A Novel NanoMIP-SPR Sensor for the Point-of-Care Diagnosis of Breast Cancer

Citation: Erol, K.; Hasabnis, G.; Altintas, Z. A Novel NanoMIP–SPR Sensor for the Point-of-Care Diagnosis of Breast Cancer. Micromachines 2023,14, 1086. https://doi.org/10.3390/mi14051086 Academic Editors: Adil Denizli and Ye¸seren Saylan Received: 24 April 2023 Revised: 15 May 2023 Accepted: 18 May 2023 Published: 21 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). micromachines Article A Novel NanoMIP–SPR Sensor for the Point-of-Care Diagnosis of Breast Cancer Kadir Erol 1,2, Gauri Hasabnis 1and Zeynep Altintas 1,3,* 1Institute of Materials Science, Faculty of Engineering, Kiel University, 24143 Kiel, Germany; [email protected] (K.E.); [email protected] (G.H.) 2 Environmental Health Program, Department of Medical Services and Techniques, Vocational School of Health Services, Hitit University, Corum 19030, Turkey 3Kiel Nano, Surface and Interface Science (KiNSIS), Kiel University, 24118 Kiel, Germany *Correspondence: [email protected]; Tel.: +49-(0)431-880-6198 Abstract: Simple, fast, selective, and reliable detection of human epidermal growth factor receptor 2 (HER2 ) is of utmost importance in the early diagnosis of breast cancer to prevent its high prevalence and mortality. Molecularly imprinted polymers (MIPs), also known as artificial antibodies, have recently been used as a specific tool in cancer diagnosis and therapy. In this study, a miniaturized surface plasmon resonance (SPR)-based sensor was developed using epitope-mediated HER2-nanoMIPs. The nanoMIP receptors were characterized using dynamic light scattering (DLS), zeta potential, Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), energydispersive X-ray spectroscopy (EDX), and fluorescent microscopy. The average size of the nanoMIPs was determined to be 67.5 ±12.5 nm. The proposed novel SPR sensor provided superior selectivity to HER2 with a detection limit (LOD) of 11.6 pg mL −1 in human serum. The high specificity of the sensor was confirmed by cross-reactivity studies using P53, human serum albumin (HSA), transferrin, and glucose. The sensor preparation steps were successfully characterized by employing cyclic and square wave voltammetry. The nanoMIP–SPR sensor demonstrates great potential for use in the early diagnosis of breast cancer as a robust tool with high sensitivity, selectivity, and specificity. Keywords: SPR sensor; human epidermal growth factor receptor 2 (HER2); peptide imprinting; nanoMIPs; breast cancer diagnosis 1. Introduction Breast cancer (BC) is considered one of the leading causes of death in females, and the BC-caused mortality rate has been increasing annually [ 1 , 2 ]. Despite the high prevalence and mortality caused by BC, its early recognition and diagnosis can significantly increase the survival rate. Hence, it is necessary to develop susceptible innovative methods for preventive and therapeutic measures to increase the survival rate of patients with BC [ 2 ]. Human epidermal growth factor receptor-2 (HER2) is a transmembrane tyrosine kinase receptor and plays a vital role in regulating average cell growth, differentiation, and survival [ 3 , 4 ]. The overexpression of HER2 is associated with a molecular anomaly in 15–25% of patients with BC. Accordingly, HER2 is considered a prognostic and predictive biomarker for the detection and monitoring of breast cancer [5]. The expression of HER2 has been recently assessed on the basis of a set of invasive techniques, such as immunohistochemistry (IHC) and biopsy using fluorescent in situ hybridization (FISH) [ 6 – 9 ]. The most important disadvantages of these methods are difficulties due to their complex nature and multi-step procedures [ 10 ], high cost, the necessity of the long-term assessment of high-quality tissue samples [ 11 , 12 ], and the requirement of trained personnel. Enzyme-linked immunosorbent assay (ELISA) is generally used to detect HER2 in the serum. However, ELISA has some drawbacks, such as suppressing thermodynamic/kinetic studies of antibody–antigen interaction and using labeled molecules to Micromachines 2023,14, 1086. https://doi.org/10.3390/mi14051086 https://www.mdpi.com/journal/micromachines Micromachines 2023,14, 1086 2 of 16 induce false-positive responses [ 13 ]. Therefore, there is still a strong need to develop a rapid, easy-to-use, noninvasive, inexpensive, and ultra-sensitive method for detecting the HER2 biomarker with the purpose of minimizing the technical impediments of conventional methods. Such a method may also accurately identify HER2 in the bloodstream [1]. To date, various electrochemical, SPR, piezoelectric, and FRET-based optical biosensors that are cheaper and more sensitive than serological methods have been reported for the detection of HER2 [ 1 ]. These biosensors enable the selective and fast detection of HER2 with a low detection limit [ 14 ]. However, the materials used as bio-receptors, including aptamers [ 14 ], antibodies [ 15 ], and peptides [ 16 ], possess some disadvantages, such as being unstable and expensive and having a short shelf-life, leading to difficulties in integrating biosensor systems. Recently, molecularly imprinted polymers (MIPs), possible alternatives to natural recognition elements, have increasingly been used in biosensing and have outstanding advantages, such as bearing highly specific, sensitive, stabile biorecognition cavities on the sensor surfaces; robustness; resistance to extreme physical conditions; and being synthesized via relatively simple, cheap, and scaleable protocols [ 17 , 18 ]. In the molecular imprinting field, epitope imprinting is generally preferred over whole-protein imprinting because of the problems arising from the large, complex structure of proteins as well as the changes in their conformation during the imprinting process [ 19 , 20 ]. Epitope-imprinted MIPs are able to recognize both the epitope and whole macromolecules (e.g., proteins, viruses, and bacteria) and bind to them specifically [ 21 ]. The solid-phase synthesis approach for obtaining nanoMIPs results in the formation of high-affinity and -sensitivity receptors even in aqueous media, which is the natural environment of biological molecules. In addition to this, it allows low detection limits for developed sensors [ 22 ]. NanoMIPs are promising alternatives to natural antibodies in diagnostic and in vivo applications due to their cost efficiency, high affinity, and stability [23–25]. To our knowledge, in this study, a miniaturized SPR-based sensor was developed for the first time for the detection of HER2 using epitope-mediated nanoMIPs. The nanoMIP receptors were characterized using dynamic light scattering (DLS), zeta potential, Fouriertransform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), energydispersive X-ray spectroscopy (EDX), fluorescence microscopy analyses, and electrochemical methods. The nanoMIP–SPR sensor could detect HER2 with high selectivity, specificity, and sensitivity. 2. Materials and Methods 2.1. Reagents and Chemicals N-isopropylacrylamide (NIPAm), N,N 0 -methylenebisacrylamide (BIS), N-(3-aminopropyl) methacrylamide hydrochloride (APMA), acrylic acid (AAc), N-tert-butylacrylamine (TBAm), N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED), ammonium persulphate (APS), ethanol (absolute), methanol (ACS reagent, ≥ 99.8%), acetone (ACS reagent, ≥ 99.5%), toluene (anhydrous), glutaraldehyde (GA), ethanolamine, Tween 20 (polyoxyethylenesorbitan monolaurate), phosphate-buffered saline (PBS), sulfuric acid, hydrogen peroxide, 11-mercaptoundecanoic acid (MUDA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), sodium hydroxide (NaOH), sodium borohydride (SBH), transferrin from human blood plasma, albumin from human serum (HSA), and tumor protein p53 were purchased from Sigma Aldrich Chemical Co. (Hamburg, Germany). 3-Aminopropyltriethoxysilane (APTES) was obtained from Fisher Scientific (Schwerte, Germany). Methacryloxyethyl thiocarbamoyl rhodamine B was provided by Polysciences Europe (Bergstrasse, Germany). HER2-ECD (10004-HCCH) and the peptide (163–175: DTNRSRACHPCSP) chosen for imprinting were provided by Sino Biological (Eschborn, Germany) and GenScript Biotech (Nanjing, China), respectively. Glass beads (0.75–1.0 mm), glucose, and syringe filters (Rotilabo PTFE, 0.45 µ m and 0.22 µ m) were supplied by Carl Roth (Karlsruhe, Germany). Without additional purification, all compounds and solvents were of analytical or HPLC quality. A 0.22 µ m syringe filter was used to filter a phosphate-buffered saline + 0.05% Tween Micromachines 2023,14, 1086 3 of 16 (PBS/T) buffer. Double-distilled water (DDW, produced by Ion Ex Mischbettpatrone, Arno Willers, Hamburg, Germany) was used to prepare all solutions in the aquatic environment. 2.2. Preparation of Glass Beads for NanoMIP Synthesis First, the glass beads, the firm support of the solid-phase synthesis, were prepared for the silanization process. For this, 60 g of glass beads was weighed in a beaker and activated by boiling in 2 M NaOH solution for 15 min. Then, the glass beads were washed five times with DDW, four times with PBS (pH: 7.4), and five times with DDW. After the last wash with excess water, the pH value of the wash water was determined to be 7.0–7.5, and the process was continued by washing the glass beads twice with acetone. The beads were then dried in a nitrogen atmosphere and incubated overnight in 2% v/v APTES solution (in anhydrous toluene) in a sealed container. After this step, the silanization part of the process was completed. The glass beads were removed from the APTES solution and washed four times with acetone and methanol. Beads dried with nitrogen gas were incubated in a solution of 7% GA prepared in PBS (pH: 7.4) for 120 min. This is an intermediate step so the peptide can be covalently attached to the glass beads. Following this step, 10 mg of the cold peptide solution dissolved in 40 mL PBS was added to the glass beads, which were washed five times with DDW. This is an essential step in the process, and overnight incubation was allowed to ensure that the peptide was covalently bound to the beads. After incubation, the peptide solution was poured out, and the glass beads were washed five times with DDW. Afterward, the glass beads were treated with SBH solution (1 mg mL −1 ) prepared in PBS (pH: 7.4) for 30 min. SBH is an effective aldehyde-blocking agent with a strong reducing structure [ 22 ]. Glass beads, repeatedly washed with an excess of DDW, interacted with 50 mL of 0.1 mM ethanolamine (pH: 7.4 in PBS) solution for 15 min to avoid non-specific interactions and self-reaction of unconjugated free GA groups. After the formed interaction, the ethanolamine solution was poured out, and the glass beads were washed five times with DDW, dried in a nitrogen atmosphere, and placed in the reaction flask. After this step, the glass beads were ready for nanoMIP synthesis (Figure 1). 2.3. Synthesis of Target NanoMIPs The necessary monomers for polymerization were first mixed. In brief, 39 mg of NIPAm, 2 mg of BIS, 54 mg of APMA, and 2.2 µ L of AAc were added to 98 mL of PBS ( pH: 7.4 ) solution in a reaction flask. In addition, 38 mg of TBAm and 3 mg of methacryloxyethyl thiocarbamoyl rhodamine B monomers were separately dissolved in 1 mL of absolute ethanol and added to the monomer mixture in the reaction flask. The monomer mixture was stirred under a magnetic stirrer for 30 min. Afterward, the mixture was sonicated (Transsonic Digital S, Elma Schmidbauer GmbH, Singen, Germany) for 20 min, and then nitrogen gas was passed through the mixture for 20 min. The process was continued by adding peptide-coupled glass beads to the reaction flask, and nitrogen gas was re-passed via the mixture for 2 min. Then, 800 µ L of APS solution (initiator, 60 mg mL −1 , in water) and 24 µ L of TEMED (activator) were instantly added to the monomer mixture, nitrogen gas was passed through the mouth of the reaction flask for 30 s, and the flask was tightly closed with a screw cap. Polymerization was completed in 1 h, the bottle was opened, and the cold wash and hot wash stages were carried out one after the other. Hot and cold washing steps were applied in a polypropylene SPE tube (including polyethylene frits: 20 µm pore size). The cold wash process was performed to remove unreacted monomers and low-affinity nanoMIPs from the polymerization medium. For this step, three consecutive washes were carried out with 20 mL of DDW (5 ◦ C). Then, the SPE column containing the glass beads was kept in a water bath (GFL Shaking Water Bath 1083, Burgwedel, Germany) for 15 min to prepare for hot washing. In the next step, seven washes were performed with 20 mL of DDW (65 ◦ C) each to collect the high-affinity (target) nanoMIPs (Figure 2). The collected nanoMIP pool (140 mL) was stored at 4 ◦ C for the subsequent experimental studies. Three samples of 10 mL were taken to calculate the yield from the obtained nanoMIP suspension. These samples were placed in the predetermined weight glass vials and dried Micromachines 2023,14, 1086 4 of 16 using freeze-drying (ALPHA 2–4LD Plus freeze-dryer, Christ, Osterode am Harz, Germany) for 48 h. After completely removing the water through the drying process, the glass vials were re-weighed, and the weight of the nanoMIPs was determined by subtracting the tare of the vials from the obtained weight values. Micromachines 2023, 14, x FOR PEER REVIEW 4 of 17 Figure 1. The immobilization of the HER2-peptide on glass beads as the template. 2.3. Synthesis of Target NanoMIPs The necessary monomers for polymerization were first mixed. In brief, 39 mg of NIPAm, 2 mg of BIS, 54 mg of APMA, and 2.2 µL of AAc were added to 98 mL of PBS (pH: 7.4) solution in a reaction flask. In addition, 38 mg of TBAm and 3 mg of methacryloxyethyl thiocarbamoyl rhodamine B monomers were separately dissolved in 1 mL of absolute ethanol and added to the monomer mixture in the reaction flask. The monomer mixture was stirred under a magnetic stirrer for 30 min. Afterward, the mixture was sonicated (Transsonic Digital S, Elma Schmidbauer GmbH, Singen, Germany) for 20 min, and then nitrogen gas was passed through the mixture for 20 min. The process was continued by adding peptide-coupled glass beads to the reaction flask, and nitrogen gas was re-passed via the mixture for 2 min. Then, 800 µL of APS solution (initiator, 60 mg mL −1 , in water) and 24 µL of TEMED (activator) were instantly added to the monomer mixture, nitrogen gas was passed through the mouth of the reaction flask for 30 s, and the flask was tightly closed with a screw cap. Polymerization was completed in 1 h, the bottle was opened, and the cold wash and hot wash stages were carried out one after the other. Hot and cold washing steps were applied in a polypropylene SPE tube (including polyethylene frits: 20 µm pore size). The cold wash process was performed to remove unreacted monomers and low-affinity nanoMIPs from the polymerization medium. For this step, three consecutive washes were carried out with 20 mL of DDW (5 °C). Then, the Figure 1. The immobilization of the HER2-peptide on glass beads as the template. 2.4. Characterization of NanoMIPs The size distribution profiles of nanoMIPs were determined by a dynamic light scattering (DLS) device (Malvern Panalytical, Zetasizer Pro., Herrenberg, Germany). Sixty runs were performed in the backscattered mode for each recording. In addition, the zeta potential value was determined with the same device to measure the stability of the nanoMIPs in water. The sample of the nanoMIP solution, which was dropped on the glass slider and dried, was imaged with a fluorescence microscope (BZ-X800LE, Keyence, Neu-Isenburg, Germany). The microscope (BZ-PA10, Plan Apochromat 10X, NA 0.45, WD 4 mm) had a 40 W LED fluorescent light source and a power supply of 100 to 240 VAC ± 10%, 50/60 Hz . Of note, the fluorescent property of methacryloxyethyl thiocarbamoyl rhodamine B monomer (excitation max: 548 nm, concentration in the polymerization mixture: 45 µ M) gave the polymer a fluorescent nature. An FT-IR device (Cary 630 FTIR, Agilent Technologies, Santa Clara, CA, USA) was used to analyze the functional groups that existed in nanoMIPs. TEM analyses were performed on an FEI Tecnai F30 G2 STwin (300 kV, FEG) equipped with an Micromachines 2023,14, 1086 5 of 16 EDX detector (Si/Li, EDAX) to visualize the morphological structure of the nanoMIPs and analyze the elemental composition of the polymer. Micromachines 2023, 14, x FOR PEER REVIEW 5 of 17 SPE column containing the glass beads was kept in a water bath (GFL Shaking Water Bath 1083, Burgwedel, Germany) for 15 min to prepare for hot washing. In the next step, seven washes were performed with 20 mL of DDW (65 °C) each to collect the high-affinity (target) nanoMIPs (Figure 2). The collected nanoMIP pool (140 mL) was stored at 4 °C for the subsequent experimental studies. Three samples of 10 mL were taken to calculate the yield from the obtained nanoMIP suspension. These samples were placed in the predetermined weight glass vials and dried using freeze-drying (ALPHA 2–4LD Plus freeze-dryer, Christ, Osterode am Harz, Germany) for 48 h. After completely removing the water through the drying process, the glass vials were re-weighed, and the weight of the nanoMIPs was determined by subtracting the tare of the vials from the obtained weight values. Figure 2. The synthesis principle of peptide-imprinted nanoMIPs. 2.4. Characterization of NanoMIPs The size distribution profiles of nanoMIPs were determined by a dynamic light scattering (DLS) device (Malvern Panalytical, Zetasizer Pro., Herrenberg, Germany). Sixty runs were performed in the backscattered mode for each recording. In addition, the zeta potential value was determined with the same device to measure the stability of the nanoMIPs in water. The sample of the nanoMIP solution, which was dropped on the glass slider and dried, was imaged with a fluorescence microscope (BZ-X800LE, Keyence, NeuIsenburg, Germany). The microscope (BZ-PA10, Plan Apochromat 10X, NA 0.45, WD 4 Figure 2. The synthesis principle of peptide-imprinted nanoMIPs. In addition, the successful development of the nanoMIP sensor was verified using two main electrochemical techniques, including cyclic voltammetry (CV) and square-wave voltammetry (SWV). For this, a gold substrate was used as the working electrode in the electrochemical measurement setup (PalmSens4 workstation, Belltec, Lüdenscheid, Germany). All CV measurements were performed at a potential range of − 0.2 to 0.8 V and a scan rate of 0.05 V s −1 . The range of applied potentials for SWV measurements was − 0.3 to 0.8 V at an amplitude of 0.05 V and a frequency of 5 or 10 Hz. The experiments were performed at room temperature. 2.5. Optical Detection of the HER2 Biomarker The bare gold SPR chip was initially cleaned with a mixture of hydrogen peroxide (35%, 2 mL), ammonia (25%, 2 mL), and millipore water (50 mL) and boiled at 80 ◦ C for 20 min . For this process, two gold chips were placed in a chip holder and immersed in the preheated mixture. Most contaminants on the gold surface were eliminated using piranha solution (a 3:1 mixture of sulfuric acid and 30% hydrogen peroxide). After this treatment, the gold chips were washed five times with millipore water and three times with absolute ethanol. The chips were then dried with a gentle flow of nitrogen gas. Next, the gold chips were immersed in the MUDA solution (2 mM, 5 mL) prepared in absolute ethanol in a Petri dish and incubated overnight in the dark. This process created a self-assembled monolayer on the surface of the chips. After incubation, the gold chips were washed with an excess of Micromachines 2023,14, 1086 6 of 16 absolute ethanol and double-distilled water, gently dried with nitrogen gas, and stored in a fridge at 4 ◦C until the time of use. A miniaturized angular SPR device (CORGI IIF, Plasmetrix, Montreal, QC, Canada) was used for all detection studies, including HER-2 peptide and protein as the target molecules. During the sensor experiments, the solutions were allowed to pass through the chip surface with a peristaltic pump (Ismatec Reglo ICC Digital pump, 2-channel, Cole-Parmer GmbH, Wertheim, Germany), providing a flow rate of 4 µ L min −1 . MUDAcoated chips were activated with a 4 min injection of a freshly made EDC/NHS solution ( 0.4 M EDC , 0.1 M NHS) for the covalent immobilization of the nanoMIPs with the aid of amine coupling chemistry. A constant flow rate (4 µ L min −1 ) was maintained for 8 min to immobilize the nanoMIPs on the gold chip surface. To prepare the nanoMIP medium ( 500 µg mL−1 ), the suspension was prepared with degassed PBS/T and filtered through a 0.22 µm syringe filter prior to sonication for 30 min. The samples were prepared in PBS/T to promote fluid flow through the sensor and microtube channels and prevent air from becoming trapped inside the microfluidics. The nanoMIP suspension was then re-filtered with a 0.45 µ m syringe filter. The chip surface then interacted with 1.0 mM ethanolamine solution for 4 min to block possible active sites that may have remained on the surface after nanoMIP immobilization. The injection of samples, including HER2 peptide and HER2 biomarker, at varying concentrations were subsequently carried out (Figure 3). The association and dissociation times were set at 5 and 2 min, respectively. Micromachines 2023, 14, x FOR PEER REVIEW 7 of 17 Figure 3. The preparation of nanoMIP-based sensor system. 3. Results and Discussion 3.1. Size and Stability of Target NanoMIPs The hydrodynamic size of target nanoMIPs was measured in DDW at room temperature by employing DLS. The nanoMIPs were discovered to have an average hydrodynamic radius of 97.79 ± 0.53 nm and a polydispersity index (PDI) of 0.263, revealing remarkably uniform and monodisperse particles (Figure 4a,b). Additionally, the zeta potential of the nanoMIPs in DDW was tested to ascertain the stability of the solution. The average zeta potential of nanoparticles (NPs) was found to be −12.37 ± 0.32 mV (Figure 4c). It was hypothesized that the NPs forming a dispersed phase endowed the solution with a colloidal character. Figure 3. The preparation of nanoMIP-based sensor system. Micromachines 2023,14, 1086 7 of 16 For selectivity studies, a control nanoMIP was obtained by imprinting a different peptide (ISASRKLQLK). It was immobilized on the sensor surface by following the aforementioned procedure prior to the injection of target analytes for the determination of sensor selectivity. Furthermore, the specificity of the developed nanoMIP–SPR sensor was realized by studying reference molecules (i.e. P53, HSA, transferrin, and glucose). 3. Results and Discussion 3.1. Size and Stability of Target NanoMIPs The hydrodynamic size of target nanoMIPs was measured in DDW at room temperature by employing DLS. The nanoMIPs were discovered to have an average hydrodynamic radius of 97.79 ± 0.53 nm and a polydispersity index (PDI) of 0.263, revealing remarkably uniform and monodisperse particles (Figure 4a,b). Additionally, the zeta potential of the nanoMIPs in DDW was tested to ascertain the stability of the solution. The average zeta potential of nanoparticles (NPs) was found to be − 12.37 ± 0.32 mV (Figure 4c). It was hypothesized that the NPs forming a dispersed phase endowed the solution with a colloidal character. Micromachines 2023, 14, x FOR PEER REVIEW 8 of 17 (a) (b) (c) Figure 4. (a) The hydrodynamic size distribution of target nanoMIPs; (b) The correlation fit of DLS analysis; (c) The zeta potential profile of nano-polymers. 3.2. Fluorescence Microscopy and TEM Analyses For fluorescence microscopy imaging, a certain concentration (500 µg mL −1 ) of nanoMIP solutions was prepared. The fluorescence microscopy image proved that the fluorescent monomer (methacryloxyethyl thiocarbamoyl rhodamine B) used in the synthesis of nanoMIP was well incorporated into the polymeric structure (Figure 5a). Additionally, TEM images confirmed the exact size, shape, and uniformity of the nanoMIPs. (Figure 5b). The size of particles acquired by TEM was approximately 67.5 ± 12.5 nm which was smaller than those measured by DLS due to solvation and swelling of polymer particles in the latter case. Another reason is that the agglomeration of nanoMIPs in solution causes an evident size increment in DLS measurements [26]. Figure 4. ( a ) The hydrodynamic size distribution of target nanoMIPs; ( b ) The correlation fit of DLS analysis; (c) The zeta potential profile of nano-polymers. Micromachines 2023,14, 1086 8 of 16 3.2. Fluorescence Microscopy and TEM Analyses For fluorescence microscopy imaging, a certain concentration (500 µ g mL −1 ) of nanoMIP solutions was prepared. The fluorescence microscopy image proved that the fluorescent monomer (methacryloxyethyl thiocarbamoyl rhodamine B) used in the synthesis of nanoMIP was well incorporated into the polymeric structure (Figure 5a). Additionally, TEM images confirmed the exact size, shape, and uniformity of the nanoMIPs. (Figure 5b). The size of particles acquired by TEM was approximately 67.5 ± 12.5 nm which was smaller than those measured by DLS due to solvation and swelling of polymer particles in the latter case. Another reason is that the agglomeration of nanoMIPs in solution causes an evident size increment in DLS measurements [26]. Micromachines 2023, 14, x FOR PEER REVIEW 9 of 17 (a) (b) Figure 5. (a) Fluorescence microscopy and (b) TEM images of nanoMIPs. 3.3. FT-IR and EDX Analysis FT-IR analysis was performed to prove the successful synthesis of peptide-imprinted nanoMIPs (Figure 6a,b). The results revealed that the spectrum of NIPAM as a structural monomer was significantly different from the polymer (nanoMIP) spectrum and exhibited only a few expected peaks. C-H asymmetric stretching (2967 cm−1), C-H symmetric stretching (2877 cm−1), C=O amide group (1632 cm−1), N-H bending (1545 cm−1), C-N stretching (1366 cm−1), -CH2 bending (1452 cm−1), and -CH3 bending (1385 cm−1) vibrations were notable peaks in the spectrum of NIPAM. Vibrations such as C-H asymmetric–symmetric stretching (2917 cm−1 and 2848 cm−1, respectively), C=O amide group (1636 cm−1), and NH bending (1539 cm−1) also appeared in the polymer spectrum. In addition, the FT-IR analysis of the imprinted peptide revealed that no peak of the peptide was identified in the nanoMIP spectrum. This indicates that the template removal from the polymeric structure, as one of the most critical steps of the imprinting process, was effectively achieved. Furthermore, the EDX analysis was performed to analyze the elemental composition of nanoMIPs. Carbon and oxygen elements in the structure indicated the synthesis of the desired organic polymer. (Figure 6c). Figure 5. (a) Fluorescence microscopy and (b) TEM images of nanoMIPs. 3.3. FT-IR and EDX Analysis FT-IR analysis was performed to prove the successful synthesis of peptide-imprinted nanoMIPs (Figure 6a,b). The results revealed that the spectrum of NIPAM as a structural monomer was significantly different from the polymer (nanoMIP) spectrum and exhibited only a few expected peaks. C-H asymmetric stretching (2967 cm −1 ), C-H symmetric stretching (2877 cm −1 ), C=O amide group (1632 cm −1 ), N-H bending (1545 cm −1 ), C-N stretching (1366 cm −1 ), -CH 2 bending (1452 cm −1 ), and -CH 3 bending (1385 cm −1 ) vibrations were notable peaks in the spectrum of NIPAM. Vibrations such as C-H asymmetric–symmetric stretching (2917 cm −1 and 2848 cm −1 , respectively), C=O amide group (1636 cm −1 ), and Micromachines 2023,14, 1086 9 of 16 N-H bending (1539 cm −1 ) also appeared in the polymer spectrum. In addition, the FT-IR analysis of the imprinted peptide revealed that no peak of the peptide was identified in the nanoMIP spectrum. This indicates that the template removal from the polymeric structure, as one of the most critical steps of the imprinting process, was effectively achieved. 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