Enzymes have remarkable catalytic power and are highly selective and quickly respond to specific substrates under mild reaction conditions,as compared with those of chemical catalysts. Therefore,enzymes have been often employed as catalysts for the synthesis of polymers or nanomaterials ^{[1, 2, 3]}. The immobilization could not only stabilize an enzyme and prevent its denaturation,it offers reusable alternatives to the free enzyme,and also makes it convenient to design bio-devices ^[4].

Electrochemical biosensors,which incorporate a biological component with functional materials,combine the recognition and catalytic properties of enzymes with the properties of various materials,and have developed into a type of useful bio-device in the detection of biochemical molecules ^[5]. Polyaniline (PANI) is one of the most intensively studied materials in electrochemical field due to its enormous capacity for binding biomolecules,tuning their bio-catalytic properties,rapid transferring of electrons and direct production of analytical signals ^{[6, 7, 8, 9]}. Usually,PANI was prepared by electrochemical or chemical oxidation of aniline in acidic solution ^{[8, 9]}. Enzymatic-mediated syntheses of functional materials not only supplied simple synthetic methods under mild conditions,but also developed an alternative approach for enzyme entrapment ^[10],and therefore opened a new method for designing sensitive enzymatic assays ^{[10, 11, 12, 13, 14, 15, 16, 17, 18, 19]}. The enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP),were independently applied to the bio-polymerization of aniline. The former usually requires a long reaction time,and the latter could only endure a narrow acidic range ^[10]. Hemoglobin (Hb) was used to catalyze the polymerization of aniline by H₂O₂ in the presence of sodium dodecylsulfate (SDS) to form a PANI/SDS complex,and demonstrated that the Hb-biocatalyzed polymerization of aniline could be carried out in a solution over a broad pH range ^[20].

In this work,we investigated the Hb-glucose oxidase (GOD) induced polymerization of aniline on the electrode surface,and applied the bi-enzyme catalyzed system to the electrochemical detection of glucose.

GOD (>95%,10,000 U,from Amresco),Hb (MW 64,500,from Sigma). PS (carboxylate modified,diameter: 0.10μm,10% solids). Glucose stock solution was allowed to mutarotate at room temperature overnight before use. A 0.1 mol/L phosphate buffer solution (PBS,pH 7.0 and pH 6.0) made from Na₂HPO₄and KH₂PO₄ was applied as reacting or detecting electrolyte,unless otherwise stated. All other reagents were of analytical grade and used as received. All solutions were prepared with deionized water. All electrochemical experiments were carried out in a three-electrode cell controlled by a CHI 660 electrochemical workstation (Chenhua Instruments,Shanghai,China). The bare,or modified,glassy carbon electrode (GCE,3 mm of diameter) acted as the working electrode. A saturated calomel electrode (SCE) and a platinum wire served as reference and counter electrode,respectively. Scanning electron microscopy (SEM) images were recorded using a JSM-6460 SEM (JEOL,Japan). X-ray diffraction (XRD) patterns were taken with an X-ray diffraction meter (Bruker,Germany) with a Cu Kα X-ray source operating at 40 kV.

In general,the preparation of GOD-Hb/PS was based on an acylation reaction between carboxylic-acid-functionalized PS and the amine-terminated Hb and GOD. Briefly,a 10μL aliquot of 10% carboxylic-acid-functionalized PS suspension was mixed with an 80μL aliquot of 0.1 mol/L PBS (pH 7.0) containing 10 mmol/L NHS and 10 mmol/L EDC,unless otherwise stated. Following activation for 1 h at room temperature,10μL of the pH 7.0 PBS solution containing 5 mg/mL Hb and 5 mg/mL GOD was added to the mixture. The reaction was allowed to proceed for 3 h at 4℃.Prior to use,the GCE was polished by a commonly used mode ^[11]. Then 6μL of the ultimate solution was applied on the surface of the GCE and allowed to dry at 4℃. The GOD-Hb/PS/GCE was rinsed thoroughly with double-distilled water.

The polymerization of PANI under enzymatic conditions was carried out by immersing the GOD-Hb/PS/GCE in a 0.1 mol/L PBS (pH 7.0) containing 20 mmol/L aniline and various amount of glucose for 10 min. After that,the electrode,identified as PANI/ GOD-Hb/PS/GCE,was removed from the above solution and washed with 0.1 mol/L PBS (pH 6.0).

The PANI/GOD-Hb/PS/GCE was transferred to a 0.1 mol/L PBS (pH 6.0),after which,the square wave voltammogram (SWV) was recorded in the potential window of-0.1 V to 0.7 V.

It is well known that GOD could catalyze the oxidation of glucose by molecular oxygen and,produce H₂O₂.Hu et al.^[20] proved the Hb-catalyzed polymerization of aniline in the presence of H₂O₂. Reasonably,the proposed mechanism for the biocatalytic growth of PANI could be expected as follows:

The morphology of PS,the GOD-Hb/PS film and the film after enzymatic deposition of PANI were characterized by the SEM images (Fig. 1). As can be seen from Fig. 1A,the PS exists in a spherical shape of about 100 nm in diameter with a uniform size distribution and high density,just as described on the package. When the protein was immobilized,the monolayer of enzymes on the PS surface changed little after covalent linkage as shown in Fig. 1B. After the GOD-Hb/PS/GCE was immersed in solution containing glucose and aniline for 10 min (Fig. 1C),the diameters of some of the PS particles maintain their previous size,and others, represented by the labeled particles on the image,increase to 240- 360 nm after the reaction. This increase in diameter and the capsulation of new material on some of the PS were attributed to the formation of PANI by the GOD-Hb couple enzymatic reaction. Therefore,the method holds promise for the biosynthesis of core- shell PANI-PS nanomaterials.

Fig. 1

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Fig. 1. SEM images of PS (A),GOD-Hb/PS (B) and PANI/GOD-Hb/PS (C).

Since PANI are electro-active polymers,the cyclic voltammogram (CV) of GOD-Hb/PS/GCE before and after immersing in the aniline or/and glucose containing solution was recorded to further confirm the biocatalyzed polymerization. From Fig. 2A,no apparent peak was observed on the voltammogram of GOD-Hb/ PS/GCE (curve a),indicating both the proteins and PS are electroinactive in the potential range of -0.1 V to 0.6 V. After immersing the GOD-Hb/PS/GCE in a 0.1 mol/L PBS (pH 7.0) containing aniline, there is still no distinct response. But,if glucose is added to the solution,an oxidative peak at 0.28 V and a reductive peak at 0.23 V (vs.SCE) were observed at a scan rate of 100 mV/s as indicated in Fig. 2A,curve c,which should be the contribution of the formed PANI ^{[17, 18]}. It is well known that PANI exists in three welldefined oxidation states: leucoemeraldine,emeraldine and pernigraniline ^[21]. The redox peaks at near 0.25 V were attributed to the oxidation of the fully reduced polyaniline (leucoemeraldine) to the radical cations (emeraldine salt) ^{[9, 22, 23]}. This was the preliminary proof of the deposition of PANI in neutral solution. The cyclic voltammetric behavior of the deposited PANI was investigated in PBS at different pH values. In the detection solutions with pH from 4 to 8,the response current of PANI changed,but the chief traits of the voltammogram remains one pair of redox peaks. The electrochemical behavior of bio-polymerized PANI was less dependent on the acidity of the medium than that of PANI synthesized by the chemical method.

Fig. 2

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Fig. 2. (A) Cyclic voltammograms of GOD-Hb/PS/GCE in 0.1 mol/L PBS (pH 6.0) (a); after immersing the electrode in pH 7.0 PBS in the presence of 20 mmol/L aniline(b); and in the presence of 20 mmol/L aniline as well as 1.0 mmol/L glucose (c). (B) SWVs obtained in 0.1 mol/L PBS (pH 6.0) at the GOD-Hb/PS/GCE after incubation in 0.1 mol/L PBS (pH 7.0) containing 20 mmol/L aniline and different glucose concentrations for 10 min. Glucose concentration: (a) 0μmol/L,(b) 0.1μmol/L,(c) 1.0μmol/L,(d) 6.0μmol/L,(e) 8.0μmol/L,(f) 10.0μmol/L.

Keeping the concentration of aniline at 20 mmol/L,the response of PANI in SWV varies linearly with the content of glucose from 0.1μol/L to 10.0μmol/L,as shown in Fig. 2B. The detection limit was estimated to be 0.05μmol/L with the signal-to-noise ratio of 3. Compared with the previous PANI/HRP-GOD/MWNTs biosensor (0.94μA mmol^-1L) ^[17] and other bi-enzyme electrodes ^{[24, 25]},the present biosensor exhibited much higher sensitivity (50.6μA mmol^-1L) and a lower detection limit. The main reason was that most of the activity of Hb and GOD is retained at pH 7.0,while HRP and GOD lost part of their activity at a pH value of 4.3 ^{[17, 22]}. In addition,a shoulder peak would emerge gradually in the voltammograms of the chemically synthesized PANI with the increase of glucose concentration ^[9]. It was explained that the pH value of the microenvironment of PANI would decrease in the presence of gluconic acid. In this work, because the different electrochemical behavior of enzymatic and chemical polymerized PANI,the shoulder peak of PANI did not arise and hence the result is a well-defined peak shape on SWV.

The GOD-Hb induced polymerization of aniline was successfully carried out on the GCE electrode surface. This method could be an alternative method for the biosynthesis of PS-PANI core- shell nanomaterial. The electrochemical behavior of PANI/GODHb/PS/GCE could be utilized for the estimation of the concentration of glucose with a high sensitivity. Therefore,this work also further enhanced the development of the GOD-Hb based biosensors.

This work was supported by the National Natural Science Foundation of China (No. 20905061). V`