Poly(3,4-ethylenedioxythiophene) Bearing Phosphorylcholine Groups

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Poly(3,4-ethylenedioxythiophene) Bearing Phosphorylcholine Groups for Metal-Free, Antibody-Free, and Low-Impedance Biosensors Specific for C‑Reactive Protein Tatsuro Goda,* Masahiro Toya, Akira Matsumoto, and Yuji Miyahara* Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan S Supporting Information *

ABSTRACT: Conducting polymers possessing biorecognition elements are essential for developing electrical biosensors sensitive and specific to clinically relevant biomolecules. We developed a new 3,4-ethylenedioxythiophene (EDOT) derivative bearing a zwitterionic phosphorylcholine group via a facile synthesis through the Michael-type addition thiol−ene “click” reaction for the detection of an acute-phase biomarker human C-reactive protein (CRP). The phosphorylcholine group, a major headgroup in phospholipid, which is the main constituent of plasma membrane, was also expected to resist nonspecific adsorption of other proteins at the electrode/ solution interface. The biomimetic EDOT derivative was randomly copolymerized with EDOT, via an electropolymerization technique with a dopant sodium perchlorate, onto a glassy carbon electrode to make the synthesized polymer film both conductive and target-responsive. The conducting copolymer films were characterized by cyclic voltammetry, scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The specific interaction of CRP with phosphorylcholine in a calcium-containing buffer solution was determined by differential pulse voltammetry, which measures the altered redox reaction between the indicators ferricyanide/ferrocyanide as a result of the binding event. The conducting polymer-based protein sensor achieved a limit of detection of 37 nM with a dynamic range of 10−160 nM, covering the dynamically changing CRP levels in circulation during the acute phase. The results will enable the development of metal-free, antibody-free, and low-impedance electrochemical biosensors for the screening of nonspecific biomarkers of inflammation and infection. KEYWORDS: differential pulse voltammetry, conducting polymer, phosphorylcholine, thiol−ene reaction, electrochemical impedance spectroscopy

1. INTRODUCTION Conducting polymers have attracted significant attention as a new class of organic materials for use in biosensors and bioelectronics because of their good electronic and ionic conductivities in a doped state, the feasibility of adding functionality by chemical modification of the constituent molecules, their responsiveness to external stimuli, the tunability of their bulk and surface properties, noncytotoxicity, mass productivity, and workability.1−4 Enhanced conductivities produce low impedances at the interfaces between electrodes and biological systems that improve the fidelity and efficiency of electrical signal transduction for recording and stimulation.5−8 A low-impedance surface improves the signal-tobackground ratio. Thus, surface modification with conducting polymers prevents electrochemical passivation of the underlying electrode. Low impedance behavior is provided by a facilitated ionic current that flows into and out of the bulk phase of conducting polymers because organic bioelectronic materials are macromolecular clusters, assembled by weak van © XXXX American Chemical Society

der Waals forces, electrostatic forces, and hydrogen bonds, in the presence of a small amount of water molecules. The loose arrangement of macromolecules at microscopic scales is in contrast with conventional metallic and inorganic conducting materials that are composed of a rigorous arrangement of atoms through the formation of valence bands with neighboring molecules. Controlling the interaction with a biological system is essential for organic bioelectronics. Anionic bioactive dopants are incorporated into a cationic conducting polymer to tailor biological functions as well as to increase conductivity. A variety of bioactive charge carriers have been composited into conducting polymers to control protein binding,9,10 cell adhesion,11,12 cell growth,13,14 cell differentiation,15,16 and tissue responses.17,18 Enzymes have been entrapped in a matrix Received: October 2, 2015 Accepted: November 20, 2015

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DOI: 10.1021/acsami.5b09325 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic of the synthesis of EDOTPC and the functionalization of an electrode with conducting polymer for CRP biosensing. (a) Synthesis route for EDOTPC. (b) Electrochemical copolymerization of EDOT and EDOTPC in the presence of dopant NaClO4 on a glassy carbon electrode for electrochemical CRP biosensing.

Figure 2. Capacitive increases and morphological changes during electropolymerization at varying EDOTPC compositions. (a−e) Cyclic voltammograms of a glassy carbon electrode in 10 mM aqueous monomer solutions at 0 (a), 25 (b), 50 (c), 75 (d), and 100 mol % (e) EDOTPC in feed composition with 100 mM NaClO4. Counter electrode, Pt; reference electrode, Ag/AgCl (in 3.3 M KCl aq); scan rate, 0.1 V s−1. (f) Increases in the interface capacitance as a function of repeated cycles of CV scans during the electropolymerization at different feed compositions of EDOTPC. Data are shown as average ± standard deviation; n = 3. (g−k) SEM images of the surface morphology of the conducting polymer films electropolymerized on a glassy carbon electrode at 0 (g), 25 (h), 50 (i), 75 (j), and 100 mol % (k) EDOTPC in the presence of 100 mM NaClO4. Magnification: ×5000. Scale bar: 10 μm.

B


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Figure 3. Surface characterization of the conducting copolymer films on gold electrodes. (a) ATR-FTIR spectra of conducting polymers prepared at 0−100 mol % EDOTPC. (b, c) Changes in the atomic compositions determined from high-resolution XPS analysis; atomic ratios of P/(S−P) (b) and C/Au (c) as a function of EDOTPC composition during preparation. Data are shown as average ± standard deviation; n = 5. (d, e) High resolution XPS spectra of C 1s (d) and O 1s (e) of the conducting copolymer films prepared at 0−100 mol % EDOTPC. Colored solid lines indicate curve fitting results.

of conducting polymer to detect substrate molecules.19,20 However, doping a polymer with a biomolecule suffers from a lack of control in the amount of dopant incorporated. The dopant undergoes reversible release and re-entry to the bulk phase, led by the redox state of the conducting polymer. Incorporation of large biomolecular composites may also affect the surface and bulk properties. Functionalization of monomers is another way to create bioactive conducting materials. Poly(3,4-ethylenedioxythiophene (EDOT)) (PEDOT) is one of the most widely used conducting polymers in biological applications.21−24 The high degree of freedom available for designing EDOT derivatives is advantageous for introducing bioactive elements in many ways.1,25,26 EDOT derivatives bearing target capturing elements have been developed to synthesize conducting polymers for detecting DNA,27−29 proteins,30 and cancer cells.31 Although EDOT adducts may compromise the original bulk properties of a conducting polymer, they can be incorporated with EDOT at arbitrary compositions using existing polymerization techniques to optimize the properties. The current study aims to develop a new EDOT derivative that contains a zwitterionic phosphorylcholine (PC) moiety as a biomimetic ligand for human C-reactive protein (CRP). The PC group is present on the surface of plasma membranes as a headgroup of phospholipid or on polysaccharide chains. PCcontaining materials have been used in biomedical applications because they are superior in resisting nonspecific protein adsorption, noncytotoxicity, tissue compatibility, moisture retention, and lubrication.32,33 Due to its attractive properties, an EDOT derivative bearing a PC group has been used to

improve the biocompatibility toward neural cells cultured on the conducting polymer.34,35 Notably, the PC group is known to specifically recognize CRP in the presence of calcium ions.36−39 CRP is a typical biomarker for the acute-phase of inflammation and infection, because its blood level increases under these conditions by 100−1000-fold within 24 h.40 Circulating CRP binds to damaged cells and tissues to augment the classical complement system.41 Therefore, CRP biosensing is of clinical importance to quickly estimate the severity of inflammation and infection. A methacrylate monomer, 2methacryloyloxyethyl phosphorylcholine (MPC), is frequently used as a building block for synthesizing biocompatible polymers. We used the “click” thiol−ene reaction between MPC and thiol-functionalized EDOT for facile synthesis of an EDOT derivative bearing PC (EDOTPC).42−44 EDOTPC was electrochemically copolymerized with EDOT at varying compositions to tune the conductivities and CRP recognition capability of the conducting polymer films.

2. RESULTS AND DISCUSSION 2.1. Monomer Synthesis and Electropolymerization. The synthesis of EDOTPC 4 started from chloromethyl-EDOT 1 synthesized from 3,4-dimethoxythiophene according to the literature (Figure 1a).45,46 The chloromethyl group was then reacted with potassium thioacetate to form methanethioester group 2.47 EDOT-methanethiol 3 was obtained by deprotection in basic medium.48 Finally, EDOTPC 4 was synthesized by thiol−ene reaction between 3 and MPC.43,44 The 1H NMR spectra and ESI-MS data are shown in Supporting Information Figures S1 and S2. The global yield was 10%. C


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fraction of EDOTPC in the copolymer film, linearly increased to a plateau value of 0.24 when increasing the feed composition of EDOTPC over the range 0−50 mol %, indicating the incorporation of EDOTPC units was proportional to the given concentration during electropolymerization. The measured surface compositions of EDOTPC were about half of the theoretical values at 0−50 mol % EDOTPC. A possible reason for the lower S/(S−P) values was that the hydrophilic PC moiety is partially buried in the bulk phase of the polymer film under the high vacuum conditions during the XPS measurements. We also determined the C/Au ratio to estimate the completeness in the coverage of conducting polymer film electrodeposited on the gold electrode (Figure 3c). The C/Au ratio reached the highest level (∼1790) at 25 mol % EDOTPC. By considering the sensing depth of ∼10 nm at a takeoff angle of 90° in XPS, thick layers of conducting polymer were formed, with a high surface coverage, as observed by SEM images (Figure 2h). High-resolution C 1s spectra identified C−C and C−S bonds at 284.8 eV, the C−O bond at 286.2 eV, and the O−CO bond at 288.8 eV (Figure 3d).51 The peak at 284.8 eV increased and the peak at 286.2 eV decreased as the given EDOTPC composition increased. High resolution O 1s spectra identified the C−O bond at 532.6 eV, PEDOT+−ClO4− at 531.1 eV, and CO bond at 529.6 eV (Figure 3e). The peak at 532.6 eV decreased and the peak at 531.1 eV increased with increasing given EDOTPC composition. The peak at 529.6 eV was not observed at 0 mol % EDOTPC (i.e., PEDOT) because the C O group was absent. 2.3. Electrochemical Impedance Spectroscopy. Because one of the features of conducting polymers in biosensing and bioelectronic applications is their low impedance at the solid/liquid interface, we investigated the electrochemical impedance spectroscopy (EIS) of poly(EDOT-co-EDOTPC) films on the glassy carbon substrate. The Nyquist plots included a semicircular part at high frequencies, corresponding to the charge transfer-limited process, and a linear part at low frequencies, corresponding to the diffusion-limited process of the redox reaction between ferricyanide/ferrocyanide [Fe(CN)63−/Fe(CN)64−] (Figure 4a). The film prepared at 0 mol % EDOTPC (i.e., PEDOT) showed a linear response, implying a capacitor-like behavior. Data modeled by the Randle’s equivalent circuit showed increases in charge transfer resistance (Rct) with increasing EDOTPC composition (Figure 4b). Trends in the constant phase element (Ccpe) as a function of given EDOTPC composition were similar to the interface capacitance determined by CV (Figure 2a−f). A higher content of EDOTPC increased the impedance of the copolymers through the decreased conductivity caused by conformational disorder of the polythiophene main chain in the presence of the bulky zwitterionic PC moiety. Incorporation of the PC group has a trade-off relationship with the electronic and ionic conductivities. Therefore, we chose films prepared at 25 and 50 mol % EDOTPC for subsequent CRP biosensing studies. 2.4. Electrochemical CRP Biosensing. Differential pulse voltammetry (DPV) measurements were performed on the electrodes prepared at 0, 25, and 50 mol % EDOTPC after incubation with target human CRP or nontarget BSA (Figure 5). The peak current before (I0) and after (I) incubation with protein at +0.2 V (vs Ag/AgCl) in DPV was obtained by the redox reaction of the anionic indicators ferricyanide/ferrocyanide. The highest changes in the DPV signal (I/I0), by a factor of about 3, were observed for the electrodes modified with 25

The electropolymerization of EDOT and EDOTPC monomers was performed to deposit the random copolymer on a glassy carbon electrode in an aqueous solution (Figure 1b). The oxidation potential in forward scans necessary to form polymer films was at +0.9 V (vs Ag/AgCl) (Figure 2a−e). The increase in current density observed with increasing CV cycles can be explained as a characteristic of nucleation and growth processes. The interface capacitance, determined from the difference in the current density between the forward and reverse scans at +0.2 V, increased with the number of CV cycles (Figure 2f). An increase in the current density was dominant at 0−50 mol % EDOTPC, indicating the formation of a conducting polymer layer. The capacitance did not increase at 75 or 100 mol % EDOTPC, which suggests a decreased conductivity of the deposited film at high EDOTPC contents. The optimal dopant was identified as sodium perchlorate (NaClO4) in terms of the capacitance increase obtained by the electrodeposition of conducting polymers (Supporting Information Figure S3). High surface roughness of a conducting polymer is known to contribute to increased capacitance at the electrode/solution interface. Indeed, scanning electron microscope (SEM) observation confirmed changes in surface morphology of electrochemically polymerized films on planar electrodes after 10 cycles of CV scans (Figure 2g−k). The surface morphology of a film prepared at 25 mol % EDOTPC resembled cauliflower-like conformation. The films prepared at 0, 50, 75, and 100 mol % EDOTPC were composed of a threedimensional assembly of small particles that formed a rough surface topography. The zwitterionic PC moiety induced a change in hydrophobic and hydrophilic balance of the conducting copolymer and a dramatic conformational change at 25 mol % EDOTPC. An enhanced surface area is advantageous in improving the sensitivity of electrochemical biosensing. The increases in the surface roughness at 50−100 mol % EDOTPC were not consistent with the increased interface capacitance (Figure 2f), suggesting decreased electrical conductivity of the copolymers arising from the conformational disorder of π-electron conjugation in the polythiophene main chain, caused by the presence of the bulky PC group in the side chain. 2.2. Surface Characterization. Attenuated total reflectionFourier transform infrared (ATR-FTIR) spectra confirmed the formation of poly(EDOT-co-EDOTPC) films (Figure 3a). The bands at 1520 cm−1 were assigned as CC and C−C stretching vibrations of the thiophene ring.49 The peaks at 1200 and 1090 cm−1 corresponded to C−O−C bond stretching. The peaks at 980, 840, and 690 cm−1 originated from the C−S bond in the thiophene ring. Moreover, the presence of the MPC unit in EDOTPC was identified by vibration at 1240, 1090, and 970 cm−1 for P−O, at 965 cm−1 for N+− (CH3)3, and at 1720 cm−1 for CO.50 A broad band at 3000−3700 cm−1 for H2O was observed for the samples prepared at 25−100 mol % EDOTPC, because of adsorption of water molecules around the hygroscopic PC zwitterions. The elemental composition on the surface of the conducting polymer film electrochemically deposited on a planar gold electrode was determined by X-ray photoelectron spectroscopy (XPS) (Supporting Information Table S1). The atomic ratio of S/(S−P) was calculated to determine the difference between given and real compositions of the EDOTPC unit on the outermost surface of the conducting polymers (Figure 3b). The S/(S−P) ratio, which is theoretically identical to the molar D


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abundant PC groups, the redox signal was impaired. We attribute the weak signals to the decreased conductivities of the film resulting from the increased EDOTPC composition (Figure 3). The dynamic range of the biosensor prepared at 25 mol % EDOTPC was 10−160 nM [CRP]. The slope value was +1.4% [CRP]−1 (R2 = 0.98) based on regression analysis. The relative standard deviation (RSD) of I/I0 was 16% (0.49/3.04, n = 3) at 160 nM [CRP]. The limit of detection (LOD) was 37 nM (=4.3 μg mL−1) based on 3 times the value of standard deviation (SD). Although the LOD is a few order of magnitude higher than that of the latest CRP sensing systems (0.1−2 ng mL−1),52−54 it is comparative to commercial immunoturbidimetric or immuno-nephelometric methods (3−8 μg mL−1).55 In addition, the proposed sensor is as sensitive as other CRP sensors recently reported such as square wave anodic stripping voltammetry immunosensors (0.5−200 μg mL−1),56 thermally controlled piezoresistive microcantilever immunosensors (1 μg mL−1),57 electrochemical impedance immunosensors (0.04−5.8 μg mL−1),58 and magnetic multiwalled carbon nanotube-based amperometric immunosensors (0.3−100 μg mL−1).59 The sensitivity of the PC-functionalized sensor is higher than other synthetic ligand-based sensors such as RNA aptamer-based carbon nanotube sensor (1−8 μM).60 Of note, the dynamic range of the proposed CRP biosensor covered the concentration changes of circulating CRP between that in the normal condition and that in the acute phase of inflammation and infection. The conducting polymer-based biosensor was regenerated and reused repeatedly by washing with 1 wt % sodium dodecyl sulfate (SDS) solution because the synthetic PC ligand, in contrast to antibody, did not suffer from conformational denaturation by treating with detergents.

Figure 4. Low-impedance surface of conducting copolymer films on glassy carbon electrodes. (a) Nyquist plots of conducting copolymer films obtained in the buffer solution (10 mM Hepes, 100 mM NaCl, 1 mM CaCl2; pH 7.4) with 5 mM ferricyanide/ferrocyanide. Inset: closer view of the plot at the origin. Counter electrode, Pt; reference electrode, Ag/AgCl in (in 3.3 M KCl aq); DC bias, 0.18 V; AC amplitude, 50 mV; AC frequency, 10 kHz−0.1 Hz. (b) Changes in the constant phase element (CPE) and the charge transfer resistance (Rct) in the Randle’s equivalent circuit model (inset) as a function of EDOTPC composition at preparation. Data are shown as average ± standard deviation; n = 4.

3. CONCLUSIONS We describe the development of EDOTPC, a new EDOT derivative bearing the zwitterionic PC group, as a building block of PEDOT-based random copolymers to obtain conductivity, biorecognition, and nonfouling properties. Electrochemical polymerization under optimized conditions creates lowimpedance surfaces of the conducting copolymers, with enhanced surface area. Specific interaction of PC with CRP were detected directly by DPV as a result of changes in the electrochemical properties of the polymers after binding events. The biosensor possesses sensitivity and selectivity with a dynamic range that covers variations of circulating CRP levels between normal conditions and the acute-phase of inflammation and infection. Our study reveals the potential of this novel, metal-free biosensor for applications in fast screening of CRP as a general biomarker for inflammation and infection. The sensitivity of the new biosensor is comparable to conventional assays that uses extra labeling steps and optical settings.

mol % EDOTPC, following incubation with 160 and 320 nM [CRP] (Figure 5d−f). The increases in I/I0 as a function of CRP concentration were attributed to the changes in electrochemical reactions between ferricyanide/ferrocyanide as a result of calcium-mediated specific binding of CRP to the PC groups.37 Changes in I/I0 following incubation with desired concentrations of BSA were caused by nonspecific adsorption, but were extensively suppressed because the PC zwitterion is known to resist nonspecific adsorption of biomolecules at the interface.32−35,44 Notably, I/I0 remained unchanged following the incubation with human serum for the film prepared at 25 mol % EDOTPC (Figure S4). The results indicate good selectivity of the biosensor toward CRP by preventing nonspecific adsorption of other proteins. In contrast, I/I0 was not significant in the presence of both CRP and BSA on electrodes modified with conducting polymers prepared at 0 or 50 mol % EDOTPC. The I/I0 ratios of the film at 0 mol % EDOTPC changed by +9.3% and −24% at 320 nM [CRP] and [BSA], respectively (Figure 5a−c). These weak signals were caused by the nonspecific adsorption of CRP or BSA onto the conducting polymer film in the absence of the PC group. The I/I0 ratios of the film at 50 mol % EDOTPC changed by −22% and −25% at 320 nM [CRP] and [BSA], respectively. Although the film prepared at 50 mol % EDOTPC specifically recognized CRP because of the presence of

4. EXPERIMENTAL SECTION 4.1. Materials. 3,4-Dimethoxythiophene, MPC, potassium ferricyanide (III), potassium ferrocyanide (IV), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (Hepes), and human serum were purchased from Sigma-Aldrich Japan (Tokyo, Japan). 3-Chloro-1,2propanediol, thioacetic acid s-potassium salt, and sodium methoxide (5 M in methanol) were purchased from TCI (Tokyo, Japan). p-Toluene sulfonic acid monohydrate, sodium perchlorate, and recombinant human C-reactive protein were purchased from Wako Pure Chemicals (Tokyo, Japan). All the other reagents were of extra pure grade and purchased from commercial sources, and then used without further E


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Figure 5. Electrochemical sensing of CRP using the EDOTPC-based conducting copolymer on a glassy carbon electrode. DPV spectra of conducting polymer films prepared at 0 (a, b), 25 (d, e), and 50 (g, h) mol % EDOTPC following incubation with 0−320 nM CRP (a, d, g) or BSA (b, e, h). Changes in the ratio of peak currents in DPV spectra of conducting polymer films prepared at 0 (c), 25 (f), and 50 (i) mol % EDOTPC as a function of protein concentration. DPV was conducted in buffer solution (10 mM Hepes, 100 mM NaCl, 1 mM CaCl2; pH 7.4) containing 5 mM ferricyanide/ferrocyanide. Counter electrode, Pt; reference electrode, Ag/AgCl (in 3.3 M KCl aq). Data are shown as average ± standard deviation; n = 3. purification. Milli-Q water (EMD Millipore Co. Billerica, MA) was used throughout the study. 4.2. Synthesis of EDOTPC. EDOTPC was synthesized via a fourstep reaction (Figure 1). First, under a nitrogen atmosphere, toluene (14 mL), 3,4-dimethoxythiphene (0.82 g, 5.7 mmol), 3-chloro-1,2propanediol (1.3 g, 12 mmol), and p-toluene sulfonic acid monohydrate (0.08 g, 0.42 mmol) were added to a two-neck flask. The mixture was heated at 90 °C for 24 h. After that more 3-chloro1,2-propanediol (1.3 g, 12 mmol) was added, and the solution was heated at 90 °C for an additional 3 h. After evaporation of toluene, the residue was purified by column chromatography (silica gel, hexane/ dichloromethane = 8/2 v/v) to give chloromethyl-EDOT 1 as a yellow oil (yield 56%). Second, N,N-dimethylformamide (DMF, 3 mL), chloromethylEDOT 1 (0.60 g, 3.1 mmol), and thioacetic acid s-potassium salt (0.54 g, 4.7 mmol) were added to a flask. The mixture was refluxed at 50 °C for 16 h. Then solution was allowed to cool to room temperature. After evaporation, the sample was extracted with dichloromethane in a separation funnel by replacing the water three times. The organic extract was dried over anhydrous magnesium sulfate, followed by evaporation to obtain EDOT-methanethioester 2 as an orange oil (yield 68%). Third, distilled THF (40 mL), thioester-EDOT 2 (0.50 g, 2.2 mmol), and sodium methoxide (1.2 M in methanol, 4.0 mL) were added to a flask and stirred at room temperature for 4 h. After treating with HCl (5 M in water), the solution was evaporated to obtain the

combined organic residues. The residues were purified by extraction with dichloromethane in a separation funnel by replacing water three times. The organic extract was evaporated to give EDOT-methanethiol 3 as an orange oil (yield 65%). Finally, under a nitrogen atmosphere, dried chloroform (2.0 mL), EDOT-methanethiol 3 (0.20 g, 1.1 mmol), and MPC (0.31 g, 1.1 mmol) were added to a two-neck flask. After adding diisopropylamine (1.5 mg, 15 μmol), the thiol−ene reaction was run at room temperature for 24 h. After evaporation, unreacted MPC was removed by dissolving it in acetone. After filtration and evaporation, unreacted thiophene compounds were removed by adding water. The aqueous phase was collected by filtration and freeze-dried to obtain EDOTPC 4 as a light orange oil (yield 40%). 1 H NMR (500 MHz in CDCl3, Bruker Daltonics, Billerica, MA) and ESI-MS (microTOF-2focus, Bruker Daltonics) data are provided in the Supporting Information. 4.3. Electropolymerization. A commercially available glassy carbon electrode (inner diameter 3 mm, CH Instruments, Austin, TX) was carefully polished with alumina powder (0.05 μm grain size, Baikowski International, Charlotte, NC), followed by washing with water prior to use. The monomers (10 mM in total) were added to 1 mL of degassed water containing 100 mM sodium perchlorate (NaClO4) in a glass cell. A three-electrode system of a glassy carbon working electrode, a platinum disc counter electrode, and an Ag/AgCl wire reference electrode was introduced to the cell and connected to a F



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potentiostat (PGSTAT302, Metrohm Autolab, Utrecht, The Netherlands). For electropolymerization, 10 consecutive cycles of CV scans were performed over a potential window from −0.6 to +1.1 to −0.6 V at a scan rate of 0.1 V s−1 at room temperature. The interface capacitance (C, mF cm−2) per unit area at each cycle of electropolymerization was determined by the difference in current density between forward and reverse scans (ΔI, mA) at +0.2 V (vs Ag/AgCl) as

C = ΔI /vA

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for financial support from the Grantin-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (#26107705) from MEXT of Japan, the Futaba Electronics Foundation, and the Center of Innovation (COI) Program from Japan Science and Technology (JST) Agency. We acknowledge Prof. K. Ishihara at the University of Tokyo for offering the use of XPS apparatus.

(1)

where v (V s−1) and A (cm−2) represent the scan rate and geometric surface area, respectively. The average and standard deviation were obtained from three independent measurements. The copolymer composition was defined by the fraction of each constituent in moles (mol %). 4.4. Characterization. SEM images were taken using a S-3400N microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 10 kV, a deceleration voltage of 0 V, and a working distance of 9800 μm at an emission current 86 μA. Samples were treated by a carbon coater prior to measurement. ATR-FTIR spectra were obtained using a Nicolet iS50 FT-IR spectrometer (Thermo Scientific). Samples were prepared onto a planar gold electrode. Elemental compositions of the poly(EDOT-co-EDOTPC) films on planar gold electrodes were determined by XPS (AXIS-HSi165; Shimadzu-Kratos, Kyoto, Japan) equipped with a 15 kV Mg Kα radiation source at the anode. The takeoff angle of the photoelectrons was set at 90°. The curve fitting of the high-resolution spectra was performed by using Gaussian functions. The average and standard deviation were obtained from the measurements at five different positions. EIS was conducted using a PGSTAT302 potentiostat (Metrohm Autolab) with a three-electrode cell of the conducting polymerdeposited glassy carbon working electrode, an Ag/AgCl (in 3.3 M KCl aqueous solution) reference electrode, and a platinum disc counter electrode in Hepes buffer (10 mM Hepes, 100 mM NaCl, 1 mM CaCl2, pH 7.4) containing 5 mM ferricyanide/ferrocyanide over a frequency range between 0.1 Hz and 10 kHz (10 points per decade), with a 50 mV AC voltage, superimposed on a DC bias of +0.2 V. The charge transfer resistance (Rct) was determined by fitting Nyquist plots to a Randle’s equivalent circuit. The average and standard deviation were obtained from four independent measurements. 4.5. DPV Measurement. The electrodeposited glassy carbon working electrode was equilibrated in Hepes buffer (10 mM Hepes, 100 mM NaCl, 1 mM CaCl2, pH 7.4) for 10 min at room temperature and then washed with fresh buffer. The electrode then was incubated at the desired concentration of CRP or BSA in Hepes buffer for 10 min, followed by rinsing by dipping in Hepes buffer five times. An oxidative DPV scan was carried out in the Hepes buffer containing 5 mM ferricyanide/ferrocyanide from −0.4 to +0.6 V at a step potential of 10 mV, modulation amplitude of 50 mV, modulation time of 0.05 s, and interval time of 0.5 s using PGSTAT302 at room temperature. The peak current before (I0) and after (I) incubation with the proteins was obtained by correcting the data with a linear baseline using bundled software. The average and standard deviation were obtained from three independent measurements. The LOD was defined as the CRP concentration that is equivalent to three-times the value of the standard deviation.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b09325. H NMR spectra, ESI-MS data, CVs for electropolymerization with different dopants, and the atomic compositions of the conducting polymers from XPS analysis (PDF) G


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Poly(3,4-ethylenedioxythiophene) Bearing Phosphorylcholine Groups

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