Polymeric Bionanocomposite Cast Thin Films with In Situ Laccase

Mar 25, 2010 - Phone/Fax: +86 731 88865515. ... The DA substrate yielded the best biosensing performance, as compared with aniline, o-phenylenediamine...
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Polymeric Bionanocomposite Cast Thin Films with In Situ Laccase-Catalyzed Polymerization of Dopamine for Biosensing and Biofuel Cell Applications Yueming Tan, Wenfang Deng, Yunyong Li, Zhao Huang, Yue Meng, Qingji Xie,* Ming Ma, 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, P. R. China ReceiVed: January 31, 2010; ReVised Manuscript ReceiVed: March 9, 2010

We report here on the facile preparation of polymer-enzyme-multiwalled carbon nanotubes (MWCNTs) cast films accompanying in situ laccase (Lac)-catalyzed polymerization for electrochemical biosensing and biofuel cell applications. Lac-catalyzed polymerization of dopamine (DA) as a new substrate was examined in detail by UV-vis spectroscopy, cyclic voltammetry, quartz crystal microbalance, and scanning electron microscopy. Casting the aqueous mixture of DA, Lac and MWCNTs on a glassy carbon electrode (GCE) yielded a robust polydopamine (PDA)-Lac-MWCNTs/GCE that can sense hydroquinone with 643 µA mM-1 cm-2 sensitivity and 20-nM detection limit (S/N ) 3). The DA substrate yielded the best biosensing performance, as compared with aniline, o-phenylenediamine, or o-aminophenol as the substrate for similar Lac-catalyzed polymerization. Casting the aqueous mixture of DA, glucose oxidase (GOx), Lac, and MWCNTs on a Pt electrode yielded a robust PDA-GOx-Lac-MWCNTs/Pt electrode that exhibits glucose-detection sensitivity of 68.6 µA mM-1 cm-2. In addition, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) was also coimmobilized to yield a PDA-Lac-MWCNTs-ABTS/GCE that can effectively catalyze the reduction of O2, and it was successfully used as the biocathode of a membraneless glucose/O2 biofuel cell (BFC) in pH 5.0 Britton-Robinson buffer. The proposed biomacromolecule-immobilization platform based on enzyme-catalyzed polymerization may be useful for preparing many other multifunctional polymeric bionanocomposites for wide applications. Introduction Laccase (Lac) of wide fundamental and application interest is a multi copper-containing polyphenol oxidase that can catalyze the oxidation of many phenolic compounds accompanied by reduction of oxygen. Virtually, the substrate of Lac is exceedingly broad, including mono-, di-, and polyphenols; aminophenols; methoxyphenols; aromatic amines; ascorbate; and benzenethiols, providing their redox potentials are sufficiently low.1,2 Lac can also be used for the transformation of nonphenolic compounds with the aid of mediator compounds.3,4 Lac has been widely used in industrial processes, including lignin degradation, textile bleaching, decolorization of dyes, oxidation of organic pollutants in wastewater, and microbial transformation of natural products.5–10 In addition, various Lac-based biosensors have been proposed for determination of phenols and their derivatives in wastewater, blood, wine, tea, fruit juice, and oil.11–17 Because Lac can catalyze the reduction of oxygen directly to water in a four-electron transfer step without intermediate formation of hydrogen peroxide, Lac is also one of the most commonly considered enzymes for bioelectrocatalytic cathodes in biofuel cells (BFCs).18–27 The use of enzymes as catalysts for polymer synthesis (enzymatic polymerization and enzymatic polymer modification) is one of the state-of-the-art themes of great research and development interest because it provides an opportunity to conduct environmentally benign “green polymer chemistry”.28,29 In fact, all of the biopolymers (biomacromolecules) are produced in living cells with enzymatic catalysis, and totally six main * To whom correspondence should be addressed. Phone/Fax: +86 731 88865515. E-mail: [email protected].

groups classified according to the Enzyme Commission are involved in the in vivo syntheses of biomacromolecules.28 In the area of “enzymatic polymer synthesis” (the in vitro enzymatic catalysis to produce polymeric materials), so far, three groups of enzymes (i.e., oxidoreductases, transferases, and hydrolases that are relatively stable) have been employed as catalysts for enzymatic polymerization, and four enzyme groups (oxidoreductases, transferases, hydrolases, and isomerases) have been applied as catalysts for enzymatic polymer modification.28 Lac belongs to the oxidoreductases, and it mainly catalyzes the polymerization of phenolic compounds, phenol derivatives, and aromatic amines.30–38 Lac that solely requires dissolved molecular oxygen as the oxidant is often superior to another oxidoreductase commonly used for polymerization, horseradish peroxidase, which requires added H2O2 as the oxidant.39,40 Previous research reports on Lac-catalyzed polymerization chemistry have clearly described the brilliant prospect in syntheses of many polymer materials for diversified future applications; however, as we are aware, the immobilization of Lac and other biomacromolecules through Lac-catalyzed polymerization and relevant bioelectrochemistry applications have not been involved hitherto, although conventional chemical and electrochemical polymerizations to immobilize biomacromolecules have been well-established for biosensing and BFC applications.41–47 Dopamine (DA) is an important hormone and neurotransmitter of redox activity that has attracted extensive studies from electrochemists.48–52 Recently, inspired by the composition of adhesive proteins in mussels, Messersmith et al. proposed and developed DA self-polymerization as a novel and important

10.1021/jp100922t  2010 American Chemical Society Published on Web 03/25/2010

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SCHEME 1: Schematic Representation of the Formation of PDA-Lac-MWCNTs Nanocomposite Film on GCE through Lac Catalysis for HQ Biosensing

SCHEME 2: Electron Transfer Steps at the Biocathode and the Bioanode of the Glucose/O2 BFC

protocol for multifunctional coatings of thin, surface-adherent polydopamine (PDA) films onto a wide range of inorganic and organic materials.49 Electrosynthesized PDA has also been employed in our laboratory as a biocompatible matrix to immobilize biomacromolecules, such as antihuman immunoglobulin G, glucose oxidase (GOx), and hemoglobin.53–56 Many reports have clearly described the attracting properties of PDA for biotechnology-purpose applications; however, the Laccatalyzed polymerization of DA has not been examined to date. Herein, we report on the Lac-catalyzed polymerization of DA as a new and efficient platform to immobilize biomacromolecules in polymeric bionanocomposite cast thin films for biosensing and BFC applications. As shown in Scheme 1, casting the aqueous mixture of DA, Lac, and multiwalled carbon nanotubes (MWCNTs) on a glassy carbon electrode (GCE) yields a PDA-Lac-MWCNTs/GCE that can sense nanomolarscale hydroquinone (HQ). Similarly, casting the aqueous mixture of DA, GOx, Lac, and MWCNTs on a Pt electrode yields a PDA-GOx-Lac-MWCNTs/Pt electrode that exhibits high glucose-detection sensitivity. In addition, 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) is coimmobilized to yield a PDA-Lac-MWCNTs-ABTS/GCE that can effectively catalyze the reduction of O2, and it is used as the biocathode in a membraneless glucose/O2 BFC, as shown in Scheme 2. Experimental Section Instrumentation and Chemicals. All electrochemical experiments were performed on an Autolab PGSTAT30 electrochemical workstation (Eco Chemie BV, The Netherlands) with GPES 4.9 software. A GCE or Pt disk electrode of 3-mmdiameter (geometric area 0.07 cm2), a KCl-saturated calomel electrode (SCE), and a platinum plate served as the working electrode, the reference electrode, and the counter electrode,

respectively. All potentials in this work are referenced to SCE. The cell voltage (Vcell) and the cell current (Icell) of the glucose/ O2 BFC at varying external resistance loads (Re) were dynamically monitored with the electrochemical noise (ECN) module of the Autolab PGSTAT30 electrochemical workstation, the details of which were recently reported.57 Quartz crystal microbalance (QCM) studies were carried out with a computerinterfaced HP4395A impedance analyzer.58 AT-cut 9-MHz goldcoated piezoelectric quartz crystals (PQCs, 12.5-mm diameter, model JA5, Beijing Chenjing Electronics Co., LTD, China) were used. The gold electrode of 6.0-mm diameter on one side of the PQC was exposed to the solution, while the other side of the PQC faced air. The solution pH measurements were carried out on a pHS-3C pH meter equipped with a composite pH glass electrode (Shanghai Leici Scientific Instruments Inc., China), after careful pH calibration procedures. UV-vis spectra were recorded on a UV2450 spectrophotometer (Shimadzu Co., Kyoto, Japan). Scanning electron microscopy (SEM) pictures were collected on a JEM-6700F field emission scanning electron microscope. Lac (22.3 U/mg) from Trametes Versicolor, GOx (∼150 U/mg), DA, and ABTS were purchased from Sigma-Aldrich. All other chemicals were of analytical grade and were used without further purification. MWCNTs (diameter 20-40 nm) were purchased from Shenzhen Nanotech Port Co. The MWCNTs were purified prior to use by stirring them in 2 mol/L aqueous nitric acid for 20 h. Glucose stock solution was allowed to mutarotate overnight at room temperature before use. Acetate buffer solution (pH 6.0) was 0.05 or 0.1 mol/L HAc-NaAc aqueous solution. Britton-Robinson (B-R) buffer aqueous solution was prepared with equimolar amounts (0.04 mol/L) of acetic, phosphoric, and boric acids and adjusted to the required pH with NaOH. Phosphate buffer solution (PBS, pH 7.0) was prepared with 0.1 mol/L K2HPO4-KH2PO4 + 0.10 mol/L KCl. Milli-Q ultrapure water (Millipore, g18 MΩ cm) was used throughout. All experiments were conducted at room temperature (25 ( 2 °C). Procedures. The GCE was polished with 1 and 0.05 µm alumina slurry sequentially and then washed ultrasonically in water and ethanol for 15 min, respectively. Then the GCE was subjected to potential cycling (-0.2 to 1.0 V, 10 mV/s) in 0.20 mol/L aqueous HClO4 until a reproducible cyclic voltammogram was obtained. For cleaning the Pt electrode surface, one drop of H2SO4 + H2O2 (3:1, v/v) was added on the electrode surface to stay for 15 s, then the electrode was rinsed thoroughly with water and dried with a stream of pure nitrogen. The treatment was repeated three times. Prior to electrochemical experiments, the Pt electrode was subjected to continuous potential cycling (0-1.5 V, 30 mV/s) in 0.20 mol/L aqueous HClO4 until the cyclic voltammogram became reproducible.

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The PDA-Lac-MWCNTs/GCE was prepared as follows. One milligram of MWCNTs were dispersed in 1 mL of 0.05 mol/L acetate buffer solution (pH 6.0) by vigorous sonication, followed by successively adding 20 mmol/L DA and 1 mg/mL Lac. Then 3 µL of the mixture was dropped on the GCE and air-dried. The PDA-Lac/GCE was prepared similarly to the PDA-Lac-MWCNTs/GCE except for the absence of MWCNTs. Polyaniline (PANI)-Lac-MWCNTs/GCE, poly(o-phenylenediamine) (PoPD)-Lac-MWCNTs/GCE, and poly(o-aminophenol) (PoAP)-Lac-MWCNTs/GCE were prepared similarly to the PDA-Lac-MWCNTs/GCE case, except that 20 mM aniline (ANI), 20 mM o-phenylenediamine (oPD), or 20 mM oaminophenol (oAP) was used instead. Note here that 0.05 mol/L acetate buffer solution (pH 5.0) was used for preparation of PANI-Lac-MWCNTs/GCE, PoPD-Lac-MWCNTs/GCE, and PoAP-Lac-MWCNTs/GCE for convenience. A PDA-GOx-Lac-MWCNTs/Pt electrode for glucose sensing was prepared by casting 3 µL of 0.05 mol/L acetate buffer solution (pH 6.0) containing 1 mg/mL MWCNTS, 20 mmol/L DA, 1 mg/mL Lac, and 1 mg/mL GOx on Pt and then drying it in air. An ABTS-MWCNTs nanocomposite was prepared as reported previously.26 In brief, 1 mg of MWCNTs was placed in 1 mL of 2 mmol/L aqueous ABTS and magnetically stirred for two days. Then the precipitates after centrifugation were collected and redispersed in water. The centrifugation procedure was repeated two more times, and a stable colloidal suspension of ABTS-MWCNTs nanocomposite was obtained. The PDALac-ABTS-MWCNTs/GCE for catalyzing O2 reduction at the biocathode in the glucose/O2 BFC was prepared with a twostep method as follows: First, 3 µL of ABTS-MWCNTs nanocomposite was dropped on GCE, followed by casting 1 µL of 0.05 mol/L acetate buffer solution (pH 6.0) containing 1 mg/mL Lac, then air-dried. Second, 3 µL of 0.05 mol/L acetate buffer solution (pH 6.0) containing 20 mM DA and 1 mg/mL Lac was dropped on the above electrode to form a PDAcomposite film. For comparison, a PDA-free Lac-ABTSMWCNTs/GCE was prepared by dipping the MWCNTs/GCE in B-R buffer solution (pH 5.0) containing 1 mM ABTS and 2 mg/mL Lac overnight at 4 °C and then drying in air. A Nafion/ GOx-ferrocene (Fc)-MWCNTs/GCE as the bioanode in the glucose/O2 BFC was prepared by successively casting 3 µL of 1 mg/mL MWCNTs, 5 µL of 0.05 mol/L Fc acetone solution, 3 µL of 5 mg/mL GOx aqueous solution, and 2 µL of 1 wt % Nafion, and each casting was done after the previous cast had been air-dried. All Lac-based electrodes (without GOx) were stored in pH 5.0 B-R buffer solution at 4 °C, and GOx-based electrodes were stored in pH 7.0 PBS at 4 °C when not in use. During biosensing experiments, the steady-state current responses of the Lac-based electrodes at various HQ concentrations were obtained at -0.05 V by successive injections of a concentrated HQ solution into 10 mL of stirred B-R buffer solution (pH 4.0), and the electroreduction of enzymatically generated benzoquinone (BQ) was detected. The steady-state current responses of the PDA-GOx-Lac-MWCNTs/Pt at various glucose concentrations were obtained at 0.50 V by successive injections of a concentrated glucose solution into 10 mL of stirred PBS (pH 7.0), and the electrooxidation of enzymatically generated H2O2 was detected. The response current was marked with the change value between the steadystate current and the background current. The assembled BFC operated in B-R buffer solution (pH 5.0) containing 10 mM glucose, with N2 bubbling in the

Tan et al.

Figure 1. UV-vis absorption spectra of 0.05 mol/L acetate buffer solution (pH 6.0) containing 100 µM DA (a) and 100 µM DA + 2 µg/mL Lac (b-g, recorded every 2 min). Inset shows UV-vis absorption spectrum of 0.05 mol/L acetate buffer solution (pH 6.0) containing 2 µg/mL Lac.

electrolyte near the bioanode and O2 bubbling in the electrolyte near the biocathode (Scheme 2). Results and Discussion Lac-Catalyzed Polymerization of DA. Lac is used here to catalyze the polymerization of DA in aqueous solution for the first time. As shown in Figure 1S of the Supporting Information, as soon as 1 mg/mL (final concentration) Lac was added into 0.05 mol/L acetate buffer solution (pH 6.0) containing 20 mmol/L DA, the solution turned red instantly, indicating the immediate onset of DA oxidation catalyzed by Lac. The solution turned black after quiescence for 30 min, and some precipitates were observed at the bottom of the centrifuge tube after centrifugation for 10 min, resulting from the Lac-catalyzed polymerization of DA. While the Lac-free DA solution was exposed to air for 3 h (still in the liquid state), the solution color changed little, indicating that the oxidation and polymerization of DA occurred very slowly here in the absence of Lac. UV-vis spectroscopy was employed to monitor the oxidation of DA. As shown in Figure 1a, in a 0.1 mol/L acetate buffer solution (pH 6.0) containing 100 µmol/L DA, an intense band was observed at 281 nm, which is directly related to the symmetry-forbidden transitions (La-Lb) of the DA molecule.51 A broad band was observed at 280 nm for Lac (inset of Figure 1), which can be related to the symmetry-forbidden transitions of aromatic amino acid residues.59 The time-dependent spectra obtained during the Lac-catalyzed oxidation of DA are shown in Figure 1b-g. During the oxidation, the band peaking at ∼306 nm rose progressively in intensity until its overlapping with the band at 281 nm. A new band peaking at 388 nm was observed, which is the n-π* transition due to the carbonyl group attached to benzene ring and is a characteristic band for the identification of the ortho-quinone family.60 Later, an intense band peaking at 471 nm rose, which is a characteristic electronic transition of the chrome form of the oxidized state of DA after intramolecular cyclization. For clarity, the basic pathway probably responsible for the enzyme-catalyzed oxidation and polymerization of DA is given in Scheme 1S of the Supporting Information, which is deduced from the reported electrochemical oxidation and polymerization pathway for DA: namely, DA is

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Figure 2. SEM images of bare GCE (A), PDA-Lac/GCE (B), and PDA-Lac-MWCNTs/GCE (C).

oxidized to dopaminequinone (DAQ); the intramolecular cyclization of DAQ via 1,4-Michael addition leads to the more readily oxidizable leucodopaminechrome (LDAC); LDAC is oxidized to dopaminechrome (DAC); and then DAC can further undergo polymerization reactions.53,61 Lac-catalyzed oxidation of DA was also validated by electrochemical techniques. The open circuit potential of the electrochemical system in 0.1 M acetate buffer solution (pH 6.0) containing 20 mmol/L DA was 0.04 V, which was changed instantly to be 0.12 V after adding 1 mg/mL Lac. Cyclic voltammetry with open circuit potential as initial potential was conducted, as shown in Figure 2S. In the first potential cycle for 0.1 M acetate buffer solution (pH 6.0) containing 20 mmol/L DA (Figure 2SA), only a small cathodic peak was observed at -0.22 V (no corresponding anodic peak), which is probably related to oxygen reduction. In the second potential cycle, a pair of redox peaks (pa1 at 0.63 V and pc1 at 0.03 V, from the DAQ/DA couple shown in Scheme 1S) and a pair of redox peaks (pa2 at -0.14 V and pc2 at -0.27 V, from the DAC/LDAC couple shown in Scheme 1S) were observed.53 In contrast, the pair of redox peaks (pa2 and pc2) at low potential was observed even in the first potential cycle in 0.1 M acetate buffer solution (pH 6.0) containing 20 mmol/L DA + 1 mg/mL Lac (Figure 2SB), highlighting the occurrence of Lac-catalyzed oxidation of DA and the intramolecular cyclization of DAQ. The solution pH plays an important role in determining the oxidation and polymerization rate of DA. We have reported that at DA concentration >2 × 10-4 M and solution pH > 3.86, the intramolecular cyclization of DAQ via 1,4-Michael addition occurs significantly, and further isomerization and oxidation of the cyclization product lead to polymer growth finally.53 In addition, Lac from T. Versicolor usually shows its high catalytic activity toward most substrates at low pH (e5.0).16,17 At higher pH, the activity drops down due to the OH- inhibition effect on the T2/T3 site of Lac. Considering both the Lac activity and intramolecular cyclization rate of DAQ, pH 6.0 is chosen for DA polymerization in the presence of Lac. DA polymerization catalyzed by Lac was allowed to occur on the electrode for bioapplications. Simply casting the aqueous mixture of DA and Lac on an electrode yields a PDA-Lac composite film; here, the amphiphilic Lac may interact with the PDA oligomers with poor solubility in an aqueous medium via hydrophobic force and serve as the seeds for further polymerization, which is virtually beneficial for the enzyme immobilization. The SEM image (Figure 2B) shows that the PDA-Lac composite film on the GCE is composed of aggregated nanoparticles, in vivid contrast to the bare GCE surface (Figure 2A). MWCNTs of attractive structural, mechanical, and electronic properties are widely accepted for electrode modification to improve the electrochemical activity.62 MWCNTs can act as the seeds for chemical formation of polyaniline.63 Cui et al. have reported that carbon nanotubes can act as the seeds for pyrrole polymerization, aided by the oxidant hydrogen peroxide that is enzymatically generated when lactate oxidase catalyzes

Figure 3. Time courses of simultaneous responses of ∆f0 and ∆R1 during coating of 5 µL of 0.05 mol/L acetate buffer solution (pH 6.0) containing 20 mmol/L DA and 1 mg/mL Lac on the QCM electrode. Arrows shows the moment for casting the solution.

the conversion of lactate to pyruvic acid in the presence of oxygen.64 Here, we have tried to use the MWCNTs as seeds for polymerization of DA with the aid of Lac (Scheme 1). Figure 2C shows the morphology of the prepared PDA-Lac-MWCNTs nanocomposite film on a GCE, where the polymer is well-coated on MWCNTs. To better understand the casting processes of the DA solution and relevant polymerization, we have adopted the QCM to monitor the solution-casting processes on the PQC electrode. The QCM can dynamically detect minute changes in mass loading and viscoelasticity of a foreign film on the electrode surface,58,65 and as we are aware, this is the first example to use the QCM to monitor the enzyme-catalyzed polymerization in a cast solution in a real-time manner. Figure 3 shows the time course of simultaneous responses of frequency shift (∆f0) and resonant resistance change (∆R1) after casting 5 µL of 0.05 mol/L acetate buffer solution (pH 6.0) containing 20 mmol/L DA and 1 mg/mL Lac onto the electrode. The coating processes on the PQC electrode here can be divided into four stages as follows: Stage I, the phase change of PQC surface from airphase to solution-phase; stage II, DA polymerization and PDA deposition on the electrode; stage III, evaporation of the last liquid and obvious concentration on the PQC; and stage IV, full dryness of the PQC surface. The responses of ∆f0 and ∆R1 during casting DA or DA + bovine serum albumin (BSA) solution (Figure 3S) were obviously different from those of DA + Lac (Figure 3). As shown in Figure 3S, in the absence of Lac, the frequency and resistance in stage II changed little, implying that the oxidation and polymerization of DA occurred very slowly in the absence of Lac. In addition, the QCM responses to the transition from stage III to IV (the last wetness to full dryness) were not obvious in the absence of Lac, which

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Figure 4. Cyclic voltammograms at PDA-Lac-MWCNTs/GCE in deaerated (b, N2 bubbling for 30 min) and air-saturated (c) B-R buffer solution (pH 4.0) containing 1 mM HQ at 50 mV/s. Curve a shows cyclic voltammogram at PDA-Lac-MWCNTs/GCE in air-saturated B-R buffer solution (pH 4.0). Scan rate: 50 mV/s.

may result from the strong bibulous ability of unpolymerized DA monomers and related species. As expected, the responses of ∆f0 and ∆R1 in the DA + BSA system were larger than those in the BSA-free DA system. The stability of the above dry films covered on the PQCs was also examined. Resulting “dry” frequency shifts (∆f0d) and resistance changes (∆R1d) of the PQC electrodes in the casting processes are listed in Table 1S. We obtained a “dry” frequency shift (∆f0 dB) of -12.5 ( 1 kHz after casting the mixture of DA and Lac, drying in air, washing with water, and then drying with a stream of pure nitrogen, indicating that most of the DA monomers had undergone polymerization. In vivid contrast, only a “dry” frequency shift within -1 kHz was finally obtained after similar treatments in the absence of Lac, since any unpolymerized DA was washed away. The high stability of the PDA-Lac film covered on the PQC in stirred aqueous solution was further validated by the recorded very minor drifting of frequency and resonant resistance responses (Figure 4S). Moreover, the mass (or thickness) of QCM electrode-supported PDA-Lac films can be controlled by changing the amount of Lac in cast solutions (Figure 5S). Interestingly, the Lac-catalyzed polymerization of DA can occur on nonconducting inorganic (e.g., glass) and organic (e.g., plastic) materials, and the PDA films can be robustly attached to the surfaces of glass and plastic (Figure 6S), indicating that the Lac-catalyzed polymerization of DA is favorable for convenient preparation of multifunctional coatings on various substrates. Preparation of PDA-Enzyme-MWCNTs/GCE for Biosensing. HQ is selected here as a model phenolic substrate to examine the immobilization of Lac in the PDA-MWCNTs nanocomposite film for phenolic biosensing. As shown in Figure 4, no obvious redox peak was observed in air-saturated HQfree B-R buffer solution (pH 4.0), implying the negligible existence of DA after its Lac-catalyzed polymerization and water rinse of the film. We obtained a higher reduction peak and a lower oxidation peak in air-saturated B-R buffer solution (pH 4.0) containing 1 mM HQ, as compared with those recorded after deaeration by N2 bubbling for 30 min. The larger reduction peak in the air-saturated solution is attributed to the reduction of quinone regenerated by oxygen-dependent Lac catalysis on

Tan et al. the electrode surface (Scheme 1). In the catalysis cycle of Lac in an oxygen-containing solution, the reduced state of Lac (LacRed) is oxidized by dissolved oxygen, and the yielded oxidized state of Lac (LacOx) can be turned over by HQ with production of benzoquinone (BQ), and HQ can be regenerated at the electrode; thus, the whole catalysis cycle solely consumes oxygen. The peak current and potential varied with solution pH, as shown in Figure 7S. The peak potential was negatively shifted with the pH increase from pH 3.0 to 7.0, and the reduction current became the largest at pH 4.0, indicating that Lac possesses the highest catalytic activity at pH 4.0 toward HQ oxidation. According to Xu,66 the pH-dependent activity of Lac 0′ is determined mainly by OH- inhibition effect and ∆E0′ (ELac 0′ - Esubstrate, difference of formal potentials) effect. The oxidation of HQ accompanies proton transfer and is pH-dependent, 0′ is normally pH-independent, so the ∆E0′ effect is whereas ELac 0′ 0′ . Since EHQ is commonly more mainly determined by EHQ 0′ and shifts to negative upon increasing pH, negative than ELac the ∆E0′ value becomes larger at higher pH, which benefits the Lac-catalyzed oxidation of HQ. However, at higher pH, the activity decreases due to the OH- inhibition effect to the T2/ T3 site of Lac. The balance of these two opposing effects plays an important role in determining the pH activity of Lac toward different substrates. To achieve the largest enzymatic activity of Lac, the solution pH is selected at pH 4.0, and the applied potential of -0.05 V (optimized) is selected to achieve the maximum sensitivity in subsequent potentiostatic experiments for the detection of HQ. Figure 5 shows the steady-state current responses at PDA-Lac-MWCNTs/GCE or PDA-Lac/GCE at -0.05 V to the successive additions of HQ into B-R buffer solution (pH 4.0). The biosensing performance is given as follows: a linear response range (LRR) of 0.1 to 48 µM with a linearity regression equation (LRE) of ∆I (µA) ) -0.055 - 45c (mM) (r 2 ) 0.9975) and a limit of detection (LOD) of 20 nM (S/N ) 3) at PDA-Lac-MWCNTs/GCE; a LRR of 0.3-90 µM with a LRE of ∆I (µA) ) -0.018 - 22.3c (mM) (r 2 ) 0.9993) and a LOD of 50 nM at PDA-Lac/GCE. Obviously, the sensitivity at PDA-Lac-MWCNTs/GCE (643 µA mM-1 cm-2, Table 2S) is higher than that at PDA-Lac/GCE, resulting from the positive effects of added MWCNTs, such as an improved electronconducting pathway, enlarged electroactive area, and nanoenhanced adsorption capacity for Lac. The 20 nM LOD at PDA-Lac-MWCNTs/GCE is much lower than the reported values (100 nM14 and 580 nM67). Because Lac can catalyze the polymerization of various substrates, including phenolic compounds, phenol derivatives, and aromatic amines, it is interesting to compare the performance of various Lac-entrapped polymeric nanocomposite modified electrodes toward HQ biosensing. In common sense, Lac-catalyzed polymerization of different substrates should proceed with different polymerization mechanisms and polymerization rates, which may yield polymers of different thickness, porosity, and structure, which may lead to different biosensing performance. As shown in Figure 8S and Table 2S, the HQ-biosensing performance at PDA-Lac-MWCNTs/GCE is much better than that of PANI-Lac-MWCNTs/GCE, PoPDLac-MWCNTs/GCE, or PoAP-Lac-MWCNTs/GCE, highlighting that DA is an excellent monomer for preparation of Lac-entrapped polymeric nanocomposite films by Lac-catalyzed polymerization. The interesting finding of the best biosensing performance in the PDA-involved case is briefly discussed as follows: The

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Figure 5. Time-dependent current responses (A) to successive injections of HQ into stirred B-R buffer solution (pH 4.0) under air atmosphere and calibration curves (B) at PDA-Lac-MWCNTs/GCE (a) and PDA-Lac/GCE (b). Applied potential: -0.05 V vs SCE.

performance of an enzyme electrode depends mainly on the enzyme load, enzymatic specific activity, and mass transfer. (1) -12.5 ( 1 kHz PDA-Lac film could be robustly attached to the surface of Au (Table 1S), but we found that only within -8 kHz of PANI-Lac, PoPD-Lac, or PoAP film could it be attached to the surfaces of Au under our experimental conditions. The PDA films may thus immobilize Lac at a higher load than other polymers, and a higher HQ-detection sensitivity for the PDA composite is expected. (2) Our previous work has revealed that the enzymatic specific activity of the enzyme immobilized in electrosynthesized PDA films is very high,47,56 and the PDA is virtually melanin-like and highly biocompatible, so we believe that Lac-synthesized PDA can also immobilize Lac at high activity. (3) Lac-synthesized PDA is porous (Figure 2), which facilitates the mass transfer for biosensing. Experimentally, a well-defined pair of redox peaks for HQ/BQ at PDALac-MWCNTs/GCE was observed (Figure 4), indicating the facilitated mass transfer of HQ/BQ at the PDA composite film. In fact, the electrosynthesized PDA has also been proven to be a better matrix for GOx immobilization than many other conducting and nonconducting polymers (e.g., PANI, PoPD, and PoAP).44,47 Michaelis-Menten kinetic analysis is carried out using the well-known Lineweaver-Burk equation, 1/∆iss ) 1/∆imax + Kapp M /(c∆imax), where ∆iss is the steady-state current response after substrate addition, ∆imax is the maximum current response at saturated substrate concentration, c is the concentration of substrate, and Kapp M is the apparent Michaelis-Menten constant. app was 60 µM at PDA-Lac-MWCNTs/GCE, which is The KM smaller than those for the biosensors prepared by carbodiimide and glutaraldehyde co-cross-linking (70 µM)68 and glutaraldehyde cross-linking (610 µM),69 which were determined also in the presence of solution-state HQ, indicating that the present PDA-MWCNTs nanocomposite is a better matrix for Lac immobilization. Note here that O2 is the substrate of LacRed, app obtained here at and HQ is the substrate of LacOx; the KM varying HQ concentrations under air-saturated condition just reflects the bioaffinity between LacOx and HQ, rather than that between LacRed and O2 in the presence of sufficient and concentration-fixed HQ. The storage stability was conducted at PDA-Lac-MWCNTs/ GCE for sensing 10 µM HQ. The response current decreased very little in the first 5 days. After 30 days, the enzyme electrode retained 85% of its original response, indicating that the stability of the PDA-Lac-MWCNTs/GCE is good. It is interesting and important to check whether other biomacromolecules can be conveniently immobilized by Lac-

catalyzed polymerization of DA. Herein, GOx is selected as a model enzyme to validate the feasibility, and a PDA-GOx-LacMWCNTs/Pt was fabricated for glucose sensing. First, the effects of glucose-detection potential and solution pH on the current response at PDA-GOx-Lac-MWCNTs/Pt were examined, as shown in Figure 9S. When the potential was above 0.5 V, the current almost reached a plateau; thus, an applied potential was selected in subsequent experiments for glucose biosening. The best pH for glucose biosensing was found to be pH 7.0 (note at this pH that the entrapped Lac is almost inactive). Figure 6 shows the steady-state current response at PDAGOx-Lac-MWCNTs/Pt to the successive addition of glucose into PBS (pH 7.0) at 0.5 V. A LRR of 0.01 to 3.7 mM with a LRE of ∆I (µA) ) 0.489 + 4.80c (r 2 ) 0.9944), and a LOD of 0.5 µM (S/N ) 3) were achieved at PDA-GOx-Lac-MWCNTs/ GCE. The sensitivity of 68.6 µA mM-1 cm-2 at PDAGOx-Lac-MWCNTs/Pt is much higher than that at PDA-GOx/ Au (3.81 µA mM-1 cm-2) reported previously.56 In fact, the sensitivity of PDA-GOx-Lac-MWCNTs/Pt is much higher than that of most GOx-based electrodes via conventional electropolymerization, chitosan-assisted electrodeposition, or direct casting (Table 3S). The Kapp M value was 4.1 mM according app value to the above Lineweaver-Burk equation. The small KM should suggest that the immobilized GOx possessed high enzymatic activity and affinity to glucose under our experimental conditions. Additionally, the PDA-GOx-Lac-MWCNTs/Pt showed excellent anti-interfering agent ability against ascorbic acid (AA) and uric acid (UA), as shown in Figure 10S, since the nonconducting PDA film inherently possesses good permselectivity toward H2O2.56 All data above have undoubtedly indicated that GOx can also be entrapped in the PDA-Lac-MWCNTs nanocomposite film with high detection sensitivity. Therefore, the proposed method for preparation of polymer-enzyme-MWCNTs nanocomposites is highly recommended for immobilization of many other biomacromolecules for wide bioapplications, and it is also expected to be feasible to couple two appropriate enzymes with a synergetic effect for special uses. Preparation of PDA-Lac-ABTS-MWCNTs/GCE for Catalyzed Reduction of Oxygen and Construction of a BFC. A PDA-Lac-ABTS-MWCNTs/GCE was fabricated with the aforementioned two-step method and applied for bioelectrocatalytic reduction of oxygen. The ABTS-MWCNTs nanocomposite was chosen here to mediate the electron communication between Lac and the electrode, which has been recommended as a mediator system for bioelectrocatalytic

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Tan et al.

Figure 6. Time-dependent current response (A) to successive injections of glucose into stirred PBS (pH 7.0) and calibration curve (B) at PDA-GOx-Lac-MWCNTs/Pt. Inset shows an enlarged plot at time