Polyaniline Nanofibers: Synthesis, Characterization, and Application

J. Phys. Chem. C 2009, 113, 4987–4996

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Polyaniline Nanofibers: Synthesis, Characterization, and Application to Direct Electron Transfer of Glucose Oxidase Min Zhao, Xiuming Wu, and Chenxin Cai* Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and EnVironmental Science, Nanjing Normal UniVersity, Nanjing 210097, P. R. China ReceiVed: August 27, 2008; ReVised Manuscript ReceiVed: December 10, 2008

Polyaniline nanofibers are synthesized by interfacial polymerization and confirmed by scanning electron microscopy. These nanofibers are characterized by XRD, FTIR, UV-vis spectroscopy, and voltammetry. A detailed study is conducted on the influence of a variety of synthetic conditions on the morphology of the polyaniline nanostructure. These conditions include organic solvents, acid dopants, concentration of dopants, the reaction time, and the concentration of aniline monomer and oxidant. The polyaniline nanofibers are used as an electrode substrate for immobilization and facilitating the direct electron transfer (DET) of redox proteins/ enzymes with glucose oxidase (GOx) as a model. After immobilized on the nanofibers, GOx can keep its natural structure and undergoes effective DET reaction with a pair of well-defined, quasi-reversible redox peaks at -418 mV (pH 7.0, 10 mV/s). The apparent electron transfer rate constant is (6.3 ( 1.6) s-1. The GOx-nanoPANI/GC electrode displays good features in electrocatalytic oxidation of glucose; therefore, it can be used as a biosensor for detecting the substrate with a low detection limit (0.5 µM), a wide linear range (0.01 to 1 mM), a low apparent Michaelis-Menten constant (1.05 ( 0.04 mM), and acceptable stability and reproducibility. The good biocompatibility of polyaniline nanofibers enables them to become a simple and effective platform for the integration of proteins/enzymes and electrodes, which can provide analytical access to a large group of enzymes for a variety of bioelectrochemical applications. 1. Introduction Nanostructured materials have attracted much interest because they show physical properties that are significantly different from their bulk materials.1,2 Dimensionality plays an important role in determining the properties of nanomaterials and synthesis of nanostructured materials with controllable morphology, size, chemical composition, and crystal structure.3-5 A lot of 1D nanomaterials have been synthesized and extensively investigated because of their potential technological applications in electronics, optoelectronics, and memory devices, 4-13 and so forth. Such nanostructures are expected to have unusual characteristics that are amplified through quantum size and shape-specific effects. 1D conducting polymer nanostructures, including nanofibers, nanorods, and nanotubes have been extensively studied because such materials possess the advantages of both low-dimensional systems and organic conductors.14-17 Among the known conducting polymers, polyaniline is one of the mostly studied conducting polymers owing to its good environmental stability, tunable conductivity switching between insulating and semiconducting materials, facile synthesis, and potential application in many areas.18-20 The polymer has the similar electronic, magnetic, and optical properties to metals, whereas it can retain the flexibility and processibility of conventional polymers. The doping level of polyaniline can also be readily controlled through an acid-doping/base-dedoping process. It has been extensively studied for many potential applications including secondary battery electrodes,21,22 supercapacitors,23 electromagnetic shielding devices,20 anticorrosion coating,24 light-emitting diodes,25 conducting molecular wires,26 sensors,15,27 and so forth. In recent years, considerable efforts * To whom correspondence should be addressed. E-mail: cxcai@ njnu.edu.cn, [email protected].

have been made on synthesis of 1D polyaniline because nanostructured polyaniline offers the possibility of enhanced performance wherever a high interfacial area between polyaniline and its environment is important.15,26,28 Several strategies have been developed for the synthesis of polyaniline nanostructures with different morphologies. Those approaches include template or surfactant synthesis,26,29 nanofiber seeding,30 interfacial polymerization,15,31,32 oligomer-assisted polymerization,33 and dilute polymerization,34 and so forth. The advantages of chemical polymerization are easy and inexpensive means of obtaining polyaniline nanofibers. However, complicated postsynthetic treatments are usually needed to remove the template or a specific complex chemical reagent from the products to recover the nanostructured polyaniline. Electrochemical polymerization14,28,35 and some physical methods, such as electrospinning36 and mechanical stretching37 can also produce polyaniline nanofibers without templates, but the nanofibers (the size is usually at the level of ca. 100 nm in diameter) have only been made on a very limited scale, such as on an electrode surface. Among those reported approaches, interfacial polymerization is a particular method because it does not rely on templates, structural directing molecules, or specific dopants, and therefore simplifies the synthetic procedures. This method produces polyaniline nanofibers at the interface of an immiscible organic/aqueous biphasic system, thus can synthesize large quantities of the nanostructured materials. The byproducts are separated according to their solubility in the organic and aqueous phases. Although there are several papers published on synthesis of polyaniline nanofiber using the approach of interfacial polymerization,15,31,32 a systematic study on the effect of various factors on the morphology of the polyaniline nanostructures is still unavailable. Moreover, some contradicting results even exist. For example, He et al. reported that the polarity of organic

10.1021/jp807621y CCC: $40.75  2009 American Chemical Society Published on Web 03/03/2009

4988 J. Phys. Chem. C, Vol. 113, No. 12, 2009 solvents had significant effects on the morphologies of the polyaniline nanostructures.32 Their results were significantly different from those obtained by Huang et al. They found that the polarity of the organic phase had little effect on the fibrous morphology of polyaniline synthesized from the interfacial polymerization.31 To merge these differences, a systematic study on the morphology of polyaniline nanostructures synthesized at a variety of conditions with the approach of interfacial polymerization being highly desirable. Considering these, the aim of this work was to synthesize the polyaniline nanofiber by interfacial polymerization and conducted a detailed study of the effects of synthetic conditions on the morphology of the polyaniline nanostructures. Those conditions included the organic solvents, acid dopants, the concentration of dopants and aniline, the reaction time, and so forth. This work also reported the new application of the nanofibers to immobilize and facilitate the direct electron transfer (DET) of enzymes by using glucose oxidase (GOx) as a model. The characteristics of DET and electrocatalytic features of the immobilized GOx were presented. The GOx is chosen as models because it is commercially available and has a well-documented structure.38 Moreover, the electrochemical oxidation of glucose catalyzed by GOx is one of the most widely studied enzymatic reactions due to its important applications in food and fermentation analysis, in medical diagnosis, and in environmental monitoring39-41 and so forth. The use of polyaniline film or polyaniline nanofiber as the immobilization matrix for constructing the glucose biosensor has been reported. For example, Forzani et al.42 reported a glucose sensor using polyaniline film/ GOx nanojunctions. The sensing of glucose of the nanojunctions was based on the change in the conductance of polyaniline induced by the enzymatically generated H2O2. The presence of O2 in the detection solution is necessary to regenerate the oxidized form of GOx (the form of GOx(FAD)). Shi et al.43 reported that the poly(aniline-aniline boronic acid) wires, which were generated on ds-DNA templates, could facilitate the electrical communication between electrode and GOx reconstituted on the polymer wires. However, the operating potential for detection glucose at this electrode was +0.5 V (vs SCE). Many other electroactive species commonly coexisting in the biological fluids, such as ascorbic acid (AA), uric acid (UA), and 4-acetamidophenol (AP), can also be oxidized at the high potential and their electrochemical signals thus severely affect the selectivity of the sensors. Comparing with those previous reports, the present approach has advantages such as ease of construction, without using any mediators, enhanced electrocatalysis, low detection limit, efficiently preserving the activity of enzyme, and effective discrimination to the common interfering species. 2. Experimental Section 2.1. Chemicals. GOx (EC 1.1.3.4, from Aspergillus niger, ∼200 U/mg, Sigma), β-D-(+)-glucose (Sigma), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, Aldrich), N-hydroxysuccinimide (NHS, 98%, Sigma), ascorbic acid (AA), uric acid (UA), and 4-acetamidophenol (AP) were used as received. Prior to use, aniline (Shanghai Chemical Company) was distilled under reduced pressure and stored in darkness. All other chemicals were of analytical grade. Phosphate buffer solution (PBS, 0.1 M, pH 7.0) was made up from Na2HPO4 and NaH2PO4. The glucose stock solution was allowed to mutarotate for at least 24 h before use. 2.2. Synthesis of Polyaniline Nanofibers. Synthesis of polyaniline nanofibers was conducted in a 20 mL plastic tube

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Figure 1. Illustration of the interfacial polymerization of aniline in an interface of aqueous solution/toluene. The reaction time (from a to e) is 0.5, 2, 5, 10, and 30 min. The top phase is toluene containing 0.1 M of aniline and the bottom phase is 1 M of sulfuric acid solution containing 0.05 M of ammonium peroxydisulfate.

(Figure 1). Typically, 1 mmol of aniline and 0.5 mmol of ammonium peroxydisulfate (APS) were dissolved in 10 mL of toluene and 10 mL of sulfuric acid aqueous solution (1 M), respectively. The organic solution was added to the aqueous solution gently and with minimal agitation along the sides of the tubes. The resulting two-phase system was left undisturbed at the ambient temperature for a desirable time. Subsequently, the polyaniline nanofibers were collected by filtering, thoroughly washed with water and ethanol thrice respectively, and finally dried under vacuum at room temperature. To study the influence of the synthetic conditions on the morphology of the polyaniline nanostructures, benzene, n-hexane, n-hexanol, carbon tetrachloride, and chloroform were used as the organic phase, respectively. A variety of dopant acids was used, including hydrochloric, phosphoric, and perchloric acid. The concentration of dopant acid was varied from 0.5 to 2 M, the concentration of APS was varied from 0.01 to 0.4 M, and the reaction time varied from 1 to 12 h. When one parameter was changed, others remained invariant. 2.3. Fabrication of nanoPANI/GC Electrode and Immobilization of GOx. Several parameters were optimized to obtain the best voltammetric response. For the fabrication polyaniline nanofibers modifying electrode, 5 mg of the nanofibers were suspended in 1 mL of aqueous solution, and 2 µL of the suspension was deposited onto the surface of glassy carbon electrode (GC, 3 mm in diameter, CH Instruments), which was pretreated using the procedures reported previously.44 The solvent was allowed to evaporate before use. The electrode was denoted as nanoPANI/GC electrode. The GOx-nanoPANI/GC electrode was fabricated by covalently attaching GOx on the surface of the polyaniline nanofibers. This was completed by treating the nanoPANI/GC electrode with a freshly made EDC-NHS mixture,45 which contained 0.4 M EDC, 0.1 M NHS, and 10 mg/ml of GOx. The electrode was kept in the mixture at 4 °C overnight. The -NH2 group of the nanofibers would react with the activated -COOH of GOx. This reaction led to covalently link GOx with the nanofibers forming the GOx-nanoPANI/GC electrode. The electrode was washed thoroughly with water and stored at 4 °C when it was not in use. The GOx/GC electrode was prepared by casting 2 µL of GOx aqueous solution (10 mg/ml, in 0.1 M PBS, pH 7.0) on the surface of the pretreated GC electrode. The solvent was allowed to evaporate before use. Its electrochemical characteristic was compared with that of the GOx-nanoPANI/GC electrode. 2.4. Apparatus and Procedures. SEM and TEM images were recorded using a JEOL JSM-5610LV scanning electron microscope and a JEOL-2010 transmission electron microscope, respectively. Energy-dispersive X-ray spectroscopy (EDS) was obtained from an Oxford Link ISIS energy-dispersive spectrometer fixed on the microscope. XRD patterns were recorded on a Rigaku/Max-3A X-ray diffractometer with Cu KR radiation

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(λ ) 0.15418 nm). UV-vis spectra were recorded using a Cary 5000 UV-vis-NIR spectrophotometer (Varian, USA) by dispersing the nanofibers in aqueous solution (at the level of 2 mg/ml). FTIR spectrum was recorded on a Nexus 670 FT-IR spectrophotometer (Nicolet Instruments Co., USA) using KBr disk at a resolution of 4 cm-1. Circular dichroic (CD) measurements were made on JASCO Model J-810 dichrograph (Japan Spectroscopic Co. Ltd., Japan) in a 1 cm quartz cuvette. The final spectra were the mean of 10 accumulated scans and were corrected for the unspecific dichroic absorbance of the medium by computer manipulation. The electrochemical experiments were performed with a CHI 660B electrochemical workstation (CH Instruments). A coiled Pt wire and a saturated calomel electrode (SCE) were used as the counter electrode and the reference electrode, respectively. The buffer was purged with high-purity nitrogen for at least 30 min prior to each electrochemical measurement and a nitrogen environment was kept over solution to prevent the solution from oxygen. The electrocatalytic activity of the GOx-nanoPANI/ GC electrode to the oxidation of glucose was evaluated by amperometry at a constant potential of -0.35 V. All electrochemical experiments were performed at room temperature (22 ( 2 °C). 3. Results and Discussion 3.1. Synthesis and Characterization of Polyaniline Nanofibers. The typical synthetic process was carried out at the interface of toluene (containing 0.1 M of aniline) and 1 M of sulfuric acid (containing 0.05 M of APS). The organic phase was carefully spread onto the aqueous phase forming an aqueous/organic interface. After a very short induction period, polyaniline nanostructures appeared at the interface, and migrated into the aqueous phase (part a of Figure 1) and finally filled the entire aqueous layer (parts b-e of Figure 1). The induction period is usually 5 to 30 s, suggesting the reaction proceeds rapidly. As the reaction proceeds, the color of the aqueous phase becomes dark-green (part e of Figure 1) and finally stops changing, indicating the reaction completion. Usually, a reaction time of 6 h is sufficient. The morphology of the products was imaged using electron microscope. Highly uniform nanofibers are observed under SEM (part A of Figure 2). It is clear that these nanofibers are interconnected to form networklike structures. The diameters of the nanofibers range from 40 to 60 nm, and the lengths are up to several micrometers, therefore the aspect ratio is high. The EDS was recorded to verify the composition of the nanofibers. The results shown in part B of Figure 2 reveal the presence of carbon (0.27 keV), nitrogen (0.39 keV), sulfur (0.16 keV, the peak cannot be observed in part B of Figure 2 because it is too small and covered by the large peak of carbon), and oxygen (0.52 keV). However, the amount of oxygen (0.25% in atomic ratio) and sulfur (0.16%) is very little. Carbon and nitrogen are observed with an estimated atomic ratio of 6:1 (85.25 to 14.33). This result is in good agreement with that expected by theory. The structure of the nanofibers was characterized by UV-vis spectroscopy as depicted in part A of Figure 3. The spectrum (curve a) shows that polyaniline nanofibers exhibit three absorption peaks at 340, 430, and 790 nm, which are the characteristic absorption peaks of the emeraldine oxidation state of polyaniline.46,47 The absorption peaks at 340 and 430 nm are attributed to π*-π transition of benzenoid rings and polaronic peak, reflecting protonation of backbone of the nanofibers.46 The peak at 790 nm represents π-polaron transition,

Figure 2. SEM image (A) and the energy-dispersive X-ray spectroscopy (B) of polyaniline nanofibers made by interfacial polymerization in an interface of 1 M sulfuric acid solution and toluene.

indicating that the nanofibers are in a conductive state.47 After the nanofibers were washed with 0.1 M of ammonium hydroxide, which can cause dedoping of polyaniline and produces the emeraldine base form of polyaniline,15 the features of the UV-vis spectrum changed significantly (curve b). Two absorption peaks at 330 and 625 nm, which are the characteristics of dedoped polyaniline,14 are observed. These absorption characteristics are identical with those reported previously for polyaniline film.23 The FTIR spectrum of the nanofibers is shown in part B of Figure 3. The peaks at 1563 and 1490 cm-1 belong to the CdC stretching of the quinoid ring and benzenoid ring, respectively. Those at 1297 and 1145 cm-1 are attributed to C-N stretching of the secondary aromatic amine. The peak at 820 cm-1 is ascribed to the out-of-plane bending of C-H on the 1,4disubstituted ring. The peak at 1244 cm-1 can be attributed to various stretching and bending associated with C-C bond. These characteristics are in good agreement with those reported for polyaniline,48 indicating the formation of polyaniline nanofibers. The polyaniline nanofibers were also examined by XRD measurements as shown in part C of Figure 3. The sample exhibits the XRD patterns with the diffraction peaks at 2θ ) 6.3, 19.0, 20.7, and 25.8°, respectively. The features of the XRD patterns are similar to those for polyaniline film.49 The welldefined peaks indicate that the crystallinity of the synthetic nanofibers is high. The electrochemical characteristics of the synthetic nanofibers were studied by voltammetry as shown in Figure 4. The cyclic voltammogram of the nanoPANI/GC electrode in 0.1 M of H2SO4 solution displays two pairs of well-defined redox peaks with the formal potential (E0′) of ca. 90 and 700 mV (at 100 mV/s) respectively, which were calculated by averaging the values of the anodic and cathodic potentials. These values are similar to those reported for polyaniline film.50 Therefore, the

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Figure 5. TEM images of polyaniline nanofibers synthesized by interfacial polymerization in an interface of 1 M sulfuric acid aqueous solution and organic solvent of toluene (A), benzene (B), n-hexane (C), n-hexanol (D), carbon tetrachloride (E), and chloroform (F). The aqueous phase contains 0.05 M of ammonium peroxydisulfate and the organic phase contains 0.1 M of aniline.

Figure 3. UV-vis (A), FTIR (B), and XRD patterns (C) of polyaniline nanofibers made by interfacial polymerization in an interface of 1 M sulfuric acid solution and toluene. The curve (a) and (b) are the UV-vis spectrum of the nanofibers as synthesized (a) and washed using 0.1 M of ammonium hydroxide (b), respectively.

Figure 4. Cyclic voltammetric response of polyaniline nanofibers in 0.1 M of H2SO4 solution at a scan rate of 100 mV/s. The nanofibers were confined on the surface of a glassy carbon electrode.

redox pair with E0′ of 90 mV corresponds to the reversible transition between leucoemeraldine (reduced form of polyaniline) and emeraldine (half-oxidized form of the polymer),51 and the redox pair with E0′ of 700 mV is ascribed to reversible transition between emeraldine and pernigraniline

(fully oxidized form of the polymer).52 The polyaniline nanofibers on GC electrode surface are fairly stable because the features of the cyclic voltammogram of the nanoPANI/GC electrode remain unchangeable after the electrode was scanned continuously for a long time (∼100 cycles, at 100 mV/s) in the potential range of interest. In addition, no obvious change is detected after the electrode has been stored in solution for two days. These characteristics are important when the electrode is used as a matrix for immobilizing enzymes and sensing substrates. 3.2. Effects of Organic Solvents. The polyaniline nanofibers can be synthesized with a variety of organic solvents as the organic phase. For example, very uniform nanofibers are observed using toluene, benzene, n-hexane, n-hexanol, carbon tetrachloride, and chloroform as the organic phase. The shape and size of the nanofibers do not appear to be affected by the organic solvent (parts A-F of Figure 5). The average diameter of these nanofibers is around 50 nm. This result indicates that the types and the polarity of the organic solvent have no significant effects on the fibrous morphologies of the synthetic polyaniline nanofibers. This is potentially a great advantage because a large variety of organic solvents can be selected as the organic phase. Huang and Kaner pointed out that the key to synthesizing polyaniline nanofibers is preventing secondary growth.53 The interface between the immiscible aqueous/organic phase does not contribute directly to the formation of nanofibers, it simply separates nanofibers formation from secondary growth. In interfacial polymerization, aniline and APS are separated by the boundary between the aqueous and the organic phase, polymerization occurs only at this interface where all of the components needed for polymerization come together. Polya-

Polyaniline Nanofibers niline nanofibers are then formed. The newly formed nanofibers are hydrophilic and can rapidly move away from the interface and diffuse into the aqueous phase. In this way, as the nanofibers form they are continuously withdrawn from the reaction front, thus avoiding secondary growth and allowing new nanofibers to grow at this interface. This effect explains why nanofibers are obtained no matter which solvent is used as the organic phase in interfacial polymerization. The result of this work is in agreement with that obtained by Huang et al. They also found that the polarity of the organic phase had little effect on the fibrous morphology of polyaniline synthesized from the interfacial polymerization.31 However, this result is quite different from that obtained by He et al. They showed that the morphologies of the synthetic polyaniline were significantly affected by the polarity of the organic phase.32 The polyaniline was granular particles when a high polarity solvent was used, whereas the product was nanobelts when a lowpolarity solvent was used. These differences might result from the different synthesis conditions used in the present work and in He’s work. They selected a weak acid (acetic acid) as a dopant, which might be a main reason causing the different morphologies upon the polarity of the organic solvent. This conclusion is also supported by the results of Huang et al., whose results indicated that the morphologies of polyaniline were particles when medium or weak acids, such as tartaric acid or pyrrolodone-5-carboxylic acid and so forth were used as a dopant, and even those acids were at high concentrations.31 3.3. Effects of Acid Dopants. The effects of acid dopants on the morphologies of the synthetic polyaniline nanostructures were studied by fixing toluene as an organic phase. A variety of acids was used as a dopant, including sulfuric acid, hydrochloric acid, phosphoric acid, and perchloric acid (all at a level of 1 M). The fibrous morphologies of the synthetic polyaniline appear to be insensitive to the acid dopants. Polyaniline nanofibers are observed no matter what acid dopant is used in the polymerization (Figure 6). This is significantly different from the traditional chemical polymerization in a homogeneous phase, which usually generates granular polyaniline.54 The diameter of the polyaniline nanofibers is slightly affected by the dopants used in the polymerization. The average diameters of the nanofibers produced in sulfuric acid are 40-60 nm, those made in hydrochloric acid are about 20-30 nm, and those synthesized in phosphoric acid are around 50 nm. The diameter of the nanofibers synthesized in perchloric acid approaches 100 nm. The exact reason that the dopant acids affect the size of the nanofibers is still unclear. It may probably be due to the different size of the anion of the acids and the dopant structure. Polyaniline needs to be doped with an anion of the acids to maintain the charge balance, therefore, the size and the structure of anion inevitably influence the size of the synthetic polyaniline nanofibers. 3.4. Effects of Concentration of Acid and the Reaction Time. The effects of the concentration of acid on the size of the polyaniline nanofibers were investigated by employing toluene as an organic phase and sulfuric acid solution as an aqueous phase. The morphologies of polyaniline are affected by the concentration of acids. High concentration (more than 0.5 M) of acids is preferable to producing the polyaniline nanofibers. At low concentration (lower than 0.1 M), the synthetic polyaniline has a morphology of particles. At high concentration, the size of nanofibers is almost independent of the acid concentration (Figure 7). For example, the average diameter of the nanofibers synthesized at the concentration of sulfuric acid of 0.5, 1.0, 1.5, and 2.0 M is about 37, 50, 45, and

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Figure 6. TEM images of polyaniline nanofibers synthesized by interfacial polymerization in an interface of toluene and 1 M sulfuric acid (A), hydrochloric acid (B), phosphoric acid (C), and perchloric acid aqueous solution (D). The aqueous phase contains 0.05 M of ammonium peroxydisulfate and the organic phase contains 0.1 M of aniline. (E) shows the dependence of the diameter distribution of polyaniline nanofibers on the dopant acid.

35 nm, respectively. We tentatively explain the effects of the concentration of dopant acids on the morphologies of the synthetic polyaniline as follows. According to the mechanism proposed by Mattoso et al.,55 the elongation process during the oxidative chemical polymerization of aniline in an acidic medium involves reactive intermediate species rearranged from protonated aniline. When the concentration of dopant acid is low or a weak acid is used as a dopant, the concentration of protonated aniline is also low due to the low concentration of H+. The low concentration of protonated aniline is not beneficial to rearrangement and elongation of reactive intermediate species. This result leads to the formation of polyaniline with morphologies of particles. The high concentration of protonated aniline results in the formation of nanofibers. The effects of the reaction time on the size and the synthetic nanofibers were also studied with the system of the toluene (containing 0.1 M aniline) and 1 M of sulfuric acid solution (containing 0.05 M of APS). The diameter of the nanofiber increases with the reaction time when the time is lower than 6 h. At the longer reaction time, the diameter of the nanofibers remains practically invariant (Figure 8) and keeps at the value of around 50 nm. Therefore, the reaction time is usually selected to be 6 h, which is sufficient for producing a high quality of the nanofibers. 3.5. Effects of Aniline Concentration. The morphology of the polyaniline seems to be significantly affected by the aniline concentration in organic phase. The concentration of APS in the aqueous phase (1 M sulfuric acid solution) was kept at 0.05

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Figure 7. TEM images of polyaniline nanofibers synthesized by interfacial polymerization in an interface of toluene and 0.5 M (A), 1.0 M (B), 1.5 M (C), and 2.0 M sulfuric acid (D). The aqueous phase contains 0.05 M of ammonium peroxydisulfate and the organic phase contains 0.1 M of aniline. (E) shows dependence of the diameter distribution of polyaniline nanofiers on the concentrations of the dopant acid.

M. The concentration of aniline in organic phase (toluene) was varied from 0.1 to 1 M. When the polymerization reaction was carried out at the monomer concentration of 0.1 M, the nanofibers were produced (part A of Figure 9). With the increasing of the aniline concentration, the quantity of granular particles starts to increase in the products. For example, 0.5 M of aniline produces the mixture of the nanofibers and particle agglomerations (part B of Figure 9). When the concentration of aniline is increased to 1 M, the products are completely particle agglomerations, and no fiber structure can be observed from the SEM image (part C of Figure 9). The effects of the aniline concentration on the morphology of polyaniline can be explained as follows. In the interfacial polymerization, nanofibers are formed at the interface and then migrate into aqueous phase (Figure 1). When a low concentration of aniline is used (0.1 M), the amount of aniline distributed into aqueous phase is very little and can be neglected. The polymerization should be terminated once the nanofibers enter into the aqueous phase. Therefore, the polyaniline nanofibers are not subject to further polymerization in the aqueous phase, producing almost exclusively nanofibers as shown in part A of Figure 9. However, the amount of aniline in aqueous cannot be neglected when a high concentration of aniline was used. Therefore, the polyaniline nanofibers in the aqueous phase are still surrounded by aniline monomer and oxidant species. The nanofibers will likely become the nucleation centers for secondary growth of polyaniline and finally grow into irregularly

Zhao et al. shaped granular particles. Therefore, the synthetic polyaniline has morphology of mixture of nanofibers and particles at medium concentration of aniline (part B of Figure 9), whereas only granular particles form of polyaniline is obtained at a high concentration of aniline (part C of Figure 9). 3.6. Effects of APS Concentration. The effects of the concentration of APS on the morphologies of the synthetic polyaniline were studied by employing toluene (containing 0.1 M aniline) as an organic phase and 1 M sulfuric acid solution as an aqueous phase. When the concentration of APS is lower than 0.5 M, polyaniline nanofibers are obtained and the diameters of the synthetic nanofibers keep almost invariant with the increase of the concentration of APS in aqueous phase (parts A-C of Figure 10). These nanofibers have average diameter of 40-60 nm. However, the product is in the form of particulates with an average particle diameter of 40-50 nm when the concentration of APS is higher than 0.5 M (for example 1 M, part D of Figure 10). The effects of the concentration of APS on the morphologies of the synthetic polyaniline can be explained. As the polymerization begins, the initiator molecules (APS) induce the formation of nanofibers by rapidly polymerizing aniline monomers. Therefore, when a low concentration of APS is used, all of the initiator molecules are consumed to form polyaniline nanofibers, thus suppressing the secondary growth of polyaniline. The product is polyaniline nanofibers as presented in parts A-C of Figure 10. When a high concentration of APS is used, the nanofibers formed at an early stage of polymerization process become scaffolds for secondary growth of polyaniline and finally turn into irregularly shaped agglomerates (part D of Figure 10). 3.7. Direct Electron Transfer of GOx at the Surface of Polyaniline Nanofibers. Having characterized polyaniline nanofibers, we next present the potential application of the nanofibers in facilitating DET of GOx. GOx is a homodimer with a molecular weight of 150-180 kDa containing two tightly bound flavine adenine dinucleotide (FAD) cofactors.38 DET from the redox center (i.e., FAD/FADH2) of the enzyme to electrode is difficult to observe because the active site is deeply embedded within a protective protein shell. One effective approach to improve the communication between active site and electrode is to immobilize the enzyme on the surface of nanomaterialbased electrodes that can enhance the electron-transfer kinetics of the enzyme.56-59 For example, it was reported that carbon nanotube (CNT) can promote the DET of GOx.56,59 Here, DET characteristics of GOx at the surface of the nanoPANI/GC electrode were presented. Before the DET characteristics of GOx were presented, CD spectra were recorded to make sure GOx could still retain its native conformation after it was covalently immobilized because the covalently attaching GOx on the surface of the nanoPANI might change the conformation of the enzyme and led to the denaturation of the enzyme. The spectra in the far-UV region (200 to 250 nm) correspond to the peptide n f π* electronic transition. The spectrum in this region is sensitive to the changes of the conformation of the enzyme.60 The CD spectra of GOx in solution are characterized by two negative peaks at ca. 208 and 218 nm (part a of Figure 11). These peaks almost remain invariable after GOx was immobilized on the surface of polyaniline nanofibers (part b of Figure 11), indicating that the secondary structure and conformation of GOx after confined on the surface of polyaniline nanofibers are essentially the same as that of the native one. Moreover, the CD spectra of GOxnanoPANI (part d of Figure 11) in the near-UV region (250 to 300 nm) are also the same as that of GOx in solution (part c of

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Figure 8. TEM images of polyaniline nanofibers synthesized by interfacial polymerization in an interface of toluene and 1.0 M sulfuric acid at the reaction time of 1 (A), 2 (B), 6 (C), 9 (D), and 12 h (E). The aqueous phase contains 0.05 M of ammonium peroxydisulfate and the organic phase contains 0.1 M of aniline. (F) shows dependence of the diameter distribution of polyaniline nanofibers on reaction time.

Figure 11). These results suggest that the microenvironment of the active site of GOx after immobilized on the surface of nanoPANI also remains the same as that of the native one because no signals can be observed in this region for a denatured GOx due to the dissociation of FAD from the enzyme.60 The cyclic voltammograms of the GOx-nanoPANI/GC (a), nanoPANI/GC (b), and GOx/GC (c) electrodes in PBS (0.1 M, pH 7.0) are depicted in Figure 12. No redox peaks are observed at both the nanoPANI/GC (curve b) and GOx/GC electrodes (curve c), suggesting that GOx cannot undergo the DET reaction at the bare GC electrode, and polyaniline nanofibers cannot be oxidized or reduced in this potential range. The GOx-nanoPANI/GC electrode shows a pair of welldefined and nearly symmetrical redox peaks with the anodic and cathodic peak potentials at -404 and -432 mV (at 10 mV/ s, curve a), respectively. The value of E0′ is -418 mV (at 10 mV/s), which is close to that previously reported for DET of GOx immobilized on the preanodized screen-printed carbon electrode (-420 mV)61 or CNT-based electrode (-460 mV).59 Therefore, the pair of peaks in curve b can be ascribed to the DET reaction (the conversion of FAD/FADH2 center) of GOx. The separation of peak potentials (∆Ep) is 28 mV, indicating that GOx on polyaniline nanofibers displays a quasi-reversible electrochemical reaction despite its large molecular structure.

Both the anodic and cathodic peak currents increase linearly with the scan rate (not shown here), suggesting that the redox process is confined to the surface of the electrode. This result confirms that the immobilized state of GOx is stable. The apparent heterogeneous electron transfer rate constant (ks) is estimated to be (6.3 ( 1.6) s-1 from the dependence of ∆Ep on the scan rates using the procedures developed by Laviron.62 This value is much higher than that reported previously by adsorbing GOx on the surface of CNT (1.5-1.7 s-1),56,59 suggesting that DET of GOx on polyaniline nanofibers has a good reversibility. These results show that polyaniline nanofibers can effectively facilitate DET between GOx and the electrode. Moreover, GOx immobilized on the surface of the nanofibers can keep its natural structure. This result is in good agreement with that obtained from CD spectra. More importantly, the GOx-nanoPANI on GC electrode surface is fairly stable since the features of the cyclic voltammogram of GOx-nanoPANI/GC electrode remain almost invariable after the electrode was scanned continuously for a long time (∼100 cycles, at 10 mV/s). In addition, no obvious change is detected after the electrode has been stored in PBS at 4 °C for 1 week. These characteristics are useful when the electrode is further used as a biosensor for sensing substrates (such as glucose).

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Figure 10. TEM images of polyaniline synthesized by interfacial polymerization in an interface of toluene and 1.0 M sulfuric acid. The organic phase contains 0.1 M of aniline and the aqueous phase contains 0.01 (A), 0.05 (B), 0.5 (C), and 1 M (D) of ammonium peroxydisulfate.

concentration, suggesting that the electrode can respond rapidly to the change of the substrate concentration. The response current increases with the concentration of glucose. These results verify that the immobilized GOx does not undergo the denaturation at the surface of polyaniline nanofibers and still keeps its intrinsic and good electrocatalytic activities toward its substrates. This conclusion is consistent with that obtained from cyclic voltammertic data. The electrocatalytic processes at the electrode surface can be depicted as following:

GOx(FAD) + glucose f GOx(FADH2) + gluconolactone (1) GOx(FADH2) f GOx(FAD) + 2e-+ 2H+

Figure 9. Effects of the concentration of aniline on the morphology of polyaniline nanostructures. Polyaniline was synthesized by interfacial polymerization in an interface of 1 M sulfuric acid solution and toluene. The concentration of aniline in toluene is 0.1 (A), 0.5 (B), and 1 M (C).

The electrocatalytic characteristics of the GOx-nanoPANI/ GC electrode toward the oxidation of glucose are evaluated by amperometry. The suitable operating potential for amperometric experiments was chosen from the hydrodynamic voltammogram of the GOx-nanoPANI/GC electrode in PBS (0.1 M, pH 7.0) containing 2 mM glucose. The hydrodynamic voltammogram (the inset of Figure 12) shows the electrocatalytic response for glucose at the GOx-nanoPANI/GC electrode starts at ca. -0.5 V and the electrocatalytic current levels off at about -0.35 V. Therefore, -0.35 V is selected as the operating potential in the amperometric measurements to ensure that the response was diffusion-controlled. Figure 13 shows the responses of the GOx-nanoPANI/GC electrode toward the change of concentration of glucose in solution. The response current reaches its steady-state value within about 5-10 s at each glucose

(2)

The inset of Figure 13 represents the calibration curve with a linear range spans from 0.01 to 1 mM of glucose with a detection limit estimated to be ca. 0.5 µM. The response deviates from the linearity at higher concentration (>2 mM) representing a typical characteristic of Michaelis-Menten kinetics. The apparent Michaelis-Menten constant (KM′) is evaluated to be (1.05 ( 0.04) mM from the Lineweaver-Burk plot. This value is similar to that reported for GOx immobilized on preanodized screen-printed carbon electrode (1.07 mM)61 and is slightly lower than that for GOx immobilized on nile blue-CNT composite (4.2 mM).39 However, the value is much lower than those for GOx entrapped in sol-gel/chitosan composite (21 mM),58 bound to self-assemble monolayer electrode (20 mM),63 immobilized on the surface of nano-CaCO3 (21.4 mM),64 and for GOx in solution (22 ( 2 mM).65 These results show that the GOx-PANI/GC electrode possesses higher biological affinity to glucose. It is thus expected that the GOx molecules do not undergo denaturation. Additional experiments were conducted to test the reproducibility and stability of the GOx-nanoPANI/GC electrode toward the oxidation of glucose. The RSD (relative standard deviation) is 2.2% estimated from the response of five different and freshly prepared electrodes to the reduction of 0.5 mM of glucose. The assay precision of the electrode was examined by 10 determinations at a glucose concentration of 0.5 mM. An RSD of 2.8%

Polyaniline Nanofibers

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Figure 11. CD spectra of GOx in aqueous solution (a and c) and immobilized on the surface of nanoPANI (b and d) in the far-UV (A) and near-UV (B) region, respectively. Curves (a) and (c) are displayed +2 units for clarity.

Figure 12. Cyclic voltammograms of the GOx-nanoPANI/GC (a), nanoPANI/GC (b), and GOx/GC (c) electrodes in PBS (0.1 M, pH 7.0) at 10 mV/s. The inset shows the effect of applied potential on the steadystate current for 2 mM glucose in PBS (0.1 M, pH 7.0) at the GOx-nanoPANI/GC electrode.

Figure 13. Chronoamperometric responses of the GOx-nanoPANI/ GC electrode to the oxidation of glucose in 0.1 M phosphate buffer solution (pH 7.0). The concentration of glucose (from curve a to e) is 0, 0.125, 0.25, 0.5, and 1 mM. The applied potential is -0.35 V (vs SCE). The inset shows calibration curve of glucose detected by the GOx-nanoPANI/GC electrode.

is obtained. These results reveal an acceptable reproducibility and precision in the construction of the electrode. No obvious change in the response is found after the electrode was immersed in solution and stored at 4 °C for 10 h. The response of the electrode can keep ca. 93% of its initial one within 2 weeks. These results indicate that the stability of the electrode is acceptable.

Figure 14. Chronoamperometric responses of the GOx-nanoPANI/ GC electrode upon addition of glucose (1 mM), AA (0.2 mM), UA (0.2 mM), and AP (0.2 mM) successively in 0.1 M phosphate buffer solution (pH 7.0). The applied potential is -0.35 V (vs SCE).

In real samples, there are some coexisting electroactive species, for example, AA, UA, and AP and so forth, might affect the biosensors response. The selectivity and anti-interference advantages of the GOx-nanoPANI/GC electrode were demonstrated (Figure 14). After the response of 1 mM of glucose at the GOx-nanoPANI/GC electrode reached a steady-state value, a relevant physiological level of AA, UA, and AP (0.2 mM,66 respectively) was added into the detection system. The results indicate that addition of AA, UA, or AP does not affect the response of glucose, and the response of AA, UA, and AP at the electrode is negligible. This feature is largely attributed to the low operating potential used in the detection. Hence, a highly selective response to glucose is obtained without the use of perm-selective membrane or enzymatic preoxidation.67 This is another advantage of the proposed biosensor over those reported previously. All of these features show that GOx immobilized on polyaniline nanofibers keeps its intrinsic and good electrocatalytic activities toward its substrates. The biocompatibility of the nanofibers enables them to become a good biosensing platform for realizing DET and electrocatalysis of redox proteins/ enzymes. 4. Conclusions Polyaniline nanofibers have been synthesized by interfacial polymerization and characterized by electron microscope (SEM and TEM), spectroscopy (UV-vis, FTIR, and XRD), and voltammetry. The effects of synthetic conditions on the morphology of the polyaniline nanostructure have been studied in details. The nanofibers have been employed as an electrode

4996 J. Phys. Chem. C, Vol. 113, No. 12, 2009 substrate for immobilization and direct electrochemistry of glucose oxidase (GOx). The electrochemical characteristics of the GOx-nanoPANI/GC electrode have been studied by voltammetry. After immobilized on the surface of the nanofibers, GOx can still retain its natural structure and undergoes effective direct electron transfer reaction with a pair of well-defined, quasi-reversible redox peak in its cyclic voltammogram. The electrode displays good features in electrocatalytic oxidation of glucose and thus can be used as a biosensor for detecting substrates with good reproducibility and stability. This study should stimulate the exploration of the application of the nanofibers especially in the fields of such as biosensors, electrocatalysis, and composite biomaterials. Acknowledgment. This work is supported by the National Natural Science Foundation of China (20673057, 20773067, 20833006) and the Program for New Century Excellent Talents in University (NET-06-0508). References and Notes (1) Lee, K. J.; Oh, J. H.; Kim, Y.; Jang, J. Chem. Mater. 2006, 18, 5002. (2) Liu, J.; Li, X.; Dai, L. AdV. Mater. 2006, 18, 1740. (3) Xia, Y.; Yang, P.; Sun, Y.; Wu, Y.; Mayers, B.; Gates, B.; Yin, Y.; Kim, F.; Yan, H. AdV. Mater. 2003, 15, 353. (4) Tang, C.; Bando, Y.; Golberg, D.; Ma, R. Angew. Chem., Int. Ed. 2005, 44, 576. (5) Shi, Y.; Zhou, B.; Wu, P.; Wang, K.; Cai, C. J. Electroanal. Chem. 2007, 611, 1. (6) Inguanta, R.; Piazza, S.; Sunseri, C. Electrochem. Commun. 2008, 10, 506. (7) Liu, Y.; Zhang, J.; Hou, W.; Zhu, J. J. Nanotechnology 2008, 19, 135707. (8) Zhang, L. Biosens. Bioelectron. 2008, 23, 1610. (9) Bu, W.; Hua, Z.; Chen, H.; Shi, J. J. Phys. Chem. B 2005, 109, 14461. (10) Xing, Y.; Li, M.; Davis, S. A.; Mann, S. J. Phys. Chem. B 2006, 110, 1111. (11) Yu, L.; Song, H.; Liu, Z.; Yang, L.; Lu, S.; Zheng, Z. J. Phys. Chem. B 2005, 109, 11450. (12) Fang, Y.-P.; Xu, A.-W.; Song, R.-Q.; Zhang, H.-X.; You, L.-P.; Yu, J. C.; Liu, H.-Q. J. Am. Chem. Soc. 2003, 125, 16025. (13) Hwang S. O.; Kim, C. H.; Myung, Y.; Park, S.-H.; Park, J.; Kim, J.; Han, C.-S.; Kim, J.-Y. J. Phys. Chem. C 2008, 112, 13911. (14) Guo, Y.; Zhou, Y. Eur. Polym. J. 2007, 43, 2292. (15) Huang, J.; Virji, S.; Weiller, B. H.; Kaner, R. B. J. Am. Chem. Soc. 2003, 125, 314. (16) Marie, E.; Rothe, R.; Antonietti, M.; Landfester, K. Macromolecules 2003, 36, 3967. (17) Huang, K.; Wan, M. Chem. Mater. 2002, 14, 3486. (18) MacDiarmid, A. G. Angew. Chem., Int. Ed. 2001, 40, 2581. (19) Huang, W. S.; Humphrey, B. D.; MacDiarmid, A. G. J. Chem. Soc., Faraday Trans. 1986, 82, 2385. (20) MacDiarmid, A. G. Synth. Met. 1997, 84, 27. (21) Varela, H.; Bruno, R. L.; Torresi, R. M. Polymer 2003, 44, 5369. (22) Mirmohseni, A.; Solhjov, R. Eur. Polym. J. 2003, 39, 219. (23) Mi, H.; Zhang, X.; Yang, S.; Ye, X.; Luo, J. Mater. Chem. Phys. 2008, 112, 127. (24) Wang, J.; Torardi, C. C.; Duch, M. W. Synth. Met. 2007, 157, 851.

Zhao et al. (25) Hou, L. T.; Hou, Q.; Mo, Y. Q.; Peng, J. B.; Cao, Y. Appl. Phys. Lett. 2005, 87, 243504. (26) Martin, C. R. Acc. Chem. Res. 1995, 28, 61. (27) Liu, H. Q.; Kameoka, J.; Czaplewski, D. A.; Craighead, H. G. Nano Lett. 2004, 4, 671. (28) Liu, J.; Lin, Y.; Liang, L.; Voigt, J. A.; Huber, D. L.; Tian, Z. R.; Coker, E.; McKenzie, B.; McDermott, M. J. Chem.sEur. J. 2003, 9, 605. (29) Li, X.; Tian, S. J.; Ping, Y.; Kim, D. H.; Knoll, W. Langmuir 2005, 21, 9393. (30) Zhang, X.; Goux, W. J.; Manohar, S. K. J. Am. Chem. Soc. 2004, 126, 4502. (31) Huang, J.; Kaner, R. B. J. Am. Chem. Soc. 2004, 126, 851. (32) He, Y.; Lu, J. React. Polym. 2007, 67, 476. (33) Li, W.; Wang, H. L. J. Am. Chem. Soc. 2004, 126, 2278. (34) Chiou, N. R.; Epstein, A. J. AdV. Mater. 2005, 17, 1679. (35) Liang, L.; Liu, J.; Windisch, C. F.; Exarhos, G. J.; Lin, Y. H. Angew. Chem., Int. Ed. 2002, 41, 3665. (36) MacDiarmid, A. G.; Jones, W. E.; Norris, I. D.; Gao, J.; Johnson, A. T.; Pinto, N. J.; Hone, J.; Han, B.; Ko, F. K.; Okuzaki, H.; Llaguno, M. Synth. Met. 2001, 119, 27. (37) He, H. X.; Li, C. Z.; Tao, N. J. Appl. Phys. Lett. 2001, 78, 811. (38) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (39) Du, P.; Zhou, B.; Cai, C. J. Electroanal. Chem. 2008, 614, 149. (40) Liu, S.; Yin, Y.; Cai, C. Chin. J. Chem. 2007, 25, 439. (41) Kafi, A. K. M.; Lee, D.-Y.; Park, S.-H.; Kwon, Y.-S. J. Nanosci. Nanotechnol. 2006, 6, 3539. (42) Forzani, E. S.; Zhang, H.; Nagahara, L. A.; Amlani, I.; Tsui, R.; Tao, N. Nano Lett. 2004, 4, 1785. (43) Shi, L.; Xiao, Y.; Willner, I. Electrochem. Commun. 2004, 6, 1057. (44) Du, P.; Liu, S.; Wu, P.; Cai, C. Electrochim. Acta 2007, 52, 6534. (45) Yeh, J. I.; Lazareck, A.; Kim, J. H.; Xu, J.; Du, S. Biosens. Bioelectron. 2007, 23, 568. (46) Jana, T.; Nandi, A. K. Langmuir 2000, 16, 3141. (47) Athawale, A. A.; Kulkarni, M. V.; Chabukswar, V. V. Mater. Chem. Phys. 2002, 73, 1. (48) Kan, J. Q.; Lv, R. G.; Zhang, S. L. Synth. Met. 2004, 145, 37. (49) Chaudhari, H. K.; Kelkar, D. S. J. Appl. Polym. Sci. 1996, 62, 15. (50) Mazˇeikiene`, R.; Malinauskas, A. Synth. Met. 2001, 123, 349. (51) Wang, Y. G.; Li, H. Q.; Xia, Y. Y. AdV. Mater. 2006, 18, 2619. (52) Hu, C. C.; Chu, C. H. Mater. Chem. Phys. 2000, 65, 329. (53) Huang, J.; Kaner, R. B. Angew. Chem., Int. Ed. 2004, 43, 5817. (54) Qiang, J.; Yu, Z.; Wu, H.; Yun, D. Synth. Met. 2008, 158, 544. (55) Mattoso, L. H. C.; MacDiamid, A. G.; Epstein, A. J. Synth. Met. 1994, 68, 1. (56) Guiseppi-Elie, A.; Lei, C.; Baughman, R. H. Nanotechnology 2002, 13, 559. (57) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408. (58) Chen, X.; Jia, J. B.; Dong, S. Electroanalysis 2003, 15, 608. (59) Cai, C.; Chen, J. Anal. Biochem. 2004, 332, 75. (60) Zolda´k, G.; Zubrik, A.; Musatov, A.; Stupa´k, M.; Sedla´k, E. J. Biol. Chem. 2004, 279, 47601. (61) Yang, T.-H.; Huang, C.-L.; Ke, J.-H.; Zen, J.-M. Electrochem. Commun. 2008, 10, 1094. (62) Laviron, E. J. Electroanal. Chem. 1979, 101, 19. (63) Murthy, A. S. N.; Sharma, J. Anal. Chem. 1998, 363, 215. (64) Shan, D.; Zhu, M.; Xue, H.; Cosnier, S. Biosens. Bioelectron. 2007, 22, 1612. (65) Wang, B.; Li, B.; Deng, Q.; Dong, S. Anal. Chem. 1998, 70, 3170. (66) Zhou, M.; Shang, L.; Li, B.; Huang, L.; Dong, S. Biosens. Bioelectron. 2008, 24, 442. (67) Scuvalio, O.; Soldatkin, O.; Lefebvre, A.; Cespuglio, R.; Soldatkin, A. Anal. Chim. Acta 2006, 573, 110.

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