Simultaneous and Sequential Synthesis of Polyaniline-g-poly

Article pubs.acs.org/Macromolecules

Simultaneous and Sequential Synthesis of Polyaniline‑g‑poly(ethylene glycol) by Combination of Oxidative Polymerization and CuAAC Click Chemistry: A Water-Soluble Instant Response Glucose Biosensor Material Tugrul Cem Bicak,† Mindaugas Gicevičius,‡ Tugba Ceren Gokoglan,§ Gorkem Yilmaz,† Arunas Ramanavicius,‡ Levent Toppare,*,§,∥,⊥,# and Yusuf Yagci*,†,7 †

Department of Chemistry, Istanbul Technical University, 34469, Maslak, Istanbul, Turkey Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko st. 24, LT-03225, Vilnius, Lithuania § Department of Chemistry, Middle East Technical University, Ankara 06800, Turkey ∥ Department of Biotechnology, Middle East Technical University, Ankara 06800, Turkey ⊥ Department of Polymer Science and Technology, Middle East Technical University, Ankara 06800, Turkey # The Center for Solar Energy Research and Application (GUNAM), Middle East Technical University, Ankara 06800, Turkey 7 Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia ‡

ABSTRACT: A novel approach for the in situ synthesis of conjugated polyanilinepoly(ethylene glycol) graft copolymer (PA-g-PEG) by the combination of oxidative polymerization and copper catalyzed azide−alkyne cycloaddition (CuAAC) click reaction is described. The method pertains to the reduction of the CuBr2 catalyst during the oxidative copolymerization of aniline and aminophenyl propargylether to Cu(I) species, which catalyze the CuAAC reaction between thus formed polyaniline with pendant alkyne groups and independently prepared azide functional PEG in both simultaneous and sequential manner. The obtained water-soluble PA-g-PEG was used for the construction of glucose biosensor by a simple one-step approach. Combined electrostatic polyanionpolycation and hydrogen bond interactions between PA-g-PEG and glucose oxidase provided a suitable immobilization matrix for the enzyme resulting in excellent analytical parameters. PA-g-PEG based glucose biosensor exhibited a remarkable response time, producing an instant signal upon addition of analyte, making this sensor an attractive alternative for the existing devices.

1. INTRODUCTION

mers can be classified as alternative materials widely used in various biosensor applications.24,25 Glucose sensing is extensively used for detecting numerous metabolic disorders, mostly in diagnosis and therapy of diabetes and also controlling various food and biotechnological processes.26 In addition, glucose monitoring is important for both chronic diabetes as well as nondiabetic acute care patients as large variations in blood glucose level may cause complications resulting from surgery or acute illness.27,28 Thus, intensive research activity has focused on the design of various sensing materials and methodologies for accurate, comparatively noninvasive and continuous glucose detection.27 Various analytical methods utilizing glucose oxidase (GOx) have been developed.29−32 The usually applied methodology

Conjugated polymers have been one of the most widely applied class of materials for the construction of biosensors1 due to their ease of synthesis, great electrical properties, functionality and surface characteristics.2−5 Among conducting polymers, polypyrrole (PPy),6 polyaniline (PA),7,8 polythiophene (PT),9 and polyphenylene (PP)10−14 have received tremendous interest owing to their high electrical conductivity, tunable structure and surface morphology, interesting optical properties and great electrochemical stability.15−17 PA is particularly attractive for biosensor applications due to its richness of possible oxidation states,18 multiple color transitions,19 high environmental and chemical stability,20 adjustable electrochemical behavior by choosing doping level, pH, and morphology. Additionally, the ability of PA to form countless number of functional composites and blends with organic,21 inorganic,22 and biomaterials23 makes it suitable for employing in different transducer systems. Molecularly imprinted poly© XXXX American Chemical Society

Received: January 18, 2017 Revised: February 17, 2017

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and DCM was evaporated. Residual solid material was recrystallized from ethanol and put into vacuum oven (yield = 53%; 1H NMR (CDCl3(ppm)) 3.42 (s, 1H, acetylenic), 4.62 (s, 2H, O−CH2−), 4.90−5.02 (broad, 2H, NH2), 6.70−6.93 (m, 4H, aromatic)). 2.3. Synthesis of Azide-Functional Poly(ethylene glycol) (PEG-N3). PEG (20 g, 10 mmol), DCM (60 mL), triethylamine (2.75 mL, 20 mmol), and a magnetic stirring bar were added to a 500 mL three-necked round-bottom flask. 4-Toluenesulfonyl chloride (6.15 g, 30 mmol) in DCM (115 mL) was added dropwise at room temperature into the mixture in 10 min, and stirring was continued for 24 h at room temperature. DCM was evaporated and the white residue was dissolved in DCM (20 mL), and then precipitated in cold diethyl ether (700 mL). The precipitate was collected by filtration and washed again with cold diethyl ether. Finally, white solid residue was put into vacuum oven for overnight. The tosylated PEG was placed into a 250 mL erlenmeyer flask and NaN3 (3.25 g, 50 mmol) in DMF (80 mL) was added. The mixture was stirred at room temperature for 24 h and then precipitated in 700 mL cold diethyl ether. The solid material was collected by vacuum filtration and washed several times with cold ether. Finally, the product was dried in vacuum for overnight (yield = 72%; Mn,GPC = 2150 g·mol−1; Mw/Mn = 1.12). 2.4. Sequential Oxidative Polymerization and Click Reactions. 2.4.1. Synthesis of Propargyl Functional Polyaniline by Oxidative Polymerization. Copolymerization of aniline with aminophenyl propargyl ether (APE): Aniline (7.2 mL, 79 mmol), APE (0.6 g, 4 mmol), copper(II) bromide (0.06 g, 0.27 mmol), DMF (25 mL) and water (25 mL) were put into a 250 mL beaker and stirred at room temperature for 24 h. Then, the reaction mixture was poured into a 200 mL of cold water, and the precipitate was filtered off and washed several times with cold water. The filtrate was then dried and dissolved in DCM (10 mL) and precipitated in 100 mL n-hexane. The filtrate was dried in a vacuum oven overnight (yield = 38%; Mn,GPC = 15200; Mw/Mn = 4.26). 2.4.2. Synthesis of PA-g-PEG by CuAAC. The alkyne functionalized polyaniline (A) (1 equiv) was reacted with PEG-N3 (0.1 equiv) for 24 h. The mixture was extracted with DCM and the organic layer was dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. The residual dark solid was dissolved in 3 mL DCM and twice precipitated in cold diethyl ether. The precipitate was then collected by filtration and dried in vacuum oven overnight (yield = 95%; Mn,GPC = 26600; Mw/Mn = 4.12). 2.4.3. One Pot Synthesis of PA-g-PEG by the Combination of Oxidative Polymerization and CuAAC. Aniline (5 mL, 54 mmol), 2aminophenyl propargyl ether (0.4 g, 2.7 mmol), PEG-N3 (9 g, 4.5 mmol), CuBr2 (0.06 g, 0.27 mmol), water (20 mL), DMF (20 mL), and a magnetic stirring bar were added to a 250 mL beaker. Stirring was continued for 24 h and the mixture was poured into a separation funnel by adding 150 mL of DCM to extract the polymer. Organic phase was separated and dried over anhydrous MgSO4, and then DCM was evaporated. Remaining product was dissolved in 5 mL of DCM and precipitated in cold ether. Precipitate was collected by filtration and washed three times with cold ether. Dark residue was dried in vacuum oven overnight (yield = 29%; Mn,GPC = 29900; Mw/Mn = 4.58). 2.5. Pretreatment of Electrodes. Graphite rods (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05 mm diameter and 13% porosity) were hand-polished with fine emery paper, washed with distilled water, and left to dry at ambient conditions. 2.6. Biosensor Fabrication. Solubility in water of all biosensor constituents enabled simple one-step approach for the construction of the biosensor. First of all, 1.0 mg of GOx was dissolved in 1.5 μL of PBS (pH 6.5) followed by addition of 5 μL PA-g-PEG solution (0.125 mg·mL−1) in PBS (pH 6.5) and 3.5 μL of 1% glutaraldehyde aqueous solution. The resulting 10 μL solution was casted onto a previously cleaned graphite electrode and left to dry for 90 min at ambient conditions. Prior to use, the modified electrode was rinsed with distilled water to remove unbounded species. 2.7. Surface Characterization. Surface morphology of modified electrodes was characterized using scanning electron microscope (SEM) (JEOL JSM-6400 model) at 5 kV, 5000× magnification.

involves their immobilization into various stable polymer matrixes.33 PA has been commonly suggested in the architecture of biosensors as immobilization matrixes due to the number of advantages such as long-term environmental stability,34 biocompatibility, charge transfer properties, their easy preparation and enzyme incorporation.35−38 However, the absolute majority of conducting polymers are insoluble in water, and intensive research effort has been given to produce hydrophilic PA derivatives.39−41 Among various Click reactions,42 the Cu(I)-catalyzed azide− alkyne cycloaddition (CuAAC) reaction43−45 is the most widely used coupling process for the synthesis of various complex macromolecular architectures46 such as functional polymers,47 block,48−50 graft51,52 and star copolymers,53,54 and bioconjugates55,56 due to its fast, simple to use, easy to purify, versatile, regiospecific characteristics giving high product yields. Moreover, they can be externally stimulated57−60 and combined with various controlled polymerization processes.51,61−65 In this study, we report a novel procedure for the synthesis of PEG grafted PA polymers (PA-g-PEG) for the first time by the in situ combination of Cu(II)-induced redox polymerization and CuAAC reaction. The synthesis involves the reduction of Cu(II) salt for oxidative copolymerization of aniline derivatives (aniline and propargyl aniline) and “click” grafting of azido functional PEG, catalyzed by the generated Cu(I) species thereof. Thus, obtained water-soluble PA-g-PEG polymers were utilized as a glucose biosensor. Achieved solubility in water enables simple and cost-effective one-step assembly procedure, while the structure of the polymer in synergy with cross-linking agent provides suitable immobilization matrix for the enzyme molecules.

2. MATERIALS AND METHODS 2.1. Chemicals. Glucose oxidase from Aspergillus niger (17 300 units per gram solid), glucose and glutaraldehyde were of analytical grade and obtained from Sigma-Aldrich(Germany). Phosphate buffer solutions (50 mmol L−1) (PBS) were prepared by 50 mmol L−1 of Na2HPO4 and 50 mmol L−1 of NaH2PO4 (purum, Fluka, Germany) and adjusting pH with HCl or NaOH solutions. Aniline (99.5%, Aldrich), poly(ethylene glycol) monomethyl ether ((PEG) Mn ∼ 2,000, Aldrich), copper(II) bromide (98%, Aldrich), sodium azide (99.5%, Aldrich), 2-nitrophenol (98%, Aldrich), sodium dithionite (85.0%,Aldrich), dichloromethane (DCM, 99.8%, Aldrich), and N,Ndimethylformamide (DMF, 99.8%, Aldrich) were used as received. Glucose stock solution (1.0 mol L−1) was prepared weekly in phosphate buffer, pH 7.0, and left for at least 2 h to mutarotate before any use. All solutions were prepared in distilled water and stored at 4 °C while not in use. 2.2. Synthesis of 2-Aminophenyl Propargyl Ether (APE). 2Nitrophenol (4.4 g, 32 mmol), methanol (40 mL), and a magnetic stirring bar were added to a 250 mL flask. Sodium hydroxide (1.3 g, 32 mmol) in methanol (15 mL) was added dropwise into the mixture while stirring. Addition was completed in 20 min, and then methanol was evaporated off. The residue was dissolved in DMF (30 mL), and propargyl bromide (3.2 mL, 40 mmol) was added to the mixture in an ice bath. Addition was completed in 30 min and the mixture was stirred at 80 °C for 2 h. The mixture was precipitated in cold water and the precipitate was collected by filtration. Then, the product was dried in a vacuum oven overnight. Thus, the obtained 2-nitrophenyl propargyl ether (4.6 g, 26 mmol) was dissolved in ethanol (30 mL). To this mixture was added sodium dithionite (6 g, 34 mmol) in water (30 mL) dropwise. After the addition was completed, a condenser was added to the reaction flask, and the mixture was heated to 100 °C for 2.5 h. Ethanol was removed by evaporator, and the residue was extracted with DCM. Organic phase was dried with anhydrous MgSO4 B

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Macromolecules 2.8. Amperometric Measurements. Amperometric measurements were carried out in a glass cell containing 10 mL PBS (pH 6.5) solution under mild magnetic stirring. Amperometric detection was performed at constant −0.7 V potential which corresponds to the reduction of O2 to H2O2 at the surface of working electrode. After the background current reached steady state, certain amount of glucose was injected into the reaction cell and the analytical response of the biosensor was recorded as the decrease in current between two equilibrium states, which corresponds to the consumption of oxygen due to enzymatic reaction of GOx. A schematic view of PA-g-PEG biosensor construction and working principle are presented in Figure 1.

functionality by a simple two-step reaction. Tosylation of PEG with p-toluene sulfonyl chloride gave PEG-Ts polymer, which was then quantitatively converted into PEG-N3 in the presence of NaN3/DMF. In order to prove the realization of the two concomitant processes, we first attempted to conduct oxidative polymerization and click reaction sequentially (Scheme 2). For this purpose, aniline and APE was copolymerized by using CuBr2 in DMF/water mixture for 24 h at room temperature. The resulting PA with alkyne side groups was subsequently coupled with PEG-N3 in the presence CuBr by the conventional CuAAC reaction. The obtained PA-g-PEG was characterized and compared with that obtained by simultaneous oxidation and CuAAC processes (see vide inf ra). Then, the structurally same graft copolymer was synthesized in a one-step manner by concurrent redox polymerization and click chemistry (Scheme 3). The sequential and simultaneous methods yielded essentially structurally same polymers with similar molecular weight characteristics (see Materials and Methods). The 1H NMR spectra of the propargyl functionalized PA and PA-g-PEG were compared in Figure 2. Naturally, the NMR data of PA-g-PEG produced by either method display the same characteristic signals of both macromolecular segments (Figure 2b). It is important to note that the side chain propargyl functionality of the initial copolymer completely disappeared after CuAAC functionalization. Moreover, the triazole proton at 8.47 ppm, further confirms the success of the synthetic strategy in both cases. The FT-IR spectrum of PA-g-PEG is shown in Figure 3. Clearly, it shows the characteristic bands of both PA and PEG segments. In addition, no stretching band around 2100 cm−1 was observed, which further confirms the success of the CuAAC reaction and absence of any unreacted PEG-N3. 3.2. Biosensor Optimization Studies. 3.2.1. pH Optimization. Optimization studies were carried out in order to improve the performance of biosensor. For the optimization of pH, buffer solutions with different pH in the range of pH 4 to pH 9 were prepared. Amperometric measurements of the biosensor were carried out at room temperature in each reaction cell containing 10 mL of buffer solutions with different pH values (50 mM, pH 4−9) at −0.7 V with respect to Ag wire electrode. Under the constant potential, the current change due to enzymatic reaction was measured. After the background current reached a steady state, a certain amount of glucose was injected in the each reaction medium with different buffer solutions and the current change was recorded as the biosensor response. The constructed biosensor produced the highest signal at pH 6.5 (Figure 5A). This corresponds well to other studies where optimum pH for GOx activity was reported.16,27 Because of the fact that aniline is a weak base, polyaniline (PA) at slightly acidic pH is partly protonated and therefore carries multitude of positive charges, which leads to the formation of a noncovalent protein−polymer complex based on electrostatic interactions. The sensor forms through hydrogen bondings and polar static interactions of PEG and PA with GOx. Schematic visualization of the interactions is given in Figure 4. 3.2.2. Polymer Amount Optimization. The amount of PA-gPEG for the biosensor construction was optimized by preparing several polymer solutions with varying concentrations. A 5 μL aliquot of each solution was taken for the design of the biosensors. Sufficient amount of polymer is required to provide a stable support for the enzyme molecule to attach itself to the

Figure 1. Fabrication and working principle of PA-g-PEG based glucose biosensor. All experiments were carried out at ambient conditions. All data are given as the mean of three measurements. Error bars indicate relative standard deviation (RSD) of three measurements. 2.9. Instrumentation. 1H NMR spectra were recorded in CDCl3 with tetramethylsilane as an internal standard at 500 MHz on a Agilent VNMRS 500 spectrometer at room temperature. FT-IR spectra were recorded on a PerkinElmer FT-IR Spectrum One spectrometer. Molecular weights were determined by using Viscotek GPCmax Autosampler system, consisting of a pump, three ViscoGEL GPC columns, a Viscotek differential refractive index (RI) detector with a THF flow rate of 1.0 mL min−1 at 30 °C. The detector was calibrated with polystyrene standards having narrow molecular weight distribution. Data were analyzed using Viscotek OmniSEC software.

3. RESULTS AND DISCUSSION 3.1. Design and Synthesis of PA-g-PEG. The strategy for obtaining the desired water-soluble PA-g-PEG refers to the reduction of Cu(II) catalyst during the oxidative polymerization of aniline to generate Cu(I) species for the subsequent CuAAC click reaction. Thus, the components of the two processes were independently synthesized. First, the alkyne functional aniline monomer, namely 2-aminophenyl propargyl ether (APE) was synthesized by simple etherification process followed by the reduction of nitro groups (Scheme 1). Azide functional poly(ethylene glycol) (PEG-N3) was obtained by converting hydroxyl group of PEG into azide Scheme 1. Synthesis of 2-Aminophenyl Propargyl Ether (APE)

C

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Macromolecules Scheme 2. Synthesis of PA-g-PEG by Sequential Oxidative Copolymerization and CuAAC

Scheme 3. Synthesis of PA-g-PEG by Concurrent Oxidative Copolymerization and CuAAC

surface of the electrode, while the excessive polymer may hinder diffusion of the enzyme substrate. It was found that only a negligible amount of PA-g-PEG (5 μL of 0.125 mg mL−1) is needed to achieve the strongest amperometric response. It is apparent from Figure 5B that the response of the biosensor increases rapidly up to optimal concentration of the polymer from which point a slight reduction in the measured signal was observed probably owing to the water solubility of the polymer. It is likely that only those polymer molecules which either participate in polyanion−polycation interaction or are crosslinked with glutaraldehyde remain on the surface of the electrode. The excess of the PA-g-PEG is either washed away or dissolves into the solution until it reaches the amount close to the determined optimal value. Therefore, it can be claimed that

Figure 3. FT-IR spectrum of PA-g-PEG.

this biosensor architecture exhibits some self-tuning and/or self-adjusting capabilities. 3.2.3. Enzyme Load Optimization. To optimize the amount of GOx needed for the construction of biosensor, five different electrodes with different amounts of enzyme ranging from 0.2 to 2.0 mg (3.5−34.6 U) were prepared. The optimal biosensor response was obtained with 1.0 mg (17.3 U) of GOx (Figure 5C). Owing to the fact that glucose oxidase operates extracellularly, it contains structurally embedded carbohydrate chains called glycans, consisting of mannose, galactose and glucosamine monosaccharide units.66 PA-g-PEG contains a

Figure 2. 1H NMR spectra of propargyl functionalized PA (a) and PA-g-PEG (b). D

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glutaraldehyde would result in the polymer and enzyme leaching, causing the loss of signal strength. On the other hand, the excess of cross-linking agent would obstruct reagent diffusion to the surface of the electrode and decrease electric conductivity of the composite. 3.3. Analytical Characterization. The calibration curve of the PA-g-PEG biosensor for glucose is given in Figure 6. The

Figure 4. Schematic depiction of electrostatic interactions between GOx and PA-g-PEG. Bottom: polyanion−polycation interaction. Arithmetic signs represent full charges at pH 6.5. Top: oligosaccharide-PEG hydrogen bond network. Magenta-colored parts indicate enzyme−oligosaccharide units.

flexible PEG chain, which can wrap around the enzyme molecule, forming a network of hydrogen bond interactions with glycans and functional groups on the side chains of GOx, therefore providing even stronger noncovalent immobilization and extra stabilization. A possible schematic interpretation of this interaction is presented in Figure 4. 3.2.4. Optimization of Amount of Cross-Linking Agent. In this study glutaraldehyde was used as the cross-linking agent. It forms covalent bonds between primary amino groups that are present on the side chains of GOx and terminal PA-g-PEG monomer units. Glutaraldehyde amount was optimized by adjusting the volume share of 1% glutaraldehyde in a total of 10 μL drop casting solution. The optimal amount of glutaraldehyde was found to be 3.5 μL, which can be observed in the optimization curve (Figure 5D). Insufficiency of the amount of

Figure 6. Calibration curve for glucose of PA-g-PEG based biosensor (pH 6.5, 50 mmol·L−1 PBS, 25 °C, −0.7 V). The inset represents a typical response of the biosensor to the addition of glucose.

biosensor response exhibited linearity between 0.05 to 1 mmol L−1 of glucose and fits with a linear equation y = 3.191x + 0.1941 (R2 = 0.9971). The limit of detection (LOD) was determined to be 0.02 mmol L−1 according to the signal-tonoise ratio S/N = 3 criterion. Michaelis−Menten constant KMApp = 0.97 mmol L−1 and Imax = 5.43 μA were calculated from a Lineweaver−Burk plot. The sensitivity of the sensor was found to be 47.72 μA mM−1 cm−2. The typical response of the biosensor is given as an inset in Figure 6, where characteristic instant response can be observed. Calculated analytical parameters were compared with recently reported studies in

Figure 5. Dependence of the amperometric signal of the PA-g-PEG biosensor on (A) pH, (B) polymer concentration, (C) GOx amount, and (D) 1% glutaraldehyde by volume. E

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Table 1. Comparison of Analytical Parameters of Polymer-Based Glucose Biosensors Reported in the Literature and Examined in This Study biosensor rGO- GOx/PPGE/graphite GOx/p-MAA/Pt polyCo/GOx/GCE GOx/AuNPs/G/MWCNTs/GCE GOx/poly(2,6-DAP)/MWNT-mod. GCE poly(TTP)/GOx/GCE PDA-GOx-GN-Au PA-g-PEG/GOx/graphite a

KMApp (mmol L−1)

linear range (mmol L−1)

sensitivity (μA mM−1 cm−2)

LOD (mmol L−1)

ref

a

0.01−1.0 0.009−8.26 0.1−1.4 0.3−2.1 0.9−8.0 0.5−20.15 0.001−4.7 0.05−1.0

9.60 11.98 47.1 29.72 52.0 0.0561 28.4 47.72

0.0058 0.01 0.0828 0.0048 1.3 × 10−4 0.1 0.0001 0.02

17 26 32 33 34 35 36 this study

NR NR 0.69 2.09 0.20 26.13 6.77 0.97

NR: not reported.

the literature. The data for comparison is presented in Table 1. It can be seen that PA-g-PEG based glucose biosensor exhibits excellent sensitivity compared to other sensors of similar kind. 3.3.1. Biosensor Stability. Shelf life and operational stability of the biosensor were determined. Ten successful measurements were performed in a period of 2 h and the relative standard deviation (RSD) of the measurements was found to be 1.59%. It indicates that PA-g-PEG based glucose biosensor is capable to generate a reproducible output while performing series of measurements. No activity loss was observed within 12 days while storing biosensor at 4 °C in air. Even after 20 days the biosensor retained full signal strength (Figure 7), though the decrease in the ability to perform consecutive measurements could be observed.

Figure 8. PA-g-PEG based biosensor amperometric response to glucose, urea, and ascorbic acid.

Figure 7. Evaluation of amperometric PA-g-PEG based glucose biosensor signal with time.

3.3.2. Selectivity and Interference. The effect of possible interferents was investigated using urea, ascorbic acid and different sugars. Only a slight negative biosensor signal has been observed after the injection of 1 mmol·L−1 of ascorbic acid while the injection of the same concentration of urea into the reaction cell did not alter the electrochemical response of the biosensor at all (Figure 8). Moreover, the selectivity of the biosensor toward the substrate was tested by injecting different sugars. No signal was detected after the injection of 1 mmol·L−1 of galactose, mannose, fructose or sucrose. This shows that the biosensor exhibits excellent selectivity and performs well in the presence of different compounds. 3.4. Surface Characterization. The morphology of the prepared composites was studied using scanning electron microscopy (SEM). The morphology of pristine PA-g-PEG drop-casted onto graphite electrode can be seen in Figure 9A. The polymer forms dispersed grain-like particles uniformly spread across the electrode surface. Surface images of GOx and PA-g-PEG/GOx composites on the electrode are provided in

Figure 9. SEM micrographs of (A) PA-g-PEG, (B) GOx cross-linked with glutaraldehyde, (C) PA-g-PEG and GOx composite, and (D) optimized PA-g-PEF/GOx/glutaraldehyde on the graphite electrode.

parts B and C of Figures 9, respectively. The change in the surface morphology after the introduction of conjugated polymer can be clearly seen as the texture changes from planar to more ordered and rougher brush-like structure. This change can be attributed to successful interaction between polymer and enzyme molecules. The surface morphology of the biosensor under optimized conditions is presented in Figure 9D. It can be observed that the presence of glutaraldehyde significantly contributes to the change in morphology by making the surface smoother and therefore, eliminating the barriers for diffusion and resulting in the enhanced performance of the biosensor. Notably, the negligible sizes of holes can be excluded, as there is no significant effect on the homogeneity. F

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4. CONCLUSIONS In conclusion, synthesis and application as biosensor of a novel water-soluble conjugated PA-g-PEG polymer is reported. It was shown that a useful combination of concurrent or sequential oxidative polymerization and CuAAC processes by means of which graft copolymers of PA with PEG can ultimately be formed by use of the high reactivity of copper catalyst in both processes. The applicability of this polymer as a GOx enzyme immobilization matrix has been discussed. Water solubility of PA-g-PEG greatly simplified the manufacturing process of the biosensor by allowing it to be performed in one-step. Moreover, it was shown that only a tiny amount of PA-g-PEG (5 μL of 0.125 mg mL−1 solution) is needed to construct a biosensor with an optimum performance, minimizing the cost of production. Successful immobilization of GOx on the surface of the electrode has been confirmed by SEM. The optimized biosensor demonstrated the following analytical characteristics: linear range of 0.05−1.0 mmol L−1(R2 = 0.9971), and sensitivity 47.72 μA mM−1 cm−2; KMApp and Imax were determined as 0.97 mmol L−1 and 5.43 μA, respectively. These results compare well with other recently reported studies. Moreover, PA-g-PEG biosensor demonstrated a rapid reaction upon the addition of glucose, therefore resulting in a very fast response time. The sensor was shown to possess a good shelf-time stability of 12 days and capability of performing reproducible analysis in a series of measurements with a very low deviation. These results show that PA-g-PEG has a great potential to be used as a supporting material in the development of glucose biosensors.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.) E-mail: [email protected]. *(L.T.) E-mail: [email protected]. ORCID

Yusuf Yagci: 0000-0001-6244-6786 Notes

The authors declare no competing financial interest.

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Article

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DOI: 10.1021/acs.macromol.7b00073 Macromolecules XXXX, XXX, XXX−XXX