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This is to certify that D. Walvin Ramiro Córdova has developed a personal work corresponding to the proposal “Concerned nanotechnology: Screen printed electrodes (SPE) to detect nanoparticles in natural media" whose final report is presented here as Final Master Project of the “Máster Universitario en Materiales Nanoestructurados para Aplicaciones Nanotecnológicas” This work has been developed in the labs of the “Grupo de Espectroscopía Analíca y Sensores (GEAS)”, IUCA under my supervision Zaragoza, September 4th, 2014 Dr. Juan Carlos Vidal Ibáñez This work has been supported by a Spanish competitive project CTQ 2012-38091. Acknowledgements Special thanks to the “Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, IUCA” by the scholarship “Apoyo a la Investigación” awarded for the current academic year and also for allowing me to realize my final master project in its facilities. I also want to thank the research group “Grupo de Espectroscopia Analítica y Sensores” to allow me to work with them in its laboratories and with its equipments, as well as the “Departamento de Química Analítica” for its support. Thank my tutor, Dr. Juan Carlos Vidal for collaboration and guidance provided in the development of this project. I also want to thank Dra. Gemma Cepriá Pamplona for her assistance in these difficult times. Thanks to Dr Mario Pérez Riera and Dr Francisco José Ruíz Blasco of the “Departamento de Matemáticas, Área de Análisis Matemático” who had helped me with Fourier transform smoothing of the chronoamperograms, without their help no collision events could have been ever seen in our lab. Many thanks to my colleagues in laboratory for their patience and support, special thanks to Dr. Laura Sanchez for allow me use their STEM data. TABLE OF CONTENTS 1. ABBREVIATIONS .............................................................................................. 1 2. SUMMARY .......................................................................................................... 3 3. INTRODUCTION ................................................................................................ 5 3.1. SILVER NANOPARTICLES (AgNPs)...................................................................... 7 3.2. CERIUM OXIDE NANOPARTICLES (CeO2NPs OR NANOCERIA) ...................... 9 4. OBJECTIVES .................................................................................................... 13 5. VOLTAMMETRY OF IMMOBILIZED PARTICLES (VIP) ......................... 15 5.1. INTRODUCTION ................................................................................................... 15 5.2. MATERIALS .......................................................................................................... 15 5.3. PROCEDURE ......................................................................................................... 17 5.3.1. Samples of AgNPs on Glassy Carbon electrode (GC) ................................... 17 5.3.2. Samples of AgNPs on a modified SPCE. ...................................................... 17 5.4. CALCULATION ..................................................................................................... 18 5.4.1. Samples of AgNPs on Glassy Carbon electrode (GC) ................................... 18 5.4.2. Samples of AgNPs on a modified SPCE. ...................................................... 19 5.5. RESULTS ............................................................................................................... 19 5.5.1. Samples of AgNPs on Glassy Carbon electrode (GC) ................................... 19 5.5.2. Samples of AgNPs on a modified SPCE. ...................................................... 21 5.6. CONCLUSIONS ..................................................................................................... 22 5.6.1. Samples of AgNPs on Glassy Carbon electrode (GC) ................................... 23 5.6.2. Samples of AgNPs on a modified SPCE. ...................................................... 23 6. CHARACTERIZATION OF NANOPARTICLES BY PARTICLE COLLISION (PC) ..................................................................................................... 25 6.1. INTRODUCTION ................................................................................................... 25 6.2. BACKGROUND NOISE ......................................................................................... 27 6.3. MATERIALS .......................................................................................................... 28 6.4. PROCEDURE FOR SILVER NANOPARTICLES (AgNPs) ................................... 28 6.4.1. Direct .......................................................................................................... 28 6.4.2. Electrocatalysis ............................................................................................ 29 6.5. CALCULATION FOR SILVER NANOPARTICLES (AgNPs) ............................... 29 6.5.1. Direct detection of AgNPs ........................................................................... 29 6.5.2. Electrocatalytic detection of AgNPs ............................................................. 30 6.6. RESULTS FOR SILVER NANOPARTICLES (AgNPs) .......................................... 30 6.6.1. Direct detection of AgNPs ........................................................................... 30 6.6.2. Electrocatalytic detection of AgNPs ............................................................. 30 6.7. CONCLUSIONS FOR SILVER NANOPARTICLES (AgNPs) ............................... 31 6.8. PROCEDURE FOR CERIA NANOPARTICLES (CeO2NPs) .................................. 32 6.8.1. Direct particle collision ................................................................................ 32 6.8.2. Particle collision with catalytic reduction. .................................................... 33 6.8.3. Study UV-visible of 10-20nm nanoceria ...................................................... 36 6.9. CALCULATION FOR CERIA NANOPARTICLES (CeO2NPs) ............................. 37 6.9.1. Nanoceria by Direct Particle Collision ......................................................... 37 6.9.2. Nanoceria by catalytic reduction particle collision. ....................................... 37 6.10. RESULTS FOR CERIA NANOPARTICLES (CeO2NPs) ........................................ 38 6.10.1. Nanoceria by Direct Particle Collision ......................................................... 38 6.10.2. Nanoceria by catalytic reduction particle collision. ....................................... 39 6.10.3. Study UV-visible of 10-20nm nanoceria ...................................................... 40 6.11. CONCLUSIONS FOR CERIA NANOPARTICLES (CeO2NPs) .............................. 40 6.11.1. Nanoceria by Direct Particle Collision ......................................................... 40 6.11.2. Nanoceria by catalytic reduction particle collision. ....................................... 41 6.11.3. Study UV-visible of 10-20nm nanoceria ...................................................... 41 7. GENERAL CONCLUSIONS ............................................................................ 43 7.1. VOLTAMMETRY IMMOBILIZED PARTICLES (VIP) ........................................ 43 7.2. PARTICLE COLLISION (PC) ................................................................................ 43 8. ANNEXES .......................................................................................................... 45 A0 Paper of previous work based in AgNPs on GC and SPCE. Reference [27]. ........... 45 A1 Samples of AgNPs on Glassy Carbon electrode (GC). ............................................ 52 A2 Samples of AgNPs on a modified Screen Printed Carbon Electrode (SPCE). .......... 55 A3 Photograph of the electrodes used in Particle Collision. ........................................ 58 A4 Study of silver nanoparticles (AgNPs) by Direct Particle Collision. ....................... 59 A5 Study of silver nanoparticles (AgNPs) by Electrocatalytic Particle Collision. ......... 60 A6 Study of ceria nanoparticles (CeO2NPs or nanoceria) by Direct Particle Collision (PC). .............................................................................................................................. 61 A7 Study of nanoceria by catalytic reduction particle collision. ................................... 62 A8 Plots of peaks in nanoceria 4nm by direct PC. ....................................................... 65 A9 Plots of peaks in 4nm nanoceria by catalytic reduction particle collision. .............. 66 A10 Radii of 4nm nanoceria by catalytic reduction particle collision. ........................... 68 A11 UV spectra of 10-20nm nanoceria with 1, 2, 4, 6 and 8mM Ascorbic Acid (AA). ..... 70 A12 Results of peaks in nanoceria by catalytic reduction particle collision..........72 A13 Peaks in 10-20nm nanoceria by catalytic reduction particle collision. .................... 73 A14 TEM images and size distribution of 4nm nanoceria. ............................................. 74 9. REFERENCES ................................................................................................... 75 1 1. ABBREVIATIONS AA Ascorbic Acid AE Auxiliary Electrode AFM Atomic Force Microscopy AP10 Argentum Plus 10ppm AP25 Argentum Plus 25ppm DLS Dynamic Light Scattering FFF Field-Flow Fractionation GC Glassy Carbon GC-ME Glassy Carbon Microelectrode HRTEM High Resolution Transmission Electron Microscopy ICP-MS Inductively Coupled Plasma Mass Spectrometry ME Microelectrode NPs Nanoparticles PC Particle Collision PCS Photon Correlation Spectroscopy PEN Project on Emerging Nanotechnology PFM Peak Fitting Module Pt-ME Platinum Microelectrode RE Reference Electrode ROS Reactive Oxygen Species SD Standard deviation SEM Scanning Electron Microscopy SN30 Source Naturals 30ppm SOD Superoxide Dismutase Activity SPCE Screen Printed Carbon Electrode TEM Transmission Electron Microscopy VIP Voltammetry Immobilized Particles WE Working Electrode XPS X-ray Photoelectron Spectroscopy 8 Antibacterial property of AgNPs is affected by the particle properties such as size, shape, coating, surface charge, etc. because the mechanism of Ag(I) release depends on them. For example, AgNPs of smaller size may exert higher toxicity due to their higher specific surface area and associated faster Ag(I) release rate compared to larger AgNPs.[9] Researchers have also found evidence of toxicity for mammalian cells. Among the affected species we can mention zebrafish, fruit fly, mices, rats and also humans. Table 1 shows some examples of mammalian cell affected by the toxicity of AgNPs.[2] The field of science which aims to standardize physical measurements at the nanometer scale is called nanometrology.[8] The characterization and quantification of nanoparticles is a difficult analytical process due to its high dependence on particle size and nature, concentration in the sample, physico-chemical characteristics of the solution and fundamental principles of analytic technique. The most common techniques used for size characterization include laser techniques, such as photon correlation spectroscopy (PCS), electron microscopes (scanning electron microscopy, SEM, transmission electron microscopy, TEM), also atomic force microscopy (AFM) and size separation techniques (field-flow fractionation, FFF, hydrodynamic chromatography and size-exclusion chromatography).[10] Table 1. Evidence of AgNPs toxicity for mammalian cells. (Extracted from reference [2]) 9 3.2. CERIUM OXIDE NANOPARTICLES (CeO2NPs or nanoceria) In contrast to the silver, cerium chemistry is not yet fully known and need to know more about their chemical reactivity. Cerium is a rare earth element which can form two main crystal structure, cerium(IV) oxide (CeO2) and cerium(III) oxide (Ce2O3). The most important for their applications is CeO2 nanoparticles or nanoceria. Today, nanoceria is being used in a variety of fields such as an active catalyst in vehicle emission system for oxidation of pollutant gases[11], in biomedicine as an antioxidant in treatments produced by ROS, also are used as an electrolyte material for solid oxide and proton exchange membrane fuel cells among others. Potential use of nanoceria is due to its crystal structure. CeO2 is not a stequiometric compound because the outermost layers of the nanoparticle have some defects. These defects are responsible that the most external atoms to be less coordinated that internal. To compensate these defects, the oxygen is released reversibly and produces the reduction of the oxidation state of some atoms of Ce(IV) to Ce(III). This facility to change the oxidation state from Ce(IV) to Ce(III) and vice versa as a function of environmental requirements is responsible for the reactivity of nanoceria. The inner core of the nanoparticle has an almost perfect crystal structure of CeO2 where all spaces are occupied by oxygen and cerium. As mention above, the most external layers of particles are responsible for the reactivity. If nanoceria are smaller, the reactivity will be higher because the size reduction involves an increase in the number of external vacancies relative to total atoms. Figure 4 shows the cubic structure of CeO2.[12] Figure 4. Typical cubic structure of CeO2 (Red spheres: Oxygen atoms; Cream spheres: Cerium atoms). (Extracted from reference [12]) 10 Nanoceria acts as a catalytic antioxidant in biological model systems. These nanoparticles show two mimetic activity, superoxide dismutase activity (SOD) to convert superoxide anion to hydrogen peroxide and catalase mimetic activity to convert hydrogen peroxide into water and molecular oxygen. These two mimetic activities are highly dependent on the Ce(III)/Ce(IV) ratio, so SOD activity is less efficient with high concentrations of Ce(IV) and catalase activity is more efficient with low levels of Ce(III).[13-15] Furthermore, these nanoparticles are able to recover its catalytic activity and therefore may have pseudoinfinite half-life.[13, 16] The most common synthesis method is oxidation at room temperature of salts of Ce(III), Ce(NO3)3 for example, by adding strong bases or ammonium salts. In any case it is an alkaline-based precipitation of Ce(III).[12] The main techniques for the characterization of nanoceria are electronic microscopy techniques (TEM and HRTEM), X-ray photoelectron spectroscopy (XPS), Raman, Inductively coupled plasma mass spectrometry (ICP-MS), UV spectroscopy, dynamic light scattering (DLS) and zeta potential.[12,13,16] Regarding the toxicological risk of nanoceria, we can mention that these nanoparticles can acts as a producer of ROS or as an antioxidant, antagonistic effects. The behavior depends on the organism, environment, dose and physical characteristics of nanoceria, for example: - In a study of two photosynthetic organisms, cyanobacteria and green algae, was found that nanoceria significantly inhibits photosynthesis in cyanobacteria, while having a dual effect on green algae because it produces a slight stimulation of growth at low concentrations and strong inhibition at high concentrations.[17] - In another example, the dose and time of exposure to nanoparticles is studied on human lens epithelial cells. The results show that 100μg/mL is acceptable for an exposure time of 48h. However, exposure time plays a more important role that the concentration indicating a potential genotoxicity for higher exposures.[18] Finally, the following table (Table 2) shows some effects, beneficial and adverse, of exposure to nanoceria[19] 11 Table 2. Reports of nanoceria effects. (Extracted from reference [19]) 12 13 4. OBJECTIVES GENERAL OBJECTIVES  The student should develop the ability to work in a research project that requires the use of many different equipment and technologies in a responsible way and never forget he is not alone and the meaning of working within a team.  He should be able to recognize his deficiencies and consequently he should find the way to acquire the knowledge he lacks.  The student will be aware of the necessity of being responsible about what it is done in the laboratory and how much it can affect life and environment. PARTICULAR OBJECTIVES  Full literature reviewing to know the state of the art of the subject.  Adequate manipulation of the instrumentation required for the research project.  Developing an analytical procedure that enables the size determination of nanoparticles in a suspension.  Mathematical treatment of the data to obtain the pursued information: FFT filtering of the signal, deconvolution algorithms, peaks adjustment procedures.  Interpretation of the data to explain the phenomena that can be observed in suspension and on the electrode surface. 14 15 5. VOLTAMMETRY OF IMMOBILIZED PARTICLES (VIP) 5.1. INTRODUCTION Voltammetry of immobilized particles (VIP) was studied and developed in the 90s by Prof. Scholz.[20-22] It is based on a simple technique of immobilization of solid particles and liquid droplets on the electrodes.[23] Then the drop is dried and voltammetry was performed in the appropriate electrolyte. There are theoretical and experimental articles that deal with VIP.[24-26] The radius of nanoparticles could be obtained by VIP experiments because there is a relationship between size and peak potential. This is related with the characteristic Gibbs free surface energy of nanoparticles which differs from energy for macroobjects. So, the standard electrode potential ( ) is lower than the redox standard potential for bulk metal ( ) and the following equation was used by Redmond et. al. to describe this phenomena.[24] Another study reveals that the degree of dispersion of the sample affects the shape of the voltammograms. So, if we have small nanoparticles and more dispersion, the range of oxidation potentials increases.[25] Therefore, we can use the electrochemical data to assess the degree of dispersion of nanoparticles assemblies and the average particle radius. Indeed, our research group has conducted a previous study of AgNPs using VIP[27] which will be used later. The paper of this work is found in the final annexes. (annex A0) 5.2. MATERIALS Three different samples were purchased from specialized health care shops. The names of them are collected in Table 3. Table 3. Names, concentration and abbreviations of the samples used in this work Name Concentration Abbreviation Source Naturals 30ppm SN30 Argentum Plus 10ppm AP10 Argentum Plus 25ppm AP25 16 In some experimental processes, some salts as KCl, KClO4 and FeCl3 were necessary. In addition, the polymer Nafion was necessary to modification of SPCE. All solutions and dilutions of AgNPs were prepared with ultrapure water (Mili-Q Advantage). These last are stored in the dark to avoid the oxidation of nanoparticles. Each nanoparticle solution was sonicated in ultrasonic bath for just one minute before their use. Voltammetric measurements were carried out with Autolab PGSTAT-12 potentiostat (Utrecht, The Netherlands) controlled by computer. Two different approaches have been used. The different electrodes used are detailed in the following lines and a general scheme is included in Figure 5: 1. 20mL voltammetric cell with Ag/AgCl/3M NaCl reference electrode, Pt wire auxiliary electrode and glassy carbon (GC) working electrode with 3mm of diameter. 2. Screen Printed Carbon Electrode (SPCEs) with working and auxiliary electrodes printed from carbon-based ink (Gwent) and the pseudoreference electrode based on silver ink. The diameter of working electrode is 4mm. Figure 5. The figure shows a diagram of the two approaches used in this section. 17 5.3. PROCEDURE 5.3.1. Samples of AgNPs on Glassy Carbon electrode (GC) The sample AP25 was diluted twice, SN30 was diluted five times and AP10 was used without dilution. The sample or its dilution is sonicated for 1 minute. Then 3μL of sample were drop cast on the graphite disk of the GC electrode. The solution should not reach the polymeric substrate. Then the drop was dried under nitrogen flow avoiding its displacement and the loss of material and aggregation. 10mL of 0.1M KClO4 was used as the supporting electrolyte in the electrochemical cell. When the drop is dried, the electrodes are introduced into the electrochemical cell and start to measure the voltammograms. The working electrode is withdrawn from the cell and washed twice with mili-Q water. Then it can be air dried, or with nitrogen flow to accelerate the drying process, and then the process is repeated. The electrochemical measurements were performed in triplicate for each sample. Measurement parameters: Linear sweep voltammetry was used in all measures with the following parameters: Begin potential (V) 0.0 End potential (V) 0.8 Step potential (V) 0.00412 Scan rate (V/s) 0.02 5.3.2. Samples of AgNPs on a modified SPCE. All samples were diluted to 1ppm. As in the previous case, the solutions are sonicated for 1 minute just prior to their use; in any case, they are stored in the dark. We need stable and reproducible measurements to relate the peak potential with a certain diameter. The pseudoreference electrode (RE), based on Ag ink, is not very stable. Therefore, its modification is required to obtain useful measurements of peak potential. 3μL of 1M FeCl3 was deposited over the RE for 1 minute to form a layer of AgCl. The excess of FeCl3 was washed with mili-Q water and air dried. Finally, 3μL of 5% Nafion in ethanol saturated with KCl were deposited on the modified RE and allowed to dry at room temperature.[27] At the end, the modified RE is composed by layers of Ag/AgCl/KCl. For sample measurements, 3μL of each sample are deposited on the working electrode of the modified SPCE. The drop is dried under nitrogen flow and then 70μL of 0.1M 24 distribution observed using TEM was broad[27] for all samples, meaning that there is a broad dispersion of particles of different size in all cases.  AP10: As in the case of GC, the values obtained are lower than those given in the article. No populations of different sizes are appreciated, it may mean that the sample has been oxidized during this time.  AP25: The values obtained are higher and lower than those given in the article. This can be explained by the agglomeration of larger particles and allow us to observe the populations of smaller size.  SN30: In this particular case, each voltammogram gives populations of different sizes. A possible explanation may be that composition itself of the sample is which facilitates the agglomeration. We can not forget that the substrate used is different and can be a possible cause of agglomeration. 25 Preview information: nanoceria standards. As it was already mentioned, the chemistry of silver is very advanced, and therefore we have a good control on the AgNPs synthesis process, stabilizers, dispersion medium, etc. Consequently, AgNPs standards of different sizes and shapes are commercially available.[31-33] However, the chemistry of cerium and its oxides is not well known yet. Besides, we cannot find nanoceria standards of different sizes in the market. The absence of standards makes difficult the use of the method employed for AgNPs. That is, we cannot obtain a calibration plot that allows us to interpolate a certain sample. In view of this difficulty, we are forced to seek alternatives for the characterization of nanoceria. One alternative that can be useful in this task is the Particle Collision (PC). 6. CHARACTERIZATION OF NANOPARTICLES BY PARTICLE COLLISION (PC) 6.1. INTRODUCTION Initial studies about individual electrochemical signals corresponding to small particles were developed by Heyrovský et. al. in 1995.[34-37] Later, Bard and his group are the first to study the particle collision. They studied the impact of different nanoparticles, mainly Pt nanoparticles, with catalysis.[38-41] Subsequently, another studies on the electrochemical signals generated by the impacts of different nanoparticles on the electrodes are developed by G. Compton et. al. between 2011 and 2012.[42-46] Even particles of clays, such as montmorillonite, have been characterized with this technique.[47] PC is based on measuring the Faradaic current associated to the electrode reaction that takes place when a nanoparticle (NP) hits the surface of a microelectrode (ME). Each collision is observed as a transient signal with an approximate duration of milliseconds, if the nanoparticle does not stick to the electrode, that is the case a step is observed whose height depends on the size of the nanoparticle. This method can be divided in three different approaches (Figure 8) according to the electrochemical reaction produced to obtain the signal:  Direct: The signal is obtained from the direct reaction between the nanoparticle(NP) and microelectrode(ME).[43](Fig.8A) 26  Electrocatalysis: The signal is obtained more easily by the presence of electrocatalytic active species whose reaction is catalyzed by the NP. These species do not react, or its rate is very low, directly on the ME[39] (Fig.8B) This approach is mandatory when the NP is not electroactive or in the case of the destruction of the nanoparticle should be avoided. It is also the way to increase the sensitivity of the procedure because the signal depends on the concentration of the reactive whose electrode reaction is catalized by the NP and the number or electrons exchanged by this reactive. Another way to accomplish these goals is depicted in the next point.  Tag-redox: Nanoparticles are covered or “tagged” with redox species. The signal is obtained from the reaction between tagged-NP and ME (Fig.8C).This is a non-destructive method.[48] This procedure is used when the nanoparticle is not electroactive or just to avoid interferences. Figure 8. Three different approaches of PC: (A) Direct, (B) Electrocatalysis, (C) Tagredox. A B C 27 Figure 9. (A) Electronic noise, (B) Instability of background by vibrations, (C) Low background signal (original data are shown as black dots and treated data are represented as blue dots). 6.2. BACKGROUND NOISE The “electronic background noise” is produced by electronic interference namely potentiostat itself, computer or power supply. This noise is a relevant problem indeed in this case because the signals generated by the nanoparticles have low intensities (pA) and can be lost in the background noise. Therefore the data must be treated to remove the majority of the electronic noise. The electronic noise is observed as different superimposed waves when the data are drawn in a different software that the used in the potentiostat, so it can be seen in Figure 9A. The same data after being treated are shown also in Figure 9A. In the first experiments we could see other noise related with the environment work. In these experiments, the potentiostat was in an area with high traffic of people and vibrations produced by people affect the stability of the background signal. Examples of vibrations can be seen in Figure 9B. The solution to this noise was to move the work area to a place with less movement of people. If the noises are wiped, it is possible to obtain very low and good backgrounds. An example is observed in Figure 9C where you can see that the original data did not differ greatly from the treated data as in the case of Figure 9A. The data processing is also important because it helps to see hidden peaks under the noise. 28 6.3. MATERIALS Chronoamperometry measurements were carried out with Eco Chemie Autolab PGSTAT-10 with low intensities module and controlled by computer. Two different WE were used: Glassy Carbon microelectrode (GC-ME) with 11 ± 2μm of diameter and Platinum microelectrode (Pt-ME) with 10μm of diameter. The reference electrode was Ag/AgCl/3M NaCl and auxiliary electrode was a Pt wire (image of these electrodes can be found in annex A3). Standard of AgNPs 100nm, (2,73.1018NP L-1 - 20ppm) were purchased from Sigma-Aldrich. Source Naturals 30ppm (SN30, 3,03.1014NP L-1) sample was also used in this section. CeO2 5% wt aqueous nanoparticle suspension (4nm diameter, 2,03.1020NP L-1) and CeO2 20% wt in 2,5% acetic acid nanoparticle suspension (10-20nm diameter, 7,61.1018NP L-1) were purchased from PlasmaChem. The different chemical reagents used are: Anhydrous sodium dihydrogen citrate, NaCl, NaClO4, NaBH4 and L-ascorbic acid. The liquids reagents were: H2O2 30% and HCl 35% from Scharlau Chemie. All solutions and dilutions of NPs were prepared with ultrapure water (Mili-Q Advantage) 6.4. PROCEDURE FOR SILVER NANOPARTICLES (AgNPs) The study was conducted using two of the three approaches mentioned on introduction section: Direct and Electrocatalysis. Platinum microelectrode was used as working electrode in both approaches 6.4.1. Direct Different procedures were followed for the direct analysis. The results obtained in this approach were not promising in any procedure. Electrolyte or characteristics can be found in the annex A4. The parameters used are collected in Table 6. General procedure: 10mL of electrolyte solution are deposited in an electrolytic cell. Then, a nitrogen flow is bubbled through the solution for 15-20 minutes to displace oxygen and prevent oxidation of the NPs. At this point, measurements Table 6. Measurement parameters used in Direct PC E (V) Measurement duration (s) Measuring time (s) 0,5 10 0,001 0,5 5 0,0005 29 of blank were performed on the electrolyte alone. The AgNPs 100nm solution, previously sonicated for 1 minute, is injected into the cell and the circuit is completed with working, reference and auxiliary electrodes. The three electrodes are clamped using parafilm to hold them in position and prevent fluctuations in the measurements. Once the circuit is closed, the nitrogen flow is extracted from the solution but not from the cell, thus maintaining a positive pressure inside and prevents the entry of oxygen. Measurement parameters are set and start measuring. The total measurement time is between 3-4 hours but can be extended to higher values. The higher total measurement time, the probability to observe collisions is higher. 6.4.2. Electrocatalysis In this case, the peaks generated are the results of the electrocatalytic reduction of H2O2 on the surface of AgNPs. As in the previous case, several tests were made and best results were obtained compared with direct detection. The experimental parameters or features are listed in annex A5. The parameters used are collected in Table 7. The general procedure is the same that in the previous case. The main differences are related with potential and electrolyte solution. The useful results were found followed the parameters or features in annex A5i. 6.5. CALCULATION FOR SILVER NANOPARTICLES (AgNPs) The data were treated with Origin 8.0. The first thing is to clean the data of eddy currents. In order to clean the data, the data at frequencies of 50Hz and 150Hz were extracted to the original data and the final data were used to obtain the area of peaks. 6.5.1. Direct detection of AgNPs In case of direct oxidation, the charge transferred for each impact (Q) is related to the number of atoms capable of being oxidized (N) by the equation (I). Table 7. Measurement parameters used in Electrocatalytic PC. E (V) Measurement duration (s) Measuring time (s) -0,9 10 0,001 -0,9 5 0,0005 30 where is the electronic charge.[43,46] Therefore, we can obtain quantitative information of nanoparticles in this case. Furthermore, the charge can also be expressed in terms of the NP radius by the equation (II). where is the number of electrons transferred per atom ( = 1 for silver), is the bulk metal density (10,49 g ml-1), the Faraday constant (96485 C mol-1), is the atomic weight of the metal (107, 8683 g mol-1), the initial NP radius and the final NP radius. In this case, the potential used is positive enough to the NP will be completely oxidized and therefore = 0.[43,46] 6.5.2. Electrocatalytic detection of AgNPs The variation in the charge (Q) depends on H2O2 concentration. Assuming full 2electron reduction of every H2O2 molecule, single spherical nanoparticle and purely diffusion-limiting current for the duration of an impact; we can obtain an equation which relates Q, [H2O2] and radius of nanoparticle, r, through the equation (III).[49] Where [H2O2] is the hydrogen peroxide concentration (2,80.10-27 mol nm-3), is the number of electrons (2 in H2O2 reduction), is the Faraday constant (96485,3399 C mol-1), is a diffusion coefficient (assuming a typical value of 10-5 cm2 s-1, 109 nm2 s) and is the peak duration in seconds. So, using the charge passed per peak you can get the radius of AgNPs in nm. 6.6. RESULTS FOR SILVER NANOPARTICLES (AgNPs) 6.6.1. Direct detection of AgNPs In direct Particle Collision, we did not get any reliable or valid result and therefore we can not show any numerical result. 6.6.2. Electrocatalytic detection of AgNPs Electrolytic particle collision shows results but they are not very useful for the sample characterization. We can find the radius of NP by this catalysis method with H2O2 using equation (III). In this experience, we obtained two peaks (Figure 10) and the radii of these AgNPs are collected below (Table 8). 31 6.7. CONCLUSIONS FOR SILVER NANOPARTICLES (AgNPs) The results generated in the Electrocatalytic approach are not sufficient to get a valid conclusion. The experimental radius is approximately four times lower that the theoretical value. One possible cause of these results is that the impacts correspond to these AgNPs sizes or their size has decreased by oxidation. However, one conclusion that we can get is the catalysis seems to offer better results than direct PC. It has to be pointed out that the AgNP concentration used in this experiment is high, higher that it is supposed to be in waste waters or in the environment. Beside the time required to obtain a useful number of events is long, up to several hours. It is necessary to increase the number of collisions even with lower concentration of nanoparticles by sizing down the volume of the cell or charging the geometry of the working electrode. Figure 10. Peaks of AgNPs (100nm) observed by Electrocatalytic PC. Table 8. AgNPs radius obtained by Electrocatalytic PC. Peaks Q (C) AgNPs radius (nm) 1st 4,67.10-12 25 2nd 1,52.10-12 17 32 6.8. PROCEDURE FOR CERIA NANOPARTICLES (CeO2NPs) The study was performed using two approaches. One approach is Direct particle collision and the other is catalytic reduction when the collision of a nanoparticle takes place. Glassy carbon and Platinum microelectrode were used as working electrode. 6.8.1. Direct particle collision Several nanoparticles have been detected by Direct PC, however we have not found any reference on the detection of nanoceria by PC. For this reason, this approach was tested in nanoceria in the first place. The characteristics or parameters used in the procedures can be found in the annex A6. The general procedure is the same as for the study of AgNPs by PC. It can be described as: 10mL of electrolyte solution are deposited in an electrolytic cell. Then, a nitrogen flow is bubbled through the solution for 15-20 minutes to displace oxygen. The presence of oxygen can influence the reactivity of the nanoceria because it strongly depends on the environment. Then, measurements of blank were performed on the electrolyte alone to control the noise level and be sure that no electromagnetic interferences are present. A low, pA, and stable base line is necessary prior the nanoparticles experiment. Nanoceria suspension, previously sonicated for 1 minute, is injected into the cell and the circuit is completed with working, reference and auxiliary electrodes. The three electrodes are clamped using parafilm to hold them in position and prevent fluctuations in the measurements. Once the circuit is closed, the nitrogen flow is extracted from the solution but not from the cell, maintaining a positive pressure inside and preventing the reequilibrium with oxygen. Measurement parameters are set and the measurement is started. The total measurement time can be 2-4 hours but it can be longer. The higher the total measurement time, the higher probability of observing more collisions. Valid results were obtained following the general procedure with parameters collected in A6ii. Forty-three peaks were extracted in 52 measurements and some of these peaks are shown in annex A8. The parameters of this procedure are collected in Table 9. 33 6.8.2. Particle collision with catalytic reduction. From the literature we know that nanoceria modified through a treatment with hydrogen peroxide can be followed by PC due to the catalytic reduction of the peroxo and superoxo groups that are bonded on its surface.[50] Hydrogen peroxide (H2O2) is used to modify the surface of nanoceria using their property as free radical scavenging.[11] (Figure 11) Several tests were made and best results were obtained compared with direct approach. The experimental parameters or features are listed in annex A7. This approach also was performed successfully, besides the results were useful to characterize nanoceria since a size distribution was obtained. The general procedure is the same that in the previous case but some differences have to be Figure 11. Scheme and reaction produced in particle collision with catalytic reduction (surface modification in nanoceria). Table 9. Parameters and features used in the procedure which gave successful result by Direct PC. E (V) -0,5 Measurement duration (s) 10 Measuring time (s) 0,001 Electrolyte 10mL 1M HCl Working electrode Glassy carbon ME Nanoceria diameter 4nm [CeO2NPs] 1000ppm NP L-1 3,9.1018 40 6.10.3. Study UV-visible of 10-20nm nanoceria The spectra measured of each AA concentration to intervals of 5 minutes are listed in annex A11. Figure 15 shows an overall graph of the initial spectra at each concentration of AA. 6.11. CONCLUSIONS FOR CERIA NANOPARTICLES (CeO2NPs) 6.11.1. Nanoceria by Direct Particle Collision The experimental value is around 30-40nm, which is far from the theoretical value of 4nm. There are three factors that have to be considered: First, the size distribution is made with 43 data and the minimum amount required are 100 data. Second, we have assumed that whole nanoparticle is responsible for the charge exchange with the electrode, that should not be completely true. Beside not all cerium is as Ce(IV), so the theoretical value of total charge exchanged per particle might be overvalorated. Third, the optimal ratio nanoparticle diameter/working electrode diameter should be 1/1000. It makes extremely difficult to detect a 4nm particle impact on a 10μm electrode. Consequently, it is easier to detect the biggest particles present in the suspension, that is to say 30 to 40nm as it happened in our experiment unless an increase of sensibility is attained. That is why we turned to a catalytic reaction coupled with particle collision. Figure 15. UV-visible spectra of 10-20nm nanoceria with different concentrations of AA. 0 0,5 1 1,5 2 2,5 3 3,5 190 240 290 340 390 440 490 540 590 640 690 740 790 Absorbance (a.u.) Wavelength (nm) UV of CeO2NPs with AA 8mM AA 1410ppm CeO2 NPs + 1mM AA NPs + 2mM AA NPs + 4mM AA NPs + 6mM AA NPs + 8mM AA 41 6.11.2. Nanoceria by catalytic reduction particle collision. A good nanoceria size distribution was obtained with this approach due to the large amount of data. The PC results give a distribution that is between 2 and 7nm with the highest value of 3nm. If we compare the result (3-4nm) with the value provided by the manufacturer (4nm), we can see that the difference is very small. Another comparison can be made with data from TEM images. Images from TEM and distribution are collected in annex A14. The size distribution obtained from TEM gives a size around 1 and 5nm with the highest value at 3nm. The result of TEM (2-3nm) can also validate the result of PC (3-4nm). A second size distribution around 10nm can be seen in figure 14. This second distribution may be the result of an agglomerate. Distribution obtained by TEM does not give the second distribution of 10nm because the preparation of the TEM sample is made in their medium. In our case, nanoceria are dispersed in 1M HCl (pH 0) which modifies the surface and could be possible that NPs agglomerates. The supporting electrolyte in which are suspended the nanoparticles as well as the capping agent influence decisively the effectiveness of the chronoamperometric procedure. For this reason, the results obtained for 10-20nm nanoceria were unsuccessful. Direct particle collision events can be detected if the number of Ce(III) atoms is increased using strong reducing agents. The measuring time affects the noise. The longer the measuring time the better. But this parameter should be short enough to allow the detection of a single particle collision event to be detected. A compromise situation should be accepted 6.11.3. Study UV-visible of 10-20nm nanoceria Nanoceria shows two characteristic regions: one is between 240-290nm and the other regions is between 290-390nm. These peaks are related with Ce(III) and Ce(IV), respectively.[15] However, nanoceria reacted with AA shows another peak around 400500nm. This peak can be related with agglomeration. AA reacts with the nanoparticles and the products of the reaction can be adsorbed on the surface causing agglomeration. 42 43 7. GENERAL CONCLUSIONS 7.1. VOLTAMMETRY IMMOBILIZED PARTICLES (VIP) VIP has proven to be useful and reliable technique when the reaction processes on the electrode surface are known. The technique has great progress and a lot of articles have been published using this technique. The procedure is simple and the relationship between the analytical signal and the size of the nanoparticle is well documented and studies by several groups. It also allows the study of aggregation/agglomeration processes on the electrode surface, that depends on the nature of this surface. Using a deconvolution algorithm it is possible to know the diameter of the agglomerates. But it requires many standards of nanoparticles with well characterized diameter. 7.2. PARTICLE COLLISION (PC) Particle collision has proven to be a useful tool to characterize the size of the nanoparticles in a concentrated suspension of nanoceria. The technique provides a view from inside the solution, detecting the formation of agglomerates/aggregates depending on the media conditions (redox potential, ionic strength, pH). PC itself is not a sensitive technique, it requires a high concentration of nanoparticles which limits its use to characterize the behavior of nanoparticles in controlled media for theoretical studies rather than to study them in real samples where concentrations are far below this limit. The sensitivity of the technique can be improved by coupling PC with catalytic reduction. 44 45 8. ANNEXES A0 Paper of previous work based in AgNPs on GC and SPCE. Reference [27]. This work is based on characterization of AgNPs on real samples by linear voltammetry. 46 47 48 49 56 AP25 on modified SPCE 57 SN30 on modified SPCE 58 A3 Photograph of the electrodes used in Particle Collision. (1) is reference electrode of Ag/AgCl/3M NaCl, (2) is Glassy Carbon microelectrode (GC-ME) with 11 ± 2μm of diameter, (3) is Platinum microelectrode (Pt-ME) with 10μm of diameter and (4) is Pt wire as auxiliary electrode. 59 A4 Study of silver nanoparticles (AgNPs) by Direct Particle Collision. The procedures, parameters or features used are described in this annex. i) 10mL of 10mM citrate† and 90mM NaCl as supporting electrolyte. Solution of AgNPs 100nm was used. The measurements parameters are: The concentration of AgNPs in 10mL of electrolyte was 2,73.1013NP L-1. Twenty measurements of 10s and twenty measurements of 5s were saved but without results. ii) A second experiment was performed with the same parameters of the previous experiment but the twice of AgNPs (100nm) were injected into the cell. The concentration of AgNPs in 10mL of electrolyte was 5,46.1013NP L-1. Twenty measurements of 10s and twenty measurements of 5s were saved but neither show results. iii) In this experiment, we tried to increase the number of NPs per liter to 1016 and the measurement parameters were the same. Twenty measurements of 10s and twenty measurements of 5s were saved but neither show results. iv) The supporting electrolyte was changed (90mM NaCl was changed by 90mM NaClO4) because Clcan acts as glue and aggregate the AgNPs. Six experiments were performed with the new electrolyte. Ten measurements of 10s and ten measurements of 5s were saved but neither show results. v) With these parameters and last electrolyte, other test were performed with standards of 10nm (1,83.1016NP L-1 - 0,1ppm) and 40nm (5,68.1013NP L-1 – 0,02ppm). Sixty measurements of 10s and sixty measurements of 5s were saved and not gave results for AgNPs 10nm. Twenty measurements of 10s and twenty measurements of 5s were saved but neither show results for AgNPs 40nm. In direct approach, any procedure used gave valid results. E (V) Measurement duration (s) Measuring time (s) 0,5 10 0,001 0,5 5 0,0005 †Citrate is a compound that easily deteriorates, therefore, must be prepared at the time of use in each experiment. 60 A5 Study of silver nanoparticles (AgNPs) by Electrocatalytic Particle Collision. The procedures, parameters or features used are described in this annex. i) 10mL of 0,1M NaClO4 and 2,8mM H2O2 as supporting electrolyte. Solution of AgNPs 100nm was used. The measurements parameters are: The concentration of AgNPs in 10mL of electrolyte was 2,73.1013NP L-1. Twenty measurements of 10s and twenty measurements of 5s were saved. This procedure gave valid results for measurements of 10s (Two peaks were extracted). ii) The same above experiment was repeated without success. Then, the experiment was repeated injecting a double volume of AgNPs (100nm). The concentration of AgNPs in 10mL of electrolyte was 5,46.1013NP L-1. Ten measurements of 10s and ten measurements of 5s were saved but not collision events were detected. iii) Another solution was tried. The sample SN30 undiluted was injected and neither obtains results. The concentration of AgNPs (SN30) in 10mL of electrolyte was 3,03.1014NP L-1. Ten measurements of 10s and ten measurements of 5s were saved but not gave results. iv) One (3,03.1014NP L-1), two (6,06.1014NP L-1) and three (9,09.1014NP L-1) volumes of SN30 sample in three different experiments with the electrolyte without hydrogen peroxide (no electrocatalytic) were also tested. The parameters were the same. Twenty measurements of 10s in each experiment were saved and any of them gave collision events. v) 10mL of 0,1M NaClO4 and 10mM citrate (without H2O2) as electrolyte. The concentration of AgNPs 100nm in 10mL of electrolyte was 2,73.1013NP L-1. The same parameters were used. Twenty measurements of 10s were saved but not collision events were observed. vi) 10mL of 0,1M NaClO4, 10mM citrate and 2,8mM H2O2 as electrolyte. The concentration of AgNPs 100nm in 10mL of electrolyte was 2,73.1013NP L1. The same parameters were used. Twenty measurements of 10s were saved but not collision events were observed. vii) 10mL of 0,1M NaClO4, 10mM citrate and 4,9mM H2O2 as electrolyte. The concentration of AgNPs 100nm in 10mL of electrolyte was 2,73.1013NP L1. The same parameters were used. Twenty measurements of 10s were saved but neither collision events were observed. E (V) Measurement duration (s) Measuring time (s) -0,9 10 0,001 -0,9 5 0,0005 61 A6 Study of ceria nanoparticles (CeO2NPs or nanoceria) by Direct Particle Collision (PC). The procedures, parameters or features used or tested are described in this annex. i) 10mL of 1M HCl is used as supporting electrolyte. GC-ME and Pt-ME are used as WE. The measurements parameters are: The concentration of 4nm nanoceria in 10mL of electrolyte was 5000ppm (1,95.1019NP L-1). Twenty measurements with GC-ME and twenty measurements with Pt-ME were collected but without results. ii) 10mL of 1M HCl is used as supporting electrolyte. GC-ME is used as WE. The measurements parameters are: The concentration of 4nm nanoceria in 10mL of electrolyte was 1000ppm (3,9.1018NP L-1). Fifty-two measurements were saved with valid results (43 peaks were extracted). iii) In order to obtain the same results as in A6ii, the last experience with the same parameters was repeated. One hundred ten measurements were obtained in three repetitions but any result was obtained. iv) The same conditions and parameters were used with 10-20nm nanoceria. The concentration of 10-20nm nanoceria in 10mL of electrolyte was 1000ppm (3,12.1016NP L-1). One hundred measurements were saved in two different experiences but no collision events were detected. v) Valid procedure (A6ii) was tested with standard of 10-20nm nanoceria. The concentration of 10-20nm nanoceria in 10mL of electrolyte was 1410ppm (3,86.1018NP L-1). Twenty measurements were saved in two different experiences but without collision events. E (V) Measurement duration (s) Measuring time (s) 0 10 0,001 E (V) Measurement duration (s) Measuring time (s) -0,5 10 0,001 62 A7 Study of nanoceria by catalytic reduction particle collision. The procedures, parameters or features used are described in this annex. i) 10mL of 1M HCl and 10mM H2O2 is used as supporting electrolyte. GCME and Pt-ME are used as WE. The measurements parameters are: The concentration of 4nm nanoceria in 10mL of electrolyte was 5000ppm (1,95.1019NP L-1). Twenty measurements with GC-ME and twenty measurements with Pt-ME were saved but without results. ii) 10mL of 1M HCl and 10mM H2O2 is used as supporting electrolyte. GCME is used as WE. The measurements parameters are: The concentration of 4nm nanoceria in 10mL of electrolyte was 1000ppm (3,9.1018NP L-1). Thirty-five measurements were saved but without results. iii) 10mL of 1M HCl and 10mM H2O2 is used as supporting electrolyte. PtME is used as WE. H2O2 and nanoceria were in contact for 1½ hours before measuring. The measurements parameters are: The concentration of 4nm nanoceria in 10mL of electrolyte was 990ppm (3,86.1018NP L-1). Pt-ME was immersed in 10mM NaBH4 solution for 15 minutes just before measuring (Activation of WE). One hundred measurements were saved with valid results (285 peaks were extracted). iv) The same conditions and parameters were used with 10-20nm nanoceria. The concentration of 10-20nm nanoceria in 10mL of electrolyte was 1000ppm (3,12.1016NP L-1). One hundred measurements were saved in two different experiences but no collision events were observed. v) 10mL of 2,5% acetic acid is used as supporting electrolyte because the 1020nm nanoceria are suspended in this electrolyte. The others conditions and parameters were the same. Concentration was increased to increase the probability of observing a collision event. The concentration of 10-20nm nanoceria in 10mL of electrolyte was 5000ppm (1,56.1017NP L-1). One hundred measurements were saved in two different experiences but without results. vi) 5mL of 0,1M HCl and 20mM H2O2 is used as supporting electrolyte. PtME is used as WE. H2O2 and nanoceria were in contact for 1 hour. The measurements parameters are the same that the valid procedure (A7iii). The concentration of 10-20nm nanoceria in 5mL of electrolyte was E (V) Measurement duration (s) Measuring time (s) 0 10 0,001 E (V) Measurement duration (s) Measuring time (s) -0,5 10 0,001 E (V) Measurement duration (s) Measuring time (s) -0,2 10 0,001 63 1410ppm (3,86.1018NP L-1). Twenty measurements were saved but without results. vii) 5mL of 1M HCl and 20mM H2O2 is used as supporting electrolyte. Pt-ME is used as WE. H2O2 and nanoceria were in contact for 30 minutes. The measurements parameters are the same that the valid procedure (A7iii). The concentration of 4nm nanoceria in 5mL of electrolyte was 1890ppm (7,37.1018NP L-1). Pt-ME was activated with the same previous procedure. Forty measurements were saved in two different experiences but just one peak was observed. The procedure is not useful. viii) In this case, we followed the procedure of reference [50] which indicates the use of centrifuge to remove excess of hydrogen peroxide. 5mL of 0,1M HCl and 10mM H2O2 is used as supporting electrolyte. Pt-ME is used as WE. H2O2 and nanoceria were in contact for 30 minutes. After this time, 710mL of 3M NaCl are added to precipitate nanoceria. To remove the excess of H2O2, the solution was centrifuged (5000rpm for 10 minutes), supernatant is discarded, add 5mL of 0,1M HCl and again centrifuged, supernatant is discarded and finally the nanoceria are redispersed in 5mL of 0,1M HCl to measure. The measurements parameters are the same that the valid procedure (A7iii). The concentration of 4nm nanoceria in 5mL of electrolyte was 1890ppm (7,37.1018NP L-1). Pt-ME was activated with the same previous procedure. Forty measurements were saved in two different experiences but just one peak was observed. The procedure is not useful. ix) Latter procedure was repeated but nanoceria concentration was changed. The concentration of 4nm nanoceria in 5mL of electrolyte was 990ppm (3,86.1018NP L-1). The measurements parameters are the same that the valid procedure (A7iii). Twenty measurements were saved but without results. x) In this procedure, we look a new redox reaction and tried with hydrogen and sodium borohydride. Hydrogen is obtained “in situ” by the reaction between NaBH4 and acid media ( ). H2 is conduced to the cell with nitrogen and it is bubbled on the nanoceria solution. 0,4372g of NaBH4 in 30mL of 0,1M HCl is used as source of hydrogen. The measurements parameters are the same that the valid procedure (A7iii). Twenty measurements were saved but without results. xi) Another way to reduce nanoceria is the direct use of NaBH4. NaBH4 is a strong reducing agent and the reduction can be excessive. 5mL of 0,1M HCl is the electrolyte. Pt-ME is used as WE. The concentration of 4nm nanoceria in 5mL of electrolyte was 1410ppm (5,5.1018NP L-1). 0,2mg of NaBH4 is added on the cell. NaBH4 and nanoceria were in contact for 30 minutes and then nitrogen is bubbled to displace the excess of hydrogen. WE is chemically activated as the previous cases. Thirty measurements were saved with the same parameters that A7iii (11 peaks were observed). 64 xii) 5mL of 0,1M HCl is the electrolyte. Pt-ME is used as WE. The parameters used were: The concentration of 10-20nm nanoceria in 5mL of electrolyte was 1410ppm (3,86.1018NP L-1). 0,2mg of NaBH4 is added on the cell. NaBH4 and nanoceria were in contact for 30-60 minutes and then nitrogen is bubbled to displace the excess of hydrogen. WE is chemically activated as the previous cases. Thirty-three measurements were saved with valid results (53 peaks were extracted). xiii) The above procedure (xii) is repeated for 10-20nm nanoceria with the same parameters and features. One hundred ninety measurements were saved with valid results (10 peaks were extracted). E (V) Measurement duration (s) Measuring time (s) -0,2 20 0,002 65 A8 Plots of peaks in 4nm nanoceria by direct PC. Some peaks obtained by direct PC following the general procedure and parameters collected in A6ii are shown. Eight graphics with peaks were superimposed in each plot. 72 A12 Results of peaks in nanoceria by catalytic reduction particle collision. Some peaks obtained by direct modified PC following the general procedure and parameters collected in A7xi are shown. 73 A13 Peaks in 10-20nm nanoceria by catalytic reduction particle collision. Some peaks detected following the general procedure and parameters collected in A7xi are shown. Five graphics with peaks were superimposed in both plots. Peak oxidation obtained in this procedure and attributed to the oxidation of Ce0. 74 A14 TEM images and size distribution of 4nm nanoceria. This annex has been written with permission and data provided by Dra. Laura Sanchez. STEM equipment (Tecnai G2-F30 Field Emission Gun microscope) was working at 300kV with a super-twin lense and 0.2 nm point-to-point resolution and 0.1 line resolution. Nanoceria 4nm were prepared by depositing and airdrying a droplet on the carbon-coated 300 mesh copper grid. Images were obtained by GATAN CCD camera and subsequently analyzed with ImageJ 1.47 v. software. 100 NPs per image were analyzed in full five images. Some STEM images and size distribution obtained from them are shown below in figures A14a and A14b respectively. Figure A14a. STEM image of 4nm nanoceria. Figure A14b. Size distribution obtained from image analysis of 4nm nanoceria. 0 50 100 150 200 250 300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Frequency radius (nm) CeO2NPs 4nm by STEM 75 9. 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