hydrophilic substrates. In this context, multifunctional waxes, including magnetic, dielectric and conductive, were also developed as an alternative to the commercially passive materials used in printing technologies. This work represents a contribution to the transition of PADs and µPADs from the laboratory into the user’s hands, presenting the development of simple and low-cost systems, with integrated active characteristics, that can be produced in existing mass production facilities, allowing to accelerate their successful commercialization. 1.2. Microfluidics The behaviour, precise control, and manipulation of fluids that are geometrically restricted to a tiny scale (usually sub-millimeter) at which surface forces dominate volumetric forces is referred as microfluidics [16]. Microfluidics is an interdisciplinary field that connects fluid dynamics, physics, and chemistry, among others [16]. The first advances in this technology were introduced around the 1950s. Particle separation and analysis techniques, such as gas and liquid chromatography were optimized in this period [17, 18]. It was discovered that with a reduction in channel size, it was possible to achieve better particle separation. Years later, the technique of capillary electrophoresis emerged to replace the initial techniques. It consists in the use of an agarose gel for the separation of electrically charged particles [19]. With the advances in microanalytical methods, the interest in combining them with microfluidic platforms also emerged. Another factor that contributed to the substantial advances in microfluidics was the need of the United States of America (USA) government to develop advanced defense programs, around the 1990s (the end of the Cold War) [16, 20]. One of the areas explored by this program was the development of microfluidic devices capable of detecting potential chemical and biological threats. Chapter 1 – Introduction 5 The last, but perhaps the most important factor in the progress of this technological area was the emergence of microelectronics. Associated with this area appeared lab-on-a-chip [21] and organ-on-achip [22] applications, small devices capable of incorporating various laboratory or biological functions into one single system [23]. Early applications of microfluidics arose associated with analyte separation and analysis due to the inherent advantages of miniaturization, including but not limited to the small amounts of sample and reagents required, short analysis times, the ability to integrate several components in one device and their low cost [21, 24]. Among the various specific characteristics of microfluidics systems, some prove to be of great importance for biomedical research. They will be discussed in the next sub-chapters. Diminutive reagent utilization A clear advantage of microfluidic devices when compared to other alternatives is that small volumes of reagents are required due to their typically small-scale channel in the microor even nanometer sizes. This advantage presents great significance when high-cost reagents, which requires complex processes of isolation and purification, are to be used [25, 26], or samples are limited in availability. High surface to volume ratio Associated with low reagent consumption arises the advantage of the extremely high surface to volume ratios provided by microfluidics systems. It allows fast mass and heat transfer, which is essential for several applications, as it is the case of biotransformation, selective temperature reactions and nanoand microparticle separation [26, 27]. High-throughput applications With low working volumes and the capability to parallelize the reaction areas, these systems are suitable for high-throughput applications. Unlike conventional methods such as fluorescent activated cell sorting, microfluidic devices have outstanding capabilities for high parallelization reactions due to their reduced dimensions. Thus, a large amount of simultaneous experiments can be performed in a small system and provide proper statistically sound conclusions from a single run [29]. Chapter 1 – Introduction 6 Portable systems One of the main objectives of the development of microfluidics was to build a device that integrates all the instrumentation and the analysis capabilities of a laboratory in hand-held size to deliver a fully portable system. Reduced dimensions allow, to a certain extent, experiments at different locations of interest for true POC testing [30]. 1.3. Conventional materials to fabricate microfluidic devices One of the most important parameters to consider before developing a microfluidic system is the selection of the materials. Each material has certain characteristics as well as associated manufacturing techniques that influence the performance and price of each device. The most relevant will be described in the next sub-chapters along with their main advantages and limitations. Silicon and glass The first materials used in this area were silicon and glass which are usually processed by manufacturing techniques well known in the semiconductor industry such as bulk micromachining using chemical corrosion and dry corrosion, or surface micromachining [31]. Bulk micromachining produces structures within the substrate by selectively corroding it using photolithography to transfer a pattern from a mask to the surface. In turn, surface micromachining creates structures on top of the substrate, meaning that thin layers of silicon are deposited successively using chemical deposition techniques [31]. While silicon is transparent to infrared light but not to visible light, glass is optically transparent in the visible spectral range. Interesting properties of silicon and glass come from their high thermal and chemical stability. Also, like silicon, the chemical modifications of glass are based on silanol, allowing chemical surface modifications for specific applications [32]. However, the hardness of silicon and glass, its high cost and time to manufacture, as well as the difficulty in sealing the microfluidic structure and the integration of functional units, together with the non-permeability of gases, preclude its use in many applications [32]. Consequently, the development of microfluidic systems using other materials that can be easily manufactured and that are compatible with a wider range of applications has become a necessity [32]. Chapter 1 – Introduction 7 Polymers Polymer-based microfluidic systems are an interesting alternative because they are mostly inexpensive, easily mass-produced and chemically modifiable. An added benefit is the wide range of polymers available that offers great flexibility in the selection of materials with specific properties. According to their physical properties, polymers can be classified into thermosetting, thermoplastics and elastomers [33]. Heat-hardeners such as SU-8 resin are usually stable even at high temperatures, resistant to most solvents, biocompatible and show optical transparency and good mechanical properties [34]. SU-8 allows the manufacture of microstructures with a high height/width ratio using photolithography. When properly heated and exposed to ultraviolet light of a specific wavelength using inverse pattern high resolution photomasks, the unexposed parts, unlike the exposed parts, are soluble and thus removed after the process [34, 35]. SU-8 has been used directly as a structural material for the manufacture of functional units (such as electromechanical systems) and often as a template for the manufacture of PDMS based microfluidic systems, the most popular elastomer used for microfluidics [33]. During the process called replica molding, PDMS is poured into a thermally cured mold (at a temperature ranging from 40 to 100 °C) and easily peeled off due to its low surface tension. To seal open microfluidic channels, PDMS can be reversibly bonded to PDMS, glass or other substrates by simple contact or irreversibly using oxygen plasma surface treatments or a thin layer of uncoated PDMS as glue [20]. PDMS shows very interesting properties for the manufacture of functional units (such as valves and pumps) and/or for biotechnological applications because of its biocompatibility, high elasticity and low cost or for being a gas permeable porous matrix [36, 37]. However, nonspecific binding of hydrophobic molecules to the channel walls due to the high hydrophobicity of the PDMS surface along with the evaporation of water through the channels can cause a change in fluid concentration and composition. Several strategies, such as chemical surface modification or the use of continuous flows can be approached to overcome these limitations. With respect to thermoplastics, due to their wide use in the industry, their thermoforming processing is very well established [35]. In this case, thousands of structures can be produced at a high rate and lowcost using metal or silicon molds and high temperatures. However, the manufacturing of this type of mold is time consuming and expensive and is therefore not widely used in research and prototyping [35]. Typical approaches to seal microchannels include high temperature bonding and glue [38]. Thermoplastics have the ability to be remodeled several times after reheating. Polymethyl methacrylate (PMMA), polystyrene, polyethylene terephthalate (PET) and polyvinyl polychloride (PVC) are typical thermoplastics used in the manufacturing of microfluidic systems [39]. Although these materials show Chapter 1 – Introduction 8 slightly better solvent compatibility than PDMS, they have poor gas permeability and their rigidity makes them difficult to manufacture as functional units. In turn, although their melting temperatures are high (above 280 °C), perfluorinated polymers such as perfluoroalkoxy alkane (known as Teflon PFA) and polytetrafluoroethylene (known as Teflon PTFE) show good gas permeability, sufficient elasticity to make functional units, they are chemically inert to solvents and chemicals, they show low adsorption of non-specific proteins compared to PDMS and they have good cellular biocompatibility and good optical transparency [40]. Despite the positive characteristics presented above, their expensiveness and complex manufacturing methods are a barrier to their implementation and commercialization of devices. This becomes even more evident for applications that require disposable systems to avoid sample contamination and allow for in situ measurements [41]. As a result of these aspects, it has been a need to implement other materials with simpler, less expensive manufacturing processes that can be used by anyone, anywhere. Paper Cellulose is a linear polymer constituted of D-glucose units linked by β-(1,4) glycosidic bonds [42]. It is a renewable polymer, produced by trees, plants and some non-pathogenic bacteria, and is available at lowcost around the world [42]. It has a number of characteristics that makes it particularly attractive for the development of microfluidic devices, particularly for diagnostics and monitoring of pathologies [43]. Coupled with the advantage of a rapid, low-cost production process, the microstructure of paper itself is a huge benefit. Its porous structure allows not only filtering (depending on pore size, particle separation is possible), but even more importantly, to passively generate flows through the capillarity phenomenon causing the fluid to move without the need for external pumps or pressure [44]. Among the several commercially available papers, one of the most 3 used in this area is Whatman filter paper no.1 [45], having a structure consisting of non-oriented flattened fibers. Currently, other types of substrates developed especially for the microfluidic industry can be found. Millipore's Hi-Flow Plus membranes comprise several substrates with slightly different pore size structures. Two examples are the HF075, which filters out entities with a size of 500 µm or larger, and HP180 which filters out entities 400 µm or larger. In addition to the characteristics presented above, paper has high biocompatibility and stability [44,46]. As a white material, it offers the necessary contrast for colorimetric detection methods. By combining these properties, it is possible to manufacture simple, portable, economic and highly efficient devices that Chapter 1 – Introduction 9 allow different biomedical applications [46]. It is thus easy to comprehend why there are several currently marketed microfluidic systems based on this raw material such as pregnancy tests [47] or for measuring the amount of glucose in blood [48], for detecting diseases such as malaria [49] or human immunodeficiency viruses [50], which are commercially available and implemented in our daily lives. Despite all positive arguments regarding the use of this material for microfluidic systems, it is important to reinforce that these paper substrates serve just as a support material and do not allow any manipulation of the fluid. Besides that, paper has some inherent physical properties that cannot be precisely controlled, such as pore size, porosity, surface area, wettability, permeability and capillary flow rate, parameters that affect the performance of the device regarding fluid flow, color uniformity, and reagent immobilization [51]. Consequently, there is an interest in exploring and introducing new active materials for microfluidic applications. Smart and bio-based polymers A smart or active polymer is one that alters, significantly and selectively, one or more properties when an external stimulus such as a change in pH or temperature or when an electric or magnetic field is applied. Within this group, piezoelectric polymers are particularly interesting. They are capable of generating electrical signals by applying a mechanical force (direct piezoelectric effect) and undergoing mechanical deformation when subjected to an electric field (inverse piezoelectric effect) [52, 53]. The direct piezoelectric effect is typically used in sensor applications, while the inverse piezoelectric effect is used in actuator applications [54]. In conjunction with the active properties of smart polymer-based materials, they can be processed in various forms depending on the manufacturing method. In fact, spheres and fibers can be obtained using electrospinning (Figure 1.1a), while porous films can be obtained by temperature-induced phase separation (TIPS) (Figure 1.1b) [54]. Hence, substrates can be processed with structures and morphologies similar to paper substrates. Chapter 1 – Introduction 10 Figure 1.1. a) Schematic representation of electrospinning technique; b) Schematic representation of the preparation of porous films by TIPS [54]. However, synthetic polymers such as poly(L-lactic acid) (PLLA), polyvinylidene fluoride (PVDF), poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHVB) or poly(D,L-lactide-co-glycolide) (PDLG) or even natural polymers such as silk fibroin show a highly hydrophobic nature, hampering their direct use as substrates for microfluidic applications. To overcome this limitation, approaches such as introducing hydrophilic entities (such as zeolites) into the polymer matrix, hydrolysis or plasma surface treatments may be used [55, 56]. These methodologies have already proven to be effective by increasing the surface energy of the polymers and simultaneously reducing their surface hydrophobicity. Other natural polymers such as chitosan, lignin or collagen could potentially require a less prolonged surface treatment and be presented as alternatives to the materials mentioned above. However it would be necessary to evaluate their mechanical resistance during the printing process and again during the analytical tests, as the majority of the natural polymers tend to have poorer mechanical strength when compared to synthetic options [57]. Nevertheless, some of them could be used as an alternative to paper-based substrates, mimicking their excellent printability and capillarity characteristics, but also allowing to obtain substrates with a wide range of tailorable morphologies, capillary flow rates, biodegradabilities and mechanical resistances, taking also the advantage of the active properties which, at a later stage, could be used to improve the performance of the assays. As referred previously, this work report on the development of alternative/complementary materials substrates to the conventional µPADs. For instance, given the hydrophilic nature of these substrates, hydrophobic boundaries with specific patterns are often required to restrict the fluid flow of the samples and reagents to specific pathway of the hydrophilic substrates. To address these goals, different strategies have been used such as photolithography, wax printing, inkjet printing and screen-printing. Folding several layers of patterned paper, origami inspired, has also been employed to obtain three-dimensional (3D) systems. Each of those approaches has its own benefits and limitations, so the choice is often based on Chapter 1 – Introduction 11 cost, fabrication time, equipment availability and also specific application demands. In this way, there will be described the most common printing techniques used to fabricate portable analytical devices. 1.4. Portable analytical devices applications PADs have been applied in many areas such as medicine, environmental monitoring, biology and pharmaceuticals [36]. Some of the most attractive features of PADs arise from their ability to provide lowcost, robust, portable and user-friendly platforms for (bio)analytical assays. These devices can be produced on a large scale and be easily distributed around the world without the need for complex infrastructures or specialized technicians. These devices have versatile functionalities, which can be applied in fields as varied as biomedical, food and environmental quality control (Figure 1.2). Thus, in the next sections PADs developed for these purposes are presented. Figure 1.2. Schematic representation of different PADs areas of application. Although the potential of PADs and the µPADs to be implemented in different applications has been recognized for a long time, it was more recently (when they were introduced as real alternatives to generate low cost and disposable biochemical sensing devices for multiple applications), that they have garnered more attention. Table 1.1 summarizes some relevant work carried out in different areas and their role on health diagnosis, environmental or food and beverage control. Chapter 1 – Introduction 12 Table 1.1. Some relevant and representative works on the use of microfluidic technology in different areas. Health diagnosis Analysis of biomolecules such as proteins, hormones, neurotransmitters and cells [58-60] Analysis of small molecules and metabolites [48, 61, 62] Nucleic acid analysis [63, 64] Virus and infectious diseases [65-67] Cancer biomarker detection [47, 68, 69] Blood typing and blood filtering [70, 71] Drug sensing [72, 73] Environmental diagnostics Water, soil, air analysis, and heavy metal detection [74-76] Food and Beverage Control Pesticide, foodborne detection, vegetables and wine quality analysis [77, 78] Research in medical diagnostic tools is gathering significant amount of efforts on PADs due to their critical importance and strong commercial possibilities. For instance, the commercialization of paper-based pregnancy tests, which represents one of the first lateral flow assays, was considered as one of the major breakthrough in this field [79-81]. A metabolite is any molecule involved in metabolic pathways of an organism and can be used for diagnosing diseases or monitoring homeostasis. Taking lactate as an example, its detection may be a form of diagnosing intra-abdominal sepsis. This has been achieved by fabricating screen-printed carbon microband electrodes from water-based ink containing lactate oxidase that are able to measure quantities of this metabolite [82]. Other lactate biosensors have also been developed with colorimetric approaches [83, 84]. Glucose detection in human serum has been achieved by the fabrication of an electrochemical µPADs with zinc oxide (ZnO) nanowires. Wax printing was used to implement the hydrophobic barriers Chapter 1 – Introduction 13 that delimit the reaction zones in paper, and electrodes patterned by stencil printing with conductive ink were drawn on top of these areas. Another paper layer that contains the carbon ink and the silver nanowires, where glucose oxidase is immobilized to enable the reaction that leads to the electric signal, is stacked, thus forming the complete device [85]. Similar strategy is common for glucose detection [86] or other metabolites such as creatinine, a chemical waste molecule resultant from muscle metabolism that can be found in the bloodstream, and is an indicator of good kidney function [87]. A very similar device using the same methodology has been developed for detection of biotin [88]. Another system was developed in the field of genetic science. The system allows to evaluate abnormalities in nucleotides present in a deoxyribonucleic acid (DNA) chain. In addition, this type of system has been shown to be capable of performing various laboratory functions, including DNA strand replication and strand separation simultaneously [59]. Cancer biomarker detection in PADs has also been developed. SU-8 photoresist was impregnated [89] into paper and cured, with electrodes added by screen-printing with carbon and silver (Ag) ink. Meticulously organized in layers and chambers, this device combined signal amplification strategies for the quantitative analysis of four different biomarkers. PADs approaches for cell analysis have also received attention especially in biomedical research and clinical practice. A device capable of quantifying male fertility potential by evaluating live and motile sperm concentration and sperm motility was fabricated by wax-printing a well in two different layers on top of a laminate layer (figure 1.3) [90]. In the wells, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was carried, generating a colorimetric signal; for the motility assay, another well contained a viscous buffer where the sperm must navigate in order to reach the reagent, generating a colorimetric signal [90]. Chapter 1 – Introduction 20 Inkjet printing Computer software and inkjet printer Different inks (Water based, Solvent based, UV or Hot melt) High resolution; requires only a desktop printer to produce devices and print sensing reagents Requires modified inkjet printers; different compositions for each printing; usually requires an extra heating step after deposition [122, 123] Inks and bioinks are one of the most important elements for the inkjet printing process, based on the limitation in ink characteristics in terms of viscosity and maximum particle size, among others, imposed by this technique. There are several examples of different inkjet inks used for printing portable analytical systems based on silver [127], gold [128], carbon nanotubes [129, 130], paper-sizing agent alkyl ketene dimer [131], poly-L-arginine ink [132], glycerol and Triton X-100 [132] or polyaniline (PANI) [133], among others. For instance, using inkjet printing, Maattanen et al. printed electrodes to work as glucose biosensors using Ag and Au inks. A commercial inkjet printer was used to pattern the electrodes on the substrate. Then, silver chloride (AgCl) was deposited over the silver lines to build the Ag/AgCl reference electrode. Finally, to obtain the glucose biosensor, the surface was modified with glucose oxidase [127]. Among the different techniques used to pattern hydrophilic/hydrophobic contrasts on microfluidic substrates, wax-based fabrication techniques are the most complete, as they are low-cost methods based on selective hydrophilization using non-toxic patterning reagents. Wax-based printing technologies feature several advantages, since the wax is inexpensive and usually readily available, as well as environmentally friendly. Moreover, printing can be accomplished without the use of a clean room, UV lamp, organic solvents, or sophisticated instrumentation. Another advantage over the previously mentioned methods is that it requires only a hot plate (or similar heated surface) making it ideal for the fabrication mainly for µPADs but also for PADs in developing countries. Wax printing was introduced by Lu et al. [134] in 2009. Despite being a three-step method encompassing paper design, printing and heating, it is suitable for mass production, once wax printers, are commercially available and relatively inexpensive [135]. The print head dispenses melted solid ink in the form of liquid droplets on a substrate, where they cool and solidify almost instantaneously. Common commercial solid inks used in this process are constituted of a mixture of hydrocarbons, carbamates and different dyes [10, 136]. Through this process, it is possible to obtain printing patterns with a resolution of up to 2400 x 2400 dpi before curing. However, to create Chapter 1 – Introduction 21 proper hydrophobic wax barriers, a second heating step is needed to ensure a correct impregnation of the wax into the substrate cross-section [43]. During this step, lateral spreading of the melted wax may occur depending on the substrate used, reducing the resolution of the printed pattern, which results in wider barriers [45, 137]. This behavior can lead to narrower channels than designed and thus to variations in the desired capillary flow rate. Therefore, depending on the intended application, this behavior must be considered when developing systems based on this technology. Nevertheless, wax printing features several positive characteristics, as it does not require specialized facilities, the process is fast (5-10 min), inexpensive (each wax cartridge can print a large number of devices) and environmentally friendly (no use of organic solvents throughout the fabrication process). Concerning the materials used as substrates, cellulose-based filter paper is presented as the gold standard [138]. Several applications have been developed in areas, such as energy storage [139], medical diagnostic [140], and environmental sensors [141], among others. The schematic representation of the wax printing process is presented in Figure 1.6. Figure 1.6. Schematic representation of the wax printing process. However, as previously referred, wax printing uses exclusively commercial waxes that work as passive barriers to create hydrophilic/hydrophobic inert contrasts throughout the microfluidic substrate, i.e. without any action on the fluids [44]. The possibility of adding functional properties to these waxes is an exciting possibility combining commercial wax with the dispersion of different fillers such as iron-oxide magnetic nanoparticles, silver nanowires, graphene or barium titanate (BT). In fact, the conjugation of new active properties could lead to different microfluidic applications such as the detection of virus, cells infected with malaria [142] as well as bacteria detection [143], all responsive to magnetic and/or electric fields. Moreover, these active hydrophobic waxes can allow, through the printing of conductive or semi-conductive microchannels, to obtain temperature control and thus improve the hydrolysis reactions of DNA and ribonucleic acid (RNA) Chapter 1 – Introduction 22 directly in the PADs and μPADs. Other potential applications include the selective separation of pollutants according to their magnetic and/or electrical properties [144]. 1.6. Objectives and methodology The main objective of this thesis is to develop advanced materials for PADs and μPADs applications, addressing current shortcomings and aiming to reach real-world applications. This will be achieved by the development and validation of a new generation of advanced polymer-based substrates and new functional waxes. This main objective will be achieved by applying the following methodology: • Development of novel microfluidic substrates based on synthetic and bio-based polymers, including piezoelectric ones. Different morphologies and/or the introduction of fillers in the polymer matrices will be also tested to increase the potential of the new microfluidic substrates when compared to the commonly used passive paper substrates. The viability and performance of the developed substrates will also be assessed to combine both functional channels and smart substrates; • Development of hydrophobic wax-based composites with functional properties, including magnetic, conductive and dielectric waxes using specific nanofillers that can be used to define functional channels within the substrate by using printing technologies. These composites will allow active manipulation of fluids and/or compounds that flow through the substrate; • Evaluation and validation of the developed materials in magnetic and heating systems as proofof-concept applications. The ultimate purpose is to allow: i) precise control of morphology, hydrophilicity and sample capillary flow rate on the device in terms of direction and rate; Ii) higher storage stability and controllable degradation; iii) superior wet strength to increase the performance of PADs and their range of possible applications that require multiple and specific steps assays. Chapter 1 – Introduction 23 1.7. Structure of the thesis The present thesis is divided into five chapters, three of them based on published or submitted scientific manuscripts. The sequence of these chapters provides a comprehensive and logical account of the progress achieved during the present research as well as further clarifies the used methodology. A summary of the work covered in each chapter is briefly described below. Chapter 1 presents the motivation of the thesis with a general introduction to the work. The main objectives and the structure of the thesis are also presented. The state of the art on specific relevant issues for the development of the work is provided in each of the different chapters. To better understand the importance of developing new alternatives to the existent PADs and μPADs, Chapter 2 is divided into four sub-chapters based on the four published works about the application of different polymer-based substrates as new platforms for as an alternative to existing devices. In the first subchapter, Chapter 2.1, microfluidic substrates based on electrospun PLLA membranes were fabricated and the influence of fibre orientation, addition of hydrophilic additives and plasma treatment on their morphology, physicochemical properties and capillary flow rate were evaluated and compared with paperbased Whatman substrates. In Chapter 2.2, polyvinylidene fluoride-co-trifluoroethylene (P(VDF-TrFE)) membranes with tailored morphological and physicochemical properties (electrospun oriented and randomly-oriented fibers, porous or spherulitic membranes) were processed, printed using wax-printing and evaluated as new platforms for colorimetric quantification of glucose. Chapter 2.3 introduces biodegradable membranes based on poly-DL-lactide-glycolide (PDLG) that were evaluated as an alternative to conventional paper substrates, revealing advantages in terms of controlled degradation and high colorimetric precision, proving suitability to a new generation of degradable PADs. Another polymer substrate is presented in Chapter 2.4, where multi-structural silk-based substrates were used as low-cost, environmentally friendly substrates for portable analytical systems by using Bombyx mori cocoons. Further, silk fibroin was extracted from these cocoons and electrospun into oriented and randomlyoriented fiber substrates. The developed materials were then used as substrates for the colorimetric quantification of three different clinical analytes. All four works are interconnected in the pursuit of developing a new generation of devices using polymers from different origins and functional characteristics, which bring unique advantages and characteristics for a next generation PADs and μPADs testing. Chapter 3 is based on the development and functionalization of different hydrophobic inks based on wax. As in the previous chapter, it is divided into four different sub-chapters. Chapter 3.1 describes the development of a process for obtaining magnetic waxes with iron oxide (Fe3O4) NPs, and then printing Chapter 1 – Introduction 24 them on cellulose substrates using wax printing. In Chapter 3.2, the development of magnetic waxes is optimized, and a self-powered printed sensing device is presented, the piezoelectric/magnetic effect is explored via magneto-mechano-electric energy conversion. The dielectric properties of the wax reinforced with high-dielectric ceramic nanofillers such as barium titanate (BT) were studied in Chapter 3.3, and a functional hydrophobic ink suitable for paper-based microfluidics was developed, able to be combined with different ceramic materials to adapt its dielectric constant, an essential aspect in applications in printed electronics. Conductive hydrophobic inks, which can represent a critical step in the commercial expansion of PADs, are approached in Chapter 3.4, where carbon nanotube (CNT) and reduced graphene oxide (rGO)-filled wax composites were developed and proven to be suitable to be applied and printed for different PoC applications. Chapter 4 integrates several of the developed technologies on new PADs platforms, evaluated in proof-ofconcept applications. Chapter 4.1 describe the development of a fully functional magnetic POC printed device composed of two neodymium iron boron (NdFeB)/wax magnets capable of attracting and detecting magnetic nanoparticles with similar characteristics and concentrations compared to hemozoin present on malaria-infected red blood cells. Different analysis methods as energy-dispersive X-ray spectroscopy (EDX), vibrating sample magnetometry (VSM), Fourier-transformed infrared spectroscopy (FTIR-ATR); and mean grey scale evaluation on photographs taken by mobile phones, were presented as complementary analytical tools. Chapter 4.2 shows the development of multifunctional hydrophobic composites based on conductive graphene nanoplatelets (GNP) integrated into the wax matrix to allow a dual role of barrier and heater, relevant for a variety of temperature-sensitive reactions and applications in microfluidic technology. Finally, Chapter 5 compiles the main conclusions of the work presented in this thesis and proposes some suggestions for future work and possible research directions. 1.8. References 1. Nations, U., Transforming our World: The 2030 Agenda for Sustainable Development. 2015. 2. Gong, M.M. and Sinton, D., Turning the Page: Advancing Paper-Based Microfluidics for Broad Diagnostic Application. Chemical Reviews, 2017. 117(12): 8447-8480. 3. Kankaanpää, M., et al., Use of point-of-care testing and early assessment model reduces length of stay for ambulatory patients in an emergency department. Scandinavian journal of trauma, resuscitation and emergency medicine, 2016. 24(1): 1-7. Chapter 1 – Introduction 25 4. Drain, P.K., et al., Diagnostic point-of-care tests in resource-limited settings. The Lancet infectious diseases, 2014. 14(3): 239-249. 5. Kosack, C.S., Page, A.-L. and Klatser, P.R., A guide to aid the selection of diagnostic tests. 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Analyst, 2016. 141(5): 1837-1846. 36 Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems Novel microfluidic substrates based on electrospun poly(l-lactic acid) (PLLA) membranes were developed to increase the limited range of commercially available paper substrates, commonly used for the fabrication of microfluidic paper-based analytical devices (µPADs). PLLA advantageous properties include being biodegradable, biocompatible, easily processed in various tailored morphologies, and cost effective, among others. Oriented and non-oriented electrospun PLLA membranes were fabricated using electrospinning and the influence of fibre orientation, addition of hydrophilic additives and plasma treatments on the morphology, physicochemical properties and capillary flow rate were evaluated and compared with commercial Whatman paper. In addition, a proof-of-concept application based on the colorimetric detection of glucose in printed PLLA and paper-based microfluidic systems was also performed. This chapter is based on the following publication:, Ricardo Brito-Pereira, Eduardo S. Pimentel, Teresa Marques-Almeida, Clarisse Ribeiro, Filipe Vaz, Senentxu Lanceros-Mendez, Vanessa F. Cardoso, Tailoring electrospun poly(l-lactic acid) nanofibers as substrates for microfluidic applications, ACS applied materials & interfaces 12 (1), 60-69 (2019). Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 37 Introduction Microfluidic paper-based analytical devices (µPADs) represent a promising platform for the development of fast, portable, disposable and cost-effective analytical tools in clinical diagnostic, environmental monitoring and food safety, among others [1]. This motivation is supported by the potential of µPADs to address the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) criteria for the development of portable analytical devices (PADs) defined by the World Health Organization (WHO), enabling local communities in developing regions to improve healthcare, environmental safety, animal health and food quality [2]. They were first introduced in 2007 as alternative to silicon, glass and PDMS materials, commonly used for prototyping and proof-of-concept studies [3]. Paper is typically made of cellulose fibres and shows the advantages of being inexpensive, ubiquitous, biocompatible and scalable [4]. In addition, cellulose’s hydrophilicity, combined with patterned hydrophobic microfluidic barriers to create channels, generate sample flow via capillary action from an inlet to a defined reaction place for subsequent analysis [5]. Although with extensive academic developments, the entry of µPADs into real-life applications is still very limited [6]. This discrepancy may be related to the limited grade and properties of commercially available papers with low storage stability, poor wet strength, and limited control of fluid flow [7]. In this sense, various strategies have been developed for programmable and analyte manipulation (e.g. filtering, mixing, heating, separation, concentration) by the integration of functional components into µPADs, including diode valves, dissolvable sugar barriers and external electromagnets [8]. Moreover, there have been attempts to control the flow by tailoring the channels design, guiding the fluid to transport vertically and laterally through paper sheets and using materials such as threads and yarns [9]. However, the reported strategies have limitation for rapid and easy production. Thus, as an attempt to address the limitations with the existing µPADs, alternative microfluidic substrates to complement paper have been developed in the present work. PLLA, a aliphatic semi-crystalline polyester, constitutes a promising candidate for this purpose. In fact, PLLA, which is a Food and Drug Administration (FDA) approved biomaterial, has attracted wide interests, from academic research to industry, due to its biocompatibility and biodegradability, as well as environmental friendliness and lowcost [10]. In addition, PLLA is also piezoelectric, allowing to convert mechanical energy into electrical and vice versa, which has been successfully used in tissue engineering and related biomedical applications [11]. A number of methods have been reported for the production of different types of PLLA structures such as films [12], membranes [13], fibres [14] and spheres [15], among others. Fibrous membranes are Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 38 particularly interesting for microfluidic applications based on the similar morphology with commercial Whatman cellulose filter paper grades, commonly used for the manufacture of µPADs [16]. They have been produced by a variety of methods such as template synthesis, self-assembly, phase separation, wet spinning, microfluidic spinning and electrospinning [17]. Within these techniques, electrospinning is currently the only one that allows the simple and reproducible fabrication of functional fibres to be applied in microfluidics [18] with diameters from few nanometres to several micrometres by applying a high electrical field to a droplet of polymer or composite solution from natural or synthetic polymers [18,19]. By properly controlling the composition of the polymer-based solution and processing parameters, a stable process can be achieved allowing to obtain structures ranging from single fibre to ordered arrangement of fibres with tailored fibre dimension and orientation [20]. The major drawback of PLLAbased electrospun membranes is associated to its inherent hydrophobicity, poor wettability and low surface energy that must be overcome so that it can be used as substrate, similar to paper, for the fabrication of microfluidic systems. Various strategies can be used to modify the surface properties and thus tailor the polymer wettability, such as chemical grafting, self-assembly and surface hydrolysis [21]. Another reported strategy involves the integration of specific fillers (such as polyethylene glycol [22], hydrophilic zeolites [23], superhydrophilic metal-organic frameworks [24], wet-chemistry treatment [25], among others) on the polymer matrix in order to reduce the hydrophobic behaviour of polymers [26]. Nevertheless, plasma treatment is one of the most extensively used methods to tailor surface adhesion and wetting properties, by the insertion of chemically reactive functional groups on the polymer surface, modifying its composition without affecting their bulk characteristics. A proper selection of plasma atmosphere (oxygen O2, argon Ar, hydrogen H2, or nitrogen N2, among others), power and time is the key to ensure the success of the plasma treatment [27]. This approach has been used to promote surface modification in PLLA [28]. Thus, the surface modification of melt-extruded sheets of PLLA under O2, H2 and N2 plasmas has been investigated, and the obtained results demonstrated a significant increase of the surface wetting, by the incorporation of polar groups composed of carboxylic (–COOH) and hydroxyl (–OH), along with a pronounced modification of its morphology, which depends much on the type of plasma [29]. Poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA) and PLLA have been processed by electrospinning to obtain nanofibrous membranes, followed by a post-treatment by O2 plasma and in situ grafting of hydrophilic acrylic acid (AA), which results in higher ratios of oxygen to carbon, lower contact angles and the presence of –COOH groups [30]. The influence of Ar-plasma treatments on flat rigid PLLA substrates, obtained by melting of PLLA powder on glass slide [31], shows that different times of this type of plasma treatment allows to achieve controlled water contact angles, down to the Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 39 superhydrophilic regime. Similarly, the suitability of carbon dioxide (CO2) plasma treatment has been proved to decrease the hydrophobicity of PLLA surface, by the introduction of O2-containing functional groups [32]. The hydrophobicity of PLLA has been also reduced by helium (He) atmospheric pressure plasma treatment, Ar and O2 plasma treatments [33]. It is to notice that most of the aforementioned studies have been tested and applied, mostly on tissue engineering applications, in order to tailor cellbiomaterial interfaces. On the other hand, only few studies reports on the stability of plasma treatment over time. To the best of our knowledge, there is no work reporting on the processing of PLLA-based materials with stable superhydrophilic behavior, to be used as substrate, instead of paper, for the fabrication of microfluidic systems. In this work, the effect of hydrophilic additives (NaY zeolites) and plasma treatments (using O2 and Ar atmospheres) on the surface wettability and others relevant physicochemical properties of electrospun PLLA-based membranes are investigated. Randomly oriented electrospun PLLA-based membranes were first produced in an attempt to mimic the structure of commercial Whatman papers, commonly used in the fabrication of µPADs. Oriented electrospun PLLA membranes were also manufactured to study the effect of fibres orientation. Thus, microfluidic substrates based on biodegradable and biocompatible PLLA material with superhydrophilic behaviour, high storage stability, high wet strength and controlled fluid flow are obtained. Experimental procedures Materials PLLA with an average molecular weight of 217.000-225.000 g.mol-1 ( Purasorb PL18 ) and NaY zeolites (CBV 100: SiO2/Al2O3 mole ratio: 2.83; nominal cation form: sodium; Na2O weight: 13 %; unit cell size: 24.65 Å; surface area: 900 m2.g-1) were supplied by Purac and Zeolyste International, respectively. N,Ndimethylformamide (DMF) and dichloromethane (DCM) were obtained from Merck and Sigma-Aldrich, respectively. Whatman filter paper, was purchased from Sigma-Aldrich. All chemicals and solvents were used as received. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 40 Sample preparation Randomly oriented electrospun substrates Randomly oriented electrospun PLLA-based membranes were produced in an attempt to mimic the structure of commercial Whatman papers, commonly used in the fabrication of µPADs, whose microstructure consist of flat randomly oriented fibres. A 10 wt.% solution of PLLA in 3/7 vol/vol DMF/DMC mixture was prepared under magnetic stirring (IKA C-MAG HS7) at room temperature, until complete dissolution of the polymer. In the preparation of polymer solutions with zeolites, NaY was previously dispersed in the solvents under ultrasound bath for 1 h, followed by the addition and dissolution of PLLA by magnetic stirring. NaY percentages of 0, 5, 10 and 20 % relative to the polymer mass were studied. Then, the polymer solution was placed in a 10 mL plastic syringe fitted with a steel needle with an inner diameter of 500 µm and introduced into a syringe pump (Syringepump). The electrospinning procedure was conducted at 17 kV by means of a high voltage power supply (Glassman PS/FC30P04) at a solution feed rate of 0.5 mL.h-1. Finally, randomly oriented electrospun membranes were collected on a grounded static plate collector with (20 cm x 15 cm) placed 15 cm away from the needle. Oriented electrospun substrates In addition, oriented electrospun PLLA-based membranes were processed to increase the range of microfluidic substrate structures, as well as to study the influence of fibre orientation on the capillary flow rate. The sample preparation is similar to the one described in section 2.2.1, except for the use of a rotating collector. Thus, oriented PLLA-based fibres were produced using a grounded rotating drum collector at a velocity of 1500 rpm. In this case, the addition of NaY zeolites was also performed. Surface modification As previously exposed, a limitation of PLLA comparatively to cellulose-based microfluidic substrates arises from their high hydrophobicity. Plasma treatments were performed, which is a technique commonly used to alter the hydrophobicity of polymers [31]. The success of a plasma treatment is associated with the selection of the plasma atmosphere (O2, Ar, N2, among others), treatment time and applied power. Oxygen is one of the most reactive elements and can generate carboxyl groups on the surface of polymers due to the incorporation of hydrophilic functional groups. Argon, in turn, tends to lead to relevant changes in surface morphology [21,27]. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 41 Surface treatments were conducted in a plasma chamber (Zepto, Diener Electronics), equipped with a 40 kHz radio frequency plasma generator. The base pressure, before plasma ignition, was 20 Pa. Plasma treatments were performed independently with O2 and Ar as working gases, for 10 min. A plasma power of 100 W under a total pressure of 80 Pa was applied. This procedure was performed on both surfaces of the processed PLLA-based substrates. Samples characterization Physicochemical characterization A scanning electron microscope (SEM) Quanta 650 FEG from FEI was employed to characterize the morphology of the electrospun PLLA-based membranes. The samples were previously coated with a thin gold layer using a sputter coater Polaron SC502. The mean fibre diameter and corresponding standard deviation were calculated measuring approximately 50 fibres by means of ImageJ software. Surface wettability of the samples was evaluated by measuring the contact angle (CA) of 3 µL ultrapure water drop using a CA analyser Data-Physics OCA20. Six measurements were carried out in each membrane at different locations, the CA being reported as the average and standard deviation. Fourier transformed infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) measurements were performed at room temperature using a Spectrum Two™ from Perkin-Elmer, with 64 scans in the range between 400 and 4000 cm−1 and a resolution of 4 cm−1. Differential scanning calorimetry (DSC) was performed with a DSC 6000 from Perkin-Elmer. Pieces of approximately 6 mg were cut and placed into 40 µL aluminium pans. The samples were heated between 30 and 200 °C at a scanning rate of 10 C.min-1. The degree of crystallinity (∆𝑋𝐶) was calculated using the following equation ∆XC=(∆𝐻 ∆𝐻𝑚 0 ⁄ )×100, where ∆𝐻 is the area under the thermogram between 65 and 160 °C and ∆𝐻𝑚 0 is the melting enthalpy for fully crystallized PLLA samples (93.1 J.g-1) [35]. The mechanical properties were studied by means of a Shimadzu AD-IS with a load cell of 50 N. Samples with length and width of 15 and 10 mm, respectively, were prepared and stretched at a rate of 1 mm.min1. The oriented fibers were evaluated along the fiber direction. The thickness of the samples ranged between ~30 and ~100 µm. The essays were performed on dry and wet samples using in this last case 40 µL of water. Three tests were performed for each sample. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 42 Cytotoxicity assay Indirect cytotoxicity evaluation of the processed electrospun PLLA-based membranes was performed adapting the ISO 10993-5 standard test method. MC3T3-E1 pre-osteoblast cells (Riken cell bank, Japan) were cultured in 75 cm2 cell culture flask at 37 °C, 5 % CO2, in humidified environment using Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 1 g.L-1 glucose, 10 % fetal bovine serum (FBS, Biochrom) and 1 % (v/v) penicillin/streptomycin solution (P/S, Biochrom). Sterilization of the samples (13 mm diameter) were carried out by multiple immersion into 70 % ethanol for 30 min each, washing with sterile phosphate-buffered saline solution (PBS; 137 mM NaCl; 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 at pH 7.4) for 5 min and exposition to ultraviolet radiation for 1 h each side. Thus, a suspension of 2 × 104 cell.mL-1 was seeded in 96-well tissue culture polystyrene plates and incubated for 24 h at 37 °C with 5 % CO2 in humidified environment to ensure cell attachment on the plate. Simultaneously, each sample was incubated for 24 h in 24-well tissue culture polystyrene plate under the conditions described above. After the incubation time, the cell culture medium in the 96-well plates was removed and 100 µL of culture medium that was in contact with the different samples was added to each well, individually. Cell viability was then evaluated after 72 h of incubation using the MTT proliferation assay according to the manufacturer’s instructions (Sigma-Aldrich). Briefly, the medium of every well was removed and fresh medium containing MTT solution (5 mg.mL-1 of MTT dissolved in DMEM in a 1:10 ratio) was added to the cells and incubated for 2 h at 37 °C in the dark. Thus, the MTT solution was removed and the precipitated formazan was dissolved with 100 µL dimethyl sulfoxide (DMSO)/well followed by measuring the optical density at 570 nm. Cells cultured in 20 % DMSO (Sigma-Aldrich) and standard culture medium were used as negative and positive controls, respectively. All quantitative results were obtained from four replicate samples and controls and presented as the average of viability ± standard deviation. The percentage of cell viability was calculated according to the following equation 2.1.1 [36]. Cell viability (%)=Absorbance of sample Absorbance of negative control ×100 (2.1.1) Capillary flow rate tests Capillary flow rate represents the speed at which a liquid sample moves along a membrane strip in a diagnostic test strip or application. A proper control of this flow will enable to select the appropriate membrane for a specific application [37]. Thus, capillary tests were performed on all samples without Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 43 and with plasma treatments. Strips with dimensions of 2.5 cm length and 1 cm width were cut and placed vertically in contact with a water solution containing food colouring. The sample portion immersed in the solution was 0.5 cm and the time taken for the dye to travel across the sample in the “antigravity” direction was measured. These tests allow to evaluate the effect of the fibre orientation, presence of zeolites and plasma treatments with Ar and O2, in the capillary flow rate and ultimately on the ability of the processed materials to generate passive flows. Proof-of-concept Glucose essays based on colorimetric detection were performed using a glucose kit from BioLabo ReagentsTM. During the reaction, glucose is oxidized by the enzyme glucose oxidase to gluconic and hydrogen peroxide, which in conjunction with peroxidase, reacts with chloro-4-phenol and 4-aminoantipyrine to form a red quinoneimine. The intensity of the coloured complex is proportional to the concentration of glucose. As a simple proof-of-concept, calibration curves were built using the following glucose concentrations: 10, 25, 75, 150 and 500 mg.dL-1, for both commercial Whatman paper and nonoriented electrospun PLLA membranes with 10 % NaY (as representative example). A microfluidic system was designed using a computer aided design software and printed on both substrates using a Xerox ColorQube 8580 printer, as illustrated in Figure 2.1.1. After printing, the samples were placed on a heating plate at 90 C during 10 min so that the wax penetrates the entire cross-section of the membrane and thus creating hydrophobic barriers that will contain the fluid. This temperature is slightly lower than the commonly used in paper (100 C for 2 min) to maintain the integrity of the PLLA samples that undergo mechanical deformations when subjected to higher temperatures. Figure 2.1.1. Microfluidic system design used for the quantification of glucose. Each reaction chambers and reference were first functionalized with reagent. The glucose oxidase flow by capillarity from the inlet to the reaction chamber to form a red colour with intensity proportional to the concentration of glucose. The reaction chamber features an area of ~80 mm2 and the channels a width of 1.2 mm. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 44 Each microfluidic system comprises six reaction chambers for the reproducibility of the study. A total of five microfluidic systems corresponding to the five studied glucose concentrations were printed in each substrate. The reagent chambers were first functionalized using 15 µL of reagent. After approximately 5 min, the inlet of the microfluidic system was introduced into the glucose solution under study, reaching the reaction chamber by capillarity. After 10 min of reaction, a red colour with intensity proportional to the concentration of the glucose was formed. Image analysis was performed by using ImageJ software in each microfluidic system. For each reaction chamber, the grey value and corresponding standard deviation were measured and the calibration curve was built. Experimental Results and Discussion Microfluidic substrates based on electrospun PLLA-based membranes were processed as an alternative to paper filters, such as the Whatman cellulose filter paper grades, commonly used for the fabrication of µPADs [16]. The effect of PLLA fibre orientation, introduction of NaY zeolites in the polymer matrix and plasma treatments with Ar and O2 on the morphology, physicochemical properties and capillary flow rate were properly studied and compared with commercial Whatman. Physicochemical characterization Representative SEM images of commercial Whatman and non-oriented and oriented electrospun PLLAbased membranes are shown in Figure 2.1.2. The respective mean fibre diameter and standard deviation are presented in Figure 2.1.3. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 45 Figure 2.1.2. Representative SEM images of a) commercial Whatman; non-oriented PLLA fibres with NaY filler content of b) 0 %, c) 5 %, d) 10 %, e) 20 %, f) 0 % and Ar plasma treatment, g) 20 % and Ar plasma treatment, h) 0 % and O2 plasma treatment, i) 20 % and O2 plasma treatment; oriented PLLA fibres with NaY filler content of j) 0 %, k) 20 %, l) 0 % and Ar plasma treatment, m) 0 % and O2 plasma treatment. Whatman membranes consist of cellulose fibres with a flat microstructure associated with the pressing process generally employed in the manufacture of paper (Figure 2.1.2.). The fibres show sizes in the micrometre scale, with average width of ~16±7 µm. In turn, untreated electrospun PLLA-based membranes are characterized by finer and rounded fibres with smooth surface, spatially distributed both longitudinally and transversally (Figures 2.1.2b-e and Figures 2.1.2k). The dimension, orientation and distribution of the fibres are not affected by the introduction of zeolites, whose presence is confirmed by some clusters in the corresponding SEM images. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 52 Capillary flow rate tests Capillary flow tests were performed in order to validate the possible use of the processed PLLA substrates in the development of microfluidic systems, where passive flows must be generated. As previously stated, thin strips of each sample were cut and dipped vertically and partially into a water solution containing food colouring. The results presented in Figure 2.1.8 were obtained calculating the time required for the dye to travel through the strip with a length of 2 cm (plus 0.5 cm dipped in solution). Figure 2.1.8. Capillary flow rates in the “antigravity” direction of dye solution in commercial Whatman and electrospun PLLA-based membranes strips. The results demonstrate that the hydrophobic nature of the PLLA membranes, without plasma treatments, has a negative effect on the capillary flow rate, presenting no flow, regardless of zeolite concentration. In turn, because of its superhydrophilic behaviour, the plasma treated PLLA samples show capillary flow rates varying from 7.1±0.9 to 70.2±1.9 mm.min-1, being the effect superior in samples treated with O2 plasma. An interesting observation comes from the treated samples with zeolites, with capillary flow rates that tend to decrease with the presence of this additive, as demonstrated by comparing the samples with 0 % and 5 % filler content. Further, similar flow rates are observed for zeolite concentrations above 5 %, indicating that a saturation is reached with respect to the effect of the zeolite content in the flow rate. This result is justified by the high adsorption and the microporous structure of Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 53 NaY zeolites that will retain water molecules, leading to a consequent decrease in flow rate. Thus, although the presence of zeolites does not have the ability to change the surface hydrophobicity of the PLLA membranes by themselves, they demonstrate to have a relevant effect on the passively generated flow rate after plasma exposure. Another important conclusion is the capillary flow rate that increases significantly with the orientation of the fibres. Maximum values of 50±2.4 mm.min-1 and 70.2±1.9 mm.min-1 were obtained for the oriented PLLA fibres without zeolites and treated with Ar and O2 plasma, respectively, comparatively to 21.5±0.8 mm.min-1 and 24.7±1.0 mm.min-1 obtained for the non-oriented PLLA fibres also without zeolites and treated with Ar and O2 plasma, respectively. The higher fibre density and preferential orientation in the oriented PLLA samples compared to the non-oriented, as previously exposed in 2.1.2, can justify this result by allowing the dye to flow more easily, and consequently faster along the fibres of the PLLA strip. The lowest capillary flow rates were observed in the PLLA membranes constituted by non-oriented fibres with 20 % of zeolites and treated with Ar and O2 plasma, with values of 7.1±0.9 mm.min-1 and 22.4±0.8 mm.min-1, respectively. In comparison, commercial Whatman features a capillary flow rate of 44±1.13 mm.min-1. These results prove the ability of the processed PLLA substrates to generate passive flows. Controlling properly the processing parameters, namely in terms of PLLA fibres orientation, zeolites concentration and plasma treatment with Ar or O2, it is possible to adjust the capillary flow rate according to a specific application that require controlled flow times and thus controllable reaction times, for example. Proof-of-concept Colorimetric assays based on the detection and quantification of glucose were evaluated on commercial Whatman and non-oriented PLLA membranes with 10 % NaY (as representative example) to demonstrate the viability and potential of the processed substrates for the fabrication of microfluidic systems in specific applications. Digital images of the glucose essays on the printed microfluidic systems are illustrated in Figure 2.1.9 and the corresponding calibration curves are presented in Figure 2.1.10. In this case, mean grey values are presented as a function of the logarithm of the glucose concentration ranging from 10 to 500 mg.dL-1. Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 54 10 mg.dL-1 25 mg.dL-1 75 mg.dL-1 150 mg.dL-1 500 mg.dL-1 Whatman PLLA Figure 2.1.9. Digital images of microfluidic systems after glucose essays on commercial Whatman paper and non-oriented electrospun PLLA membranes with 10 % NaY (as representative example). From left to the right: glucose concentration of 10, 25, 75, 150 and 500 mg.dL-1. Figure 2.1.10. Calibration curves of glucose for a) commercial Whatman and b) non-oriented electrospun PLLA membranes with 10 % NaY (as representative example). The results are presented as mean grey value and corresponding standard deviation measured on the reaction chambers of the microfluidic substrates using ImageJ software according to the method described in [41]. Analysing first the print quality of the microfluidic systems on both substrates, it is concluded that the design loses some resolution in commercial Whatman paper, where the wax expands approximately Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 55 2 mm during the post-thermal treatment. Accordingly, this unavoidable effect must be taken into account when designing the microfluidic system since the width of the channels and, therefore, the dimensions of the entire system, is changed after the printing process, affecting not only the passive capillary flow rate but also the viability of the microfluidic system, depending on the application requirements. In turn, electrospun PLLA-based membranes feature the advantage of maintaining the dimension and definition of the microfluidic design. Regarding the colour of the reaction chambers, it is observed a gradual increase of the red colour intensity with increasing glucose concentration. The calibration curves, which present the correlations between the colour intensity, denoted as mean grey values, and the logarithm of glucose concentrations, demonstrate a good linearity with correlation coefficients R2 of 0.99, in both substrates. It should be noted that the grey values were only analysed on the reaction chambers and the calibration curves built accordingly. Thus, the reddish colour observed on the channels of the microfluidic systems, which comes from colorimetric reaction between the reagent that flows passively outside the reaction chambers during functionalization and glucose, does not interfere with the measurements. In addition, the electrospun PLLA-based membranes demonstrate an improved sensibility with a curve slope of -34.5 comparatively to -16.2 obtained with the commercial Whatman. Thus, these results demonstrate the potential of the processed electrospun PLLA-based membranes as complementary or alternative substrates to the commonly used Whatman papers for the fabrication of microfluidic systems not just for colorimetric detection, as proven in this section, but also for other relevant (bio)technological applications that can take advantages of the demonstrated beneficial physicochemical properties of the processed substrates. Conclusions The present work reports on the processing and evaluation of electrospun PLLA membranes as innovative substrates for the fabrication of microfluidic systems, complementary or alternatives to the µPADs based on paper substrates. The influence of fibre orientation, addition of hydrophilic additives (NaY zeolites) and plasma treatments (Ar and O2) on the morphology, physicochemical properties, biocompatibility and capillary flow rate were properly studied and compared with the commonly used paper substrates based on commercial Whatman. The results demonstrate that controlling properly the processing conditions, it is possible to tailor the morphology, surface hydrophilicity and capillary flow rate of the electrospun PLLAbased membranes, which is beneficial in order to reach specific applications requirements, which can be Chapter 2.1 – Tailoring electrospun poly(l-lactic acid) nanofibers as novel substrates portable analytical systems 56 difficult to obtain with the current commercially available paper substrates. 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ACS Sens. 2018, 3(7), 1416. 2.2. Fluorinated polymer membranes as advanced substrates for portable analytical systems This chapter describes the processing of fluorinated poly(vinylidene-co-trifluorethylene) , P(VDF-TrFE), membranes with tailored morphological and physicochemical properties to be used as microfluidic substrates for portable analytical systems, commonly called portable analytical devices (PADs) in the medical field. The morphology of the developed membranes includes spherulitic, porous, randomly oriented and oriented fibres. Microfluidic systems were then designed and printed by wax-printing for the colorimetric quantification of glucose. This chapter is based on the following publication:, Ricardo Brito-Pereira, AS Macedo, CR Tubio, Senentxu Lanceros-Mendez,Vanessa F. Cardoso, Fluorinated polymer membranes as advanced substrates for portable analytical systems and their proof-of-concept for colorimetric bioassays, ACS Applied Materials & Interfaces 13 (15), 18065-18076 (2021). Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 61 Introduction Portable devices for expedite and easy medical diagnosis are increasingly demanded as a suitable tool to detect various diseases. Those devices allow early diagnosis in situ and can be accessible to a large number of people without the need for complex laboratory equipment. They are particularly valuable for preliminary analysis before further evaluation or to evaluate on-going processes, allowing to improve healthcare assistance [1]. Moreover, they have been also used in other research fields such as environmental safety [2], animal health [3] or food quality [4], among others. In particular, in times of pandemic outbreaks, as the one we are experiencing nowadays, access to lowcost, easy to handle diagnostic methods that can be distributed quickly among the population is becoming essential to promote mass diagnosis and provide high quality control concerning the spread of a disease at an early stage [5]. It is particularly relevant that these tools address the ASSURED guidelines for the development of PADs, as defined by the WHO. Devices with low manufacturing cost, low sample volume and with minimal user manipulation are then best suited to satisfy these guidelines [6,7]. The µPADs, introduced by the Whitesides group in 2007 [8], are a relevant option for the development of such tools. Besides, microfluidic platforms have been fabricated from a wide array of materials such as silicon, glass or polymers that can be tailored into three-dimensional (3D) solid structures, as it is the case of PDMS and PMMA, which possess overall high mechanical strength [9]. Nonetheless, cellulose paper features economical, compact and lightweight properties for the fabrication of µPADs. Moreover, because of its superhydrophilic properties, passive capillary flow discards the need for an external pump [10]. Hydrophobic barriers can be printed to improve liquid flow through the substrate, allowing designs to guide solutions to different locations of the microfluidic platform for specific purposes. Several technologies may be used to implement these barriers, including wax and inkjet printing [9], photolithography [8], printed circuit technology [11] and screen-printing [12]. Nonetheless, commercial microfluidic substrates show disadvantages that include weak mechanical properties so the type of cellulosic paper has to be carefully selected: commercial inkjet printing paper is unsuitable due to its low porosity and surface tension; filter paper such as Whatman cellulose paper (the gold standard on point-of-care applications after its introduction [8] may possess pores that are too large, preventing proper capillary flow [6]. More recently, Hi-Flow Plus nitrocellulose substrates from Millipore have emerged as an interesting alternative for microfluidic applications, allowing controlled capillary flow rate and thus sensitivity, depending on the specific type of membrane. However, it is expensive, must be Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 68 time taken for the dye to travel across the remaining 2 cm of the sample in the antigravitational direction was measured. These tests allow an evaluation of the effect of fibre orientation, film porosity and oxygen plasma treatment on capillary flow rate, and ultimately the evaluation of the ability of the different materials to generate passive flows. Three measurements were carried out in each sample and the average and standard deviation were calculated. Proof-of-concept Glucose assays based on colorimetric detection were performed using a glucose kit (Trinder – Endpoint, FAR Diagnostic). During the reaction, glucose is oxidised by glucose oxidase to gluconic acid and hydrogen peroxide. The latter reacts with phenol and 4-aminophenazone in the presence of peroxidase, producing a coloured complex whose colour intensity is directly proportional to the glucose concentration in the samples. Calibration curves were determined for all samples using glucose concentrations of 25, 50, 75, and 100 mg.dL-1. This range of concentrations includes ranges of reference values for both new-born babies (20 to 80 mg.dL-1) and adults (70 to 110 mg.dL-1). A microfluidic system was designed using SolidWorks 2020 and hydrophobic wax was printed using a Xerox ColorQube 8870 printer, as illustrated in Figure 2.2.2. Wax-Printing, when compared to other methods commonly used to produce µPADs presents several advantages such as not requiring specialized facilities, the process is rapid, inexpensive (each wax cartridge can print a large number of devices) and environmental friendly (no use of organic solvents throughout the fabrication process) [33-35]. After printing, the samples were placed on a hot plate at 100 C for 10 min for the wax to penetrate the substrates all the way through to the opposing surface in order to fabricate the barriers able to properly contain the fluids. Figure 2.2.2. Design of the microfluidic system for the quantification of glucose. The glucose solution was placed in both reaction and reference chambers and the reagent in the inlet. The reagent flow by capillarity Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 69 from the inlet to the reaction chamber to form a red/orange colour with intensity proportional to the concentration of glucose. The reaction chamber features a diameter of 8 mm and the channels a width of 1.2 mm. Each microfluidic system comprises eight reaction chambers, with each glucose concentration being carried out in two reaction chambers separately and at the same time, as indicated in Figure 2.2.2. Three microfluidic systems were printed for each membrane in order to assess the reproducibility of the system. The reaction chambers were first functionalized using 15 µL of the glucose solution under study. After approximately 5 min, the reagent was introduced in the inlet of the microfluidic system, reaching the reaction chambers by capillarity. After 10 min of reaction, a red/orange colour with intensity proportional to the glucose concentration was obtained. The substrates were then scanned (Brother DCP-1610W) and colour analysis was performed with the aid of ImageJ software by measuring mean grey values and the corresponding standard deviation on each reaction chamber of the microfluidic systems. These results were used to build the calibration curves [36]. Results and Discussion P(VDF-TrFE) membranes with tailorable morphologies were processed by three distinct processing techniques. In the following sections, a complete physicochemical characterization of the processed P(VDF-TrFE) membranes will be presented, discussed and compared with commercial Whatman no.1 and Millipore HF090 membranes. The colorimetric quantification of glucose in the microfluidic systems printed on the membranes will also be presented. Physicochemical characterization The use of membranes as microfluidic substrates is dependent on proper porous morphology [37,38]. Representative SEM images of the processed and commercial membranes are presented in Figure 2.2.3. Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 70 Figure 2.2.3. Representative SEM images of the processed P(VDF-TrFE) membranes before and after oxygen plasma treatment together with commercial Whatman no.1 and Millipore HF090 substrates, for comparison. Commercial Whatman no.1 substrates (Figure 2.2.3a) are composed by randomly oriented cellulose microfibres with a flat structure (width in the order of tens of micrometer), which is associated with the manufacturing process of paper. On the other hand, Millipore HF090 substrates (Figure 2.2.3b) feature a spherulitic-like nitrocellulose porous structure laminated with a backing transparent material. Taking into account these microstructures, manufacturing techniques for P(VDF-TrFE)-based membranes were selected [14] in order to mimic the morphology of these commercial substrates and allowing to overcome some of their limitations, as will be discussed ahead. NIPS technique (Figure 2.2.3c) with ethanol as nonsolvent was used to produce P(VDF-TrFE) membranes with morphology similar to Millipore HF090, however without the need for a support material. TIPS technique was also applied to obtain P(VDF-TrFE) Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 71 membranes with an alternative porous microstructure characterized by well-defined round shaped pores (Figure 2.2.3e). Randomly oriented P(VDF-TrFE) fibre membranes were produced by ES (RO-ES) in order to mimic the morphology of the Whatman no.1 substrates (Figure 2.2.3g). Finally, oriented electrospun P(VDF-TrFE) fibres (O-ES) were also processed in order to evaluate the influence of fibre orientation in the physicochemical properties and passive capillary flow rate of the P(VDF-TrFE) membranes (Figure 2.2.3i). As the processed samples are hydrophobic, the P(VDF-TrFE) membranes were subjected to oxygen plasma treatment in order to tailor the polymer’s surface wettability and to allow capillary flow. Oxygen plasma treatment allows to generate carboxyl group on the polymer surface by the incorporation of hydrophilic functional groups [29]. SEM images of the plasma treated P(VDF-TrFE) membranes (Figure 2.2.3d, 2.2.3f, 2.2.3h and 2.2.3j) reveal a slight change in the morphology of the membranes compared to the untreated P(VDF-TrFE) membranes, which is attributed to local polymer melting associated with the high power (100 W) and high exposure time (10 min) [13]. For instance, the plasma-treated P(VDFTrFE) membranes obtained by NIPS (Figure 2.2.3d) present some melted and recrystallized regions in the interfaces between the spherulites, whereas the cross-section images of the plasma-treated P(VDFTrFE) membranes obtained by TIPS (Figure 2.2.3f, inset) reveal a change in pore spherical shape, presenting more deformed and flattened pores. In turn, the overall 3D fibre structure with smooth surfaces of the P(VDF-TrFE) membranes is maintained after plasma treatment, with average fibre diameter of 310 ±50 nm and 370±70 nm for the RO-ES (Figure 2.2.3g and 2.2.3h) and O-ES P(VDF-TrFE) (Figure 2.2.3i and 2.2.3j) membranes, respectively. The porosity of the membranes is presented in Figure 2.2.4a. NIPS TIPS RO-ES O-ES 0 10 20 30 40 50 60 70 Samples Porosimetry (%) 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 Whatman NIPS TIPS RO-ES O-ES 0 25 50 75 100 125 150 175 600 650 700 Young modulus (MPa) Samples Dry Wet Stress (MPa) Strain (%) Dry Whatman Wet Whatman Dry NIPS w/plasma Wet NIPS w/plasma Dry TIPS w/plasma Wet TIPS w/plasma Dry RO-ES w/plasma Wet RO-ES w/plasma Dry O-ES w/plasma Wet O-ES w/plasma a) b) Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 72 40 60 80 100 120 140 160 180 200 Heat Flow (W.g-1) Temperature (ºC) NIPS w/ plasma NIPS w/o plasma TIPS w/ plasma TIPS w/o plasma RO-ES w/ plasma RO-ES w/o plasma O-ES w/ plasma O-ES w/o plasma Endo TM 600 800 1000 1200 1400 1600 O-ES w/o plasma O-ES w/ plasma RO-ES w/o plasma RO-ES w/ plasma TIPS w/o plasma TIPS w/ plasma NIPS w/o plasma Transmittance (a. u.) Wavenumber (cm-1) NIPS w/ plasma c) d) Figure 2.2.4. a) Porosity of the P(VDF-TrFE) membranes before oxygen plasma treatment; b) Representative stress-strain curves up to 8 % of strain and corresponding Young modulus with mean and standard deviation (inset) of the plasma treated P(VDF-TrFE) membranes; c) Representative DSC curves and d) Representative FTIR-ATR spectra of the P(VDF-TrFE) membranes before and after oxygen plasma treatment. Independently of the processing method, all samples show degrees of porosity above 50 %. No significant variations occur for the surface plasma treated samples (Figure 2.2.3). In addition to the relevance of a porous morphology, membrane-based microfluidic substrates must keep adequate mechanical properties when wet during a functionalization process and/or after immersion in a solution containing the (bio)entity(ies) to be quantified. Stress-strain assays were conducted on dry and wet plasma treated P(VDF-TrFE) membranes and commercial Whatman no.1 microfluidic substrates. This experiment was not performed on Millipore HF090 substrates as they possess backing material, which provides improved mechanical stability but prevents the determination of the mechanical properties of the nitrocellulose membrane itself. The stress-strain mechanical curves are characterized by a linear elastic regime followed by a plastic regime after yielding where the material suffers permanent deformation after any increase in load or stress. By further stretching the samples, the rupture stress-strain is reached. Brittle materials, contrarily to ductile materials, typically feature little or no plastic deformation, fracturing within the linear elastic region. From the linear regime of the stress-strain curves, where materials are generally used, the Young’s modulus has been obtained for each sample by applying Hooke’s law. The characteristic stress-strain curves up to 8 % of strain along with the Young’s modulus of the samples under study are presented in Figure 2.2.4b. Commercial Whatman no.1 substrates show almost no plastic regime in both dry and wet state, contrarily to the plasma-treated P(VDF-TrFE) membranes. Dry commercial Whatman no.1 Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 73 substrates feature a more rigid behaviour with the highest Young’s modulus of 667.2 ± 38.8 MPa, strongly decreasing to 22.3 ± 6.5 MPa when wet. In turn, the P(VDF-TrFE) membranes are mechanically stable before and after wetting, with the wet samples nearly preserving the same elastic behaviour and mechanical characteristics as the dry ones. In fact, wet treated P(VDF-TrFE) membranes only suffered a slight decrease of the Young’s modulus compared to dry treated P(VDF-TrFE) membranes, being the wet treated RO-ES P(VDF-TrFE) and the dry treated O-ES P(VDF-TrFE) membranes the ones with the lowest and highest Young’s modulus of 71.4 ± 2.9 and 181.2 ± 7.1 MPa, respectively. This result shows that the orientation of the fibres directly influences the mechanical properties of the P(VDF-TrFE) membranes, where oriented fibre membranes lead to higher Young’s modules, when stretched in the direction of the fibres [39]. Similar high Young’s modules are also presented by the porous TIPS P(VDF-TrFE) membranes before and after wetted. On the other hand, comparing the RO-ES P(VDF-TrFE) membranes with the Whatman no.1 substrates, both featuring randomly oriented fibres, the former is characterized by a much higher Young’s modulus, when wet, indicating that P(VDF-TrFE) membranes possess more suitable mechanical properties than cellulose for the present application. Moreover, although it was not possible to determine the Young’s modulus of the Millipore HF090 substrates, it should be noted that besides needing a support material, the nitrocellulose membrane itself breaks up very easily when, for example, a finger is passed over and must therefore be handled very carefully, contrarily to the NIPS P(VDF-TrFE) membranes with similar spherulitic-like porous structure that present high Young’s modulus, similar to the RO-ES P(VDF-TrFE) membranes, in both dry and wet state. Combined with these excellent mechanical properties, it should be noted that P(VDF-TrFE) membranes can be repeatedly wetted and dried without losing their physicomechanical properties, allowing, therefore, cleaning and reuse. Molecular weight, degree of crystallinity and microstructure strongly influence the mechanical properties of polymer-based membranes. The degree of crystallinity, calculated (see Section 2.4.1) from the thermograms of the P(VDF-TrFE) membranes presented in Figure 2.2.4c, remains approximately constant, 26 ± 3 %, regardless of the processing conditions and plasma treatment, being within the typical range of values obtained for this copolymer [40]. Moreover, no significant variation is observed in the melting transition (TM) which occurs at 149.7 ± 1.0 °C. Thus, the variations of the mechanical properties are attributed just to the different morphological features of the P(VDF-TrFE) membranes. Finally, the crystalline phases of the P(VDF-TrFE) membranes are identified in the infrared spectra presented in Figure 2.2.4d. All membranes show the characteristic vibration modes at 839, 886 and 1402 cm-1, which identify the polymer crystallization in the trans TTT’ highly polar chain confirmation. Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 74 Because of its electroactive β-phase, these membranes can be further explored for active multifunctional microfluidic substrates, with added value compared to the passive commercial microfluidic ones [16-18]. Contact angle and capillary flow rate assays The hydrophobic nature of P(VDF-TrFE) is a major drawback that must be overcome for its use as microfluidic substrates where capillary flow is essential. In this sense, various surface modification approaches can be applied to tailor the polymer surface wettability, such as plasma treatment, defluorination-sulfonation, surface coating/deposition, blending, and electron beam radiation, among others [41,42]. Plasma treatment stands out as the most suitable method, due to their high versatility and for maintaining the main physicochemical properties of the polymer [29]. In this context, a reactive oxygen atmosphere allows to promote a stable hydrophilicity through the generation of carboxyl groups on the polymer surface by the incorporation of hydrophilic functional groups [28]. Thus, contact angle measurements over time were performed on the different substrates as a simple and effective method to quantify the wetting properties of the surfaces. A surface is commonly named hydrophobic when its water contact angle is higher than 90°, whereas values lower that 90 are associated with hydrophilic surfaces. Superhydrophobic and superhydrophilic are terms also used to characterize surfaces that features contact angles higher that 150 and lower than 10, respectively [43]. The water contact angle values of the processed P(VDF-TrFE) w/o and w/ plasma treatment, along with the ones for the commercial substrates, are presented in Figure 2.2.5a. Whatman Millipore NIPS TIPS RO-ES O-ES 0 20 40 60 80 100 120 140 Contact angle (º) Samples w/o plasma w/ plasma Whatman Millipore NIPS TIPS NO-ES O-ES 0 20 40 60 80 100 w/o plasma w/ plasma Samples Capillary flow rate (mm.min-1) a) b) Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 75 Figure 2.2.5. a) Contact angle and b) capillary flow rates in the “antigravity” direction of a dye solution for the commercial substrates and P(VDF-TrFE) membranes before and after oxygen plasma treatment; inset: Representative photograph of an experimental assay. Commercial WhatmanTM no.1 and Millipore H090 substrates are superhydrophilic. In turn, the processed P(VDF-TrFE) membranes present hydrophobic properties with an average contact angle of 122±2°, independently of the morphology. After oxygen plasma treatment, the P(VDF-TrFE) membranes become superhydrophilic and are stable for at least 2 months. Capillary flow rate assays were then performed. This experiment validates the ability of the membranes to be used as microfluidic substrates, where passive capillary flow must occur preventing the need of any external actuation systems. The results are presented in Figure 2.2.5b. As anticipated, no flow was observed in the as-processed hydrophobic P(VDF-TrFE) membranes. In turn, plasma treated P(VDF-TrFE) membranes feature capillary flow rates varying from 35.7 ± 2.5 mm.min-1 to 88.3 ± 3.7 mm.min-1, corresponding to the plasma treated RO-ES and plasma treated O-ES P(VDF-TrFE) membranes, respectively. This result demonstrates that, besides the type of porous morphology, the orientation of the fibres has a direct effect on the capillary flow rate, the highest value being obtained for the O-ES membranes due to the orientation of the fibres along the length of the strip. Comparing the plasma treated NO-ES P(VDF-TrFE) membranes and the commercial WhatmanTM no.1 substrates, with similar randomly oriented fibre morphologies, the obtained capillary flow rates are slightly higher for the WhatmanTM samples with values of 44.1 ± 1.1 mm.min-1 compared to 35.7 ± 2.5 mm.min-1 for the NO-ES P(VDF-TrFE) membranes, which can be attributed to the more compact structure of the WhatmanTM membranes that allows higher flow rates. In turn, the spherulitic-like porous structure of the plasma treated NIPS P(VDFTrFE) membranes present capillary flow rates of 46.8 ± 1.3 mm.min-1, higher than the ones obtained for Millipore HF090 membranes with similar morphology (25.0 ± 3.4 mm.min-1). This behaviour can be attributed to the polymer melted and recrystallized regions caused by the plasma treatment that increase the contact areas for the fluid to flow and thus allow faster flow rates. Finally, plasma treated porous TIPS P(VDF-TrFE) membranes feature capillary flow rates of 57.1 ± 2.9 mm.min-1, an intermediate value between all measured samples. Thus, the capillary flow rate of the P(VDF-TrFE) membranes can be adapted and tailored by tuning their morphology and/or fibre orientation, which is relevant in order to match process requirements (such as collection, separation, pre-concentration or mixing, among others) in microfluidic substrates. Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 76 Colorimetric glucose determination Colorimetric analysis allows to determine the presence or even concentration of a specific chemical entity in a solution based on a colour variation. This method is widely used in clinical analysis laboratories, for industrial purposes such as analysis of water contaminants and PADs. In the scope of this research, colorimetric assays based on the quantification of glucose were performed on commercial WhatmanTM no.1 and Millipore HF090 substrates, as well as on the plasma treated P(VDF-TrFE) membranes with different morphologies to evaluate their performance as microfluidic substrates. First, an optimized microfluidic design was printed using a wax printer for the quantification of glucose in the range of 25 to 100 mg.dL-1, as described. Scanned images of the microfluidic systems after the glucose assays are presented in Figure 2.2.6a and their corresponding calibration curves as well as the extrapolated linear fittings are shown in Figures 6b and 6c, respectively. For that, mean grey values are presented as a function of the logarithm of the glucose concentration. WhatmanTM Millipore NIPS TIPS RO-ES O-ES a) b) c) Figure 2.2.6. a) Representative scanned images of commercial substrates and plasma treated P(VDF-TrFE) membranes after glucose assays. Millipore HF090 substrates were evaluated on the baking side and the image horizontally inverted. For identification of glucose concentration see Figure 2.2.2. b) Calibration curves of glucose for commercial substrates and plasma treated P(VDF-TrFE) membranes. The results are presented as mean grey values and corresponding standard deviation measured on the Chapter 2.2 – Fluorinated polymer membranes as advanced substrates for portable analytical systems 77 reaction chambers of the microfluidic substrates using ImageJ software according to the method described in ref [36]. c) Corresponding linear fitting. With respect to the wax print quality, it is confirmed that all microfluidic substrates present good resolution, except for the Whatman no.1 substrates where the wax expands near to 1.2 mm during the thermal post-treatment. This behaviour may affect the capillary flow rate and thus attention must be paid when Whatman no.1 substrates are used in combination with wax printing, according to the applications requirements [38,44-46]. Regarding the colours generated after the colorimetric assays, they are only visible on the back side in the case of the Millipore HF090 substrates, remaining completely white on the front side. Thus, scanned images of the Millipore HF090 in Figure 2.2.6a represent the back side and the image was horizontally inverted to present colour intensities and thus glucose concentrations in the same reaction chambers as the other substrates. Figure 2.2.6a confirms that the plasma treated P(VDF-TrFE) substrates feature good wax printing resolution and colour formation on the front side. In all colorimetric assays, the grey intensity in the reaction chambers (corresponding to the red colour in Figure 2.2.6a, for proper identification) intensifies with increasing glucose concentration. However, the homogeneity of the colour generated in the reaction chambers varies depending on the substrate, which directly affects the quality of the obtained calibration curves (Figures 2.2.6b and 2.2.6c). Very good linear fittings were obtained with the Whatman no.1 substrates and plasma treated NIPS and TIPS P(VDF-TrFE) membranes with R2 higher than 0.990, followed by the plasma treated RO-ES P(VDF-TrFE) and Millipore HF090 with R2 of 0.989 and 0.985, respectively. Higher slope associated with higher sensitivity was obtained with the Whatman no.1 substrates and plasma treated NIPS P(VDF-TrFE) membranes with values of -58.4 and -56.7, respectively. This small variation demonstrates that the expansion of the wax during the thermal post-treatment in the Whatman no.1 substrates did not affect significantly the colorimetric reaction, in this specific application. The worst fitting was obtained with the plasma treated O-ES P(VDF-TrFE) membranes that may result from the orientation of the fibres were the unidirectional flow leads to the heterogeneous formation of the coloured solution in the reaction chamber that feature a circular shape. This behaviour along with the capillary flow rate presented in Figure 2.2.6b indicate that P(VDF-TrFE) membranes based on oriented fibres show high potential for unidirectional capillary flow of solutions/entities at higher rate but are not the most indicated for colorimetric quantification using more isotropic microfluidic design. Thus, except 84 2.3. Biodegradable polymer-based membranes for sustainable portable analytical devices applications In this chapter, a biodegradable aliphatic polyester poly(D,L-lactide-co-glycolide acid) lactide:glycolide (50:50), PDLG, is used to produce poreand fiber-based membranes, as alternative to conventional paper substrates. Two fabrication methods are used, with potential for industrial scale-up. These new substrates could presesent great importance for the design and manufacture of a new generation of sustainable portable analytical devices, compatible with circular economy paradigms, and a step forward to cross the challenging academia to industry barrier for their commercialization and widespread adoption. This chapter is based on the following publication:, Ricardo Brito-Pereira, Clarisse Ribeiro, Senentxu Lanceros-Méndez and Vanessa Fernandes Cardoso, Biodegradable polymer-based microfluidic membranes for sustainable point-of-care devices, Chemical Engineering Journal 448, 137639 (2022). Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 85 Introduction Advances in biomedicine through near-patient follow-up continue to play an essential role in the improvement of healthcare services in the developed and developing world [1]. In fact, access to clinical analysis laboratory equipment cannot constitute a barrier against the performance of diagnostic tests, especially in resource-limited locations worldwide [2]. This led to the development of point-of-care (POC) devices that offer new possibilities for prevention, diagnosis and monitoring of medical conditions and/or disease [3]. This portable devices should addressed the ASSURED WHO [4]. Despite the relevant developments witnessed during the last decade, the field of POC devices using traditional microfluidic materials (e.g. silicon, glass and polymers such as polydimethylsiloxane) are still associated with highcost, equipment-dependent and complicated fabrication process and environmental impact with respect to production and disposal, which has significantly limited their introduction to the market [5]. Since its introduction in 2007 by the Whitesides group, microfluidic paper-based analytical devices (µPADs) have attracted increasing attention as promising portable analytical tools not only in the medical field but also in the food industry, biodefence and environmental monitoring applications, owing to their advantages such as easy-to-use, portability, in situ measurement, minimal reagent consumption and disposability [6,7]. Moreover, the porous structure of paper composed of cellulose fibers with hydrophilic behavior allows fluid transport via capillary forces without the need for external pumping systems [8]. By properly tailoring the channels design using physical or chemical modification techniques such as wax, screen or inkjet printing, stamping, plasma treatment, photolithography or even cutting, fluids of a few microliters can be directed into specific detection zones for multiplexed assays [9]. Processing and surface treatments allow to modify specific zones of the paper substrate from hydrophilic to hydrophobic through specific hydrophobic barriers design creating channels that guide the liquid flow under the effects of capillary action [10]. The barrier type and characteristics are important and must be carefully selected considering the nature of substrate and solutions, in order to ensure compatibility. In addition, threedimensional (3D) µPADs structures using origami technique have been also reported by folding or stacking the paper in an attempt to control sample and reagent flow rate and path [11]. Analytical detection can be accomplished by measuring the intensity of a colorimetric reaction or, for improved sensitivity, by using electrochemical, fluorescent, immunological or molecular detection methods [12– 15]. Different types of paper may be employed in this scope, that depend on the processing method and the compound to be detect, such as filter paper, chromatography paper, nitrocellulose membrane paper, or Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 86 graphite paper, among others [16]. Within this range, Whatman cellulose filter paper, Advantec chromatography paper and Hi-Flow Plus nitrocellulose substrates from Millipore are among the most commonly used because of their good wicking performance and uniform thickness [16–18]. Although µPADS feature properties that make them a good choice for the fabrication of portable analytical devices, there are still at an early stage of development and significant research efforts are needed to improve their performance to reach comparable results than currently used technologies. A major challenge remains on reproducibility and sensitivity that are directly affected by the physicochemical properties of paper’s porous membranes that offer limited control over sample flow speed and direction [4,19]. To overcome this fact, advanced processing and characterization on papers are needed, similarly to those performed in previous studies with Whatman nº1 cellulose filter paper and Hi-Flow Plus nitrocellulose substrates to properly understand their behaviour as microfluidic substrates [17,20]. Last but not least, paper usually required complex processes that include wood preparation, pulping, chemical recovery, bleaching and papermaking to convert wood to the final product [21]. These processes along with the paper composition that typically include additives such as starch, minerals and synthetic polymers, turn paper a multi-component materials with a complex and varied nature, wich in turn have a crucial role on paper aging and degradation, in most of the cases difficult to interpret or unknown [22]. On the other hand, little attention has been paid to the development of new materials with tailorable physicochemical properties as potential pathway to complement the range of available paper substrates. Recently, flexible polymer membranes based on poly(vinylidene-co-trifluorethylene) and PLLA have been explored as microfluidic substrates. They both demonstrated promising results including tailorable morphology, controlled capillary flow rate, suitable mechanical properties and biocompatibility [17,20]. In this work, a copolymer of poly(D,L-lactide-co-glycolide acid) (PLGA), an aliphatic polyester approved by the FDA has been explored for portable analytical platforms [23]. PLGA has been applied in drug-delivery, surgical implants and tissue engineering applications based on its adjustable biocompatibility and biodegradability, its degradation rate being controlled by varying the relative amount between D, L – lactic acid and glycolic acid monomers [19-21]. Other parameters that affect the degradation rate include molecular weights, size of the samples, integration of additives and environmental conditions [25,26]. During degradation, the copolymer undergoes hydrolysis to produce the original monomers, which are non-toxic for mammalian cells and are metabolized to water and carbon dioxide [27]. The primary role in using biodegradable polymers in medicine is typically to provide a mechanically robust temporary framework during a required time, from which it degrades rapidly and without unwanted side effects. In the case of microfluidic substrates for portable analytical devices, the same behavior is desired, that is, Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 87 to maintain the properties of the substrate until and during the assays and ensuring its subsequent quick degradation, turning then more sustainable materials, compatible with circular economy paradigms. Material and methods PDLG membranes with various morphologies were processed by two different processing techniques, temperature induced-phase separation (TIPS) and electrospinning (ES), and their potential to be used as substrates for microfluidic applications studied. For that, physicochemical characterization, along with wettability, cytotoxicity and degradation assays of the processed membranes were performed. To evaluate their applicability as microfluidic substrates for portable analytical devices, colorimetric quantification of glucose in printed plasma-treated PDLG substrates was carried out. The following nomenclature will be used to facilitate the identification of the membranes: TIPS, ES-RO, ES-O for pore-based, randomnly oriented and oriented fiberbased membranes, respetively. The samples without and with oxygen plasma treatment will be identified by w/o.P and w/.P, respectively. Materials PURASORB PDLG 5010, a copolymer of PLGA, with an inherent viscosity midpoint of 1.0 dL.g -1 was acquired from Corbion Purac. N,N-dimethylformamide (DMF) and absolute ethanol were obtained from Merck, while chloroform (Chl) was acquired from Sigma-Aldrich. All chemicals and reagents were used as received. Membranes processing A 10 wt.% solution of PDLG was prepared dissolving the copolymer powder in a mixture of DMF/Chl 60/40 v/v solvent under magnetic stirring until a transparent, homogeneous and bubble free solution was obtained, which took no longer than 2 h. This protocol was previously optimized to guarantee a PDLG solution concentration compatible with the various processing techniques employed, described below. Accordingly, porous PDLG membranes were produced by TIPS. For that, PDLG solution was spread on clean glass substrates (10 cm × 15 cm) using a 450 µm gap hand-casting knife and placed in an oven (JP SELECTA Digitronic-TFT) at 25 °C during 72 h to ensure a controlled environment and full crystallization. Fibre-based PDLG membranes were produced by ES. A 10 mL disposable syringe with a Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 88 blunt steel needle with an inner diameter of 500 µm was filled with PDLG solution and placed in a syringe pump (New Era NE-1000). A high voltage power supply (Glassman PS/FC30P04) set at 17 kV was applied and the solution was pumped at a flow rate of 0.5 mL.h-1. RO-ES PDLG membranes were collected on a grounded 20 cm × 15 cm static plate collector placed 15 cm away from the needle. O-ES PDLG membranes were collected on a grounded rotating drum collector set at a speed of 1500 rpm. Membranes surface modification PDLG membranes present a hydrophobic behaviour after their processing, avoiding the absorption of fluids and thus their ability to spontaneously generate passive flow along the membrane. Therefore, to overcome this limitation and allow their use in the development of portable analytical devices, oxygen plasma treatment was applied to the PDLG membranes to make them (super)hydrophilic [28]. This is an established technique to effectively change the hydrophobicity of polymers by the generation of carboxyl group due to the integration of hydrophilic functional groups on the surface of the copolymer [29,30]. Accordingly, the membranes were placed in a plasma chamber (Zepto, Diener Electronics), equipped with a 40 kHz radio frequency plasma generator. A plasma power of 50 W was used under a total pressure of 80 Pa. In order to overcome overheating and morphological variations, the procedure was optimized and thus plasma treatment was performed 10 times in periods of 30 s on both surfaces of the PDLG membranes. Physicochemical characterization The surface and transversal morphologies of the PDLG membranes were obtained using a scanning electron microscope (SEM) Quanta 650 FEG from FEI. The samples were previously sputtered (Polaron SC502) with a thin gold layer. From these images, the main pore size and fibre diameter distributions were determine using the software ImageJ. SEM images were obtained in just processed membranes and in membranes kept in vacuum for 6 months, to demonstrate their stability under this storage condition. The porosity of the membranes was determined by liquid displacement technique using a pycnometer [31,32]. Ethanol was used to fill the pycnometer and the weight measured and labelled as W1. Each membrane, whose weight was Ws, was individually immersed and saturated with ethanol and additional Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 89 ethanol was added to fill the volume of the pycnometer. The pycnometer was weighted and labelled as W2. The sample filled with ethanol was then extracted from the pycnometer and the residual weight of the ethanol and the pycnometer was labelled as W3. The following equation was used to calculate the porosity of the membrane 𝜀𝑝=(𝑊2 − 𝑊3 −𝑊𝑆) (𝑊1 −𝑊3)⁄ . The data will be presented as the average of the values determined in three samples obtained after each processing technique. Absolute ethanol was employed for being a non-solvent of PDLG, penetrating the pores of the samples without inducing shrinking or swelling. The assays were performed on untreated plasma membranes, since hydrophilic membranes absorb ethanol, leading to porosity measurement errors. Stress-stress mechanical measurements were performed to assess the mechanical properties of the processed plasma-treated PDLG membranes with hydrophilic properties for microfluidic substrates. For that, the assays were carried out in the tensile mode with a Shimadzu AD-IS universal testing set up with a load cell of 50 N. 15 mm long and 10 mm wide samples were stretched at a rate of 1 mm.min-1 on dry and wet samples (to mimic the hydration that occurs in portable analytical systems), using 40 µL of PBS (pH 7.4) in the latter. The measurements were performed in triplicate and the plasma treated ES-O membranes were stretched along the direction of the fibres. The average sample thicknesses of the samples are ~108, ~87 and ~82 µm for the plasma treated TIPS, ES-RO and ES-O samples, respectively, as measured using a Fischer Dualscope MPOR. Infrared measurements (FTIR), differential scanning calorimetry (DSC) and thermogravimetry (TGA) were also performed. FTIR was carried out using a Jasco FT/IR 4100 (Jasco, Easton, Maryland, USA) apparatus in attenuated total reflectance mode (ATR) from 4000 to 600 cm−1. FTIR spectra were obtained after 64 scans with a resolution of 4 cm−1. DSC analysis was performed in a Mettler Toledo DSC822e apparatus using a heating rate of 10 °C.min-1. The samples were cut into small pieces and placed into 40 µL aluminium pans. Finally, TGA studies were performed with a thermal analyser TGA/SDTA 851e from Mettler Toledo. The samples were heated between 25 and 500 °C, at a heating rate of 10 °C.min1. Contact angle and capillary flow rate assays Water contact angle measurements were performed to study the effect of plasma treatment on the surface properties of the processed membranes, namely regarding their wettability. The assays were performed using a Data Physics OCA20 instrument by the static sessile drop method with ultrapure water. For that, water drops (3 µL and 15 µL) were dropped on the surface of the Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 90 sample and the contact angles were measured using the SCA20 software. The mean contact angle and standard deviation were calculated through the measurement at six different places of each sample. Two volumes were tested because of the different behaviour of the membranes according to the fluid volume. Capillary assays were carried out to evaluate the ability of the different samples to generate spontaneously passive flows. For that, sample strips 2.5 cm long and 1 cm wide and a solution coloured with green food dye were used. A strip segment of 0.5 cm was vertically submersed and the time taken for the coloured solution to travel across the remaining 2 cm of the sample in the antigravitational direction was measured. Three measurements were performed in each sample and the average and standard deviation were calculated. Cytotoxicity assays Adapting the ISO 10993-5 standard test method, indirect cytotoxicity evaluation of the processed PLGA membranes (after processing or stored in vacuum for 6 months) was performed. For that, L929 cells were cultured in 75 cm2 cell culture flask at 37 °C in a humidified environment and 5 % CO2, using Dulbecco’s modified Eagle’s medium (DMEM, Biochrom, Berlin, Germany) containing 4.5 g.L−1 glucose, 10 % fetal bovine serum (FBS, Biochrom, Berlin, Germany) and 1 % (v/v) penicillin/streptomycin solution (P/S, Biochrom). The different samples were cut with approximately 1.5 cm2. Then, their sterilization was carried out by exposition to ultraviolet radiation for 1 h each side of the samples and washing with sterile PBS at pH 7.4. Then, a suspension of 2 × 104 cell.mL−1 was seeded in 96-well tissue culture polystyrene plates and incubated for 24 h at the same conditions described above to ensure cell attachment on the plate. Simultaneously, each sample was incubated for 24 h in a 24-well tissue culture polystyrene plate with DMEM. After this time, the cell culture medium in the 96-well plates was removed, and 100 µL of culture medium (that was in contact with the different samples) was added to each well and allowed to incubate for 72 h in standardized culture conditions as mentioned above. A solution of 20 % dimethyl sulfoxide (DMSO) was used for positive control. The metabolic activity was then evaluated after 72 h of incubation using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS, Promega). Briefly, the medium of every well was removed, and fresh medium containing MTS solution (in a 1:5 ratio) was added to each well and incubated for 2 h. After this incubation time, the optical density was measured at 490 nm with a spectrophotometric plate reader (Biotech Synergy HT). Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 91 The results are presented as the average of viability ± standard deviation. The percentage of metabolic activity was calculated according to: Metabolic activity (%) = (Absorbance of sample/Absorbance of negative control) × 100 Degradation assays The degradation of the processed PDLG membranes was monitored by the weight loss in air and in PBS at pH 7.4. Samples from each morphology with an area of 1 cm2 were cut and placed in 12-well tissue culture polystyrene plates, in contact with air or immersed in 3 mL of PBS at 37 °C. The PBS was replaced every week to avoid variations in pH and ion concentration. The samples were studied at week 1, 3 and 6. Each sample was weight at each time and compared with the initial weight using a Sartorius Cubis® II Micro Lab balance. The samples immersed in PBS were previously washed with distilled water and placed in an oven at 37 °C during 6 h for drying and subsequent weighting. Colorimetric quantification of glucose To validate the potential of the obtained microfluidic substrates, microfluidic platforms were designed (Figure 2.3.1) and set up using plasma-treated PDLG membranes for the colorimetric quantification of glucose. The microfluidic platforms were designed using a computer-aided design software and hydrophobic wax barriers were printed using a Xerox ColorQube 8870 printer. Before printing, the samples were fixed to a paper sheet with kapton tape. Thus, the hydrophobic barriers were printed, the samples fixed to a glass and then placed in a hot plate (Prazitherm PZ 28-1) and the temperature gradually increased until reaching 95 °C for 5 s, cooled down, and the process repeated 3 times. This temperature is required for the wax to melt and penetrate through the opposite surface of the substrates and thus to contain the fluids in the channels between the hydrophobic barriers. In the scope of the present study, wax-printing technique was used for allowing reproducibility assays, for being a rapid and inexpensive process and for being environmental friendly (lack of organic solvents), besides not requiring specialized facilities [33,34]. For the experiment, a glucose kit (Trinder – Endpoint, FAR Diagnostic) was used. Calibration curves were determined using glucose concentrations of 25, 50, 75, and 100 mg.dL-1. This range of concentrations includes ranges of reference values for both new-born babies (20 to 80 mg.dL-1) and adults (70 to 110 mg.dL-1). Each microfluidic systems holds eight reaction chambers, with each Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 92 glucose concentration being quantified in two separate reaction chambers, as illustrated in Figure 2.3.1. Three microfluidic systems were printed for each plasma treated PDLG membranes in order to determine the reproducibility of the colorimetric reactions. 15 µL of glucose solution were used to functionalize the reaction chambers according to the glucose concentration. After 5 min, reagent was introduced in the inlets of the microfluidic system, flowing to the reaction chambers by capillarity. During the reaction, an orange/red colour with intensity proportional to the glucose concentration is produced, which take no more than 10 min. The substrates were then scanned (Brother DCP-1610 W) and colour analysis was performed using ImageJ software by measuring the mean grey values and the corresponding standard deviation on each reaction chambers of the microfluidic systems. Calibration curves were built according to the results obtained [35]. Figure 2.3.1. Schematic illustration of the microfluidic system for the colorimetric quantification of glucose. The glucose solution was placed in the reaction and reference chamber and the reagent was introduced in the inlet to flow by capillarity to the reaction chambers to form an orange/red color with intensity proportional to the concentration of glucose. The inlet, reaction and reference chambers present a diameter of 4 mm and the channels a width of 1.5 mm. Results and Discussion Physicochemical characterization Characterization of the PDLG membranes morphology after processing is needed to confirm their morphology and to determine their porosity, which are key properties when applied as microfluidic substrates. Thus, representative SEM images of as processed PDLG membranes obtained by TIPS and ES are shown in Figure 2.3.2. Moreover, knowing the high degradability of PDLG, ES-RO.w/.P membranes were kept in vacuum for 6 months, as representative samples, to demonstrate their stability under vacuum. Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 93 Figure 2.3.2. Representative SEM images of the processed PDLG membranes before and after oxygen plasma treatment and plasma treated randomly-oriented PDLG membrane stored in vacuum for 6 months. The results demonstrate that the expected morphologies were obtained according to the corresponding processing technique. Pore-based PDLG membranes were produced by TIPS with well-defined pores all along the cross-section. The average diameter of the pores is 10.8 and 11.5 µm for untreated and plasma treated membranes, respectively. On the other hand, fibre-based PDLG membranes were obtained by ES with smooth fiber surface and controlled orientation. ES-RO membranes show average fiber widths of 239 and 153 nm for untreated and plasma treated membranes, whereas ES-O membranes present higher average fiber widths of 496 and 455 nm, respectively. Thus, oxygen plasma treatments lead to a reduction of fibre width and to the fusion of some fibers in the case of the ES-RO membranes. These results are consistent with the literature and are attributed to the high power used during the plasma treatment that lead to a surface heating and flowing of the polymer [29,36]. It is important to emphasize that the plasma treatment protocol was optimized to minimize overheating and significant morphological variations but also to ensure the stability of the hydrophilic surface over time, as will be discussed in the next section. Further, oxygen plasma treatment allows to integrate hydrophilic functional groups by the generation of carboxyl group on the polymer surface, which is a key issue to produce hydrophilic membranes for microfluidic applications [30,37]. Finally, the SEM image of the plasma treated ES-RO membrane stored in vacuum for 6 months shows no visible variations of the fiber morphology and orientation, contrary to the membranes exposed to air or immersed in PBS after processing. The porosity of the untreated PDLG membranes was also studied and the results presented in Figure 2.3.3a. As indicated above, no experiments were performed on the plasma treated PDLG membranes since hydrophilic membranes absorb ethanol, leading to porosity measurement errors. Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 100 Proof-of-concept Colorimetric analyses are qualitative or quantitative methods commonly used in medical laboratories and for industrial purposes to determine the presence and/or concentration of a chemical compound in a solution with the aid of a specific reagent, in such a way as to produce a colour intensity proportional to the concentration of the compound being tested. This technique has been adapted for the development of portable analytical devices for the detection and/or quantification in situ of glucose, uric acid, nitrite, creatinine, drugs, HIV, SARS-CoV-2 (Covid-19), drugs, contaminants in water samples, among others [50]. In the scope of this work, the plasma-treated PDLG membranes were evaluated for the colorimetric quantification of glucose, to validate their use as microfluidic substrates for the manufacture of portable analytical devices, taking into account their tailorable physicochemical properties, superhydrophilicity, biocompatibility, biodegradability and portability. To do so, hydrophobic microfluidic channels were printed to fabricate a system composed of a geometry allowing the quantification in the same system of several glucose concentrations, as described in section 2.3.2.6. For the experiments, the samples kept in vacuum for 6 months after processing were used. The results obtained for the ES-RO and ES-O membranes are presented in Figure 2.3.7 and illustrated the mean grey values as a function of the logarithm of the glucose concentration ranging from 25 to 100 mg.dL-1, from which the calibration curves were produced. Moreover, scanned images of the microfluidic systems after the experiments are also presented as inset. The assays were not performed in the pore-based PDLG membranes, as they change to a viscous state and do not promote a proper wax adhesion during the wax printing process. Nevertheless, it is to notice that the pore-based PDLG membranes are compatible with other manufacturing processes conventionally used for the fabrication of portable analytical devices such as cutting process or screen printing, among other. Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 101 Figure 2.3.7. Calibration curves of glucose for plasma-treated ES-RO and ES-O PDLG membranes. The results are presented as mean gray values and corresponding standard deviations, which were measured on the reaction chambers of the printed PDLG microfluidic substrates using ImageJ software according to the methods described in [35]. Inset: PDLG membranes after the colorimetric reaction. Both electrospun membranes present good printing quality, as observed in the inset of the Figure 2.3.7. On the other hand, the homogeneity of the colour generated in the reaction chambers is lower in the case of the ES-O membranes, which is related to the orientation of the fiber [2]. However, this behaviour does not have a significant impact on the quality of the calibration curves that feature very good determination coefficient R 2 of 0.997 and 0.986, for the ES-RO and ES-O membranes, respectively. Nevertheless, the ES-RO membranes present a higher sensitivity with a calibration curve slope of 30.63 slightly superior to the 24.21 value obtained for the ES-O membranes. Comparing with previous studies carried out with P(VDF-TrFE) and PLLA membranes, the sensitivity of the samples for the colorimetric quantification of glucose with the same fiber-based morphologies are quite similar, being only slightly inferior to commercial paper substrates [17,20]. Nonetheless, as previously stated and demonstrated, the PDLG membranes can be degraded very quickly after their use, which is a key point for the development of sustainable portable analytical devices. Therefore, this property along with the biocompatibility, tailorable morphology and capillary flow rate, good wax printing quality and excellent mechanical properties (including in the wet state) make this material as an excellent alternative to the commonly used paper substrates, not just for colorimetric assays but also for related (bio)technological applications that can take advantages of the characteristics of PDLG [51]. Conclusions This work demonstrates the suitability of PDLG membranes for sustainable next generation portable analytical devices. Biodegradable aliphatic polyester PDLG was processed by TIPS and ES followed by oxygen plasma treatment to produce hydrophilic poreand fiber-based membranes. The processed samples were characterized in terms of physicochemical properties along with contact angle, capillary flow, biocompatibility and degradation assays. The results demonstrate the potential of PDLG membranes to be used as microfluidic substrates, by virtue of their tailorable morphologies, i.e. pore, randomly and oriented fiber-based membranes, surface hydrophilic adaptation to generate spontaneously passive flows and controllable capillary flow rates from 36.2 ± 4.2 to 84.1 ± 5.2 mm.min-1. Moreover, the membranes Chapter 2.3 – Biodegradable polymer-based membranes for sustainable portable analytical devices applications 102 show good mechanical properties even in wet state and are biocompatible. Engineered wax-based hydrophobic patterns were printed on the membranes to fabricate portable analytical platforms suitable for the colorimetric quantification of glucose. The ES-based membranes were successful in determining the glucose concentration in the ranges of clinical reference values with results comparable to commercial microfluidic substrates. Last but not least, it was proven the physicochemical stability of the plasmatreated PDLG membranes over time when kept in vacuum for at least 6 months and showing a subsequent quick degradation after use, reaching values higher than 90 % after 6 weeks when wet. 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The developed materials can be used as substrates for multiple colorimetric quantification of three commonly scrutinized clinical analytes: albumin, uric acid, and glucose. This chapter is based on following publication: Ricardo Brito-Pereira, André S. Macedo, Clarisse Ribeiro, Vanessa F. Cardoso, Senentxu Lanceros-Méndez, Natural based reusable materials for microfluidic substrates: The silk road towards sustainable portable analytical systems, Applied Materials Today 28, 101507 (2022). Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 107 Introduction Prevalence of diseases often associated with bad nutritional habits or poor lifestyles in regions that suffer from inequality of income and a lack of access to medical supervision has been rising. In Germany, by 2040, an increase ranging from 54 % to 77 % of the incidence of type 2 diabetes has been projected [1]. High serum uric acid levels have been linked to prediabetes [2], as well as an increased risk of cardiovascular diseases [3], gout and renal calculi [4]. Low blood albumin levels can be caused by liver diseases such as cirrhosis or hepatitis [5], infections, general inflammation, as well as malnutrition and malabsorption [6]. The populace of third-world countries, where resources are scarcer, especially struggle with the latter. This data demonstrates there is a strong need for low-cost, easy to use, fast and portable devices capable of not only early medical diagnosis and low-cost periodic monitoring of specific health conditions, but also for other applications such as detection of water contaminants [7] or food safety [8]. PADs are diagnostic tools suitable to assist in solving of those issues, allowing in situ preliminary evaluation that can be followed up accordingly. Microfluidic paper-based analytical devices (µPADs), first introduced by Martinez et al. [9], who created hydrophobic channel walls using photolithography on Whatman chromatography paper, are particularly appealing due to their portability, versatility, ease of use and low-cost to manufacture. Hydrophobic channels for the development of µPADs have been also implemented by plotting [10], inkjet [11], screen [12] or wax printing [13]. The latter has been used, among others, to develop µPADs able to carry out colorimetric assays, where a reaction between one or more reagents and an analyte generates a color change that may correspond to either the simple presence or a more exact quantification of the specific analyte [14]. Research is being developed to overcome the limitations of paper based substrates, such as unsuitable porosity and weak mechanical properties [15], by exploring alternative polymer-based microfluidic substrates such as PLLA [16] or P(VDF-TrFE) [17]. In the present day, together with the improving the performance of devices, one of the most urgent needs is increasing sustainability of materials and processes, in particular in the area of disposable devices [18]. In fact, the amount of waste produced per hospital patient per year varies greatly worldwide, from 0.44 kg in Mauritius to 8.4 kg in the United States of America (USA) [19], here with an additional 50000 tons generated from home healthcare annually [20]. This waste is a danger to global health and the environment, and thus the search for natural, reusable, and renewable materials, as well as the use of sustainable processing techniques and general circular economy concerns has been met with investment [21]. The development of reusable PADs based on sustainable materials represent one way to address this problem. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 108 Silk fibroin (SF), a biocompatible and biodegradable [22] fibrous protein with mechanical strength and thermal resistance [23], can be extracted from a number of arthropods including spiders and silkworms [24], most typically from Bombyx mori cocoons, as is the case of the present work. The silk filament is composed of sericin (the outer coating) and fibroin (the inner brins) [25]. Silk fibroin can be processed into a variety of formats and has been extensively used in applications as diverse as scaffolds for tissue engineering and in vitro disease models or drug delivery in the form of hydrogels, microspheres or films [27, 28], combined with polyethylene oxide to create air filtration systems [29], substrates for wearable displays [30], and often joined with graphene in electronic applications such as bio-integrated electrode systems [31], pressure sensors [32], or enzymatic biosensor transistors [33]. Electrospinning is a technique that allows the development of fiber matts by applying a high voltage electrical field to a droplet of polymeric solution, typically fed through a syringe with a metallic needle by a pump, giving rise to a jet that is collected in a metallic platform. The intensity of the electrical field, solution concentration, pumping rate and distance to the collector regulate fiber shape and size. Oriented fibers can be obtained by using a rotating collector [34]. ES silk fibroin mats have been prepared by tailoring diameter and thickness for controlled drug release [35], modified with graphene oxide to improve antibacterial activity and biocompatibility [36], or carbon nanotube composites for enhancing cardiomyocyte functionalities [37]. Further, the surface characteristics and hydrophilicity of silk fibroin fiber matts can be modified by plasma treatment, where functional groups are introduced to the surface of the exposed material changing its composition, improving the bonding of water molecules by integrating C=O bonds [38, 39]. Herein we report on silk cocoon and ES SF substrates for reusable portable analytical systems. Together with their processing and characterization, their suitability to carry out colorimetric assays for the detection and quantification of albumin, uric acid, and glucose as proofs of concept is demonstrated. Materials and Methods Materials Bombyx mori silkworm cocoons were supplied by APPACDM from Castelo Branco (Portugal). Sodium carbonate (Na2CO3), formic acid (FA, CH2O2), calcium chloride (CaCl2), absolute ethanol (C2H5OH) and Hach pH 4.01 buffer solution were obtained from Sigma-Aldrich. Distilled water was prepared in the laboratory. All reagents and solvents were used as received. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 109 Sample preparation Preparation of Bombyx mori silkworm cocoon-based substrates The cocoons were first cleaned and cut in 30 mm2 pieces and then subjected to a mechanical press (Model 4350.L Bench Top, Carver, Inc., USA) at a pressure of 5 tons for 3 h, reducing their thickness and making them flat and suitable for printing. Raw cocoons will be addressed as R-cocoons, whereas pressed cocoons will be identified as P-cocoons. Silk Fibroin extraction and purification The extraction of SF from Bombyx mori silkworm cocoons was carried out by a soap degumming method. Cocoons were cleaned, cut in 1 cm2 pieces and boiled in a 0.05 wt.% Na2CO3 solution for 30 min in a silk to water solution ratio (w/v) of 1:40. The resultant fibers of SF were thoroughly washed with distilled water and dried at room temperature for 24 h. These fibers were then dissolved in a 0.17 M solution of FA/CaCl2 with a ratio of 12:1 v/w (FA:SF). To remove impurities, this solution was centrifuged at 6000 rpm for 10 min and the supernatant SF/FA/CaCl2 solution was cast on a Petri dish and left to dry at room temperature for 24 h to allow the FA to evaporate. The resultant transparent and plastic material was once again washed in a distilled water bath to remove CaCl2 and dried at room temperature, leading to brittle, whitish solid SF. Randomly oriented and oriented electrospun fiber mats SF was dissolved in FA (8:1 v/w FA:SF) to obtain a solution suitable for ES. The SF/FA solution was transferred to a 10 mL disposable syringe fitted with a blunt steel needle, with an inner diameter of 0.41 mm, and placed in a syringe pump (New Era NE-1000). ES was conducted using a high voltage power supply (Glassman PS/FC30P04) set at 20 kV and the solution was pumped at a flow rate of 0.5 mL.h-1. The resulting randomly oriented ES SF samples were collected on a grounded 20 cm x 15 cm static plate collector placed 15 cm away from the tip of the needle. These will be referred to as RO-ES samples and substrates. Oriented ES SF samples were obtained using the same process as the one described before except for the use of a grounded rotating drum collector set at a speed of 1500 rpm. These will be referred to as O-ES samples and substrates. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 116 Figure 2.4.3. a) Stress−strain curves up to 15 % of strain; b) Elastic modulus; c) FTIR-ATR spectra and d) DSC thermograms of the oriented (O-ES), randomly-oriented (RO-ES) electrospun Bombyx mori silk fibroin samples and pressed cocoons (P-cocoons). Stress-strain curves up to 15 % reveal highly deformable structures at low forces, with the mechanical characteristics being determined by a deformation of the network structure and not of the material itself [45]. P-cocoons possess a much higher resistance to deformation when compared with the electrospun samples, due to their larger fiber diameter. The electrospun samples reveal an earlier plastic regime, with the O-ES samples possessing higher resistance than RO-ES, as the aligned fibers allow the sample to withstand higher forces. O-ES samples in dry state reach 348.23 ± 16.16 MPa (276.96 ± 12.09 MPa when wet), a value close to, but still inferior to the wet P-cocoons. The lowest values correspond to the RO-ES fibers (255.54 ± 13.46 and 198.18 ± 9.04 MPa dry and wet, respectively), concurrently revealing they possess the lowest linear regime capacity, quickly giving way to plastic regime. The fact that the stress-strain measurements were taken in the direction the fibers were spun (not perpendicularly) explain the higher resistance of O-ES samples when compared to the RO-ES sample [45]. The cocoons’ superior strength can be attributed to greater fiber-size, as well as the presence of sericin which has been shown to enhance the tensile properties of regenerated silk filament [46]. As is later proven by the proofs of 0 2 4 6 8 10 12 14 0 10 20 30 40 50 Stress (MPa) Strain (%) Dry P-cocoon w/ plasma Wet P-cocoon w/ plasma Dry RO-ES w/ plasma Wet RO-ES w/ plasma Dry O-ES w/ plasma Wet O-ES w/ plasma a) O-ES RO-ES P-cocoon 0 100 200 300 400 500 600 700 Young's modulus (MPa) Dry Wet b) 2000 1800 1600 1400 1200 1000 Absorbance (a.u.) Wavenumber (cm-1) P-cocoon P-cocoon w/ plasma RO-ES RO-ES w/ plasma O-ES O-ES w/ plasma c) Amide I Amide II Amide III 100 150 200 250 300 350 Temperature (ºC) P-cocoon P-cocoon w/ plasma RO-ES RO-ES w/ plasma O-ES O-ES w/ plasma Heat flow (a.u.) 2 W.g-1 endo d) Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 117 concept, these values show the samples are more than able to withstand the conditions necessary for carrying aqueous assays. Figure 2.4.3b shows that all samples are consistently more resistant to mechanical deformation when dry. Cocoons present the highest Young’s modulus, with values of 592.13 ± 19.83 MPa in a dry state and 377.2 ± 14.39 MPa when wetted, the highest values of the three samples. This data, in association with knowing the deformation occurs at a network structure level, shows the samples possess an ability to recuperate their initial structure. FTIR spectra (Figure 2.4.3c) of samples exposed and unexposed to plasma treatment show no relevant differences among them, indicating that the use of O2 plasma does not induce any significant chemical change. amide I (which corresponds to C=O stretching) and II (N-H in-plane bending) bands with peaks at 1616 and 1508 cm-1 are evident in the cocoons’ spectra, with a clear shift to higher wavenumbers for all RO-ES and O-ES samples, peaking at 1648 and 1527 cm-1 respectively. The bands corresponding to amide III (–N and N–H functionalities) at around 1263 and 1230 cm−1 are less apparent but may also be correlated with an alteration of the secondary structure, an increase of the relative proportion between the random coils and β-sheet conformations [44,47]. This alteration can be attributed to the slow solvent evaporation during the electrospinning process, which leads to improved polymer chain organization and consequentially a highly crystallized structure [48]. The lower peaks in the P-cocoon samples for Amide I, II and III may be due the presence of sericin which seems to have an effect of masking the fibroin and of attenuating the vibrational peaks, as sericin is present as a sheath over the fibroin core of the fiber [49]. The degree of crystallinity of the different samples was calculated from the FTIR-ATR spectra, and the results are shown in Table 2.4.1. Table 2.4.1. Crystallinity degree of the Bombyx mori cocoons and electrospun SF samples. Sample XC (%) P-cocoon 47.6 ± 1.9 P-cocoon w/ plasma 47.3 ± 1.9 RO-ES 43.2 ± 1.7 RO-ES w/ plasma 42.9 ± 1.7 O-ES 43.1 ± 1.7 O-ES w/ plasma 42.9 ± 1.7 Crystallinity degrees of approximately 47 % and 43 % were obtained for the Bombyx mori cocoons and electrospun SF samples, respectively. This difference is within experimental margin considering the natural origin of the cocoon and the typical variations between samples [50]. No variations in the degree of crystallinity of the samples are observed due to the plasma treatment, as its effect is restricted to the fiber surface [41]. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 118 Figure 2.4.3d shows the DSC thermograms of the different samples for a heating scan from 60 to 375 °C. Cocoons show the endothermic peak at 310 C associated with the heat-protective sericin layer [51]. Silk I conformation, an organized, crystalline form of fibroin, transforms into the β-sheet structure of silk II with the application of mechanical strain or other energy inputs. The presence of silk II or both I and II is correlated with the endothermic peaks at higher temperatures [52], and a larger presence of silk II crystalline domain may have been induced by the pressing of the cocoons. O-ES and RO-ES samples show the characteristic endothermic peak around 280 C due to the degradation of highly crystalline SF structures, specifically the side chains groups amino acid residues and the cleavage of peptide bonds [48, 53]. This lower temperature peak is ascribed to the sole presence of fibroin in the electrospun samples, as opposed to fibroin and sericin in the P-cocoons. There are no significant changes between the plasma-exposed and untreated samples, as the treatment only acts at the surface level. Contact angle and capillary flow rate assays Contact angle and capillary flow rate results are presented in Figure 2.4.4. Figure 2.4.4. a) Contact angle and b) capillary flow rates in the antigravity direction of a dye solution for the oriented (O-ES), randomly-oriented (RO-ES) electrospun Bombyx mori silk fibroin samples and pressed cocoons (P-cocoons). The hydrophobic nature of B. mori and ES samples is a key issue that must be overcome for their use as microfluidic substrates, where capillary flow is essential. Surface wettability is governed by surface chemistry, surface energy and morphology as well as by the properties of the solution [54]. CA is the most used method to measure these properties and provides accurate results about the wettability of materials and was used in both B. mori cocoons and SF electrospun samples [39, 44]. O-ES RO-ES P-cocoon 0 20 40 60 80 100 120 140 Contact angle (o) w/o plasma w/ plasma a) O-ES RO-ES P-cocoons 0 10 20 30 40 50 60 70 80 90 Capillary flow rate (mm.min-1) w/o plasma w/ plasma b) Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 119 Morphology and plasma treatment have a direct effect on water CA values and therefore on surface wettability (Figure 2.4.4a). Pressed B. mori samples have a CA value of 130 ± 4.5°, representing a slightly hydrophobic behavior when compared to the ES samples which have lower CA values of 117.7 ± 3.4 ° and 104.1 ± 3.8 ° for the RO-ES and O-ES SF samples, respectively. This difference can be explained by the higher surface roughness of the P-cocoons samples when compared to the ES samples, as well as differences in terms of compactness, crystallinity, and composition as cocoons still possess sericin, while the electrospun samples are only composed of fibroin [55]. The CA of all O2 plasma-treated samples, Pcocoons, O-ES and RO-ES fibers decreased to 0 °, with the water drop being completely absorbed after a 30 s exposure. The reduction of the CA value is a clear indication of the increase in wettability, based on the introduction of C-O and C=O groups [41]. Capillary flow rate evaluation ascertains the ability of the samples to generate passive capillary flow. The results for the different samples can be found in Figure 2.4.4b. The samples that were not exposed to plasma treatment show no visible capillary flow, as expected due to their hydrophobic nature. Treated cocoons and RO-ES samples are characterized by similar flow rates: 44.8 ± 3.75 and 46.5 ± 2.05 mm.min-1, respectively. The closeness of these results is related to the similar microstructural features governed by the non-oriented fibers, with the differences in fiber diameter seemingly having no effect on capillary flow. Oriented fibers allow solutions to follow a specific direction without obstruction from transversal fibers, as is verified by the higher mean value of capillary flow, 82.1 ± 4.95 mm.min-1, obtained for the O-ES samples. Albumin, uric acid and glucose colorimetric assays on silk substrates After wax-printing the pattern for the portable analytical system, colorimetric analysis for albumin was first carried out for all the silk-based substrates under study. These tests are depicted in Figure 2.4.5a. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 120 Figure 2.4.5. Colorimetric albumin bioassay proofs of concept on silk-based substrates and the corresponding calibration curves using a) pressed Bombyx mori cocoons (P-cocoons), oriented (O-ES) and randomly-oriented (RO-ES) electrospun plasma-treated. b) Colorimetric uric acid assays in reused cocoons, corresponding calibration curves and SEM image of cocoon microstructure after the washing process. c) Colorimetric glucose assays on two times reused cocoons, corresponding calibration curves and SEM image of cocoon microstructure after the second washing process. Overall, print quality and wax adhesion varies for each substrate. The plasma treated SF substrates show more defined borders in comparison to the cocoon substrates, as expected given the cocoons’ more irregular surface even after pressed. On the other hand, wax adhesion is improved in the cocoons. Electrospinning produces thinner fibers than those of the P-cocoons, which are also deposited in a much more compact manner, leading to smaller interstices. This may explain the absence of wax at certain spots in the electrospun substrates, which nevertheless did not hinder the applicability of the materials. The same principle applies to the cocoons: as the interstitial spaces between fibers are wider, vestigial Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 121 spaces that were not filled with wax during the thermal curing process may allow small leakages of fluid beyond the printed frontiers, as is observed in Figure 2.4.5a. This did not affect the assays in any of the cases, immediately apparent by the color formation which gradually grows in intensity as concentrations increase, and also confirmed by mean gray value evaluation. The calibration curves for the albumin assay show that colorimetric assays on all substrates show linear fittings, with R2 values of 0.992, 0.957 and 0.991 for RO-ES, O-ES, and Pcocoons respectively, demonstrating the strong correlation between variation in color intensity and concentration. The somewhat lower R2 value for the O-ES substrate may be due to the more heterogeneous color formation within the reaction chamber due to fiber orientation. Aligned fiber orientation may prevent less homogeneous reagent and sample mixing than occurs in the other substrates. Nonetheless, with a value of 0.957, the correlation is still evident. The cocoons exhibit a lower slope value, which is associated with a lower sensitivity to the color gradient, which may be associated with how the color tone is produced when contrasting with the substrate’s own color. This is not detrimental for the assay, as the correlation is still very strong (R2 0.991). After the assay, P-cocoons were then cleaned according to the indicated protocol (see experimental section) and uric acid colorimetric assays were performed, as illustrated in Figure 2.4.5b. In addition to the reagent and sample used to carry the albumin colorimetric assay, the cleaning process removed some of the wax from the printed zone of the cocoons, as indicated by the lighter areas observed in some samples (figure 2.4.5b). In this sense, the cleaning process can be further optimized to better preserve the wax patterns. Nevertheless, the fluids are still mostly contained within the reaction chambers and color complex formation is clear, which is confirmed by the corresponding fitting. R2 value decreased only slightly (from 0.991 to 0.983), showing that a strong correlation between concentration and color intensity is maintained. The slope value is closer to those observed in the ES substrates, higher than the albumin assay and hence an improvement towards the previous assay in the same cocoons. This may be due to a better contrast of the pigment when interacting with the substrate. Substrates were once again cleaned and the glucose assays were carried, as shown in Figure 2.4.5c. The cleaning process did not remove wax any further than in the previous assay, which suggests the amount of wax previously washed away was mostly excess from the surface. The leakage from the channels and chambers is slightly higher but the hydrophobic barriers still fulfill their role in containing the fluids. The R2 value again decreases in a very small amount from 0.983 to 0.967, showing that a strong correlation is still present for the glucose assay. The slope value is considerably higher than the result from the uric acid assay (79.02 compared to 26.4), suggesting the pigment may be even more adequate for this substrate. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 122 Conclusions Bombyx mori cocoons and ES silk fibroin were used to manufacture substrates for a next generation of sustainable portable analytical devices. The substrates consist of pressed cocoons and both oriented and randomly-oriented ES fibers prepared from purified silk fibroin extracted from the same type of cocoon. Oxygen plasma treatment was applied to these samples to make them superhydrophilic, and a thorough characterization of their physicochemical properties and overall behavior when interacting with aqueous solutions was carried out, showing mechanical stability, thermal resistance, and ability for capillary flow. Plasma treated Bombyx mori cocoons in particular show superhydrophilicity, capillary flow rates of 44.8 ± 3.75 mm.min-1, and mechanical resistance, with Young’s modulus values of 592.13 ± 19.83 MPa (dry conditions) and 377.2 ± 14.39 MPa (wet conditions). Proofs of concept were produced by wax-printing hydrophobic barriers according to a pattern composed of insertion and reaction chambers joined by channels. Although ES samples allow higher printing quality, an albumin colorimetric assay showed that all substrates performed well and a suitable quantity of fluid was contained inside the chambers, allowing formation of the colored complexes. As P-cocoons require no further processing besides pressing and plasma treatment, they were selected for subsequent cleaning and further colorimetric testing. Uric acid and glucose tests were successfully carried out after in between cleaning procedures, showing that reutilization is possible without compromising substrate and printed microstructure and, therefore, system functionality. B. mori P-cocoons as well as silk fibroin ES mats are demonstrated to be suitable substrates for the development of portable analytical devices. Natural-based materials as reusable substrates are thus proposed for a next generation of PADs with reduced environmental impact, not only for colorimetric assays, but also for other methods of detection in areas such as biomedicine, environmental monitoring, or food quality control, among others. References 1. Tönnies, T., et al., Projected number of people with diagnosed Type 2 diabetes in Germany in 2040. Diabetic Medicine. 2019; 36(10): 1217-25. 2. Van der Schaft, N., et al., The association between serum uric acid and the incidence of prediabetes and type 2 diabetes mellitus: The Rotterdam Study. PLOS ONE. 2017; 12(6): e0179482. Chapter 2.4 – Natural silk based reusable materials for portable analytical devices 123 3. Virdis, A., et al., Identification of the Uric Acid Thresholds Predicting an Increased Total and Cardiovascular Mortality Over 20 Years. Hypertension. 2020; 75(2): 302-308. 4. 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Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 228 Proof-of-concept The origami device has been submitted to functional experiments in order to prove the multifunctional detection capability: detection of the nanoparticles through EDX (Figure 4.1.7a-b); detection through VSM (Figure 4.1.7c-d); detection through FTIR (Figure 4.1.7e-f); and color detection through a mobile phone camera (Figure 4.1.7g-h). 0.0 0.2 0.4 0.6 0.8 1.0 1.0 1.5 2.0 2.5 3.0 3.5 -20k -10k 0 10k 20k -400 -200 0 200 400 0.0 0.2 0.4 0.6 0.8 1.0 -50 0 50 100 150 200 250 300 350 600 550 500 450 -10 -5 0 0.0 0.2 0.4 0.6 0.8 1.0 0 2 4 6 8 10 12 0.0 0.2 0.4 0.6 0.8 1.0 100 120 140 160 180 wt.% of Fe |Fe3O4| (mg.L-1) EDX images (Red: Fe) Origami photographs y=2.992x+1.092 R2=0.996 a) b) c) d) e) f) g) h) 0.8mg.ml-1 0.2mg.ml-1 0.05mg.ml-1 0.003125mg.ml-1 i)ii) iii)iv) i) ii) iii)iv) v) Magnetization (memu.g-1) DC magnetic field (Oe) 0.8 mg.l-1 0.2 mg.l-1 0.05 mg.l-1 0.0125 mg.l-1 0.003125 mg.l-1 0.8 mg.ml-1 0.2 mg.ml-1 0.05 mg.ml-1 0.0125 mg.ml-1 0.003125 mg.ml-1 Magnetization (memu.g-1) y=423.85x+2.67 R2=0.9988 |Fe3O4| (mg.ml-1) Absorbance (a.u.) Wavenumber (cm-1) Peak size (a.u.) |Fe3O4| (mg.ml-1) y=9.28-1.70ln(x+0.0016) R2=0.93 y=-77.08x+77.99 R2=0.9987 Mean Gray Value (a.u.) |Fe3O4| (mg.ml-1) Figure 4.1.7. a) Color map obtained by EDS in the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: iv) 0.8 mg.ml-1; iii) 0.2 mg.ml-1; ii) 0.05 mg.ml-1; and i) 0.003125 mg.ml1. b) Relation between Fe wt.% in the testing zone rectangle (obtained from EDS) and the Fe3O4 Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 229 concentration. c) Room-temperature magnetization as a function of the applied DC magnetic field for the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: 0.8 mg.ml-1; 0.2 mg.ml-1; 0.05 mg.ml-1; 0.0125 mg.ml-1; and 0.003125 mg.ml-1. d) Relation between the saturation magnetization (at 20 kOe) found on the testing zone rectangle and the Fe3O4 concentration. e) Infrared absorbance vs. wavenumber for the testing zone rectangle into which were added 50 μl of the mixture with Fe3O4 content of: 0.8 mg.ml-1; 0.2 mg.ml-1; 0.05 mg.ml-1; 0.0125 mg.ml-1, and 0.003125 mg.ml-1. f) Relation between the 530 cm−1 FTIR band (reported as representing Fe-O vibrations in Fe3O4) absorbance and the Fe3O4 concentration on the drop added to the testing zone rectangle. g) Mobile phone photographs (iPhone 12) of the origami testing platform into which were added 50 μl of the mixture with Fe3O4 content of: v) 0.8 mg.ml-1; iv) 0.2 mg.ml-1; iii) 0.05 mg.ml-1; ii) 0.0125 mg.ml-1, and i) 0.003125 mg.ml-1. h) Relation between the mean grey value (obtained through image J software) and the Fe3O4 concentration of the drop added to the testing zone rectangle. EDX analysis carried out in the testing zone square allowed to relate the Fe wt.% with the concentration of the different Fe3O4@H2O suspensions with high linearity (R2 = 0.996; Figure 4.1.7a-b). Such linearity was improved to 0.998 when VSM measurements were used to relate the saturation magnetization obtained in the testing zone square (in which they were previously added the different Fe3O4@H2O drops) with the concentration of the different Fe3O4@H2O suspensions (Figure 4.1.7c-d). Taking advantage of previous studies reporting that the FTIR band located at 565 cm−1 - 580 cm−1 is related with Fe-O stretching in highly crystalline Fe3O4 nanoparticles [61], the spectra of the different testing squares was been used to relate the absorbance of the 565 cm−1 - 580 cm−1 band with the Fe3O4 wt.% (Figure 4.1.7e-f). Such approach proved to be the one with the lowest correlation, yet it proved to be effective in detecting Fe3O4 concentrations as small as 3.125 μg.ml-1. As the Fe3O4 nanopowders introduce a brown-black color to the white substrates [62], the images taken with a mobile phone and later analysed with the image J software (mean grey value: Figure 4.1.7g-h) allowed to almost match the maximum linearity of the VSM approach. Such method is quite simple and only requires a mobile phone equipped with a camera and a photo analysis software. Thus, it is confirmed that the proposed MOPAS allows to detect magnetic nanoparticles in mixtures with low Fe3O4 wt.% (3.125 μg.ml-1) with the use of a small amount of testing liquid (50 μl), exhibiting a limit of detection lower than 156 μg of Fe3O4. Those features can be further optimized with the use of the use of surface-functionalized magnetic nanoparticles that link to biomarkers or magnetoactive disposal products, being therefore a powerful platform for the development of biomedical µPADs. Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 230 Conclusions In conclusion, this work presents the development, fabrication and evaluation of a flexible point-of-care device upgraded with in situ magnetic concentration capability. When compared with magnetic flow detectors currently found on the market and fabricated on rigid substrates, the advantages of the proposed platform are the economic viability of the fabrication and the mass production allowance as a result of the low cost, printability and lightweight of the device (less than 100 mg). The mechanical properties of the device (flexibility, foldability, and 0.5-2 GPa Young’s Modulus) and printing stability/durability, provide the required long-term stability of the POC multifunctional platform for practical applications. The content of the Fe3O4 within the analysis rectangle was determined through energy-dispersive X-ray spectroscopy, vibrating sample magnetometry, infrared spectroscopy; and color change (greyscale) in photographs taken by a mobile phone, with linearity of 0.996, 0.998, 0.930, and 0.998, respectively, for Fe3O4@H2O mixtures with Fe3O4 wt.% up to 3.125 μg.ml-1 (50 μl of Fe3O4@H2O), being determined a limit of detection lower than 156 μg of Fe3O4. The proposed device provides a state-of-the-art solution to incorporate printed magnets on a flexible µPAD platform that can serve as a platform for the implementation of the detection of a high range of diseases (with magnetoactive disposal products or with the use of biomarkers) in a new type of multifunctional biomedical device that combines X-ray spectroscopy, vibrating sample magnetometry, infrared spectroscopy; and color change sensitivities. Thus, the proposed printed magnetic origami paper represents a lightweight, sustainable, printable and low cost platform for a successful disease detection procedure. References 1. Markwalter, CF., et al., Inorganic Complexes and Metal-Based Nanomaterials for Infectious Disease Diagnostics. Chemical Reviews (2019) 119: 1456-1518. 2. Osler, W. Aequanimitas., With Other Addresses to Medical Students, Nurses and Practitioners of Medicine. (Creative Media Partners, LLC, 2018). 3. Karlikow, M., et al., Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples. Nature Biomedical Engineering (2022) 6: 246-256. 4. Deng, J., et al., Nanosensors for Diagnosis of Infectious Diseases. ACS Applied Bio Materials (2021) 4: 3863-3879. Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 231 5. 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Zhang, T., et al., A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution. Nature Biomedical Engineering 2022; 6: 957-967. 28. Oliveira, J., et al., Polymer-based smart materials by printing technologies: Improving application and integration. Additive Manufacturing 2018; 21: 269-283. 29. Zhao, TH., et al., Printing of Responsive Photonic Cellulose Nanocrystal Microfilm Arrays. Advanced Functional Materials 2019; 29: 1804531 30. Siqueira, G., et al., Cellulose Nanocrystal Inks for 3D Printing of Textured Cellular Architectures. Advanced Functional Materials 2017; 27: 1604619 31. Yu C., et al., Recent Advances in Design of Flexible Electrodes for Miniaturized Supercapacitors. Small Methods 2020;.4:1900824 32. Brito-Pereira R., et al., Reconfigurable 3D-printable magnets with improved maximum energy product. Journal of Materials Chemistry C 2020; 8: 952-958. 33. Dixon, C, Lamanna, J, Wheeler, AR. Printed Microfluidics. Advanced Functional Materials 2017; 27:1604824 34. Chowdhury NA., et al., Sustainable Recycling of Rare-Earth Elements from NdFeB Magnet Swarf: Techno-Economic and Environmental Perspectives. ACS Sustainable Chemistry and Engineering 2021; 9: 15915-15924. 35. Costis S., et al., Recovery potential of rare earth elements from mining and industrial residues: A review and cases studies. Journal of Geochemical Exploration 2021; 221: 106699 Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 233 36. Gawande, MB, Branco, PS, Varma, RS. Nano-magnetite (Fe3O4) as a support for recyclablecatalysts in the development of sustainable methodologies. Chemical Society Reviews 2013; 42: 3371-3393. 37. Nicolas-Boluda., et al., Self-Assemblies of Fe3O4 Nanocrystals: Toward Nanoscale Precision of Photothermal Effects in the Tumor Microenvironment. Advanced Functional Materials 2021;. 31: 2006824 38. Chen X., et al., Electrospinning Multilayered Scaffolds Loaded with Melatonin and Fe3O4Magnetic Nanoparticles for Peripheral Nerve Regeneration. Advanced Functional Materials 2020; 30: 2004537 39. McCoey, JM., et al., Quantum Magnetic Imaging of Iron Biomineralization in Teeth of the Chiton Acanthopleura hirtosa. Small Methods 2020; 4: 1900754 40. Knowlton, S., et al., 3D-printed smartphone-based point of care tool for fluorescenceand magnetophoresis-based cytometry. Lab on a Chip 2017; 17: 2839-2851. 41. Kim, J., et al., A soft and transparent contact lens for the wireless quantitative monitoring of intraocular pressure. Nature Biomedical Engineering 2021; 5: 772-782. 42. Martins, P., et al., Tailored Magnetic and Magnetoelectric Responses of Polymer-Based Composites. ACS Applied Materials and Interfaces 2015; 7: 15017-15022. 43. Hao, T., et al., Highly robust, transparent, and conductive films based on AgNW-C nanowires for flexible smart windows. Applied Surface Science 2021; 559:149846 44. Krainer, S., Smit, C., Hirn, U., The effect of viscosity and surface tension on inkjet printed picoliter dots. RSC Advances 2019; 9: 31708-31719. 45. He, B., et al., The roles of wettability and surface tension in droplet formation during inkjet printing. Scientific Reports 2017; 7: 11841 46. Alade, OS., et al., Novel Approach for Improving the Flow of Waxy Crude Oil Using Thermochemical Fluids: Experimental and Simulation Study. ACS Omega 2020; 5: 4313-4321. 47. Saji, VS., Wax-based artificial superhydrophobic surfaces and coatings. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2020; 602: 1251325 48. Rapp, BE., Chapter 9 - Fluids. In: Rapp BE (ed). Microfluidics: Modelling, Mechanics and Mathematics. 2017, pp 243-263. 49. Murata, H., Rheology - Theory and Application to Biomaterials. (INTECH Open Access Publisher, 2012. 50. Shakeel, A., Kirichek, A., Chassagne C., Effect of pre-shearing on the steady and dynamic rheological properties of mud sediments. Marine and Petroleum Geology 2020; 116: 104338. Chapter 4.1 - Wax printed magnetic origami paper as a platform for low cost disease multi-tool detection 234 51. M'Barki, A., Bocquet, L., Stevenson, A., Linking Rheology and Printability for Dense and Strong Ceramics by Direct Ink Writing. Scientific Reports 2017; 7: 6017 52. Lewis, JA., Direct ink writing of 3D functional materials. Advanced Functional Materials 2006; 16: 2193-2204. 53. Dabbaghi M., et al., Viscoelastic and deformation characteristics of structurally different commercial topical systems. Pharmaceutics 2021; 13: 1351 54. Xerox. Xerox® ColorQube® 8580/8880Color PrinterImprimante CouleurUser Guide. https://usermanual.wiki/Xerox/XeroxColorqube8880UsersManual684582.347452995/html#pf3; 2015. 55. Madhuranthakam, CMR., Pandiyan, S., Penlidis, A., Performance Evaluation of Nonacosan-10-olBased Polyethylene Packaging Material Using Molecular Dynamics Simulations. Polymers 2022; 14: 1779 56. Martins, P., et al., Dielectric and magnetic properties of ferrite/poly(vinylidene fluoride) nanocomposites. Materials Chemistry and Physics 2012; 131: 698-705. 57. Gonçalves, R., et al., Development of magnetoelectric CoFe2O4 /poly(vinylidene fluoride) microspheres. RSC Advances 2015; 5: 35852-35857. 58. Krishnan, KM., et al., Nanomagnetism and spin electronics: materials, microstructure and novel properties. Journal of Materials Science 2006; 41: 793-815. 59. Liu, XD., et al., Hydrothermal synthesis of superparamagnetic Fe3O4 nanoparticles with ionic liquids as stabilizer. Materials Research Bulletin 2015; 62: 217-225. 60. Houshiar, M., et al., Synthesis of cobalt ferrite CoFe2O4 nanoparticles using combustion, coprecipitation, and precipitation methods: A comparison study of size, structural, and magnetic properties. Journal of Magnetism and Magnetic Materials 2014; 371: 43-48. 61. Chipara, DM., et al., PVDF-Fe3O4 nanocomposites: spectroscopic investigations. Journal of Polymer Research 2022; 29: 48907 62. Pfaff, G., Iron oxide pigments. Physical Sciences Reviews 2021; 6: 535-548. 4.2. Wax printable conductive and low-power thermal actuator paper-based analytical device In this chapter is developed a paper-based analytical device printed with multifunctional hydrophobic composites based on conductive graphene nanoplatelets (GNP) integrated into the wax matrix to allow a dual role of barrier and heater, the latter being required in a large variety of temperature-sensitive reactions and applications of microfluidic technology. The benefits of µPADs allied to the increased functionality and performance of the developed wax, hold great promise to meet the requirements for a next generation of versatile, effective and accurate µPADs for an increasing number of applications. This chapter is based on the following work: R. Brito-Pereira, C. Ribeiro, P. Costa, V. Correia, V. F. Cardoso, S. Lanceros Mendez, Graphene based printable conductive wax for low-power thermal actuation on microfluidic paper-based analytical devices. (Submitted). Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 236 Introduction Paper has been a base material for many applications for centuries. It is based on cellulose, which is a renewable polymer, produced by trees, plants and some non-pathogenic bacteria [1]. Besides being affordable and widely available, this polymer features interesting physicochemical properties such as biocompatibility, flexibility, low weight and hydrophilicity by nature, which results in an effortless fluid flow by capillary action with no need for external pumps or pressure [2,3]. Moreover, it allows modification with a variety of functional groups to perform specific analytical assays [4]. These assets make it suitable for the development of devices, including the µPADs, a concept that was introduced in 2007 by Whitesides and co-workers when photolithography was employed to patterned paper and colorimetric analyses were implemented [5]. Although Whatman nº1 paper is the gold standard used microfluidic substrate, other versions such as Whatman nº4, nitrocellulose, bacteria cellulose nanopaper and nylon membranes are also used to develop µPADs [6]. Recently, new polymer-based materials with promising and tailorable physicochemical properties, including controllable flow rates, have been introduced and successfully tested as alternative to the commonly used microfluidic paper substrates [7–10]. µPADs have been initially developed for resources limited environments, in-field applications or for their use in private homes, as they fulfil the ASSURED criteria defined by the WHO and being therefore suitable for PADs development [4]. Thus, their potential has expanded to such an extent that nowadays this technology is used in numerous applications worldwide such as clinical [1], veterinary [11], food industry [12], agriculture [13], biodefense [14], energy [15] and environmental purposes [16], among others [17– 19]. Its popularity is due to the fact that µPADs present several advantages comparatively to traditional microfluidic devices made of silicon, glass or polymer (e.g. PDMS), as well as to the conventional laboratory techniques, including low cost, low sample volume consumption, ease of use, portability, in situ measurement, high sensitivity, and no need for sophisticated laboratory equipment and trained user [20,21]. Moreover, they can be used not just in lateral flow assays but also for multiplex analysis and complex analysis requiring multiple steps (e.g. sample transport, pre-treatment, mixing, reaction, separation) [22,23]. Thus, significant growth of academic research on paper-based analytical fabrication methods and integrated tools (e.g, optical sensors, pressure sensors, micropumps, microvalves, heaters, temperature sensors, among others [24]) has been observed for the past decade to increase their functionality and performance. This will allow the development of effective, accurate and standardize µPADs and promote their commercialization, maintaining simplicity and without compromising its cost [25-27]. Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 237 For instance, given the hydrophilic nature of paper, hydrophobic boundaries with specific patterns are often required to restrict the fluid flow of samples and reagents to specific pathways of the hydrophilic substrate [28]. To address these goals, different strategies have been used such as photolithography, wax printing, inkjet printing and screen-printing. Folding several layers of patterned paper, origami inspired, has also been employed to obtain three-dimensional (3D) µPADs. Several review articles address these techniques in detail [2,22,29–31]. Each of those approaches has its own benefits and limitations, so the choice is often based on cost, substrate, fabrication time, equipment availability and also specific application demands. Photolithography used a chemical photoresist to infiltrate the paper substrate and create the barrier by exposing the substrate to light through a photomask. This technique presents good resolution but suffer from the cost of the photoresist and organic solvent, fragile nature of the resulting devices and possible production of background signals [4]. A technique that has become popular to pattern channels in µPADs is wax printing. Although the printing resolution cannot catch up with photolithography, the production steps are simpler, faster and cheaper, being, thus, best suited for large scale production [32]. It involves a three-step method including the pattern drawing using an appropriate software, the paper printing using a commercial wax printer and the heating step to allow impregnation of the wax through the paper to the opposite surface [33]. Moreover, wax-based devices are mechanically resistant and are compatible with lamination, allowing to fabricate complex two (2D) or 3D structures for multipurpose applications [28]. Nonetheless, despite the excellent quality of wax printing that makes it one of the materials and techniques most used in the manufacture of uPADs systems, its function is passive, working only as boundaries to delineate hydrophilic channels without any action on the fluids. On the other hand, thermal actuators are commonly needed in microfluidic technology for precise temperature-sensitive reactions and applications such as colorimetric analytic analysis [9,34], cell culture [35,36], polymerase chain reaction [37,38], cell lysis [39,40], among others technological applications. However, they are still not well developed for µPADs. As instance, heaters were implemented in µPADs in the forms of non-contact inductive heater [41], laser heater [42,43], infrared heater [44], and microwave heater [45,46], among others, being resistive heaters [24,47,48] the most commonly used due to their low fabrication cost and mature fabrication process. However, they required additional processing techniques for their proper integration into µPADs. In this context, the present work reports on the implementation of active functionalities to the hydrophobic wax through the integration of conductive graphene fillers, on behalf of their electrical and thermal conductivity and good mechanical properties [49–51]. Conductive wax inks and printed patterns have Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 244 results (Figures 4.2.2c and d) and in the proofs of concepts discussed later (Section 3.2), the hydrophobic barriers needed to contain fluids are guaranteed. To evaluate how the printing process and the corresponding thermal treatment (curing process) affects the mechanical properties of the printed substrates, peeling assays (Figures 4.2.4a and b) and stressstrain measurements (Figures 4.2.4c and d) in tensile mode were carried out. Uncured prints can find application when the conductive waxes, i.e. heater, do not need to cross the entire substrate and work as fluid barriers, as occurred in the second proof-of-concept. However, it can negatively affect the mechanical properties, as it will be shown in the following, so that the need for curing must be properly evaluated according to the application. 025 50 75 100 125 0 50 100 150 200 Force (N) Time (s) 200 N 150 N 100 N 50 N 050 100 150 200 0.0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 Unc. Wax@Paper Unc. 5GNP/Wax@Paper Unc. 10GNP/Wax@Paper Unc. 15GNP/Wax@Paper Unc. 20GNP/Wax@Paper C. Wax@Paper C. 5GNP/Wax@Paper C. 10GNP/Wax@Paper C. 15GNP/Wax@Paper C. 20GNP/Wax@Paper Weight Loss (%) Pressed Force (N) a) b) 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 45 50 55 60 Paper Unc. Wax@Paper Unc. 5GNP/Wax@Paper Unc. 10GNP/Wax@Paper Unc. 15GNP/Wax@Paper Unc. 20GNP/Wax@Paper C. Wax@Paper C. 5GNP/Wax@Paper C. 10GNP/Wax@Paper C. 15GrNP/Wax@Paper C. 20GNP/Wax@Paper Stress (MPa) Strain (%) 0 200 400 600 800 1000 1200 1400 1600 1800 Young Modulus (MPa) Samples Paper Unc. Wax@Paper C. Wax@Paper Unc. 5GNP/Wax@Paper C. 5GNP/Wax@Paper Unc. 10GNP/Wax@Paper C. 10GNP/Wax@Paper Unc. 15GNP/Wax@Paper C. 15GNP/Wax@Paper Unc. 20GNP/Wax@Paper C. 20GNP/Wax@Paper c) d) Figure 4.2.4. a) Force profile of the peeling assays for the various wax printed samples as a function of time and compression force; b) respective weight loss after the peeling assays as function of the compression force; c) stress-strain curves for the various wax printed samples obtained at room temperature; d) respective Young’s modulus values obtained from a). Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 245 The force profile during the peeling assays for the printed layers (Figure 4.2.4a) shows that the lowest compression force of 50 N leads to the lowest peeling force of ≈11 N, while the highest compression force of 200 N leads to the highest peeling force of ≈25 N. Regarding Figure 4.2.4b, it is shown that increased compression forces lead to increased weight loss, regardless the sample. Moreover, the benefits of thermal treatment on the stability of the wax print is demonstrated, with less weight loss in the cured samples. A maximum of ≈1.02 % is loss for the cured 20 wt.% GNP/Wax at a compression force of 200 N (≈1.58 % for the respective uncured sample) whereas a minimum of ≈0.27 % is loss in the case of cured 5 wt.% GNP/Wax (≈0.45 % for the respective uncured), very close to the value of ≈0.23 % for the cured neat wax (≈0.44 % for the respective uncured). Although some weight loss is evidenced, which is more pronounced for high compression forces and uncured waxes, the results confirm the mechanical stability of the printed waxes, as the observed weight loss does not compromise the mechanical and functional characteristics of the samples. Further, none of the samples experienced weight loss in a second round of assays. Accordingly, the proofs of concepts will be performed using cured waxes. The characteristic stress-strain curves of the processed samples are presented in Figure 4.2.4c. From the liner regimes of the curves, the Young’s modules were obtained for each sample applying Hooke’s law and the values are presented in Figure 4.2.4d. Commercial Whatman nº1 paper features almost no plastic regime, with a fracture near the end of the linear elastic region of the curve. On the other hand, all printed paper samples present both higher stress and strain at the breaking point. This increase proves to be significantly higher when the wax is thermally cured, indicating a clear improvement of the mechanical properties. Comparable improvements are detected with the increase of GNP filler concentration in the wax. The analysis of the Young’s modules obtained in the linear mechanical regime, where the materials are generally used, allow to reinforce the previously mentioned conclusions. In fact, increasing GNP filler concentration in the wax leads to higher Young’s modules, the increase being much more significant when the wax is cured. An increase of ≈67 % is obtained in the cured wax compared to the neat paper. Moreover, the addition 20 wt.% GNP to the wax and the curing process allow an increase of ≈49 % compared the cured neat wax and ≈148 % compared to the neat paper. Regarding the relevance of thermal curing in the mechanical properties and analysing the samples composed by 20 wt.% GNP/Wax, an increase of ≈53 % is obtained after curing. The increase of the Young’s modulus can thus be explained by the impregnation of wax in the fibrillary structure of the Whatman nº1 paper in combination with the addition of GNP fillers (with a large Young’s modulus of 20-60 GPa [53]) and the corresponding filler-matrix interaction. Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 246 The sheet electrical resistance of wax and the corresponding composites up to 20 wt.% are presented in Figure 4.2.5a. 0 5 10 15 20 0 2 4 6 8 10 12 14 Conductivity (S.m-1) Gr concentration (wt.%) 0 2 4 6 8 10 12 14 16 20 30 40 50 60 70 80 90 100 110 15 mm long 1 mm width Temperature (ºC) Voltage applied (V) C. Wax@Paper (T) C. Wax@Paper (B) C. 5GNP/Wax@Paper (T) C. 5GNP/Wax@Paper (B) C. 10GNP/Wax@Paper (T) C. 10GNP/Wax@Paper (B) C. 15GNP/Wax@Paper (T) C. 15GNP/Wax@Paper (B) C. 20GNP/Wax@Paper (T) C. 20GNP/Wax@Paper (B) a) b) 0 5 10 15 20 25 30 20 30 40 50 60 70 80 90 100 110 Temperature (ºC) Time (s) 0V 1V 5V 9V 12V 15V 0 2 4 6 8 10 12 14 16 20 30 40 50 60 70 80 90 100 110 2 mm Temperatute (ºC) Voltage Applied (V) 1 mm Dry 2 mm Dry 1 mm Wet 2 mm Wet 1 mm Conductive line c) d) Figure 4.2.5. a) Electrical conductivity values of the various wax based printed samples as a function of GNP concentration. b) Temperature at the top (T) and bottom (B) of the different GNP/Wax lines printed and cured on Whatman nº1 paper as function of the applied voltage. c) Temperature variation as a function of time for various applied voltages measured at the top of the printed and cured 20 wt.% GNP/Wax lines. d) Temperature measured at the top of the substrate in the dry and wet state as function of the applied voltage, 1 and 2 mm away from the printed and cured 20 wt.% GNP/Wax lines. The electrical conductivity of the neat wax is near 2.1×10-12 S.m-1 increasing about 12 orders of magnitude with increasing filler content up to a value of ≈13.2 S.m-1 for the sample with a filler content of 20 wt.%. This increase in the electrical conductivity will have a direct effect on the generated heat capacity, as described in the following. Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 247 By means of a temperature probe, thermal measurements at the top and bottom of the different GNP/Wax lines (2x10 mm) printed and cured on Whatman nº1 paper were measured as function of the electrical voltage, after 30 s (Figure 4.2.5b). As expected, no heating is generated by neat wax, regardless the measuring surface. On the other hand, in all composite waxes, the higher the applied voltage, the higher the heating generated by the conductive lines. The temperature increase shows to be higher on the upper surface of the printed line (T) compared to the inner surface of the line (B), i.e. bottom surface of the substrate, which confirms that there is a higher accumulation of GNP fillers on the surface of the print, as previously demonstrated in the contact angle measurements (Figure 4.2.2d) and SEM images (Figures 4.2.3e-h). A maximum temperature of ≈107 C was measured at the top of the cured 20 wt.% GNP/Wax line (≈102 C below) for an applied voltage of 15 V, while a value of ≈37 C was obtained at the top of the cured 5 wt.% GNP/Wax line (≈32 °C below) for the same applied voltage. Higher voltages were not evaluated due to the thermal sensitivity of the Whatman Nº1 substrate, which is based on cellulose. However, more thermally resistant membranes, such as PVDF and their copolymers [8,54], can be an option if higher heating is required. Applying the voltage with alkaline batteries of 1.5, 9 and 12 V, temperatures of ≈31.5, 51.6 and 62.1 °C were obtained at the top of the cured 20 wt.% GNP/Wax line, respectively. These results are interesting for applications in several fields, since it is demonstrated that it is possible to adjust and control the heating generated through the proper selection of the applied electrical signal and/or the concentration of GNP fillers in the wax. Another interesting conclusion illustrated in Figure 4.2.5c is that the maximum temperature is achieved in a few seconds (≈9 s), remaining stable over time. Figure 4.2.5c reports on 20 wt.% GNP printed lines after curing. Finally, temperature was measured at a fixed distance from the conducting lines, namely at 1 and 2 mm to evaluate the heat loss through the paper. These dimensions were chosen considering that in µPADs, the channels have a very small width (mm or below) in order to allow adequate passive flows without the need for external pumps or pressure. The results are shown in Figure 4.2.5d for a conducting line of cured 20 wt.% GNP/Wax. Comparing with the results presented in Figure 4.2.5b, it is concluded that there is a small heat transfer, which is more accentuated when the paper is dry when compared to the wet state. This is explained by the fact that wet paper feature a higher thermal conductivity than dry paper, receptively [55,56]. For the maximum applied voltage of 15 V, a temperature variation of ≈4 and ≈7 °C (from the initial ≈107 °C at the top of the line) occurs at a distance of 1 mm from the line in the wet and dry states, respectively. At a distance of 2 mm, the temperature variation presents values of ≈6 and ≈10 °C in the wet and dry state, respectively. Those variations have to be taken into consideration when Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 248 precise temperature control is required and µPADs design and dimensions have to be optimized to account for those temperature gradients. Proofs of concept validation Thermochromic inks have the ability to change colour when heated above a certain temperature. This concept was employed in µPADs devices made of Whatman nº1 paper substrates and patterned using printed and cured neat wax and 20 wt.% GNP/Wax lines (Figure 4.2.6a). In this system, red and green thermochromic inks were used, changing to more translucent (light rose) and yellow at temperatures higher than 47 °C and 28 °C, respectively (Figure 4.2.6b). Different conductive lines were activated by means of a DC power supply at 12 V (Figure 4.2.6c) and 15 V (Figure 4.2.6d). At room temperature (≈25 C), the thermochromic inks feature a clear green and red colour (Figure 4.2.6b). When a voltage of 12 V is applied to the three lower conductive lines, a maximum temperature of ≈64.3 C was measured using the thermal imaging camera, near the ≈61.9 C obtained by means of the temperature probe (Figure 4.2.5b). This result in a colour change of the inks presented in the three lower channels means that the thermochromic activation temperatures are reached. On the other hand, the upper channel features a clear colour gradient from translucent red to bright red (light rose) also indicating a temperature gradient between values above and below the activation temperature. Thus, it becomes evident that there is a reduction in temperature with increasing distance between the reaction channel and the superior conductive line, as expected and previously concluded in Figure 4.2.5d. When the voltage is increased to 14 V and applied to the four lower conductive lines, all inks undergo colour changes, with a maximum temperature of ≈83.9 C measured with the thermal imaging camera, near the ≈80.1 C obtained using the temperature probe, much higher than the activation temperature of both thermochromic inks. Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 249 a) b) c) d) Figure 4.2.6. Photographs of a) µPADs system with individual hydrophilic channels delineated by printed and cured hydrophobic neat wax and hydrophobic conductive 20 wt.% GNP/Wax lines; b) two thermochromic inks at room temperature (red activated at > 47 C and green activated at >28 C); Resulting colour changes when specific conductive lines are subject to an electrical voltage of c) 12 V and d) 14V indicated by the blue arrows. Corresponding thermal images in inset. To further strengthen the capabilities of the manufactured materials as well as their suitability for µPADs applications, a second proof-of-concept was carried out using alkaline batteries of 1.5, 9 and 12 V instead of a DC power supply. For that, a 3D printed support was manufactured to include the battery inside and protect it from possible moisture and short circuit. The disposable µPADs consist of a folded Whatman nº1 paper with a rectangular heater made of 20 wt.% GNP/Wax underneath a circular testing area delimitated by neat wax, where the thermochromic ink is placed (Figure 4.2.7a). Although there was no need for post-thermal cure of the conductive wax since in this case it works only as heater, the entire system was cured to increase the mechanical stability of the printed waxes and thus of the µPADs, as concluded in Figures 4.2.4a and b. In this case, two red thermochromic inks were used with activation temperatures at 28 C (Figure 4.2.7b) and 47 °C (Figure 4.2.7c), respectively. a) Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 250 b) c) Figure 4.2.7. Photographs of a) portable printed folded Whatman nº1 paper devices, in which a 20 wt.% GNP/Wax square is connected to a battery (1.5, 9 or 12 V) placed inside the 3D printed support, in order to provide heat to the thermochromic ink contained in the circular testing area delimited by neat wax; b) thermochromic ink (activated at 28 °C) with conductive square subjected to a voltage of 1.5, 9 and 12 V and corresponding thermal images; c) thermochromic ink (activated at 47 °C) with conductive square subjected to a voltage of 1.5, 9 and 12 V and corresponding thermal images. Regarding the results presented in Figure 4.2.7b, the ink with an activation temperature of 28 °C changes colour regardless the battery used from a bright red to a more translucent red (light rose.) At 1.5 V, 9 and 12 V applied voltages, maximum temperatures of ≈32.0, 50.5 and ≈59.8 °C are measured using the thermal imaging camera, close the values of ≈31.5, 51.6 and 62.1 C obtained by means of the temperature probe (Figure 4.2.5b), respectively, which are higher than the activation temperature of the ink, which is in line with the obtained results. On the other hand, the heating generated by the 1.5 V battery is not enough to change the colour of the ink with an activation temperature of 47 °C, as shown in Figure 4.2.7c. The two batteries with higher potential allow to generate enough temperature variation for a colour transition, with the values previously described. These aforementioned proofs of concept can be further tailored according to the application, e.g. by varying µPADs design, GNP/Wax concentration, or applied voltage, but nevertheless convincingly validate the concept of multifunctional conductive wax development for portable µPAD systems, allowing to work simultaneously as hydrophobic barriers and heaters, using a simple, rapid and low cost fabrication method with reproducible response. Chapter 4.2 - Wax printable conductive and low-power thermal actuator paper-based analytical device 251 Conclusions This work reports on the development of multifunctional waxes based on the integration of conductive graphene nanoplatelets (GNP) into the wax matrix for µPAD applications. This approach allows the wax to work simultaneously as hydrophobic barriers, its commonly used property to delimit boundaries into hydrophilic paper substrates, and also as heater for temperature-sensitive applications. The process of wax patterning in paper involves a single, simple and fast printing method allowing cost-effective tailorable production. Moreover, portability is assured by the possibility of using lightweight and low-power actuation systems such as alkaline batteries. Wax prints with mechanical stability and adequate impregnation through the paper were obtained after proper post-thermal cure. Tailorable temperatures ranging from room temperature to ≈107 °C were achieved by varying GNP weight content up to 20 wt.% and applied electric potential up to 15 V. Moreover, thermochromic inks with the ability to change colour above a certain activation temperature were used in pre-designed and printed wax-based µPADs to validate heating reproducibility and low-power thermal actuation. 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Chapter 5 - Conclusions and future works 260 - Develop new polymer substrates based on natural polymers, such as lignin, collagen, chitosan or new types of cellulose, which can bring new solutions and turn PADs and µPADs more compatible with the 2030 agenda in terms of the circular economy; - Develop wax-based hydrophobic inks with embedded semiconductor particles, which can increase the variety of applications available in the current devices, towards functional printed electronic systems; - Replace synthetic-based waxes such as the Xerox commercial wax, with natural-based waxes i.e. beeswax, carnauba wax or myrica wax, with different melting temperatures; - Combine the tailored polymeric substrates and the new multifunctional waxes to take full advantage of the unique characteristics provided by the use of active components both in the support material and in the printing material. These PADs can combine colorimetry with an electrochemical response.