Highly Sensitive Glucose Biosensor Based on One-Pot Biochemical

Jan 12, 2009 - E-mail: [email protected]. † Hunan Normal .... to continuous potential cycling (0-1.5 V, 30 mV s-1) in 0.20. M aqueous HClO4, until ...
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J. Phys. Chem. B 2009, 113, 1332–1340

Highly Sensitive Glucose Biosensor Based on One-Pot Biochemical Preoxidation and Electropolymerization of 2,5-Dimercapto-1,3,4-thiadiazole in Glucose Oxidase-Containing Aqueous Suspension Yingchun Fu,† Can Zou,† Qingji Xie,*,† Xiahong Xu,‡ Chao Chen,† Wenfang Deng,† and Shouzhuo Yao†,‡ Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal UniVersity, Changsha 410081, People’s Republic of China, and State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan UniVersity, Changsha 410082, People’s Republic of China ReceiVed: August 16, 2008; ReVised Manuscript ReceiVed: NoVember 16, 2008

A novel and high-performance biosensing platform was prepared on the basis of one-pot biochemical preoxidation and electropolymerization of monomer (BPEM) for high-load and high-activity immobilization of enzymes. As representative materials here, 2,5-dimercapto-1,3,4-thiadiazole (DMcT) was used as the monomer, glucose oxidase (GOx) was used as the model enzyme, and enzymatically generated H2O2 (EGH2O2) in the presence of glucose was used as the preoxidant. In the BPEM protocol, glucose was added to a pH 7.0 phosphate buffer suspension containing ultrasonically dispersed DMcT and GOx, in order to enzymatically generate H2O2, which preoxidized DMcT to DMcT oligomers/polymer (DMcTO) and thus led to the formation of DMcTO-GOx composites with a great deal of GOx entrapped; the composites were then co-electrodeposited with poly(DMcT) on a Au electrode. For comparison, the enzyme immobilization was also conducted by a preoxidation-free conventional electropolymerization protocol (CEP), as well as a chemical preoxidation and electropolymerization of monomer (CPEM) protocol with externally added H2O2 (EA-H2O2) as the preoxidant. The glucose biosensors constructed by the BPEM and CPEM protocols exhibited detection sensitivities enhanced by 119 and 88 times, respectively, compared to that constructed by the CEP protocol, as well as limits of detection lowered by ca. 2 orders of magnitude. The higher sensitivity of the enzyme electrode prepared by the BPEM protocol compared to that prepared by the CPEM protocol is probably due to the improved proximity of biochemical preoxidation around the enzyme molecules and thus a larger enzyme load. The electrochemical quartz crystal microbalance technique was used to investigate various electrode modification processes, which also revealed that poly(DMcT) could be cathodically detached from the electrode surface, favorably enabling the electrochemical regeneration of the electrode substrate. The proposed BPEM strategy is recommended for wide biosensing and biocatalysis applications based on many other polymers/ enzymes. Introduction The effective immobilization of biomolecules definitely plays a key role for constructing high-performance biodevices including biosensors.1-3 Chemical or electrochemical polymerization has been widely used to immobilize various enzymes, and it is well-known that, compared with the chemical polymerization, electropolymerization allows for better control of the film thickness even at a micro-/nanoscale substrate. Typical conducting polymers electrosynthesized as enzyme-immobilization matrixes include poly(aniline),4-7 polypyrrole,8-10 poly(thiophene),11 and their derivatives,12-16 and typical electrosynthesized nonconducting polymers include poly(o-phenylenediamine)17-19 and poly(o-aminophenol).20-22 The nonconducting films are well-known to show high resistance against electrode fouling and high anti-interferent capability against some electroactive substances including ascorbic acid and uric acid.17-24 Generally, the enzymes immobilized in electrosynthesized films show limited enzyme load and activity,1,25 but the entrapment * To whom correspondence should be addressed. Tel./Fax: +86-7318865515. E-mail: [email protected]. † Hunan Normal University. ‡ Hunan University.

of enzymes in chemically oxidized polymers largely overcomes such limitations and leads to high-performance biosensors.25,26 However, the modification of such chemically oxidized polymer precipitates with entrapped enzymes on the electrode is usually conducted by dip-drying a microliter-scale enzyme-containing mixture on the electrode surface, which can lead to an inhomogeneous film structure, uncontrollability of film thickness, and difficulty in fine operation on micro-/nano-sized substrates. Very recently, we proposed the immobilization of enzymes through the one-pot chemical preoxidation and electropolymerization of monomer (CPEM) in enzyme-containing aqueous suspensions to develop biosensors with improved performance.23 Two preoxidants of K3Fe(CN)6 and p-benzoquinone, two dithiol monomers of 1,4-benzenedithiol and 1,6-hexanedithiol, and two enzymes of glucose oxidase (GOx) and alkaline phosphatase (AP) were used to examine the CPEM protocol. Briefly, a selected preoxidant was added to a solution containing a monomer and an enzyme, in order to allow the chemical oxidation of the monomer to oligomers, simultaneously yielding oligomer-enzyme composites with high-load and high-activity enzymes entrapped. Some of the composites were subsequently

10.1021/jp807337f CCC: $40.75  2009 American Chemical Society Published on Web 01/12/2009

Highly Sensitive Glucose Biosensor Based on DMcT

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SCHEME 1: Illustration of the Immobilization of GOx by the CPEM Protocol Involving the Preoxidant EA-H2O2 (I f IV) and by the BPEM Protocol Involving the Preoxidant EG-H2O2 (I f V f VII)

co-electrodeposited onto the electrode surface with the polymer from the electro-oxidation of the remaining monomers, finally yielding an enzyme film with high enzymatic load and activity. The basic procedures are depicted in Scheme 1 [from I to IV, as indicated by the gray arrows, but here, the preoxidant is H2O2 and the monomer is 2,4-dimercapto-1,3,5-thiadiazole (DMcT)]. The GOx-based and AP-based first-generation biosensors developed from the CPEM protocol exhibited notably improved performance compared with analogues from the preoxidationfree conventional electropolymerization (CEP) protocol. The use of biocatalyst systems, e.g., purified enzymes, is regarded as a very powerful route for synthesizing chemicals in various fields, especially in synthetic organic chemistry, with advantages of higher catalytic activity, higher enantioselectivity, mild reaction conditions, and lower energy requirements, as compared with the use of chemical catalyst systems.27,28 Whereas essential enzymatic reactions of many biocatalysts have been widely utilized to effectively transform substrates into desired products,29,30 chemical reactions initiated by the enzymatically generated products are becoming increasingly attractive. Such approaches involve mainly the enzymatic generation of reactive products in the presence of given substrates and utilization of the yielded products to react with other species. For example, the reductive enzymatic products could reduce oxidative noble metal salts to the atomic form and yield metallic nanoparticles,31-33 which have been employed to switch surface properties31 or detect enzymatic activity.33 More importantly, because the generation of enzymatic products and the reaction of products with other species takes place in the vicinity of enzyme molecules, it allows one to finely elaborate the surroundings of the enzymes, which is favored for the enzyme-assisted nanolithography32,34 and the preparation of enzyme-entrapped polymer nanocomposties.35,36 Among various enzymatically generated products, H2O2 is well-known as a good “green oxidant” for its producing only natural H2O or O2 and thus being environmentally benign.37 Dithiols and thiols have been widely used to construct highly ordered and compact self-assembled monolayers on some metal surfaces for various applications.38 DMcT and its adducts with conducting polymers were intensively studied for the development of important electrode materials for high-energy lithium batteries.39-41 However, to our knowledge, poly(DMcT) (PDMcT), as an interesting and nonconducting polymer formed via S-S linking after oxidation of DMcT, has not been used to immobilize biomolecules for bioapplications.

Herein, we report on a novel and high-performance biosensing platform based on the one-pot biochemical preoxidation and electropolymerization of monomer (BPEM) for high-load and high-activity immobilization of enzyme. In the BPEM protocol, glucose is added to a pH 7.0 phosphate buffer suspension containing ultrasonically dispersed DMcT and GOx, and the enzymatically generated H2O2 (EG-H2O2) preoxidizes DMcT to DMcT oligomers/polymer (DMcTO) and thus leads to the formation of DMcTO-GOx composites. The composites are then co-electrodeposited with PDMcT on a Au electrode, as illustrated in Scheme 1(from I directly to V and then to VII, as indicated by the black arrows). The glucose biosensor constructed by the BPEM protocol exhibits better performance than those constructed by the CPEM protocol with externally added H2O2 (EA-H2O2) as a preoxidant and the CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to track various electrode modification processes. Experimental Section Instrumentation and Chemicals. All electrochemical experiments were conducted on a CHI660C electrochemical workstation (CH Instruments Inc., Austin, TX), and a conventional three-electrode electrolytic cell was used. Scanning electron microscopy images were collected on a JEM-6700F field-emission scanning electron microscope. A computerinterfaced HP4395A impedance analyzer was applied in the EQCM experiments. The microbalance measurements were performed under conditions of 41 points, a frequency span of 10 kHz covering the piezoelectric quartz crystal resonant frequency, an intermediate-frequency bandwidth (IF BW) value of 10 kHz, and a source power value of 0.5 dBm (ca. 3.16 mW). AT-cut 9 MHz piezoelectric quartz crystals (model JA5, Beijing Chenjing Electronics Co., Ltd., Beijing, China) with a 12.5mm wafer diameter were used in the experiments. A Au electrode with a 6.0-mm diameter (keyhole configuration, area ) 0.29 ( 0.01 cm2) on one side of the piezoelectric quartz crystal was exposed to the solution and served as the working electrode, whereas that on the other side faced air. The reference electrode was a KCl-saturated calomel electrode (SCE), and a carbon rod served as the counter electrode. All potentials reported in this article are cited versus the standard calomel electrode (vs SCE), unless otherwise specified. GOx (EC 1.1.3.4; type II from Aspergillus niger, activity ≈ 150 kU g-1) was purchased from Sigma. DMcT was a product

1334 J. Phys. Chem. B, Vol. 113, No. 5, 2009 of Alfa Aesar. Glucose was obtained from Shanghai Chemicals Station. A pH 7.0 phosphate-buffered saline (PBS) solution, 0.1 M KH2PO4-K2HPO4 + 0.1 M K2SO4, was used. All other chemicals were of analytical grade or better quality, and used as received. Milli-Q ultrapure water (Millipore, g 18 MΩ cm) was used throughout. All experiments were performed at room temperature around 25 °C. Fabrication and Characterization of the Enzyme Electrodes. To clean the Au electrode surface, one drop of fresh prepared concentrated H2SO4 + H2O2 (3:1, v/v) was placed on the electrode surface for 20 s, after which the electrode was rinsed thoroughly with water and then dried with a stream of pure nitrogen. The treatment was repeated three times. Prior to the electrochemical experiments, the Au electrode was subjected to continuous potential cycling (0-1.5 V, 30 mV s-1) in 0.20 M aqueous HClO4, until the cyclic voltammogram indicating a clean Au electrode surface became reproducible. As illustrated in Scheme 1, the BPEM- and CPEM-based biosensors were prepared as follows: To an ultrasonically dispersed aqueous suspension of 0.5 mg mL-1 DMcT in 1.0 mL of PBS was added 1.0 mg GOx (final concentration ) 1.0 mg mL-1) under stirring, followed by 10 µL of 1.0 M glucose (or H2O2). The mixture was slowly stirred for 20 min to allow the reaction to form DMcTO-GOx-EG-H2O2 (or DMcTOGOx-EA-H2O2) composites, and then the codeposition of some DMcTO-GOx-EG-H2O2 (or DMcTO-GOx-EA-H2O2) composites with PDMcT on a bare Au electrode was performed potentiostatically at 1.3 V to achieve a ca. 6.0-kHz film (PDMcT-DMcTO-GOx-EG-H2O2 or PDMcT-DMcTOGOx-EA-H2O2), as monitored by the EQCM. The electrode was then thoroughly rinsed with Milli-Q water to remove physically adsorbed species. In the CEP case, 1.0 mg of GOx was added to 1.0 mL of PBS suspension containing 0.5 mg mL-1 DMcT, and the mixture was subjected to 1.3 V potentiostatic electropolymerization to grow ca. 2.0-kHz polymer (PDMcT-GOx; note the lower deposition rate here than for the BPEM and CPEM protocols), with EQCM monitoring; then, the resulting electrode was thoroughly rinsed in water. When not in use, the prepared GOx electrodes were stored in pH 7.0 PBS at 4 °C (refrigerator). Bare Au electrodes were characterized in PBS containing 1.0 mM K4Fe(CN)6 and 1.0 mM K3Fe(CN)6 by cyclic voltammetry (at 100 mV s-1) and electrochemical impedance spectroscopy (EIS), which was generally performed after each modification step for comparisons among and better understanding of various surfaces. For the EIS measurements, the working electrode potential was fixed at the formal potential of the Fe(CN)63-/4couple after being conditioned at this potential for 100 s. Amperometric Biosensing Measurements. Measurements of the prepared enzyme electrodes were carried out under solution-stirred conditions in pH 7.0 PBS, and the response current was marked with the change value between the steadystate current after addition of a substrate and the initial background current without the substrate. Results and Discussion Electropolymerization of DMcT in Neutral Aqueous Medium. The electrochemistry of DMcT as an interesting electrode material for high-energy lithium batteries has been studied intensively. Most of these studies were conducted in organic media such as acetonitrile,39-41 whereas fewer have used alkaline aqueous media in which DMcT is acceptably soluble.42 To our knowledge, this is the first time that the electrochemistry

Fu et al. and electropolymerization of DMcT have been examined in a neutral aqueous medium. First, we studied the self-assembly of DMcT and the adsorption of DMcT precipitates onto a Au electrode surface, which are virtually unavoidable steps for DMcT electropolymerization and should be assessed. As shown in Figure 1A, the time courses of the frequency and resonant resistance of the piezoelectric quartz crystal, as well as opencircuit potential during the self-assembly of DMcT on Au in a stirred PBS suspension were recorded. After the injection of DMcT, the frequency decreased and the resistance increased sharply in the first 10 s, which can be ascribed to a rapid selfassembly process of DMcT on the Au electrode, although the poor solubility of DMcT in the PBS suspension and the adsorption of the insoluble DMcT aggregates might complicate the data to some degree. A final frequency decrease (-∆f0) of 183 Hz and a final resistance increase (∆R1) of 3.5 Ω were obtained, and the -∆f0/∆R1 ratio was 52.3 Hz Ω-1, being larger than 10 Hz Ω-1 that is characteristic of a net viscous effect for a Newtonian liquid19,23,24,43,44 and thus demonstrating the high rigidity of the obtained self-assembled layer. The limited frequency response observed here might indicate that the adsorption of the insoluble DMcT aggregates was not significant enough to interfere with the self-assembly process. The simultaneously recorded open-circuit potential (Eoc) gave an abrupt decrease to -0.18 V, followed by a rapid increase to ca. -0.07 V, and then a very slow increase finally to -0.05 V, indicating a fast homogenization effect after the addition of reductive DMcT to the stirred medium. When another aliquot of DMcT was added after the adsorption experiment, negligible responses of frequency, resistance, and Eoc were observed (not shown), implying that full self-assembly had been achieved. A potential-cycling EQCM experiment was conducted in the neutral PBS suspension (unstirred) to investigate the electrochemistry of DMcT, and the results are shown in Figure 1B. The control experiment conducted in a blank pH 7.0 PBS revealed that the Au was oxidized to gold oxides at potentials approximately from 0.7 to 1.2 V, and the reverse electrode reaction took place at potentials roughly from 0.6 to 0.35 V (peaking at 0.45 V), with concomitant and recoverable frequency changes of only 18 Hz, implying minor mass changes of the Au electrode in its oxidation and reduction processes, which are virtually equivalent to adsorption/desorption of monolayer oxygen on the Au surface.45 In the presence of 0.5 mg mL-1 DMcT, on the first positive scan from -0.08 V, an anodic peak was observed at 0.15 V. As a dibasic acid, DMcT has two aqueous pKa values of between -1.4 and 2.1 (pKa1) and 7.5 (pKa2);39,40 therefore, the DMcT in the pH 7.0 PBS medium here should mainly exist in the anionic form of DMcT-. Thus, the anodic current peaking at 0.15 V can be assigned mainly to the oxidation of DMcT- to its dimer, being similar to a previous report on the oxidation of DMcT- at 0.1 V vs Ag/AgCl/ NaCl(saturated) in 0.1 M LiClO4-acetonitrile containing 10 mM DMcT and 20 mM pyridine or 20 mM triethylamine.40 On the anodic scan, the frequency began its obvious decrease at potentials just positive of ca. 0.15 V, but in nonaqueous media, the EQCM frequency decreased significantly at potentials positive of ca. 0.5 V vs Ag/AgCl/NaCl(saturated), in response to the electropolymerization of DMcT-.40 We can ascribe the disagreement to the solubility difference of DMcT dimer in nonaqueous and aqueous media. The dimer in nonaqueous media is highly soluble and cannot deposit on the electrode until its further electropolymerization occurs. However, in the aqueous medium here, the solubility of DMcT is limited, and the electrogenerated dimer of DMcT- is less soluble and can deposit

Highly Sensitive Glucose Biosensor Based on DMcT

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Figure 1. (A) Time-dependent responses of frequency, resonant resistance, and open-circuit potential (Eoc) during self-assembly of 0.5 mg mL-1 DMcT in a PBS suspension. (B) Responses of current, frequency, and resonant resistance to potential cycling in blank PBS (dashed curves) and in a PBS suspension of 0.5 mg mL-1 DMcT (solid curves). Initial potential ) -0.08 V, scan rate ) 30 mV s-1.

SCHEME 2: Illustration of Proton Dissociation Equilibrium and Electrochemical Reactions of DMcT

on the electrode surface when its concentration becomes sufficiently high, leading to the obvious frequency decrease observed here. We also guess that some more poorly soluble oligomers might be gradually electrogenerated as surface deposits with the further positive change in the potential, which might contribute to the frequency response. Another anodic peak was observed at roughly 0.5 V, and the simultaneously recorded frequency decreased sharply, most likely resulting from the electropolymerization of the dimer and oligomers of DMcT-.46 On the negative scan, two reduction peaks were found at -0.3 and -0.7 V, which can be ascribed to the reduction of DMcTdimers and PDMcT, respectively, as reported previously for nonaqueous media.46 After experiencing oxidation at potentials positive of ca. 0.2 V, the dimers might be mostly turned into PDMcT, so the cathodic peak current was obviously larger at -0.7 V (depolymerization) than that at -0.3 V (dedimerization). In addition, the frequency changed little at -0.3 V but increased sharply at potentials negative of -0.5 V, indicating the reductive cleavage of the S-S bonds in the dimer and PDMcT and thus detachment of the surface deposits. The net frequency change after one complete potential cycle was as small as 3 Hz here

(initial and end potentials ) -0.08 V), indicating that the reductive dedimerization and depolymerization of the surface deposits were complete. The motional resistance response was observed as a complicated trace, but the notable changes were found near the peak potentials. The maximum resistance observed at -0.7 V (positive going) should indicate the most chaotic and thus most viscous state of the film during reductive depolymerization at this potential, and the net change in motional resistance after one potential cycle was also negligible. A repeated potential cycling could well reproduce the basic EQCM responses shown in Figure 1B. For clarity, the proton dissociation equilibrium and electrochemical reactions discussed above are briefly illustrated in Scheme 2.39,40,46 Fabrication and Characterization of the Enzyme Electrodes. As discussed above, the electrochemical oxidation of DMcT- involves two successive steps, i.e., the dimerization peaking at 0.15 V and the further electropolymerization peaking at 0.5 V. Although we could readily obtain a dimer film by fixing the potential at 0.15 V, such a film was rather unstable during rinsing with water and use in a stirred PBS solution.

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Figure 2. Time-dependent responses of current, frequency, and resonant resistance during potentiostatic polymerization at 1.3 V in stirred PBS containing (1) 0.5 mg mL-1 DMcT + 1.0 mg mL-1 GOx, (2) 0.5 mg mL-1 DMcT + 1.0 mg mL-1 GOx + 3.0 mM H2O2, (3) 0.5 mg mL-1 DMcT + 1.0 mg mL-1 GOx + 10 mM H2O2, or (4) 0.5 mg mL-1 DMcT + 1.0 mg mL-1 GOx + 10 mM glucose. Stirring speed ≈ 100 rpm.

Thus, the films were potentiostatically grown at 1.3 V in order to accelerate the film deposition and improve the film stability. We recorded the EQCM responses during the 1.3 V potentiostatic growth of various films in a PBS suspension containing 0.5 mg mL-1 DMcT + 1.0 mg mL-1 GOx in the presence of 0, 3.0, or 10 mM H2O2, or 10 mM glucose, as shown in Figure 2. The redox kinetics of DMcT was reported to be rather slow at room temperature,47,48 and this fact was also verified by our observation that it took as long as 20 min after addition of 10 mM H2O2 in the stirred PBS suspension containing DMcT and GOx to change the suspension color from slight yellow to brownish yellow. A relatively slow frequency decrease was observed during the electropolymerization of DMcT monomers without H2O2. Upon addition of 3.0 mM H2O2 to the suspension containing 0.5 mg mL-1 DMcT and 1.0 mg mL-1 GOx, followed by stirring for 20 min for the chemical preoxidation, the subsequent potentiostatic electropolymerization became a little faster than that in the case without H2O2, as a result of the codeposition of some DMcTO-GOx composites to some extent.23 Here, the total molar concentration of DMcT was 3.3 mM (assuming that the DMcT was fully dispersed), and thus 3.0 mM H2O2 was comparable to the total quantity of the DMcT. It should be noted that the 3.0 mM H2O2 used here might be in slight excess in terms of the stoichiometric solution-state preoxidation reaction, as the DMcT could not be completely exposed to the liquid phase because of the poor solubility of DMcT in the neutral aqueous medium. When H2O2 was in a large excess (10 mM H2O2, ca. 3-fold molar concentration of total DMcT), the film growth was largely accelerated, which is obviously different from our previous recommendation that a successful electropolymerization occurred when the preoxidant

Fu et al. was in just a slight deficiency.23 The observation here is quite interesting, and we speculate that the likely responsible factors are that (1) the relatively slow oxidation of DMcT by H2O2 required a higher concentration of H2O2 to yield plenty of DMcTO-GOx precipitates to participate in the codeposition with PDMcT and thus accelerate the film growth and (2) the 1.3 V potentiostatic treatment in the presence of the large excess of H2O2 here might yield some highly oxidative radicals from H2O2 in the vicinity of electrode surface, e.g., O2•, that can more effectively accelerate the polymerization of DMcT (or DMcT dimer/oligomers) and the film growth. It should be noted that the dissolved O2 could seemingly act as a preoxidant too; however, the chemical preoxidation of DMcT by O2 might be kinetically very sluggish, as an effective preoxidation of 0.5 mg mL-1 DMcT in 1 mL of PBS suspension solely by the O2 in air required over 12 h under our experimental conditions. In the BPEM protocol, 10 mM glucose was added to a PBS suspension containing 0.5 mg mL-1 DMcT and 1 mg mL-1 GOx, and the mixture was then stirred for 20 min to yield sufficient EG-H2O2 and achieve the enzymatically catalyzed preoxidation. By imposing a 1.3 V potentiostatic treatment, a film-growth rate similar to that in the case of 10 mM EA-H2O2 (curves 3) was observed, as shown in Figure 2 by curves 4, demonstrating that the enzymatically generated H2O2 can also be effective for preoxidation and rapid film growth. The values of -∆f0/∆R1 for the films prepared in the presence of 0, 3.0, and 10 mM H2O2, and 10 mM glucose were 40, 47, 21, and 21 Hz Ω-1, respectively, demonstrating that all of the polymers were highly rigid.19,23,24,43,44 The smaller -∆f0/∆R1 ratios for the deposited films obtained by the CPEM protocol in the presence of 10 mM H2O2 (curves 3) and by the BPEM protocol in the presence of 10 mM glucose (curves 4) suggest that these films should be more porous, which are supported by the following scanning electron microscopy images and electrochemical characterizations. The morphologies of the bare Au electrode surface and the polymer films on it were examined, as shown in Figure 3. The PDMcT-GOx film prepared from the CEP protocol showed a rugged surface with polymer nanoparticles, and the BPEM and CPEM protocols led to thicker and obviously more porous films, resulting mainly from the participation of the DMcTO-GOx composites. Cyclic voltammetry and electrochemical impedance spectroscopy experiments with the K3Fe(CN)6/K4Fe(CN)6 probe revealed that the BPEM- and CPEM-based polymers were more porous as indicated by the high observed electroactivity of the probe, as shown in Figure 4. The findings agree well with those in our previous report.23 The more porous structures of the enzyme films in the BPEM and CPEM cases should facilitate the mass transfer of reactants to grow a thick film,49 and the mass transfer of the substrates and the enzymatically generated products during biosensing experiments based on these enzymeimmobilization matrices should be largely benefited as well. Condition Optimization. To obtain the best sensitivities for the glucose assay, various conditions, including the concentrations of monomer, GOx and preoxidants (EA-/EG-H2O2), the film thickness (expressed as the absolute values of frequency shifts in this work, for convenience), detection potential, and detection pH were investigated by varying the examined parameter while holding the others fixed. The condition optimization for enzymatic film construction is shown in Figure 5, and the condition optimization for biosensing experiments is shown in Figure 6. The current response increased when the concentration of DMcT was increased from 0.1 to 0.5 mg mL-1. However,

Highly Sensitive Glucose Biosensor Based on DMcT

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Figure 3. Scanning electron microscopy images of (1) the bare Au electrode and the Au electrode modified with (2) PDMcT-GOx, (3) PDMcT-DMcTO-GOx-EA-H2O2, and (4) PDMcT-DMcTO-GOx-EG-H2O2. Scale bar ) 100 nm. In each case, 0.5 mg mL-1 DMcT and 1.0 mg mL-1 GOx were used, and 10 mM H2O2 or glucose was used when needed.

Figure 4. (A) Cyclic voltammetry and (B) electrochemical impedance spectroscopy in a PBS solution (pH 7.0) containing 1.0 mM K4Fe(CN)6 + 1.0 mM K3Fe(CN)6 on the (1) PDMcT-GOx-, (2) PDMcT-DMcTO-GOx-EA-H2O2-, and (3) PDMcT-DMcTO-GOx-EG-H2O2-modified Au electrodes. Scan rate ) 100 mV s-1. 100 kHz-0.05 Hz, 10 mV rms, 0.21 V vs SCE.

clearly visible precipitates were produced after the chemical or biochemical preoxidation if the concentration was further increased, and the sensitivity of the thus-prepared biosensor was also suppressed, so 0.5 mg mL-1 was selected as the best DMcT concentration. The value of 1 mg mL-1 for GOx was fixed as the optimum enzyme concentration. As mentioned above, the suitable concentration of H2O2 was of particular importance for the successful preoxidation and sequent electropolymerization, so the concentration of EA-H2O2 or glucose for EG-H2O2 was carefully examined. The biosensors achieved their highest sensitivity when the concentration of EA-H2O2 was 10 mM and larger. The optimized concentration of glucose for EG-H2O2 was found to be 10 mM or slightly more, being consistent with

that for EA-H2O2. According to our previous study,23 the activity of the solution GOx was estimated to be 79.1 U mg-1 (where 1 U of enzyme activity is defined as enzymatic generation of 1 µmol of H2O2 in 60 s under our experimental conditions), which means that 1 mg of GOx in suspension could catalyze the oxidation of 79.1 µmol of glucose, and the reoxidation of the reduced GOx by the soluble O2 could produce 79.1 µmol of H2O2 in 1 min. When this reaction occurred in 1 mL of suspension, the theoretical maximum concentrations of glucose that 1 mg of GOx could catalyze and of EG-H2O2 in 60 s are both 79.1 mM, being obviously larger than the glucose concentration of 10 mM applied in our experiments, implying that 10 mM EG-H2O2 could be readily obtained for preoxidation

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Fu et al.

Figure 5. Optimization of (A,O) GOx concentration, (A,b) DMcT concentration, (B,b) added glucose concentration, (C,b) time for preoxidation, and (C,O) film thickness for PDMcT-DMcTO-GOx-EG-H2O2, as well as (B,O) added H2O2 concentration for PDMcT-DMcTO-GOx-EAH2O2. The parameters utilized here were 0.5 mg mL-1 for DMcT, 1 mg mL-1 for GOx, 10 mM for EA-H2O2 or glucose, and ca. 6.0 kHz for film thickness, except for the film-thickness optimization experiments. The response currents for 2.0 mM glucose (∆i) were obtained in pH 7.0 PBS at 0.7 V vs SCE.

Figure 6. Optimization of detection potential (b) and detection pH (O, adjusted by changing the K2HPO4/KH2PO4 ratio). The enzyme electrode was fabricated under optimized conditions, and 2.0 mM glucose in pH 7.0 PBS was used.

and electropolymerization. On the other hand, the concentration of dissolved O2 in the suspension should be an important factor for the enzymatically catalyzed preoxidation, which was reported to be 1.3 mM in water,50 being smaller than the 10 mM concentration of added glucose. As a result, the enzymatic catalysis on the glucose conversion might last for several minutes, as part of the O2 needed for the enzymatic reaction relied on the replenishment of dissolved O2 via continuous dissolution of air-O2 from open air to the stirred solution. To achieve effective preoxidation, the time for preoxidation was examined (with the case of EG-H2O2 chosen as an example), and the value corresponding to the best sensitivity

was 20 min. When the time period for the preoxidation was not sufficient, the preoxidation was not allowed to react appropriately, whereas when the time was too long, fewer DMcT monomers/dimers were reserved in the liquid suspension, and the electropolymerization would be so slow that it even stopped, because the large DMcTO-GOx composites could not be solely polymerized onto the electrode surface in the absence of DMcT monomers/dimers, which is similar to our previous study.23 An optimal film thickness of 6.0 kHz was found. It should be noted that, although the PDMcT film is nonconductive, a film of ca. 9 kHz could be grown, probably because the porous structure of the composite films in the EA-H2O2 and EG-H2O2 cases largely facilitated the mass transfer of the reactants.49 However, the facile control of film thickness was usually achieved when the film was not thicker than 6 kHz. In addition, we found the optimal detection potential to be 0.7 V and the optimal pH of the detection solution to be pH 7.0, as shown in Figure 6. Performance of Enzyme Electrodes. Under the optimized conditions, the performance of the prepared enzyme electrodes was examined. The amperometric response and calibration curves are shown in Figure 7, and the values of sensitivity, linear detection range, and limit of detection are listed in Table 1. The enzyme electrodes prepared by the CPEM protocol (PDMcT-DMcTO-GOx-EA-H2O2) and by the BPEM protocol (PDMcT-DMcTO-GOx-EG-H2O2) presented notably increased sensitivities, with factors of 88 and 119 improvements compared to that prepared by the CEP protocol (PDMcT-GOx), again demonstrating that the protocols involving the preoxidation and electropolymerization of monomer are indeed promising for constructing performance-enhanced biosensors, mainly because of the largely promoted enzyme load and activity, as concluded in our previous study.23 Also, these sensitivities are obviously superior to those for most other first-generation glucose biosensors, which have been reported to be generally lower than 20 µA cm-1 mM-1 (mostly