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A new glucose sensor based on encapsulated glucose oxidase within organically modified sol–gel glass
Sensors and Actuators B 60 Ž1999. 83–89 www.elsevier.nlrlocatersensorb
A new glucose sensor based on encapsulated glucose oxidase within organically modified sol–gel glass P.C. Pandey ) , S. Upadhyay, H.C. Pathak Department of Chemistry, Banaras Hindu UniÕersity, Varanasi 221005, India Received 15 April 1999; received in revised form 19 April 1999; accepted 23 April 1999
Abstract A non-mediated glucose biosensor is reported based on encapsulated glucose oxidase ŽGOD. within the composite sol–gel glass, which is prepared using optimum concentrations of 3-aminopropyltriethoxy silane, 2-Ž3, 4-epoxycyclohexyl.-ethyltrimethoxy silane, GOD dissolved in double distilled water and HCl. A white, smooth film of sol–gel glass with controlled thickness is also prepared at the surface of a Pt disk electrode without GOD to study the electrochemistry of ferrocene monocarboxylic acid at the surface of the modified electrode. The electrochemistry of ferrocene monocarboxylic acid at composite sol–gel glass electrode with varying thickness is reported. The GOD-immobilized film over the Pt disk surface shows a yellow colour. The new sol–gel glass in the absence and the presence of GOD is characterized by scanning electron microscopy ŽSEM.. The enzyme-immobilized film of different thickness is made using varying concentrations of soluble sol–gel components applied to the well of the Pt disk electrode. The enzyme is cross-lined with the 3-aminopropyltriethoxysilane, one of the composite component of sol–gel glass using glyoxal at 48C for 4 h. The response of non-mediated enzyme sensor is studied based on cyclic voltammetry and amperometric measurements. A typical amperometric response of the enzyme sensor having varying thickness of the modified sol–gel glass film is reported. The variation of the response time as a function of the film thickness is reported. The stability of cross-linked GOD to sol–gel glass is found to be more than a month without loss of enzymatic activity when the enzyme sensor is stored at 48C. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Glucose; Sol–gel; Glucose oxidase
1. Introduction The recent reports on the synthesis of sol–gel glasses w1–6x have received widespread attentions because of its application in various directions. One of the potential application of such materials is in the development of sensors particularly for attaching the sensing material to the surface of physico-chemical transducers. A number of publications are available in the literature on the applications of sol–gel glass for the development of optical and electrochemical sensors. The development of electrochemical biosensor involves the coupling of biological components with the polarizable or non-polarizable electrodes. The use of sol–gel glass for the development of electro-
) Corresponding author. Tel.: q91-542-317-745; fax: q91-542-317074; E-mail: [email protected]
chemical biosensors have received great attention because of its possible applications in commercialization. The development of such biosensors based on sol–gel glass is currently restricted mainly due to two major problems: Ž1. the requirement of controlled gelation of soluble sol–gel components at ambient conditions, Ž2. preparation of sol– gel glass of smooth surface, controlled thickness and porosity. Additionally, the stability of biological element within the sol–gel network is another need to develop such sensors at commercial scale. Apparently, the synthesis of suitable bio-compatible sol–gel glass of desired thickness and porosity is of considerable interest. The soluble materials leading to the formation of sol–gel glasses are the derivatives of alkoxysilane. These alkoxysilanes in acidic and some time in basic medium generate a solid network whose physical structure can be comparable to conventional glass. However, research is needed to synthesize such sol–gel glasses suitable for the better performance as sensors and reactors
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P.C. Pandey et al.r Sensors and Actuators B 60 (1999) 83–89
of practical significance. The application of these glasses in sensors requires controlled synthesis of solid-state network with desired porosity and thickness. Additionally, the availability of a suitable group within the solid-state network provides an advantage for the cross-linking of the sensing element to the solid-state network. Recently, we reported w3x a glucose biosensor based on sol–gel glass using trimethoxysilane and 3-glycidoxypropyltrimethoxysilane with and without glucose oxidase ŽGOD.. The sol–gel glass without protein shows a nice network with symmetrical distribution of the pores whereas the introduction of GOD results irregular distribution of the protein throughout the gel network together with the least porosity. The present investigation is attributed to the fabrication of such sol–gel glass using a mixture of two silanes, 3-aminopropyltriethoxysilane and 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silane. These silanes under optimum conditions generate the solid-state network of desired configuration for the construction of biosensors and is reported in the present research work. The availability of the amino-group in one of the composite sol–gel glass components provides an extra event for cross-linking with proteinrenzyme using bifunctional reagent ŽGlyoxal.. Two types of the composite sol–gel glass have been developed in the present investigation: Ž1. sol–gel glass of controlled thickness and porosity without enzyme ŽGOD., Ž2. sol–gel glass of controlled thickness and porosity with enzyme GOD. The electrochemistry of ferrocene monocarboxylic acid is studied on system 1. The performance of system 2 as a glucose biosensor is studied. The performance of non-mediated glucose sensors based on cyclic voltammetry and the typical amperometric responses of the biosensors having varying thickness and porosity are reported.
2. Experimental 2.1. Materials and methods 3-Aminopropyltriethoxy silane was obtained from Aldrich; 2-Ž3,4-Epoxycyclohexyl.-ethyltrimethoxy silane was obtained from United Chemical Technologies, Petrarche Silanes and Silicones, Bristol, PA, USA; GOD was obtained from Sigma. All other chemical employed were of analytical grade. 2.2. Preparation of organically modified sol–gel glass electrodes The electrode body used for the preparation of composite sol–gel glass modified electrodes was similar as described in an earlier publication w7x made from Teflon
containing platinum base with a recessed depth of 2 mm. The new material is prepared using optimum concentrations of 3-aminopropyltriethoxy silane Ž70 ml., 2-Ž3,4epoxycyclohexyl.-ethyltrimethoxy silane Ž20 ml., 4 mg GOD dissolved in double distilled water Ž700 ml., 0.1 M HCl Ž5 ml.. The resulting mixture is stirred thoroughly and the desired amount of the homogeneous solution is added to the well Ž0.0314 cm2 . of the specially designed electrode body. The gelation is allowed to occur at 258C for 30 h. A smooth, very thin GOD-immobilized film of sol–gel glass appeared on the Pt surface as evidenced by SEM and electrochemical measurements. The resulting enzyme-immobilized sol–gel film is then incubated in 1% glyoxal for 4 h at 48C. The electrode obtained in this manner is washed with 0.1 M phosphate buffer, pH 7.0 several times and stored in 0.1 M phosphate buffer, pH 7.0 at 48C when not in use. Some of the glucose sensors made following such procedures were stored at room temperature Ž268C.. A control sol–gel glass without the enzyme was also prepared to study the electrochemistry of redox species Žferrocene. on the sol–gel glass modified electrode. The composition of the control sol–gel glass was similar to that of the sol–gel glass used for glucose sensor without the presence of GOD. 2.3. Electrochemical measurements The electrochemical measurements were performed with a Solartron Electrochemical Interface ŽSolartron 1287 Electrochemical Interface, UK. connected to a PC through the serial port. The electrode responses were recorded and plotted with a Printer. A one compartment cell with a working volume of 4 ml and a sol–gel glass modified working electrode, AgrAgCl reference electrode and a platinum foil auxiliary electrode were used for the measurements. The cyclic voltammetry using GOD modified sol–gel glass electrode were studied between 0 V and 1 V vs. AgrAgCl. The amperometric measurements using GOD-immobilized sol–gel modified electrode was operated at 0.70 V vs. AgrAgCl. The experiments were performed in phosphate buffer Ž0.1 M, pH 7. employing both types of modified electrodes.
3. Results and discussion 3.1. Physical characterization of organically modified sol– gel glass electrode The physical characteristics of the sol–gel glass with and has been examined using various composition of 3-aminopropyltriethoxy silane, 2-Ž3,4-epoxycyclohexyl.ethyltrimethoxy silane, enzyme dissolved in water and
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istics of the sol–gel matrix. The colour of the enzyme-immobilized sol–gel film is yellow whereas the colour is white in the absence of enzyme. The smooth sol–gel film without cracking and having better performance when used as sensor was obtained using the optimum concentrations reported in Section 2. Another important requirement of the sensors based on such design is the coupling of modified sol–gel film and Pt surface which contribute significantly to the performance and storage stability of the biosensors. The present modified sol–gel film has been found to be strongly attached and is not easily removable from the Pt surface. The scanning electron micrographs of these two types of organically modified sol–gel glasses Žone in the absence of GOD and other in the presence of GOD. are shown in Fig. 1a, b, c. Fig. 1a shows the SEM of the sol–gel glass without enzyme at 1000 magnification. The surface structure shows a smooth feature with relatively better homogeneous distribution of sol–gel domains. Fig. 1b and c show the SEM of GOD-encapsulated sol–gel glass at 500 and 2000 magnification, respectively. The surface structure shows a smooth feature with relatively better distribution of GOD within organically modified sol–gel glass network. 3.2. Electrochemistry of ferrocene monocarboxylic acid on unmodified and control sol–gel glass modified electrodes A control sol–gel glass without the immobilized enzyme was made to study the electrochemical behavior of ferrocene monocarboxylic acid at its surface. The cyclic voltammetry of ferrocene monocarboxylic acid was studied on following three electrodes: Ži. plane cleaned Pt disk electrode, Žii. sol–gel modified Pt disk electrode made
Fig. 1. SEM of organically modified sol–gel glass; Ža. in the absence of enzyme; Žb. in the presence of enzyme at 500 magnification; Žc. in the presence of enzyme at 2000 magnification.
HCl. The content of 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silane significantly affects the physical character-
Fig. 2. Cyclic voltammograms of 5 mM ferrocene monocarboxylic acid in 0.1 M phosphate buffer ŽpH 7.0. on: Ž1. plane Pt electrode, Ž2. sol–gel glass modified electrode without enzyme made using 14 ml of soluble gel components and Ž3. sol–gel glass modified electrode without enzyme made using 8 ml of soluble gel components, at 258C at a scan rate of 5 mVrs.
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Fig. 3. Cyclic voltammogram of GOD-immobilized composite sol–gel glass in the absence Ž1. and the presence Ž2. of 50 mM glucose acid in 0.1 M phosphate buffer pH 7.0 at the scan rate of 5 mVrs.
using 14 ml of soluble sol–gel component into the well of Pt disk electrode and Žiii. sol–gel modified Pt disk using 8 ml of soluble sol–gel components into the well of the Pt disk electrode. The colour of sol–gel without enzyme was white, however, the porosity under these two conditions was different. Fig. 2 shows the cyclic voltammograms of cleaned platinum disk electrode in the presence of 5 mM ferrocene Žcurve 1.. Curve 2 of Fig. 1 shows the control sol–gel glass film, prepared using 14 ml of homogeneous solution without enzyme into the well of the Pt disk electrode, in 5 mM ferrocene solution. Curve 3 shows the electrochemistry of ferrocene solution made using 8 ml of homogenous solution of the sol–gel glass components without enzyme into the well of the electrode body. Curve 1 shows the well-defined reversible electrochemistry of ferrocene as described in the literature. Whereas curves 2 and 3 show that although voltammograms are reversible in nature, however, there is a diffusion-limited condition prevailing at the sol–gel glass interface. The porosity in the sol–gel glass films made using 14 ml and 8 ml of homogeneous solution of sol–gel components can also be analyzed from these two curves. The film made using 8 ml of the sol–gel components showing curve 3 is relatively thicker than the film 2 made using 14 ml of sol–gel components resulting curve 2 ŽFig. 2.. When chemical modifier such as metal dispersion, water-soluble polymers and proteins are added to the materials, the resulting electrodes became more hydrophilic and subsequently, alter the water contact angle which manifests the wettability. It has been reported w2x that a blank sol–gel electrode without any hydrophilic modifier shows a highest water contact angle Ž808. and in turn, lowest wettability Žnot amenable for nitrogen adsorption analysis., whereas the sol–gel electrode with all hydrophilic modifiers Žcarbon, polyŽethylene glycol, protein and Pd-GOD or even water itself. shows the lowest water contact angle Ž428. and highest wettability Ž42 m2rg.. An increase in wetted area
increases the wetted conductive surface accessible to the solution and also the corresponding electrochemically active area and capacitive current. On the other hand, the unwetted area does not contribute to the capacitive or faradic currents. Although the composition of both films made using 14 ml and 8 ml of sol–gel glass precursors are the same, however, after the gelation for strictly the same period under a similar surface area might bring about a change in water wettability condition due to a change in the thickness along with hydrophilic Žwater. content. The relatively better wetted surface area of the film made using 8 ml of sol–gel precursors provide a relatively better diffusion-limited condition as compared to sol–gel glass electrode made using 14 ml of the sol–gel precursors. Fig. 2 was recorded using aqueous solution of ferrocene and freshly prepared organically modified sol–gel glass electrodes.
Fig. 4. Ža. Cyclic voltammograms of 5 mM ferrocene monocarboxylic acid in 0.1 M phosphate buffer pH 7.1 on GOD-encapsulated sol–gel glass modified electrode made using 700 ml of water at 258C at the scan rate of 3 mVrs, 6 mVrs, 10 mVrs, 20 mVrs, 50 mVrs, and 100 mVrs. The inset shows the plot of anodic peak current vs. the square root of scan rate. Žb. Cyclic voltammogram of GOD-encapsulated sol–gel glass made using 700 ml of water with the absence Ž1. and the presence Ž2. of 150 mM glucose in 0.1 M phosphate buffer pH 7.1 containing 5 mM ferrocene monocarboxylic acid at the scan rate of 5 mVrs.
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3.3. Electrochemistry of glucose oxidase-immobilized sol– gel glass modified electrodes The GOD-immobilized sol–gel glasses of two different compositions were made which differ only in water content. The first one is made with 4 mg of GOD dissolved in 500 ml of double distilled water whereas the second one is made using same amount of enzyme dissolved in 700 ml water. The cyclic voltammograms of the sol–gel glass made using 700 ml of water in the absence Ž1. and the presence Ž2. of 60 mM glucose is shown in Fig. 3. The large increase in anodic current after the addition of glucose shows the performance of the glucose sensor based on sol–gel glass. The increase in anodic current is attributed to the performance of non-mediated glucose sensor based on the oxidation of hydrogen peroxide. We have also studied the electrochemistry of ferrocene monocarboxylic acid at the surface of GOD-encapsulated sol–gel glass made using 700 ml of water. Before taking the electrochemical measurements, the enzyme-encapsulated sol–gel glass was incubated overnight in 5 mM aqueous ferrocene solution Ž0.1 M phosphate buffer pH 7.1.. Fig. 4a shows the cyclic voltammograms of enzyme-encapsulated sol–gel glass electrode at the scan rates of 3 mVrs, 6 mVrs, 10 mVrs, 20 mVrs, 50 mVrs and 100 mVrs in 0.1 M phosphate buffer pH 7.0. There is reversible electrochemistry of ferrocene with a peak separation of 86 mV. The inset shows the plot of peak current vs. the square root of scan rate. A linear relation is recorded and the straight line does not pass through the origin which suggests that the system is poorly diffusion controlled. In order to study the mediated response between sol–gel glass encapsulated GOD and soluble ferrocene, we studied the cyclic voltammograms of enzyme electrode in 5 mM ferrocene monocarboxylic solution in 0.1 M phosphate buffer ŽpH 7.1. in
Fig. 5. Typical response curves of the sol–gel glass modified electrode together with GOD with varying water contents; Ž1. 3- Aminopropyltriethoxy silanes 70 ml; 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silanes 20 ml; 4 mg GOD dissolved in 500 ml water; 0.1 M HCls 5 ml Žinset. and Ž 2 . 3-A m inopropyltriethoxy silane s 70 m l; 2- Ž 3,4epoxycyclohexyl.-ethyltrimethoxy silanes 20 ml; 4 mg GOD dissolved in 700 ml water; 0.1 M HCls 5 ml at 0.70 V vs. AgrAgCl.
Fig. 6. Typical response curve of the GOD-immobilized sol–gel glass modified electrode made with 700 ml water. The results show the subsequent addition of increasing concentrations of glucose. The inset shows a typical response curve.
the absence and presence of 150 mM glucose ŽFig. 4b.. There is an increase in anodic current in the presence of glucose showing the heterogeneous-mediated enzyme catalyzed reaction. 3.4. Amperometric response of enzyme-immobilized sol–gel glasses based glucose sensor The typical chrono-amperometric responses at 0.70 V vs. AgrAgCl of the glucose biosensors which differ in water contents are recorded in Fig. 5. Curve 1, as recorded in the inset, shows the responses of the biosensor made with 500 ml water on the subsequent addition of two concentrations of glucose Ž0.6 mM,; 1.6 mM., whereas curve 2 represents the response of the glucose sensor, made using 700 ml of water on the addition of glucose Ž0.6 mM.. The glucose biosensors made under these two conditions although both show the low background current to the order of - 100 nA, however, these sensors differ in the following aspects: Ž1. the response time of the biosensor with low water content is ) 3 min with a relatively small magnitude of the anodic current; Ž2. the response time of the biosensor made with high water content is - 1 min with large magnitude of anodic current on the addition of same amount of glucose. These results clearly suggest that the sol–gel film is relatively thicker and least porous made using low water content which can again be analyzed by water contact angle and wettability of the sol–gel glass as discussed above. Additionally, the incorporation of protein within the sol–gel matrix ŽFig. 1b and c. also increases the water wetted area within which electrochemical reactions take place. Another important conclusion from these differences on amperometric responses can be made the kinetically controlled situation prevailing at the sensor interface. The amperometric response depends on the production of hydrogen peroxide and its subsequent electrochemical oxidation at 0.7 V vs. AgrAgCl. It is
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strate concentrations. The inset in Fig. 7 shows the linear range of calibration curve. The stability of the new GOD-encapsulated sol–gel glass network is determined under two conditions. In the first case, the enzyme electrode was stored in 0.1 M phosphate buffer pH 7.0 at 48C whereas in the second case, the enzyme electrode was stored at room temperature in the same buffer. The stability of the enzyme electrode stored under the first condition is relatively good without loss of the amperometric response after 2 months. Under the second storage condition, the response is consistent without loss for 20 days. Fig. 7. Calibration curve for glucose analysis calculated from the data recorded in Fig. 6 in phosphate buffer pH 7.0 at 0.7 V vs. AgrAgCl. The inset shows the linear range of the calibration curve.
important to understand the concentration of oxygen at the site of the enzymatic reaction. The formation of hydrogen peroxide depends on the diffusion of glucose through the sol–gel glass within which GOD is distributed. Along with the substrate diffusion, the dissolved oxygen in buffer and the glucose solution is only available oxygen at the site of enzymatic reaction. Since sol–gel is rigid and has the least porous structure within which the enzyme is caged, the possibility of restricted diffusion of oxygen within the sol–gel is expected. The typical response as shown in Fig. 6 indicates the kinetic limitation of the oxygen-based enzymatic reaction. The lesser the porosity, the lesser the rate of oxygen diffusion within the sol–gel glass. Although the concentration of active enzyme within the sol–gel matrix is relatively high as compared to the response recorded in curves 1 and 2 ŽFig. 5.. Additionally, it can also be concluded from the data recorded in Fig. 6 that there is a relatively large increase in anodic current on the small addition of glucose justifying this conclusion since the enzyme present at the surface does not require diffusion-limited condition of oxygen. On the other hand, subsequent additions show a relatively less response as compared to the concentrations of glucose keeping constant the reported value of the K m for GOD. Hence, the present sensor shows a linear response at low concentrations of glucose under which the surface enzyme does not get saturated and the subsequent response becomes kinetically controlled as a result of restricted diffusion of oxygen. A typical response curve of the enzyme sensor made using 700 ml content of the sol–gel glass is shown in Fig. 6. The calibration curve for the glucose analysis prepared from the data recorded in Fig. 6 is shown in Fig. 7. The detection limit of the sensor is - 0.1 mM. The sensitivity of the analysis is found to be 0.78 " 0.01 mArmM. The nature of the response curve ŽFig. 7. also supports the diffusion-limited condition of oxygen-based enzymatic reaction since non-linearity is started at relatively low sub-
4. Conclusion We report the fabrication of a new composite sol–gel glass using 3-aminopropyltriethoxysilane and 2-Ž3,4epoxycyclohexyl.-ethyltrimethoxysilane, in the presence of distilled water and HCl. The resulting silane provides a very smooth surface with a rigid porous structure. The enzyme-modified sol–gel glass is reported to construct an amperometric biosensor for glucose. Under optimum composition of the sol–gel glass ingredients, a very smooth and thin stably adhered to the Pt surface was obtained with better porosity and regular distribution of enzyme within the solid-state network which contributed to the better performance of the biosensors as discussed above.
Acknowledgements The authors are thankful to UGC, New Delhi for financial assistance.
References w1x V. Glezer, O. Lev, Sol–gel vanadium pentaoxide glucose biosensor, J. Am. Chem. Soc. 115 Ž1993. 2533. w2x S. Sampath, O. Lev, Inert metal-modified, composite ceramic-carbon, amperometric biosensors: renewable, controlled reactive layer, Anal. Chem. 68 Ž1996. 2015. w3x U. Narang, P.N. Prasad, F.V. Bright, K. Ramanathan, N.D. Kumar, B.D. Malhotra, S. Chandra, Glucose biosensor based on a sol–gel derived plateform, Anal. Chem. 66 Ž1994. 3139. w4x P.C. Pandey, S. Singh, B. Upadhyay, H.H. Weetall, P.K. Chen, Reversal in the kinetics of M-state decay of D96N bacteriorhodopsin; probing of enzyme catalyzed reaction, Sensors and Actuators B 35–36 Ž1996. 470. w5x Y. Tatsu, K. Yamashita, M. Yamaguchi, S. Yamamura, H. Yamamoto, S. Yoshikawa, Entrapment of glucose oxidase in silica gel by sol–gel method and its application to glucose sensor, Chem. Lett., 1992, p. 1615. w6x P.C. Pandey, S. Upadhyay, H.C. Pathak, A new glucose biosensor based on sandwich configuration of organically-modified sol–gel glass, Electroanalysis 11 Ž1999. 59. w7x P.C. Pandey, R. Prakash, Polyindole modified potassium ion sensor using dibenzo-18-crown-6 mediated PVC membrane, Sensors and Actuators 46 Ž1998. 61.
P.C. Pandey et al.r Sensors and Actuators B 60 (1999) 83–89 Prem Chandra Pandey received his MSc degree in Chemistry in 1980 and PhD in 1986, both from Gorakhpur University. Since 1988, he is a lecturer in the Analytical Chemistry division at Banaras Hindu University and subsequently worked as Reader since 1996 in the same division. He is a member of the Bioelectrochemical society and the Indian Chemical society. He has worked as a visiting scientist at Ecole des Mines, France and the National Institute of Standards and Technology, Gaithersburg, USA. His current interests include mediated, non-mediated and electrocatalytic biosensors, optical biosensors, ion-selective electrodes, chemically modified electrodes, electropolymerization, re-chargeable batteries, dynamics of membrane processes both in linear and non-linear regime, flow injection analysis and photo-chemistry.
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Sanjay Upadhyay obtained his PhD degree in 1993 from Banaras Hindu University on ‘‘liquid membrane transport’’. Since 1994, he has been working as Research Associate at the Department of Chemistry. He is working on electrochemical biosensors in the group of Dr. P.C. Pandey.
Harish C. Pathak received his MSc in Chemistry from Poorvanchal University in 1994. Since 1996, he has been a student of Banaras Hindu University and has been working for his PhD degree in the group of Dr. P.C. Pandey. His current interests include electrochemical biosensors and photophysical properties of bacteriorhodopsin molecule.
A new glucose sensor based on encapsulated glucose oxidase within organically modified sol–gel glass P.C. Pandey ) , S. Upadhyay, H.C. Pathak Department of Chemistry, Banaras Hindu UniÕersity, Varanasi 221005, India Received 15 April 1999; received in revised form 19 April 1999; accepted 23 April 1999
Abstract A non-mediated glucose biosensor is reported based on encapsulated glucose oxidase ŽGOD. within the composite sol–gel glass, which is prepared using optimum concentrations of 3-aminopropyltriethoxy silane, 2-Ž3, 4-epoxycyclohexyl.-ethyltrimethoxy silane, GOD dissolved in double distilled water and HCl. A white, smooth film of sol–gel glass with controlled thickness is also prepared at the surface of a Pt disk electrode without GOD to study the electrochemistry of ferrocene monocarboxylic acid at the surface of the modified electrode. The electrochemistry of ferrocene monocarboxylic acid at composite sol–gel glass electrode with varying thickness is reported. The GOD-immobilized film over the Pt disk surface shows a yellow colour. The new sol–gel glass in the absence and the presence of GOD is characterized by scanning electron microscopy ŽSEM.. The enzyme-immobilized film of different thickness is made using varying concentrations of soluble sol–gel components applied to the well of the Pt disk electrode. The enzyme is cross-lined with the 3-aminopropyltriethoxysilane, one of the composite component of sol–gel glass using glyoxal at 48C for 4 h. The response of non-mediated enzyme sensor is studied based on cyclic voltammetry and amperometric measurements. A typical amperometric response of the enzyme sensor having varying thickness of the modified sol–gel glass film is reported. The variation of the response time as a function of the film thickness is reported. The stability of cross-linked GOD to sol–gel glass is found to be more than a month without loss of enzymatic activity when the enzyme sensor is stored at 48C. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Glucose; Sol–gel; Glucose oxidase
1. Introduction The recent reports on the synthesis of sol–gel glasses w1–6x have received widespread attentions because of its application in various directions. One of the potential application of such materials is in the development of sensors particularly for attaching the sensing material to the surface of physico-chemical transducers. A number of publications are available in the literature on the applications of sol–gel glass for the development of optical and electrochemical sensors. The development of electrochemical biosensor involves the coupling of biological components with the polarizable or non-polarizable electrodes. The use of sol–gel glass for the development of electro-
) Corresponding author. Tel.: q91-542-317-745; fax: q91-542-317074; E-mail: [email protected]
chemical biosensors have received great attention because of its possible applications in commercialization. The development of such biosensors based on sol–gel glass is currently restricted mainly due to two major problems: Ž1. the requirement of controlled gelation of soluble sol–gel components at ambient conditions, Ž2. preparation of sol– gel glass of smooth surface, controlled thickness and porosity. Additionally, the stability of biological element within the sol–gel network is another need to develop such sensors at commercial scale. Apparently, the synthesis of suitable bio-compatible sol–gel glass of desired thickness and porosity is of considerable interest. The soluble materials leading to the formation of sol–gel glasses are the derivatives of alkoxysilane. These alkoxysilanes in acidic and some time in basic medium generate a solid network whose physical structure can be comparable to conventional glass. However, research is needed to synthesize such sol–gel glasses suitable for the better performance as sensors and reactors
0925-4005r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 2 4 6 - 4
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P.C. Pandey et al.r Sensors and Actuators B 60 (1999) 83–89
of practical significance. The application of these glasses in sensors requires controlled synthesis of solid-state network with desired porosity and thickness. Additionally, the availability of a suitable group within the solid-state network provides an advantage for the cross-linking of the sensing element to the solid-state network. Recently, we reported w3x a glucose biosensor based on sol–gel glass using trimethoxysilane and 3-glycidoxypropyltrimethoxysilane with and without glucose oxidase ŽGOD.. The sol–gel glass without protein shows a nice network with symmetrical distribution of the pores whereas the introduction of GOD results irregular distribution of the protein throughout the gel network together with the least porosity. The present investigation is attributed to the fabrication of such sol–gel glass using a mixture of two silanes, 3-aminopropyltriethoxysilane and 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silane. These silanes under optimum conditions generate the solid-state network of desired configuration for the construction of biosensors and is reported in the present research work. The availability of the amino-group in one of the composite sol–gel glass components provides an extra event for cross-linking with proteinrenzyme using bifunctional reagent ŽGlyoxal.. Two types of the composite sol–gel glass have been developed in the present investigation: Ž1. sol–gel glass of controlled thickness and porosity without enzyme ŽGOD., Ž2. sol–gel glass of controlled thickness and porosity with enzyme GOD. The electrochemistry of ferrocene monocarboxylic acid is studied on system 1. The performance of system 2 as a glucose biosensor is studied. The performance of non-mediated glucose sensors based on cyclic voltammetry and the typical amperometric responses of the biosensors having varying thickness and porosity are reported.
2. Experimental 2.1. Materials and methods 3-Aminopropyltriethoxy silane was obtained from Aldrich; 2-Ž3,4-Epoxycyclohexyl.-ethyltrimethoxy silane was obtained from United Chemical Technologies, Petrarche Silanes and Silicones, Bristol, PA, USA; GOD was obtained from Sigma. All other chemical employed were of analytical grade. 2.2. Preparation of organically modified sol–gel glass electrodes The electrode body used for the preparation of composite sol–gel glass modified electrodes was similar as described in an earlier publication w7x made from Teflon
containing platinum base with a recessed depth of 2 mm. The new material is prepared using optimum concentrations of 3-aminopropyltriethoxy silane Ž70 ml., 2-Ž3,4epoxycyclohexyl.-ethyltrimethoxy silane Ž20 ml., 4 mg GOD dissolved in double distilled water Ž700 ml., 0.1 M HCl Ž5 ml.. The resulting mixture is stirred thoroughly and the desired amount of the homogeneous solution is added to the well Ž0.0314 cm2 . of the specially designed electrode body. The gelation is allowed to occur at 258C for 30 h. A smooth, very thin GOD-immobilized film of sol–gel glass appeared on the Pt surface as evidenced by SEM and electrochemical measurements. The resulting enzyme-immobilized sol–gel film is then incubated in 1% glyoxal for 4 h at 48C. The electrode obtained in this manner is washed with 0.1 M phosphate buffer, pH 7.0 several times and stored in 0.1 M phosphate buffer, pH 7.0 at 48C when not in use. Some of the glucose sensors made following such procedures were stored at room temperature Ž268C.. A control sol–gel glass without the enzyme was also prepared to study the electrochemistry of redox species Žferrocene. on the sol–gel glass modified electrode. The composition of the control sol–gel glass was similar to that of the sol–gel glass used for glucose sensor without the presence of GOD. 2.3. Electrochemical measurements The electrochemical measurements were performed with a Solartron Electrochemical Interface ŽSolartron 1287 Electrochemical Interface, UK. connected to a PC through the serial port. The electrode responses were recorded and plotted with a Printer. A one compartment cell with a working volume of 4 ml and a sol–gel glass modified working electrode, AgrAgCl reference electrode and a platinum foil auxiliary electrode were used for the measurements. The cyclic voltammetry using GOD modified sol–gel glass electrode were studied between 0 V and 1 V vs. AgrAgCl. The amperometric measurements using GOD-immobilized sol–gel modified electrode was operated at 0.70 V vs. AgrAgCl. The experiments were performed in phosphate buffer Ž0.1 M, pH 7. employing both types of modified electrodes.
3. Results and discussion 3.1. Physical characterization of organically modified sol– gel glass electrode The physical characteristics of the sol–gel glass with and has been examined using various composition of 3-aminopropyltriethoxy silane, 2-Ž3,4-epoxycyclohexyl.ethyltrimethoxy silane, enzyme dissolved in water and
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istics of the sol–gel matrix. The colour of the enzyme-immobilized sol–gel film is yellow whereas the colour is white in the absence of enzyme. The smooth sol–gel film without cracking and having better performance when used as sensor was obtained using the optimum concentrations reported in Section 2. Another important requirement of the sensors based on such design is the coupling of modified sol–gel film and Pt surface which contribute significantly to the performance and storage stability of the biosensors. The present modified sol–gel film has been found to be strongly attached and is not easily removable from the Pt surface. The scanning electron micrographs of these two types of organically modified sol–gel glasses Žone in the absence of GOD and other in the presence of GOD. are shown in Fig. 1a, b, c. Fig. 1a shows the SEM of the sol–gel glass without enzyme at 1000 magnification. The surface structure shows a smooth feature with relatively better homogeneous distribution of sol–gel domains. Fig. 1b and c show the SEM of GOD-encapsulated sol–gel glass at 500 and 2000 magnification, respectively. The surface structure shows a smooth feature with relatively better distribution of GOD within organically modified sol–gel glass network. 3.2. Electrochemistry of ferrocene monocarboxylic acid on unmodified and control sol–gel glass modified electrodes A control sol–gel glass without the immobilized enzyme was made to study the electrochemical behavior of ferrocene monocarboxylic acid at its surface. The cyclic voltammetry of ferrocene monocarboxylic acid was studied on following three electrodes: Ži. plane cleaned Pt disk electrode, Žii. sol–gel modified Pt disk electrode made
Fig. 1. SEM of organically modified sol–gel glass; Ža. in the absence of enzyme; Žb. in the presence of enzyme at 500 magnification; Žc. in the presence of enzyme at 2000 magnification.
HCl. The content of 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silane significantly affects the physical character-
Fig. 2. Cyclic voltammograms of 5 mM ferrocene monocarboxylic acid in 0.1 M phosphate buffer ŽpH 7.0. on: Ž1. plane Pt electrode, Ž2. sol–gel glass modified electrode without enzyme made using 14 ml of soluble gel components and Ž3. sol–gel glass modified electrode without enzyme made using 8 ml of soluble gel components, at 258C at a scan rate of 5 mVrs.
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Fig. 3. Cyclic voltammogram of GOD-immobilized composite sol–gel glass in the absence Ž1. and the presence Ž2. of 50 mM glucose acid in 0.1 M phosphate buffer pH 7.0 at the scan rate of 5 mVrs.
using 14 ml of soluble sol–gel component into the well of Pt disk electrode and Žiii. sol–gel modified Pt disk using 8 ml of soluble sol–gel components into the well of the Pt disk electrode. The colour of sol–gel without enzyme was white, however, the porosity under these two conditions was different. Fig. 2 shows the cyclic voltammograms of cleaned platinum disk electrode in the presence of 5 mM ferrocene Žcurve 1.. Curve 2 of Fig. 1 shows the control sol–gel glass film, prepared using 14 ml of homogeneous solution without enzyme into the well of the Pt disk electrode, in 5 mM ferrocene solution. Curve 3 shows the electrochemistry of ferrocene solution made using 8 ml of homogenous solution of the sol–gel glass components without enzyme into the well of the electrode body. Curve 1 shows the well-defined reversible electrochemistry of ferrocene as described in the literature. Whereas curves 2 and 3 show that although voltammograms are reversible in nature, however, there is a diffusion-limited condition prevailing at the sol–gel glass interface. The porosity in the sol–gel glass films made using 14 ml and 8 ml of homogeneous solution of sol–gel components can also be analyzed from these two curves. The film made using 8 ml of the sol–gel components showing curve 3 is relatively thicker than the film 2 made using 14 ml of sol–gel components resulting curve 2 ŽFig. 2.. When chemical modifier such as metal dispersion, water-soluble polymers and proteins are added to the materials, the resulting electrodes became more hydrophilic and subsequently, alter the water contact angle which manifests the wettability. It has been reported w2x that a blank sol–gel electrode without any hydrophilic modifier shows a highest water contact angle Ž808. and in turn, lowest wettability Žnot amenable for nitrogen adsorption analysis., whereas the sol–gel electrode with all hydrophilic modifiers Žcarbon, polyŽethylene glycol, protein and Pd-GOD or even water itself. shows the lowest water contact angle Ž428. and highest wettability Ž42 m2rg.. An increase in wetted area
increases the wetted conductive surface accessible to the solution and also the corresponding electrochemically active area and capacitive current. On the other hand, the unwetted area does not contribute to the capacitive or faradic currents. Although the composition of both films made using 14 ml and 8 ml of sol–gel glass precursors are the same, however, after the gelation for strictly the same period under a similar surface area might bring about a change in water wettability condition due to a change in the thickness along with hydrophilic Žwater. content. The relatively better wetted surface area of the film made using 8 ml of sol–gel precursors provide a relatively better diffusion-limited condition as compared to sol–gel glass electrode made using 14 ml of the sol–gel precursors. Fig. 2 was recorded using aqueous solution of ferrocene and freshly prepared organically modified sol–gel glass electrodes.
Fig. 4. Ža. Cyclic voltammograms of 5 mM ferrocene monocarboxylic acid in 0.1 M phosphate buffer pH 7.1 on GOD-encapsulated sol–gel glass modified electrode made using 700 ml of water at 258C at the scan rate of 3 mVrs, 6 mVrs, 10 mVrs, 20 mVrs, 50 mVrs, and 100 mVrs. The inset shows the plot of anodic peak current vs. the square root of scan rate. Žb. Cyclic voltammogram of GOD-encapsulated sol–gel glass made using 700 ml of water with the absence Ž1. and the presence Ž2. of 150 mM glucose in 0.1 M phosphate buffer pH 7.1 containing 5 mM ferrocene monocarboxylic acid at the scan rate of 5 mVrs.
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3.3. Electrochemistry of glucose oxidase-immobilized sol– gel glass modified electrodes The GOD-immobilized sol–gel glasses of two different compositions were made which differ only in water content. The first one is made with 4 mg of GOD dissolved in 500 ml of double distilled water whereas the second one is made using same amount of enzyme dissolved in 700 ml water. The cyclic voltammograms of the sol–gel glass made using 700 ml of water in the absence Ž1. and the presence Ž2. of 60 mM glucose is shown in Fig. 3. The large increase in anodic current after the addition of glucose shows the performance of the glucose sensor based on sol–gel glass. The increase in anodic current is attributed to the performance of non-mediated glucose sensor based on the oxidation of hydrogen peroxide. We have also studied the electrochemistry of ferrocene monocarboxylic acid at the surface of GOD-encapsulated sol–gel glass made using 700 ml of water. Before taking the electrochemical measurements, the enzyme-encapsulated sol–gel glass was incubated overnight in 5 mM aqueous ferrocene solution Ž0.1 M phosphate buffer pH 7.1.. Fig. 4a shows the cyclic voltammograms of enzyme-encapsulated sol–gel glass electrode at the scan rates of 3 mVrs, 6 mVrs, 10 mVrs, 20 mVrs, 50 mVrs and 100 mVrs in 0.1 M phosphate buffer pH 7.0. There is reversible electrochemistry of ferrocene with a peak separation of 86 mV. The inset shows the plot of peak current vs. the square root of scan rate. A linear relation is recorded and the straight line does not pass through the origin which suggests that the system is poorly diffusion controlled. In order to study the mediated response between sol–gel glass encapsulated GOD and soluble ferrocene, we studied the cyclic voltammograms of enzyme electrode in 5 mM ferrocene monocarboxylic solution in 0.1 M phosphate buffer ŽpH 7.1. in
Fig. 5. Typical response curves of the sol–gel glass modified electrode together with GOD with varying water contents; Ž1. 3- Aminopropyltriethoxy silanes 70 ml; 2-Ž3,4-epoxycyclohexyl.-ethyltrimethoxy silanes 20 ml; 4 mg GOD dissolved in 500 ml water; 0.1 M HCls 5 ml Žinset. and Ž 2 . 3-A m inopropyltriethoxy silane s 70 m l; 2- Ž 3,4epoxycyclohexyl.-ethyltrimethoxy silanes 20 ml; 4 mg GOD dissolved in 700 ml water; 0.1 M HCls 5 ml at 0.70 V vs. AgrAgCl.
Fig. 6. Typical response curve of the GOD-immobilized sol–gel glass modified electrode made with 700 ml water. The results show the subsequent addition of increasing concentrations of glucose. The inset shows a typical response curve.
the absence and presence of 150 mM glucose ŽFig. 4b.. There is an increase in anodic current in the presence of glucose showing the heterogeneous-mediated enzyme catalyzed reaction. 3.4. Amperometric response of enzyme-immobilized sol–gel glasses based glucose sensor The typical chrono-amperometric responses at 0.70 V vs. AgrAgCl of the glucose biosensors which differ in water contents are recorded in Fig. 5. Curve 1, as recorded in the inset, shows the responses of the biosensor made with 500 ml water on the subsequent addition of two concentrations of glucose Ž0.6 mM,; 1.6 mM., whereas curve 2 represents the response of the glucose sensor, made using 700 ml of water on the addition of glucose Ž0.6 mM.. The glucose biosensors made under these two conditions although both show the low background current to the order of - 100 nA, however, these sensors differ in the following aspects: Ž1. the response time of the biosensor with low water content is ) 3 min with a relatively small magnitude of the anodic current; Ž2. the response time of the biosensor made with high water content is - 1 min with large magnitude of anodic current on the addition of same amount of glucose. These results clearly suggest that the sol–gel film is relatively thicker and least porous made using low water content which can again be analyzed by water contact angle and wettability of the sol–gel glass as discussed above. Additionally, the incorporation of protein within the sol–gel matrix ŽFig. 1b and c. also increases the water wetted area within which electrochemical reactions take place. Another important conclusion from these differences on amperometric responses can be made the kinetically controlled situation prevailing at the sensor interface. The amperometric response depends on the production of hydrogen peroxide and its subsequent electrochemical oxidation at 0.7 V vs. AgrAgCl. It is
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strate concentrations. The inset in Fig. 7 shows the linear range of calibration curve. The stability of the new GOD-encapsulated sol–gel glass network is determined under two conditions. In the first case, the enzyme electrode was stored in 0.1 M phosphate buffer pH 7.0 at 48C whereas in the second case, the enzyme electrode was stored at room temperature in the same buffer. The stability of the enzyme electrode stored under the first condition is relatively good without loss of the amperometric response after 2 months. Under the second storage condition, the response is consistent without loss for 20 days. Fig. 7. Calibration curve for glucose analysis calculated from the data recorded in Fig. 6 in phosphate buffer pH 7.0 at 0.7 V vs. AgrAgCl. The inset shows the linear range of the calibration curve.
important to understand the concentration of oxygen at the site of the enzymatic reaction. The formation of hydrogen peroxide depends on the diffusion of glucose through the sol–gel glass within which GOD is distributed. Along with the substrate diffusion, the dissolved oxygen in buffer and the glucose solution is only available oxygen at the site of enzymatic reaction. Since sol–gel is rigid and has the least porous structure within which the enzyme is caged, the possibility of restricted diffusion of oxygen within the sol–gel is expected. The typical response as shown in Fig. 6 indicates the kinetic limitation of the oxygen-based enzymatic reaction. The lesser the porosity, the lesser the rate of oxygen diffusion within the sol–gel glass. Although the concentration of active enzyme within the sol–gel matrix is relatively high as compared to the response recorded in curves 1 and 2 ŽFig. 5.. Additionally, it can also be concluded from the data recorded in Fig. 6 that there is a relatively large increase in anodic current on the small addition of glucose justifying this conclusion since the enzyme present at the surface does not require diffusion-limited condition of oxygen. On the other hand, subsequent additions show a relatively less response as compared to the concentrations of glucose keeping constant the reported value of the K m for GOD. Hence, the present sensor shows a linear response at low concentrations of glucose under which the surface enzyme does not get saturated and the subsequent response becomes kinetically controlled as a result of restricted diffusion of oxygen. A typical response curve of the enzyme sensor made using 700 ml content of the sol–gel glass is shown in Fig. 6. The calibration curve for the glucose analysis prepared from the data recorded in Fig. 6 is shown in Fig. 7. The detection limit of the sensor is - 0.1 mM. The sensitivity of the analysis is found to be 0.78 " 0.01 mArmM. The nature of the response curve ŽFig. 7. also supports the diffusion-limited condition of oxygen-based enzymatic reaction since non-linearity is started at relatively low sub-
4. Conclusion We report the fabrication of a new composite sol–gel glass using 3-aminopropyltriethoxysilane and 2-Ž3,4epoxycyclohexyl.-ethyltrimethoxysilane, in the presence of distilled water and HCl. The resulting silane provides a very smooth surface with a rigid porous structure. The enzyme-modified sol–gel glass is reported to construct an amperometric biosensor for glucose. Under optimum composition of the sol–gel glass ingredients, a very smooth and thin stably adhered to the Pt surface was obtained with better porosity and regular distribution of enzyme within the solid-state network which contributed to the better performance of the biosensors as discussed above.
Acknowledgements The authors are thankful to UGC, New Delhi for financial assistance.
References w1x V. Glezer, O. Lev, Sol–gel vanadium pentaoxide glucose biosensor, J. Am. Chem. Soc. 115 Ž1993. 2533. w2x S. Sampath, O. Lev, Inert metal-modified, composite ceramic-carbon, amperometric biosensors: renewable, controlled reactive layer, Anal. Chem. 68 Ž1996. 2015. w3x U. Narang, P.N. Prasad, F.V. Bright, K. Ramanathan, N.D. Kumar, B.D. Malhotra, S. Chandra, Glucose biosensor based on a sol–gel derived plateform, Anal. Chem. 66 Ž1994. 3139. w4x P.C. Pandey, S. Singh, B. Upadhyay, H.H. Weetall, P.K. Chen, Reversal in the kinetics of M-state decay of D96N bacteriorhodopsin; probing of enzyme catalyzed reaction, Sensors and Actuators B 35–36 Ž1996. 470. w5x Y. Tatsu, K. Yamashita, M. Yamaguchi, S. Yamamura, H. Yamamoto, S. Yoshikawa, Entrapment of glucose oxidase in silica gel by sol–gel method and its application to glucose sensor, Chem. Lett., 1992, p. 1615. w6x P.C. Pandey, S. Upadhyay, H.C. Pathak, A new glucose biosensor based on sandwich configuration of organically-modified sol–gel glass, Electroanalysis 11 Ž1999. 59. w7x P.C. Pandey, R. Prakash, Polyindole modified potassium ion sensor using dibenzo-18-crown-6 mediated PVC membrane, Sensors and Actuators 46 Ž1998. 61.
P.C. Pandey et al.r Sensors and Actuators B 60 (1999) 83–89 Prem Chandra Pandey received his MSc degree in Chemistry in 1980 and PhD in 1986, both from Gorakhpur University. Since 1988, he is a lecturer in the Analytical Chemistry division at Banaras Hindu University and subsequently worked as Reader since 1996 in the same division. He is a member of the Bioelectrochemical society and the Indian Chemical society. He has worked as a visiting scientist at Ecole des Mines, France and the National Institute of Standards and Technology, Gaithersburg, USA. His current interests include mediated, non-mediated and electrocatalytic biosensors, optical biosensors, ion-selective electrodes, chemically modified electrodes, electropolymerization, re-chargeable batteries, dynamics of membrane processes both in linear and non-linear regime, flow injection analysis and photo-chemistry.
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Sanjay Upadhyay obtained his PhD degree in 1993 from Banaras Hindu University on ‘‘liquid membrane transport’’. Since 1994, he has been working as Research Associate at the Department of Chemistry. He is working on electrochemical biosensors in the group of Dr. P.C. Pandey.
Harish C. Pathak received his MSc in Chemistry from Poorvanchal University in 1994. Since 1996, he has been a student of Banaras Hindu University and has been working for his PhD degree in the group of Dr. P.C. Pandey. His current interests include electrochemical biosensors and photophysical properties of bacteriorhodopsin molecule.