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Preparation of Ag–Au nanoparticle and its application to glucose biosensor
Sensors and Actuators B 110 (2005) 358–363
Preparation of Ag–Au nanoparticle and its application to glucose biosensor Xiangling Ren, Xianwei Meng, Fangqiong Tang ∗ Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, Beijing 100101, PR China Received 29 October 2004; received in revised form 29 December 2004; accepted 6 February 2005 Available online 3 March 2005
Abstract In this paper, Ag–Au nanoparticles are produced in sodium-bis(2-ethylhexyl)-sulfosuccinate (AOT)–cyclohexane reverse micelle system. The properties of the obtained nanoparticles are characterized with transmission electron microscope (TEM) and UV–vis absorption spectrophotometer. Glucose biosensors have been formed with glucose oxidase (GOx) immobilized in Ag–Au sol. GOx are simply mixed with Ag–Au nanoparticles and crosslinked with a polyvinyl butyral (PVB) medium by glutaraldehyde. Then a platinum electrode is coated with the mixture. The effects of the various molar ratios of Ag–Au particles with respect to the current response and the stability of the GOx electrodes are studied. The experimental results indicate the current response of the enzyme electrode containing Ag–Au sol increase from 0.32 to 19 A cm−2 in the solution of 10 mM -d-glucose. In our study, the stability of enzyme electrodes is also enhanced. © 2005 Elsevier B.V. All rights reserved. Keywords: Ag–Au nanoparticles; Immobilization enzyme; Glucose oxidase; Biosensor
1. Introduction Glucose biosensor is one of the most important biosensors. The ability to obtain rapid, accurate and precise glucose measurement is essential to the appropriate administration of insulin therapy [1]. Glucose biosensors would offer the possibility of carrying out analytical and diagnostic procedures simply and rapidly [2]. They allow greater ease of use with opportunities for self-monitoring by user groups such as diabetics. With improved glucose control, it is possible that some of the long-term diabetic syndromes, such as retinal and kidney damage, would be avoided. In biosensor the biological substances are used as recognition elements, which can convert the analytic substances concentration with the measurable electrical signals. The immobilization of the enzymes is one of the crucial factors in biosensor preparation. Many methods have been used to immobilize enzymes and to improve the enzymatic activity [3–9]. Miniaturization is one of the important developments ∗
Corresponding author. Tel.: +86 10 64888064; fax: +86 10 64879375. E-mail address: [email protected] (F. Tang).
0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.02.016
in biosensor technology [10]. Miniaturization, however, may result in low current. To overcome this problem, nanoparticles have been introduced to the immobilization of the enzymes. Metal nanoparticles have many unique properties: large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability. They have possible applications in many areas such as nonlinear optical switching [11], immunoassay labeling [12], and Raman spectroscopy enhancement [13]. The immobilization of proteins in nanoparticles sol has been reported in Refs. [14–17]. Recently, Xiao et al. [18] report the reconstitution of an apo-flavoenzyme, apo-glucose oxidase, on a 1.4 nm gold nanocrystal functionalized with the cofactor flavin adenine dinucleotide and integrated into a conductive film that yields a bioelectrocatalytic system with exceptional electrical contact with the electrode support. Their work shows that electron transfer through the Au nanoparticles is much faster than electron transfer to O2 . In this paper, Ag–Au nanoparticle is introduced to the research of the glucose electrode. We use Ag–Au nanoparticle because silver is the best conductor among metals and gold has good biocompatibility. Different from other papers,
X. Ren et al. / Sensors and Actuators B 110 (2005) 358–363
we use the Ag–Au nanoparticle prepared in reverse micelle. Numerous articles have given the proof that enzymes have higher activity in reverse micelles than in aqueous systems [19–21]. Our experiments show that these Ag–Au particles can significantly enhance the current sensitivity of GOx enzyme electrodes.
2. Experimental 2.1. Chemicals and reagents Glucose oxidase (GOx) was extracted from Aspergillus niger (100 U mg−1 ; Toyobo Co. Ltd., Japan). Sodiumbis(2-ethylhexyl)-sulfosuccinate (AOT) was obtained from Nacalai Tesque (Kyoto Inc., Japan). -d-Glucose was from Sigma. Silver nitrate (AgNO3 ), chlorauric acid (HAuCl4 ) and sodium citrate were obtained from Beijing ShiJi Company. All the chemicals were used without further purification. All solutions were prepared with redistilled water. 2.2. Preparation of Ag–Au sol The preparation of the Ag–Au nanoparticles was achieved by mixing two sets of reverse micelle solutions (0.2 mol/kg AOT/cyclohexane), with AgNO3 and HAuCl4 ([AgNO3 ] + [HAuCl4 ] = 0.8 mM) solubilized in one set and sodium citrate ([Na3 C6 H5 O7 ] = 2 mM) as the reduction agent in the other set. The micelles in cyclohexane were stabilized by the surfactant. The concentration of the AOT and the micellar size were the same in both sets. These reversed micelles were then stirred thoroughly at room temperature until the solution turned to red. Varying the molar ratios of HAuCl4 and AgNO3 aqueous solutions, we could achieve different nanoparticles. 2.3. Characterization of the metal colloids The samples for transmission electron microscopy (TEM) were prepared by putting one drop of the Ag–Au colloid on a formvar-coated copper grid followed by drying in a desiccator. Electron micrographs were taken with a NEC JEM100CX electron microscope, operating at 100 kV. Absorption spectra were recorded using a Hitachi U-2001 diode array spectrophotometer at 1 cm path length. 2.4. Preparation of enzyme electrode In order to simplify production process of the enzyme electrode, the self-assembly method was used to prepare GOx electrode. Platinum wire with a diameter of 1 mm was polished and boiled in nitric acid for 5 min, washed in redistilled water and boiled in redistilled water. When it became cool, platinum wires was washed in acetone and redistilled water by ultrasound.
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The aqueous solution of six units GOx was added to a 10 ml beaker. An amount of 200 l Ag–Au nanoparticle sol was added to the GOx solution. Several minutes later, 2 ml PVB solution (2%) in anhydrous alcohol and 100 l of glutaraldehyde solution (1%) were added to the beaker. After the mixer was uniform, the platinum wire was dipped into the solution to a depth of 1 cm for several minutes and dried in air. A thin membrane was formed on the electrode. The electrode was stored in a refrigerator at 277 K. The enzyme electrode without Ag–Au nanoparticle, which was used as compare, was prepared by dipping platinum wire into the mixture containing GOx, PVB and glutaraldehyde. This method and amount were as same as described above. 2.5. Cyclic voltammetry Cyclic voltammetry studies used a PARC EG&G Model 283 potentiostat and three electrode cells equipped with a Pt-wire as counter electrode and a Ag/AgCl electrode as reference electrode. The applied scan rate was 50 mV/s. The cyclic voltammograms was taken at 33 mmol/L glucose concentrations. 2.6. Amperometric measurement The sensitivity of the glucose biosensor was tested by measuring the current response. The experiment was carried out using a two-electrode cell consisting of an enzyme working electrode and a reference electrode of Ag/AgCl [22]. Measurements were conducted in a 5 ml phosphate buffer (KHPO4 −NaOH, pH 6.8) cell at 308 K. A fixed potential of 0.4 V was applied to this electronic cell. Firstly, working electrode and reference electrode were put into a phosphate solution at 308 K. When background current reached a constant value, different concentrations (from 2.7 to 33 mM) of -d-glucose solution were added. Then response current was noted down, and background current was deducted, and the correlation between response currents and different concentrations of glucose solution was obtained.
3. Results and discussion 3.1. Preparation of Ag–Au particles The Ag–Au particles were prepared with the various mixtures of HAuCl4 and AgNO3 aqueous solutions. Except the pure silver, the color of the other solutions turned red at last. Transmission electron micrographs of the Ag–Au nanoparticles are in Fig. 1. Fig. 2 shows the UV–vis absorption spectra of Ag–Au composite particles. A remarkable shift in the absorption band at 530 nm was observed for silver (75)–gold (25); the number in metal colloids indicate molar percent of the metal salt incorporated. But with increasing molar ratio of HAuCl4 , the absorption peak
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Fig. 1. Transmission electron micrographs of Ag–Au nanoparticles: (a) silver (75)–gold (25), the average diameter of these particles is about 3.3 ± 0.8 nm; (b) silver (50)–gold (50), the average diameter of these particles is about 9.8 ± 1.5 nm; (c) silver (25)–gold (75), the average diameter of these particles is about 10.5 ± 1.7nm.
at 530 nm became broad and slightly shifted to longer wavelength. It is known that silver and gold are miscible in all proportions due to the almost identical lattice constants. Currently the formation of Ag–Au particles has three possibilities: (1) core-shell particles, silver core with gold shell or vice versa; (2) alloy particles; (3) phase-separated composites, which are made partly of silver and partly of gold. If core-shell particles were formed, they would consist of a gold core with a silver shell, because the reduction rate for gold is much faster than that for silver. The observed spectra are different from the theoretical spectra of the gold core with a silver shell obtained using the equations in the form given by
Bohren and Huffman [23]. The theoretical spectra show that the single plasmon band splits into two peaks, one of which is located at a shorter wavelength than that of silver particles and the other is located at a slightly shorter wavelength than that of gold particles with increasing molar fraction of silver. Torigoe and co-workers [24,25] have given the theoretical spectra of the alloy particles using the same way. Recently, Mallin and Murphy [26] have succeeded in making Ag–Au alloy nanoparticles and given the UV–vis absorption spectra of those. These spectra show that the plasmon band remains in a single peak and shifts continuously from 400 to 520 nm with increasing molar ratio of gold. They are different from Fig. 2. Although this disagreement in the spectra is not interpreted adequately at the present time, it seems that the Ag–Au nanoparticles in our study are neither coreshell particles nor alloy particles. So the Ag–Au composite particles prepared in this study are probably phase-separated composites. 3.2. Ag–Au nanoparticles used in enzyme electrode
Fig. 2. UV–vis absorption spectra of Ag–Au nanoparticles with various molar ratios of Ag and Au.
3.2.1. The cyclic votammograms for the electrode The cyclic votammograms for the electrode in the glucose were shown in Fig. 3. The bare platinum electrode has good electric conductivity, so it exhibits peaks (curve 1). After the electrode modified by PVB, an obvious decrease in the peaks were observed (curve 2). The reason is that the PVB membrane can act as the inert electron and mass transfer blocking layer, and it hinders the diffusion of electron toward the electrode surface. When Ag–Au nanoparticles were
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Fig. 3. Cyclic voltammograms of the electrodes immersed in 33 mmol/L glucose solution; (1) bare Pt electrode; (2) the electrode with PVB film; (3) the electrode with PVB film added GOx and Ag–Au nanoparticles; a Ag/AgCl electrode used as reference electrode; scan rate 50 mV/s.
embedded in the PVB membrane, the electrode shows peaks again (curve 3). This may be due to Ag–Au nanoparticles were distributed throughout the sol–gel network and formed a continuous array of Ag–Au nanoparticles on the electrode. It is evident from the figure that Ag–Au nanoparticles have a marked influence on the barrier property of PVB-modified electrodes and the Ag–Au nanoparticles can assist the electron transfer between the enzyme and the bulk electrode surface. 3.2.2. The current response curves of the immobilized GOx electrode with Ag–Au nanoparticles Fig. 4 shows the current response curves of GOx electrodes without and with Ag–Au nanoparticles on the current response of glucose. When glucose concentration is 10 mM, the current density of the electrode without nanoparticle is 0.32 A cm−2 , and that of the electrode with Ag–Au particle is 19 A cm−2 . It can be seen that nanoparticles can significantly enhance the current response of the elec-
Fig. 4. Calibration curves of the electrodes containing (a) no particles and (b) Ag–Au nanoparticles.
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trode. It is mainly due to the large surface area and the high surface free energy of Ag–Au nanoparticles, which can make GOx inevitably adsorbed on the surface of nanoparticles. Our previous research about SiO2 nanoparticles [16] has given the proof. From cyclic voltammograms, we know Ag–Au nanoparticles can assist the electron transfer between the GOx and the platinum wire surface. The capability of the GOx enzyme electrode can be improved. The Ag–Au nanoparticle we prepared is in reverse micelle. Fig. 5 shows the schematic representation of the Ag–Au particle’s formation. It is very probable that the GOx solution can be solubilized into the reverse micelle. Enzyme molecule has a hydrated shell. It is the requirement to keep the activity of enzyme [27]. The organic reagents can make GOx lose its hydrated shell. The reverse micelle provides good reactors for the enzyme adsorption on the nanoparticles’ surface, which can decrease the contact of GOx molecules with organic reagents and protect the hydrated shell. On this condition there is very little free water. It can decrease the precipitation of PVB and avoid the severe action between enzyme and PVB, therefore increase the activity and stability of immobilized GOx. The electrode made from Ag–Au nanoparticles and GOx has not only the good ability of electron transfer, but also the property of strong adsorption and good microenvironment provided by reverse micelle. So the current response of the electrode is much larger than that of the electrode with equal amount of enzyme but without nanoparticle. The current response curves of GOx electrodes with various molar ratios of Ag–Au composite particles shown in Fig. 6. From Fig. 6 we can see the current response of silver (75)–gold (25) (the number in metal colloids indicate molar percent of the metal salt incorporated) is the lowest. It may be due to the diameter of the silver (75)–gold (25) about 3 nm. They can absorb a large number of GOx molecules. But the adsorbed GOx molecules on the surface of Ag–Au particles may overlap with each other, and the enzyme molecules would be deformed severely, which would block the way of substrate and product diffuse between the electrode and the solution. So the current response was low. The current responses of silver (50)–gold (50) and the silver (25)–gold (75) were higher. Based on the structure of GOx reported by Chi et al. [28] and Losic et al. [29], GOx molecule is like a butterfly, which is about 7 nm. The scale of these particles is about 10 nm, which is about the same magnitude of the GOx molecule. They can enhance the current response of the electrode most significantly. We have studied on the stability of the immobilized GOx electrode with Ag–Au nanoparticles (Fig. 7, curve b). In 1 month, the measurements were conducted every day, and the current response of the last measurement was 90% that of the first measurement, while it was only 50% that of the first measurement using the electrode without Ag–Au nanoparticles (curve a).
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Fig. 5. Schematic representation of the formation process of the Ag–Au nanoparticle.
Fig. 6. Calibration curves of the electrodes containing various molar ratios of Ag–Au composite particles.
Ag–Au composite particles were probably phase-separated one. In the research of enzyme electrode, the current response of the glucose biosensor containing Ag–Au nanoparticles was much higher than that without nanoparticles. This was due to the strong adsorption property of Ag–Au nanoparticles and the good electrical conducting property of Ag and Au. The cyclic voltammograms prove that the Ag–Au nanoparticles can assist the electron transfer between the enzyme and the bulk electrode surface. Reversed micelles could provide a natural surrounding for the immobilization enzyme. So the Ag–Au particles could enhance the current response of the electrode strongly. In the various molar ratios of the Ag–Au composite particles, the current response of the electrode with silver (50)–gold (50) and silver (25)–gold (75) were higher than that of the electrode with silver (75)–gold (25), because the particle were about the same magnitude of the GOx molecule. After introducing the nanoparticles, the stability and sensibility of glucose biosensors were improved.
Acknowledgement This work has been supported by the National Natural Science Foundation of China (Project nos. 60372009 and 20301015).
References
Fig. 7. The stability of the electrode containing (a) no particles and (b) Ag–Au nanoparticles.
4. Conclusions Hydrophobic Ag–Au nanoparticles were prepared in reversed micelle. The UV–vis absorption spectra showed that
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Biographies Xiangling Ren received her master’s degree in 2004 from Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Currently she is an Assistant Professor in Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Her research programs involve preparation of controllable technology of metal nanoparticles and the development of their application to biosensors. Xianwei Meng received his PhD in 2001 from China. Currently he is an Associate Professor in Physics and Chemistry of Chinese Academy of main fields of interest are development of enzyme materials.
Si Chuan University, Technical Institute of Sciences, China. His biosensor using nano-
Fangqiong Tang received her bachelor degree in chemistry in 1970 from Beijing Normal University, China. Currently she is a Professor in Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Her main research interests are preparation and application of controllable technology of nano-materials, in particular the development of their application to biosensors.
Preparation of Ag–Au nanoparticle and its application to glucose biosensor Xiangling Ren, Xianwei Meng, Fangqiong Tang ∗ Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, Beijing 100101, PR China Received 29 October 2004; received in revised form 29 December 2004; accepted 6 February 2005 Available online 3 March 2005
Abstract In this paper, Ag–Au nanoparticles are produced in sodium-bis(2-ethylhexyl)-sulfosuccinate (AOT)–cyclohexane reverse micelle system. The properties of the obtained nanoparticles are characterized with transmission electron microscope (TEM) and UV–vis absorption spectrophotometer. Glucose biosensors have been formed with glucose oxidase (GOx) immobilized in Ag–Au sol. GOx are simply mixed with Ag–Au nanoparticles and crosslinked with a polyvinyl butyral (PVB) medium by glutaraldehyde. Then a platinum electrode is coated with the mixture. The effects of the various molar ratios of Ag–Au particles with respect to the current response and the stability of the GOx electrodes are studied. The experimental results indicate the current response of the enzyme electrode containing Ag–Au sol increase from 0.32 to 19 A cm−2 in the solution of 10 mM -d-glucose. In our study, the stability of enzyme electrodes is also enhanced. © 2005 Elsevier B.V. All rights reserved. Keywords: Ag–Au nanoparticles; Immobilization enzyme; Glucose oxidase; Biosensor
1. Introduction Glucose biosensor is one of the most important biosensors. The ability to obtain rapid, accurate and precise glucose measurement is essential to the appropriate administration of insulin therapy [1]. Glucose biosensors would offer the possibility of carrying out analytical and diagnostic procedures simply and rapidly [2]. They allow greater ease of use with opportunities for self-monitoring by user groups such as diabetics. With improved glucose control, it is possible that some of the long-term diabetic syndromes, such as retinal and kidney damage, would be avoided. In biosensor the biological substances are used as recognition elements, which can convert the analytic substances concentration with the measurable electrical signals. The immobilization of the enzymes is one of the crucial factors in biosensor preparation. Many methods have been used to immobilize enzymes and to improve the enzymatic activity [3–9]. Miniaturization is one of the important developments ∗
Corresponding author. Tel.: +86 10 64888064; fax: +86 10 64879375. E-mail address: [email protected] (F. Tang).
0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.02.016
in biosensor technology [10]. Miniaturization, however, may result in low current. To overcome this problem, nanoparticles have been introduced to the immobilization of the enzymes. Metal nanoparticles have many unique properties: large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability. They have possible applications in many areas such as nonlinear optical switching [11], immunoassay labeling [12], and Raman spectroscopy enhancement [13]. The immobilization of proteins in nanoparticles sol has been reported in Refs. [14–17]. Recently, Xiao et al. [18] report the reconstitution of an apo-flavoenzyme, apo-glucose oxidase, on a 1.4 nm gold nanocrystal functionalized with the cofactor flavin adenine dinucleotide and integrated into a conductive film that yields a bioelectrocatalytic system with exceptional electrical contact with the electrode support. Their work shows that electron transfer through the Au nanoparticles is much faster than electron transfer to O2 . In this paper, Ag–Au nanoparticle is introduced to the research of the glucose electrode. We use Ag–Au nanoparticle because silver is the best conductor among metals and gold has good biocompatibility. Different from other papers,
X. Ren et al. / Sensors and Actuators B 110 (2005) 358–363
we use the Ag–Au nanoparticle prepared in reverse micelle. Numerous articles have given the proof that enzymes have higher activity in reverse micelles than in aqueous systems [19–21]. Our experiments show that these Ag–Au particles can significantly enhance the current sensitivity of GOx enzyme electrodes.
2. Experimental 2.1. Chemicals and reagents Glucose oxidase (GOx) was extracted from Aspergillus niger (100 U mg−1 ; Toyobo Co. Ltd., Japan). Sodiumbis(2-ethylhexyl)-sulfosuccinate (AOT) was obtained from Nacalai Tesque (Kyoto Inc., Japan). -d-Glucose was from Sigma. Silver nitrate (AgNO3 ), chlorauric acid (HAuCl4 ) and sodium citrate were obtained from Beijing ShiJi Company. All the chemicals were used without further purification. All solutions were prepared with redistilled water. 2.2. Preparation of Ag–Au sol The preparation of the Ag–Au nanoparticles was achieved by mixing two sets of reverse micelle solutions (0.2 mol/kg AOT/cyclohexane), with AgNO3 and HAuCl4 ([AgNO3 ] + [HAuCl4 ] = 0.8 mM) solubilized in one set and sodium citrate ([Na3 C6 H5 O7 ] = 2 mM) as the reduction agent in the other set. The micelles in cyclohexane were stabilized by the surfactant. The concentration of the AOT and the micellar size were the same in both sets. These reversed micelles were then stirred thoroughly at room temperature until the solution turned to red. Varying the molar ratios of HAuCl4 and AgNO3 aqueous solutions, we could achieve different nanoparticles. 2.3. Characterization of the metal colloids The samples for transmission electron microscopy (TEM) were prepared by putting one drop of the Ag–Au colloid on a formvar-coated copper grid followed by drying in a desiccator. Electron micrographs were taken with a NEC JEM100CX electron microscope, operating at 100 kV. Absorption spectra were recorded using a Hitachi U-2001 diode array spectrophotometer at 1 cm path length. 2.4. Preparation of enzyme electrode In order to simplify production process of the enzyme electrode, the self-assembly method was used to prepare GOx electrode. Platinum wire with a diameter of 1 mm was polished and boiled in nitric acid for 5 min, washed in redistilled water and boiled in redistilled water. When it became cool, platinum wires was washed in acetone and redistilled water by ultrasound.
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The aqueous solution of six units GOx was added to a 10 ml beaker. An amount of 200 l Ag–Au nanoparticle sol was added to the GOx solution. Several minutes later, 2 ml PVB solution (2%) in anhydrous alcohol and 100 l of glutaraldehyde solution (1%) were added to the beaker. After the mixer was uniform, the platinum wire was dipped into the solution to a depth of 1 cm for several minutes and dried in air. A thin membrane was formed on the electrode. The electrode was stored in a refrigerator at 277 K. The enzyme electrode without Ag–Au nanoparticle, which was used as compare, was prepared by dipping platinum wire into the mixture containing GOx, PVB and glutaraldehyde. This method and amount were as same as described above. 2.5. Cyclic voltammetry Cyclic voltammetry studies used a PARC EG&G Model 283 potentiostat and three electrode cells equipped with a Pt-wire as counter electrode and a Ag/AgCl electrode as reference electrode. The applied scan rate was 50 mV/s. The cyclic voltammograms was taken at 33 mmol/L glucose concentrations. 2.6. Amperometric measurement The sensitivity of the glucose biosensor was tested by measuring the current response. The experiment was carried out using a two-electrode cell consisting of an enzyme working electrode and a reference electrode of Ag/AgCl [22]. Measurements were conducted in a 5 ml phosphate buffer (KHPO4 −NaOH, pH 6.8) cell at 308 K. A fixed potential of 0.4 V was applied to this electronic cell. Firstly, working electrode and reference electrode were put into a phosphate solution at 308 K. When background current reached a constant value, different concentrations (from 2.7 to 33 mM) of -d-glucose solution were added. Then response current was noted down, and background current was deducted, and the correlation between response currents and different concentrations of glucose solution was obtained.
3. Results and discussion 3.1. Preparation of Ag–Au particles The Ag–Au particles were prepared with the various mixtures of HAuCl4 and AgNO3 aqueous solutions. Except the pure silver, the color of the other solutions turned red at last. Transmission electron micrographs of the Ag–Au nanoparticles are in Fig. 1. Fig. 2 shows the UV–vis absorption spectra of Ag–Au composite particles. A remarkable shift in the absorption band at 530 nm was observed for silver (75)–gold (25); the number in metal colloids indicate molar percent of the metal salt incorporated. But with increasing molar ratio of HAuCl4 , the absorption peak
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Fig. 1. Transmission electron micrographs of Ag–Au nanoparticles: (a) silver (75)–gold (25), the average diameter of these particles is about 3.3 ± 0.8 nm; (b) silver (50)–gold (50), the average diameter of these particles is about 9.8 ± 1.5 nm; (c) silver (25)–gold (75), the average diameter of these particles is about 10.5 ± 1.7nm.
at 530 nm became broad and slightly shifted to longer wavelength. It is known that silver and gold are miscible in all proportions due to the almost identical lattice constants. Currently the formation of Ag–Au particles has three possibilities: (1) core-shell particles, silver core with gold shell or vice versa; (2) alloy particles; (3) phase-separated composites, which are made partly of silver and partly of gold. If core-shell particles were formed, they would consist of a gold core with a silver shell, because the reduction rate for gold is much faster than that for silver. The observed spectra are different from the theoretical spectra of the gold core with a silver shell obtained using the equations in the form given by
Bohren and Huffman [23]. The theoretical spectra show that the single plasmon band splits into two peaks, one of which is located at a shorter wavelength than that of silver particles and the other is located at a slightly shorter wavelength than that of gold particles with increasing molar fraction of silver. Torigoe and co-workers [24,25] have given the theoretical spectra of the alloy particles using the same way. Recently, Mallin and Murphy [26] have succeeded in making Ag–Au alloy nanoparticles and given the UV–vis absorption spectra of those. These spectra show that the plasmon band remains in a single peak and shifts continuously from 400 to 520 nm with increasing molar ratio of gold. They are different from Fig. 2. Although this disagreement in the spectra is not interpreted adequately at the present time, it seems that the Ag–Au nanoparticles in our study are neither coreshell particles nor alloy particles. So the Ag–Au composite particles prepared in this study are probably phase-separated composites. 3.2. Ag–Au nanoparticles used in enzyme electrode
Fig. 2. UV–vis absorption spectra of Ag–Au nanoparticles with various molar ratios of Ag and Au.
3.2.1. The cyclic votammograms for the electrode The cyclic votammograms for the electrode in the glucose were shown in Fig. 3. The bare platinum electrode has good electric conductivity, so it exhibits peaks (curve 1). After the electrode modified by PVB, an obvious decrease in the peaks were observed (curve 2). The reason is that the PVB membrane can act as the inert electron and mass transfer blocking layer, and it hinders the diffusion of electron toward the electrode surface. When Ag–Au nanoparticles were
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Fig. 3. Cyclic voltammograms of the electrodes immersed in 33 mmol/L glucose solution; (1) bare Pt electrode; (2) the electrode with PVB film; (3) the electrode with PVB film added GOx and Ag–Au nanoparticles; a Ag/AgCl electrode used as reference electrode; scan rate 50 mV/s.
embedded in the PVB membrane, the electrode shows peaks again (curve 3). This may be due to Ag–Au nanoparticles were distributed throughout the sol–gel network and formed a continuous array of Ag–Au nanoparticles on the electrode. It is evident from the figure that Ag–Au nanoparticles have a marked influence on the barrier property of PVB-modified electrodes and the Ag–Au nanoparticles can assist the electron transfer between the enzyme and the bulk electrode surface. 3.2.2. The current response curves of the immobilized GOx electrode with Ag–Au nanoparticles Fig. 4 shows the current response curves of GOx electrodes without and with Ag–Au nanoparticles on the current response of glucose. When glucose concentration is 10 mM, the current density of the electrode without nanoparticle is 0.32 A cm−2 , and that of the electrode with Ag–Au particle is 19 A cm−2 . It can be seen that nanoparticles can significantly enhance the current response of the elec-
Fig. 4. Calibration curves of the electrodes containing (a) no particles and (b) Ag–Au nanoparticles.
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trode. It is mainly due to the large surface area and the high surface free energy of Ag–Au nanoparticles, which can make GOx inevitably adsorbed on the surface of nanoparticles. Our previous research about SiO2 nanoparticles [16] has given the proof. From cyclic voltammograms, we know Ag–Au nanoparticles can assist the electron transfer between the GOx and the platinum wire surface. The capability of the GOx enzyme electrode can be improved. The Ag–Au nanoparticle we prepared is in reverse micelle. Fig. 5 shows the schematic representation of the Ag–Au particle’s formation. It is very probable that the GOx solution can be solubilized into the reverse micelle. Enzyme molecule has a hydrated shell. It is the requirement to keep the activity of enzyme [27]. The organic reagents can make GOx lose its hydrated shell. The reverse micelle provides good reactors for the enzyme adsorption on the nanoparticles’ surface, which can decrease the contact of GOx molecules with organic reagents and protect the hydrated shell. On this condition there is very little free water. It can decrease the precipitation of PVB and avoid the severe action between enzyme and PVB, therefore increase the activity and stability of immobilized GOx. The electrode made from Ag–Au nanoparticles and GOx has not only the good ability of electron transfer, but also the property of strong adsorption and good microenvironment provided by reverse micelle. So the current response of the electrode is much larger than that of the electrode with equal amount of enzyme but without nanoparticle. The current response curves of GOx electrodes with various molar ratios of Ag–Au composite particles shown in Fig. 6. From Fig. 6 we can see the current response of silver (75)–gold (25) (the number in metal colloids indicate molar percent of the metal salt incorporated) is the lowest. It may be due to the diameter of the silver (75)–gold (25) about 3 nm. They can absorb a large number of GOx molecules. But the adsorbed GOx molecules on the surface of Ag–Au particles may overlap with each other, and the enzyme molecules would be deformed severely, which would block the way of substrate and product diffuse between the electrode and the solution. So the current response was low. The current responses of silver (50)–gold (50) and the silver (25)–gold (75) were higher. Based on the structure of GOx reported by Chi et al. [28] and Losic et al. [29], GOx molecule is like a butterfly, which is about 7 nm. The scale of these particles is about 10 nm, which is about the same magnitude of the GOx molecule. They can enhance the current response of the electrode most significantly. We have studied on the stability of the immobilized GOx electrode with Ag–Au nanoparticles (Fig. 7, curve b). In 1 month, the measurements were conducted every day, and the current response of the last measurement was 90% that of the first measurement, while it was only 50% that of the first measurement using the electrode without Ag–Au nanoparticles (curve a).
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Fig. 5. Schematic representation of the formation process of the Ag–Au nanoparticle.
Fig. 6. Calibration curves of the electrodes containing various molar ratios of Ag–Au composite particles.
Ag–Au composite particles were probably phase-separated one. In the research of enzyme electrode, the current response of the glucose biosensor containing Ag–Au nanoparticles was much higher than that without nanoparticles. This was due to the strong adsorption property of Ag–Au nanoparticles and the good electrical conducting property of Ag and Au. The cyclic voltammograms prove that the Ag–Au nanoparticles can assist the electron transfer between the enzyme and the bulk electrode surface. Reversed micelles could provide a natural surrounding for the immobilization enzyme. So the Ag–Au particles could enhance the current response of the electrode strongly. In the various molar ratios of the Ag–Au composite particles, the current response of the electrode with silver (50)–gold (50) and silver (25)–gold (75) were higher than that of the electrode with silver (75)–gold (25), because the particle were about the same magnitude of the GOx molecule. After introducing the nanoparticles, the stability and sensibility of glucose biosensors were improved.
Acknowledgement This work has been supported by the National Natural Science Foundation of China (Project nos. 60372009 and 20301015).
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Fig. 7. The stability of the electrode containing (a) no particles and (b) Ag–Au nanoparticles.
4. Conclusions Hydrophobic Ag–Au nanoparticles were prepared in reversed micelle. The UV–vis absorption spectra showed that
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Biographies Xiangling Ren received her master’s degree in 2004 from Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Currently she is an Assistant Professor in Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Her research programs involve preparation of controllable technology of metal nanoparticles and the development of their application to biosensors. Xianwei Meng received his PhD in 2001 from China. Currently he is an Associate Professor in Physics and Chemistry of Chinese Academy of main fields of interest are development of enzyme materials.
Si Chuan University, Technical Institute of Sciences, China. His biosensor using nano-
Fangqiong Tang received her bachelor degree in chemistry in 1970 from Beijing Normal University, China. Currently she is a Professor in Technical Institute of Physics and Chemistry of Chinese Academy of Sciences, China. Her main research interests are preparation and application of controllable technology of nano-materials, in particular the development of their application to biosensors.