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Spectroelectrochemical Nanosensor for the Determination of Cystatin C in Human Blood Waleed A. Hassanain, Emad L. Izake, and Godwin A Ayoko Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02121 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018
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Spectroelectrochemical Nanosensor for the Determination of Cystatin C in Human Blood Waleed A. Hassanain1, Emad L. Izake1*, and Godwin A. Ayoko1 1
Nanotechnology and Molecular Science Discipline, School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology, 2 George Street, Brisbane 4000, Australia. * Corresponding author E-mail:
[email protected]; Phone: +61731382501
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ABSTARCT The detection of protein biomarkers for the clinical diagnosis of diseases requires selective, and sensitive methodologies and biosensors that can be easily used at pathology labs and points of care. An ideal methodology would be able to conduct multimode screening of low and high concentrations of proteins in biological fluids using recyclable platforms. In this work, we demonstrate a novel nanosensing methodology for the dual detection of cystatin C (CST-C), as a protein biomarker model, in blood plasma by surface enhanced Raman spectroscopy and electrochemistry. The new methodology utilizes the thiol chemistry of biomolecules to develop target-specific and recyclable extractor chip for the rapid isolation of protein biomarkers from blood plasma. This is followed by the rapid reduction of the disulfide bonds within the isolated protein to influence its oriented immobilization onto conductive gold coated silicon nanopillar substrate via stable gold–sulphur (Au-S) bonds. The oriented immobilization led to reproducible surface enhanced Raman spectroscopy (SERS) measurements of the reduced protein (RSD =3.8%) and allowed for its direct electrochemical determination. After the SERS measurement, differential pulse voltammetry (DPV) was used to desorb the analyte from the substrate and generate a reduction current that is proportional to its concentration. CST-C was determined down to 1 pM and 62.5 nM by SERS and DPV respectively which satisfy the requirements for monitoring Alzheimer’s and kidney failure diseases. The new dual nanosensing methodology has strong potential for miniaturization in a lab-on-a-chip platform for the screening of many protein biomarkers that have disulfide bond structure.
KEYWORDS: Dual nanosensing, Recyclable chip, Molecular diagnostics, kidney & Alzheimer diseases.
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INTRODCUTION There is a growing demand within the molecular diagnostics market for rapid and cost effective sensing methodologies that can be miniaturized and used for reliable screening of biomarkers in biological fluids at pathology/research labs and points of care. Electrochemical biosensors have been demonstrated in the literature for the detection of protein biomarkers in biological fluids.1 Despite their simplicity, the selectivity of these sensors still requires improvements to allow for their miniaturization and utilization at pathology labs and points of care.2,3 Surface enhanced Raman spectroscopy (SERS) is a powerful analytical tool that can provide molecular structure identification of proteins at ultra-trace concentrations.4 In SERS, the Raman bands intensities are significantly enhanced when the analyte molecules become adsorbed at the gaps between the nanostructures of a noble metal surface where the localised surface plasmon resonance (SPR) on the neighbouring nanostructures couple and generate a large electromagnetic field that is experienced by the analyte molecules.5,6 In addition, when the analyte molecules become adsorbed on the metal substrate, their HOMO and LUMO energy levels interact with the Fermi energy level of the metal surface and a charge transfer complex is formed. When the substrate interacts with incident light of energy that matches the charge transfer transition energy, electron transition occurs between the molecular orbitals of the analyte and the Fermi level of the substrate. This causes the molecular polarization of the analyte molecules to change and produces a chemical enhancement.7 The conductive property of metallic SERS substrates qualifies them as combined SERS / electrochemical platforms to acquire spectral information about the molecular structure and concentration of analytes.8,9 Despite the high sensitivity of SERS, the measurements often suffer from low reproducibility due to the non-uniform distribution of hotspots, and the random orientation of the analyte molecules on the SERS substrate.10-12 To address this problem, we demonstrated in a previous work a highly patterned SERS substrate that has uniform hotspots distribution for the detection of proteins by a handheld Raman spectrometer. By using a raster orbital scanning mode (ROS), the handheld device was able to acquire an average SERS signal from the analyte molecules on the substrate’s surface and reduce the SERS signal variability between repeated measurments.13-15 However, the SERS signal variability that is caused by the random orientation of the adsorbed analyte molecules was not addressed. Therefore, in this work, we were motivated to develop a dual biosensing method for protein biomarkers that takes advantage of the attractive characteristics of SERS and electrochemistry and 3 ACS Paragon Plus Environment
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address the problem of SERS signal variability due to random orientation of analyte molecules. CST-C is a low molecular weight protein that belongs to type 2 cystatin gene family and is used as a biomarker for kidney failure disease.16,17 CST-C is also be a biomarker for cardiovascular diseases, Alzheimer's disease, cancer and type-2 diabetes.18-22 In addition, CST-C levels have been reported to change in patients with thyroid dysfunction and glucocorticoid therapy.23,24 A number of methods have been reported for the detection of CST-C in the biological fluids such as: chromatography, ELISA, particle enhanced turbidimetric immunoassay (PETIA), particle-enhanced nephelometric immunoassay (PENIA), radio immunoassay, fluorescence, NMR and NIR methods.25-36 Despite their sensitivity, the reported methods suffer from disadvantages that limit their use for the rapid screening of CST-C.36,37 Mi et al reported a sensitive photo-electrochemical indirect detection method for the quantification of CST-C in serum where they monitored the change in the photocurrent of a nanobody-TiO2 nanotube array after its immunoreaction with CST-C.38 However, this method required lengthy procedures for the functionalization of the TiO2 array and the binding of the protein (~14 hours). In addition, the method did not directly detect CST-C and the developed immunosensor was not recyclable for repeated use. To the best of our knowledge, no Raman or electrochemical studies have been performed for the direct detection of CST-C. In this work, a SERS/electrochemistry dual biosensing method was developed to detect CST-C by handheld devices for the clinical diagnosis of many diseases such as renal, heart and Alzheimer’s diseases.39,40 EXPRIMENTAL Synthesis of antibody fragments and functionalization of the extractor chip F(ab')2 antibody fragments were synthesized by digesting anti-CST-C antibody using Pierce Mouse IgG1 F(ab')2 preparation Kit. 90 µL of the produced F(ab')2 fragments were mixed with an equal volume of 0.2 mM neutral TCEP to reduce the disulfide bonds in their hinge region and produce thiol-ended Fab' fragments (R-SH). The mixture was loaded onto the gold coated silicon nanopillar chip and left for 40 min at room temperature to bind thiolended Fab' fragments to the gold surface of the chip via Au-S bonds. The chip was then washed six times with 200 µL of 1x PBS to remove any unbound Fab' fragments. To backfill the remaining bare sites on the chip surface, 20 µL of 10-6 M butanethiol (dissolved in 1x PBS, pH 7.4) were loaded and left for 1 hour at 4 °C to form Au-S bonds between the bare 4 ACS Paragon Plus Environment
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gold sites and the alkanethiol. The chip was then washed six times with 200 µL of 1x PBS to remove the excess butanethiol. After washing, the chip was suspended in 200 µL of 1x PBS (pH 7.4) and stored at 4 °C for future use. Isolation and purification of CST-C from blood plasma For the selective capture of CST-C from biological fluids by the extractor chip, a 200 µL of a human blood plasma sample was loaded onto the chip and left to stand for 15 min. The chip was then washed 5 times with 200 µL of 1x PBS to remove any weakly adsorbed molecules from its surface. To release the protein from the extractor chip, it was immersed in 100 µL of glycine.HCl buffer (pH 2.5) for 5 min. The chip was then removed and re-used to capture CST-C from a new human blood plasma sample. To remove the glycine buffer from the released protein sample, it was loaded onto size exclusion column and the CST-C protein eluted within 5 min using 500 µL of 1x PBS (pH 7.4) as the eluent. Control tests Since CST-C exists naturally in blood, it was not possible to carry out positive and/or negative control tests using blood plasma matrix. Therefore we carried out the interference study using an aqueous solution of L-cysteine in BSA, blank PBS buffer (pH 7.4), insulin and phenylalanine. For positive control test, 200 µL of L-cysteine in 0.1% BSA (pH 7.4) were loaded onto a clean extractor chip for 15 min. The chip was then washed 5 times with 200 µL of 1x PBS and immersed in 100 µL of glycine. HCl buffer (pH 2.5) for 5 min. After removing the chip, the liquid phase was loaded onto a size exclusion column to remove the buffer. Finally the column was eluted using 500 µL of 1x PBS (pH 7.4) and the collected eluate screened by SERS. For the negative control test, 20 µL of 1x PBS solution was loaded onto a clean extractor chip, the above procedures carried out and the eluate from the size exclusion column screened by SERS. For the insulin and the phenylalanine tests, the protein or amino acid solution (10-5 M in PBS, pH 7.4) was loaded onto the extractor chip and the capture/release procedures carried out. After removing the chip, the liquid phase was loaded onto a size exclusion column to remove the buffer. Finally the column was eluted using 500 µL of 1x PBS (pH 7.4) and the collected eluate screened by SERS.
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Determination of CST-C in human blood plasma by SERS and DPV To determine CST-C in biological fluids, the protein was isolated from blood plasma sample using the extractor chip. 20 µl of the isolated protein were chemically reduced using TCEP onto a gold coated silicon nanopillar substrate and left to stand for 10 min. The substrate was then washed 5 times with 200 µL of 1x PBS and screened by SERS and he intensity of the band at 1363 cm-1 was monitored to determine the concentration of the protein using the developed SERS calibration curve. For the electrochemical quantification of the protein in blood, 200 µL of a human blood plasma sample was extracted using the extractor chip and reduced using TCEP. 20 µl of the isolated protein were reduced by TCEP onto a clean gold coated silicon nanopillar substrate and left to stand for 10 min. The substrate was then washed 5 times with 200µL of 1x PBS and the protein quantified by DPV. Recycle of functionalized extractor chip by cyclic voltammetry (CV) To recycle the extractor chip, the inactive antibody fragments were electrochemically desorbed from the gold coated silicon nanopillar substrate by CV. The extractor chip was utilized as the working electrode in a three electrode cell. 0.1 M KOH solution was used as an electrolyte. The cell potential was swept from 0.1 V to -1.4 V using a potential step of 0.004 V and a scan rate of 0.1 VS-1. 130 CV cycles were carried out for the removal of the antibody fragments. The recycled chip was then washed 3 times with 1x BPS buffer, re-functionalized with fresh thiol-ended Fab' fragments and screened by SERS to confirm the attachment of the fragments. RESULTS AND DISCUSSION SERS spectrum of authentic and reduced CST-C The molecular structure of human CST-C contains 120 amino acids with four cysteine residues connected by two disulfide bonds.41 To analyse CST-C by Raman spectroscopy, we acquired, for the first time, its Raman spectrum where authentic samples of the protein were loaded onto gold coated silicon nanopillar substrates and screened by a benchtop and handheld Raman spectrometers in the raster orbital scanning mode (Figures S1, and 1a respectively).42 The background noise and florescence were automatically corrected by the spectrometer software.13 As indicated by the figures, the Raman spectrum collected by the benchtop instrument showed significant fluorescence background while that collected by the 6 ACS Paragon Plus Environment
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handheld device has less resolution. However due to its portability, and ability to rapidly acquire an average SERS signal from the entire SERS substrate, the handheld device was utilized for the rest of this study.13, 43 The Raman vibration modes of CST-C, as recorded by the handheld Raman spectrometer, are depicted in Table S1.44-57 As indicated by Figure 1b, the repeated SERS screening of CST-C on the gold coated silicon nanopillar substrate showed noticeable variations especially in the wave number regions of 1528-1590, 1261 and 1197 cm-1 vibration modes. These variations are attributed, in part, to the random orientation of the protein molecules onto the substrate. 10-12 For the determination of proteins by SERS, the substrate should have uniform distribution of hotspots, the analyte molecules should take a unified orientation on the substrate and the SERS measurement should represent the entire sample loaded on the substrate.11,
12, 58
Therefore to screen CST-C by SERS, we utilized a highly ordered gold
coated silicon nanopillar substrate that has uniform hotspot distribution (Figure S2).15 We also carried out the measurements using the handheld Raman spectrometer in the raster orbital mode to collect an average SERS signal. To orient the protein molecules in one direction on the substrate, we modified the molecular structure of human CST-C. Cystatin C contains two disulfide bonds between four cysteine residues. Therefore, we reduced the disulfide bonds in the protein structure to generate free SH terminal groups that form Au-S bonds with the gold surface of the substrate (Figure S3) and assemble the protein molecules in an upward orientation onto the substrate surface.59 The SERS spectrum of the reduced protein showed general agreement with that of the unreduced protein (Figures 1c and 1a), with new bands appearing at 1363, 1179, 931, and 467 cm-1 (Table S1). The new band at 467 cm-1 indicates the immobilization of the C-S bonds of the reduced protein onto the plasmonic gold surface of the substrate, thus experiencing strong electromagnetic enhancement.60 The SERS measurements of the reduced protein, on seventeen independent gold coated silicon nanopillar substrates, were highly reproducible (Figure 1d), as compared to those of the unreduced protein (Figure 1b). Manufacture of CST-C extractor chip To manufacture an extractor chip for CST-C, thiol-ended Fab' fragments (R-SH) of anti CST-C antibody were synthesized and immobilized onto a gold coated chip via Au-S bonds.61 This allows the binding sites of the immobilized antibody fragments to align in an upward orientation and, maximizes the capturing efficiency of the extractor chip towards the 7 ACS Paragon Plus Environment
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antigen by alleviating the steric hindrance that prevents the binding process.13,15,59,62,63 In addition, the small size of the Fab' fragments increases their surface loading on the substrate and increases the overall capturing capacity of the extractor chip.64 Following its functionalization with the antibody fragments, the remaining bare sites on the chip surface were backfilled with butanethiol to prevent nonspecific binding events by foreign molecules. The formation of Au-S bonds between the alkane thiol and the extractor chip alters the charge of its gold surface. This change and leads to a charge transfer between HOMO/LUMO energy levels of butanethiol and the Fermi energy level of the gold.7 The change in the surface charge also leads to the displacement of a few antibody fragment molecules from the gold surface and the re-orientation of the lying down antibody fragment to assume an upward position.59,65 This mechanical switch of the lying-down antibody fragments boosts the capturing efficiency of the functionalized chip towards the target protein. Isolation of CST-C from blood plasma and control tests The extractor chip was used to selectively capture CST-C from blood plasma. We attempted to directly determine CST-C by SERS (in situ SERS detection) after its binding to the antibody fragments on the extractor chip. However, the binding of the protein caused minor changes to the SERS spectrum of antibody fragments. This may be attributed to the fact that the bound protein is lying on top of the antibody fragment at a far distance from the plasmonic surface of the extractor chip and therefore experience only weak SERS enhancement. In addition, the antibody fragments and the protein have some common amino acid building blocks and therefore it is difficult to reliably quantify the protein concentration by in situ SERS measurements. This observation has been also reported by Hassanain et al for the determination of microcystin LR by SERS.13 Therefore, for the direct detection of CST-C by SERS, we attempted the release and purification of the protein from the chip using glycine buffer (pH 2.5).66 The protein was then reduced and screened by SERS. As indicated by Figure 2i, the acquired spectrum of the reduced protein was in general agreement with that of the reduced CST-C standard in Figure 1c. This result indicates the selective capture of the target protein from the biological matrix. To further demonstrate the selectivity of the extractor chip towards CST-C, positive and negative control tests were carried out using L-cysteine in 0.1% BSA (positive control) and blank PBS (negative control) samples. We also tested the selectivity of the extractor chip 8 ACS Paragon Plus Environment
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against insulin as another protein that has have disulfide bond structure and against phenylalanine as hydrophobic amino acid that exists in biological fluids. After carrying out the capture and release processes, the samples were screened by SERS. As indicated by Figure 2(ii-v), no Raman spectra were detected from the positive and negative control samples as well as the insulin and phenylalanine molecules which confirms the inability of the extractor chip to bind biomolecules other than CST-C. SERS quantification of CST-C For the SERS quantification of CST-C (in the reduced form), the intensity of the Raman band at 1363 cm-1 in the reduced protein spectrum was monitored at different concentrations and found to monotonically increase with the protein concentration in the working range of 1x10-7 M to 1x10-12 M (Figure 3a). The relationship between the intensity at 1363 cm-1 Raman band and log the concentration of the reduced CST-C was found to follow the linear regression equation y = 93.122x + 1495.3 (Figure 3b). The correlation coefficient (R2) was found to be 0.9986 while the LOQ of the SERS quantification was 1 pM. The RSD in the SERS measurements was calculated using the intensity of the Raman band at 1363 cm-1 and found to be 3.8% (n = 15). The RSD in the SERS measurements between days was 7.45%. The wide working range and excellent sensitivity of the SERS screening confirms its potential value in monitoring CST-C blood levels in renal, heart and Alzheimer’s diseases.39,67 The uniform distribution of hotspots on the nanopillar substrate surface has been reported to lead to RSD values of ≤ 8% in SERS measurements that were carried out on substrate different batches.15 Therefore, the low RSD of 3.8% in the SERS measurements of CST-C is attributed to the oriented immobilization of the reduced CST-C molecules onto the uniform hotspots of the substrate. Electrochemical quantification of CST-C The electrochemical desorption of alkanethiols from a gold substrate can be achieved by reducing the Au-S bonds between the thiol compound and the gold at a negative potential. The magnitude of the DPV current is proportional to the concentration of the alkanethiol molecules on the substarte.68 The immobilized reduced CST-C molecules on the conductive nanopillar substrate represent an alkanethiol. Therefore, we used DPV for their reductive desorption and electrochemical quantification after the SERS measurement. As indicated by Figures 4a, b, the electrochemical desorption of the reduced protein resulted in a negative potential at -0.92 V of a magnitude that is proportional to the reduced protein concentration in 9 ACS Paragon Plus Environment
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the range 6.25x10-8 M to 1x10-6 M. The relationship between the DPV current and the concentration of reduced CST-C followed the linear regression equation y = 4.3657x + 9.6945, R2 = 0.9932 (Figure 4c). The LOQ of the electrochemical quantification of CST-C was found to be 62.5 nM which is suitable for the determination of high levels of the protein in patients at a high risk of developing kidney failure disease.67 The sensitivity of the electrochemical measurement of CST-C is attributed to the high population of gold nanostructures on the surface of the nanopillar substrate where they act as active sites for the electrochemical measurement of the reduced protein. The LOQ of the SERS / DPV determinations are compared to other CST-C detection methods in Table S2.29,30,32-36,38,69,70 As indicated by the table the developed dual sensing method compares favourably to other detection methods in terms of simplicity, wide working range, recyclability of the used sensors, and capacity to provide molecular structure information of the screened biomolecule similar to Mass detection. Due to their 0.4x0.4 cm small surface area, the extractor chip and SERS substrate can be miniaturized in “a lab on a chip” platform for molecular diagnostics where a few microliters of blood plasma can be injected onto the extractor chip and the captured protein released to a detection zone where it is screened onto the conductive SERS substrate by commercial handheld Raman and potentiostat devices. Determination of CST-C in human plasma To demonstrate the potential of the new dual biosensing for molecular diagnostics, it was applied for the quantification of CST-C in human blood by SERS and DPV. The protein was extracted from 20 µL of human blood plasma sample, reduced and quantified by SERS on the nanopillar substrate. The concentration of the protein in the human plasma sample was found to be 9.66x10-8 M (n=6). Another aliquot of the blood plasma sample was rescreened by ELISA for the cross validation of the SERS measurement. The concentration of CST-C in the sample by found to be 9.86x10-8 M (n=6) by the ELISA method (Figure S4). Therefore, the average % agreement between the SERS and ELISA determinations was 97.97 % which indicates the high capture efficiency of the extractor chip. For the electrochemical quantification of CST-C, the protein was captured from a second human blood plasma sample (20 µL), reduced and loaded onto a new nanopillar substrate. The protein concentration was first screened by SERS then desorbed by DPV. The SERS and DPV quantification of the protein in the sample were found to be 1.42x10-7 M (n = 10 ACS Paragon Plus Environment
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13) and 1.61x10-7 M (n=3) respectively. Therefore the average % agreement between the DPV and SERS quantifications was 113.38% and 111.08 % between the DPV and ELISA quantifications. Yang et al. recently reported an ultra-sensitive electrochemical immunoassay for CSTC down to 75 fM in blood.70 The method utilized 2 complementary target-specific antibodies to bind CST-C from human serum within 120 min. The sandwich immunoreaction induced proximity hybridization between two DNA strands and caused the displacement of an output DNA. The output DNA was then loaded onto a modified gold electrode and incubated for 5 hours before its electrochemical detection by DPV. Therefore, this method utilized a complex analytical protocol that involved the use of 8 different biomolecules for the indirect detection of CST-C. Considering the complexity of Yang’s method and the fact that the median concentration of CST-C in healthy subjects is ~1067 ng mL-1 (80 nM),40 the new dual nanosensing method presented in this work is satisfactory for the rapid and cost-effective detection of CST-C in healthy subjects and kidney failure/ Alzheimer’s patients. Recycle of the extractor chip To reduce the cost of protein analysis, the extractor chip was recycled by CV to electrochemically reduce the Au-S bonds and desorb the inactive antibody fragments from the gold surface of the chip.71 The electrochemical recycling process was monitored by SERS (Figure S5 a-c). As indicated by the figure, the Raman bands of the antibody fragments gradually disappeared with the progress of the CV cycles. This observation was supported by the gradual decrease in the magnitude of the reduction current of the fragments at -0.8 V (Figure S6 a, b). After the electrochemical desorption, the recycled substrate was re-functionalized with new antibody fragments and screened by the handheld Raman spectrometer. The SERS spectra of the extractor chip before recycle, after 130 CV and after re-functionalization with new antibody fragments are depicted in Figure 5 i, ii, iii respectively. The reappearance of the antibody Raman spectrum on the extractor chip after re-functionalization confirms its successful recycle. CONCLUSION In this work we presented a dual SERS/DPV biosensor for the rapid detection of protein biomarkers in human blood plasma. To isolate CST-C from blood plasma, we 11 ACS Paragon Plus Environment
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develop target-specific and recyclable extractor chip using the thiol chemistry of antibodies where thiol-ended antibody fragments were synthesised and assembled onto gold coated silicon chip. To recycle of the extractor chip after use, CV was used to electrochemically desorb the inactive antibody fragments then re-functionalize with fresh fragments. For reproducible SERS measurements, the disulfide bond structure of the isolated protein was chemically reduced and assembled, in a highly oriented monolayer, onto a gold coted silicon nanopillar substrate. This led to the sensitive quantification of CST-C in blood plasma down to 1pM and a low RSD of 3.8% which is useful for the early diagnosis of Alzheimer’s disease. The Au-S bonds between the assembled reduced protein and the gold surface of the substrate were electrochemically reduced by DPV. This led to the electrochemical quantification of CST-C down to 62.5 nM which is useful for its quantification in kidney failure disease. Due to the small size of the used nanostructures substrates and the speed of the new dual nanosensing methodology, it has strong potential for miniaturization in a-lab-on a-chip device for protein analysis at pathology labs and POC. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Included are the Raman spectrum of CST-C by the InVia Raman microscope, SEM image of the gold coated silicon nanopillar substrate, ELISA calibration curve of CST-C, immobilisation scheme of reduced CST-C onto gold coated silicon nanopillar substrate, Raman spectra of CST-C antibody fragments on gold coated silicon chip after different CV cycles, electrochemical desorption of antibody fragments from the extractor chip surface by cyclic voltammetry, table of Band assignment of the Raman spectrum of reduced and unreduced forms of CST-C on gold coated silicon nanopillar substrate, table of comparison of different methods used for the detection of CST-C. ACKNOWLDGMENTS The authors thank Queensland University of Technology (QUT) for the award of QUT Postgraduate Research Award (QUTPRA) and QUT International HDR Tuition Fee Sponsorship to W.A. Hassanain. The authors also acknowledge the support from the Science and Engineering Faculty to access the Central Analytical Research Facility (CARF) at QUT.
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Figure 1. Raman spectra of (a) standard CST-C (the red line depicts the Raman spectrum of the bare substrate), (b) different scans of standard CST-C, (c) CST-C standard after reduction with TCEP, and (d) 17 measurements of seventeen reduced CST-C samples in the concentration range 10-7 to 10-12 M on 17 independent gold coated silicon nanopillar substrates.
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Figure 2. Raman spectra of (i) extracted and reduced CST-C from human blood plasma, (ii) positive control, (iii) negative control, (iv) insulin, and (v) phenylalanine on gold coated silicon nanopillar substrate.
Figure 3. (a) Raman band intensity of reduced CST-C at 1363 cm-1 in the concentration range of 100 nM to 1 pM. (b) SERS calibration curve of reduced CST-C within the same concentration range.
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Figure 4. (a) Electrochemical desorption of CST-C by DPV (the black arrow depicts the desorption current of CST-C at -0.92 V. The red line depicts the DPV of a blank substrate). (b) DPV current of reduced CST-C in the concentration range 0.0625 µm to 1 µm. (c) electrochemical calibration curve of reduced CST-C by DPV.
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Figure 5. Raman spectra of (i) CST-C antibody fragments on the extractor chip, (ii) extractor chip after electrochemical desorption of antibody fragments (after 130 CV cycles), and (iii) fresh CST-C antibody fragments on recycled extractor chip after the electrochemical treatment (5 ii).
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