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Development of an enzyme substrate incorporating semiconducting amorphous carbons for use in biosensors
Biosensors& Bioelectronics 6 (1991) 325-332
Development of an enzyme substrate incorporating semiconducting amorphous carbons for use in biosensors S. A. Roulston, M. F. Chaplin, L. J. Dunne & A. D. Clark South Bank Polytechnic,
London.
SE1 OA& UK
(Received 4 May 1990; revised version received 23 July 1990; accepted 7 August 1990)
Abstrack Carbonaceous materials formed by the thermal decomposition of sulphanilic acid in the temperature range 700-1400 K are being studied for their potential as enzyme supports and electron mediators in biosensors. These carbons have been formulated into cylindrical pellets. Chemical analyses for the functional groups have established the presence of carboxylic. phenolic. lactonic and quinolic surface’ acidic oxides. This assignment is supported by infrared and electron spectroscopy. The enzyme glucose oxidase has been immobilised to carboxylic residues in the pellets via the carbodiimide method, with up to 8 X IO-’ units of enzyme immobilised per cm2 of the pellet (geometric area). Electrochemical investigations of the pellets have shown that the potential of the 1400 K electrode varies with pH showing approximately Nemstian behaviour. Cyclic voltammetry in I M sulphuric acid with these carbon pellets, show the existence of a single oxidation/reduction peak on carbons prepared above IOOOK. The nature of the group/redox couple responsible for this peak is not yet certain. Keywords: amorphous carbons. biosensor, cyclic voltammetry. electron spin resonance, glucose oxidase. hydrogen electrode, surface functional groups.
susceptible to thermal decomposition, the control over the decomposition conditions and the potential for post formation modifications all contribute to a rich variety in the characteristics of the carbon and allow desirable features to be selected. We have previously characterised sulphanilic acid carbons (SACS), prepared in the heat treatment temperature (HTQ range 700-l 100 K (Roulston ec al., 1990). Our aim is to incorporate these carbons into a biosensor and thus it is essential that a full understanding of the physics and chemistry of these carbons and electrodes
INTRODUCTION Carbonaceous materials formed by the thermal decomposition of organic molecules possess two significant features which lend themselves for use in biosensors. Functional groups present on the surface allow chemical immobilisation of enzymes and the nature and abundance of these groups can be manipulated by a variety of techniques. Further to this, such carbons have good electrical conductivity, facilitating possible electron transfer with the biological agent of a biosensor. The number of substances which are 325 Biosenson & Bioeltwnnics
095~5663/91/$03.50~
1991
Elsevier Science Publishers Ltd. England. Printed in Great Britain
326 which incorporate them is achieved. In this paper, we give details of the characteristics of SACS and electrodes incorporating them and outline the implications of such features for the development of biosensors.
EXPERIMENTAL The preparation of SACS follows Harker and Sherwood (1973) though we have extended the range of HIT to 700-1400 K The following experimentation on these carbons concentrates on carbons which have been formulated into cylindrical pellets but also includes some work on the particulate carbons. The pellets have a cross sectional area of I.42 cm2 and weigh about 150 mg. Although SAC is not the only constituent of these pellets, it should be noted that it is the major constituent and that it is the only chemically and electrochemically active component. No further details of pellet formation can be given here. Geometric measurements of pellet diameter and height were made at room temperature and used to derive the density of the pellets. Electrical resistance measurements of the pellets were also performed at room temperature. Scanning electron micrographs of these pellets were taken using an Hitachi S-450 SEM. Base neutralisation studies were performed on SACS following the method developed by Boehm and co-workers (Boehm et al., 1965). Here, 100 mg samples of the particulate carbon were mixed constantly for 24 h at room temperature with 25 ml of 50 mM solutions of one of four bases of differing strength. The carbon was filtered and samples of the reaction solution titrated against 50 mM HCI to a set pH and compared to standard base solutions. For electrochemical investigations and immobilisation of glucose oxidase, the pellets were mounted into the form of an electrode assembly. The initial mounting was via attachment of the pellet using paraffin wax to a hollow, polyacetate tube. The edges of the pellet were subsequently sealed with wax such that only one flat face was exposed to the solutions of interest. For electrochemical investigations, mercury was added to the hollow region of the electrode assembly and electrical connection achieved by a platinum wire. Cyclic voltammetry of these mounted pellets
S. A. Roulston et al. was undertaken in outgassed 1 M sulphuric acid at room temperature in a single compartment cell with a saturated calomel reference electrode (SCE) and a platinum foil counter electrode. Outgassing was performed by passing nitrogen through the solution for 1 h and all experiments were performed under a nitrogen atmosphere. Compensation was made for cell resistance drop but not for resistance arising from the pellets. A Princeton Model 362 scanning potentiostat was used for potential generation and interfaced to an Amstrad 1640 equipped with Condecon 300 software supplied by EG&G instruments, Sorbus House, Wokingham, Berks RGI 1 2GY, UK. Immobilisation of the enzyme glucose oxidase has been undertaken on the pelletised form of the carbons following Cho and Bailey (1978). Mounted carbon pellets were immersed in 12 ml of sodium acetate buffer (pH 5.5) containing 10 mM ethyl-dimethylaminopropyl-carbodiimidehydrochloride and allowed to react for 24 h. The pellets were washed three times with buffer to remove unbound carbodiimide and subsequently immersed in 12 ml of buffer containing glucose oxidase at 9 IU ml-’ for 48 h. The pellets were washed repeatedly in buffer, until no enzyme activity was detected in the supematant. Assay of apparent immobilised enzyme activity was achieved via a two step calorimetric assay in acetate buffer. In the first step, reaction of the immobilised glucose oxidase with an aerated solution containing excess glucose was allowed for a set length of time and the reaction stopped by removal of the pellet from the reaction solution. The second step was via a standard calorimetric assay for hydrogen peroxide involving a portion of the supernatant with the enzyme peroxidase and the chromogen azino-bis-ethylbenzthiazoline-sulphonic acid (ABTS). No glucose oxidase enzyme activity was detected in this solution. RESULTS AND DISCUSSION Figures 1 and 2 show scanning electron micrographs of pellets formed from 700 and900 K SAC. They represent cross-sectional views of the pellets with the outer surface of the pellet at the top and the inner structure below. The particulate appearance of the inside of these pellets closely resembles the irregularly shaped particles of unpelletised SACS shown previously (Roulston et al., 1990). The particles within the pellet fall into
Enzyme substrate incorporating semiconducting amorphous carbons
Fig. 1. SEA4 of 700 K sulphanilic acid carbon pellets, shown in cross-section. Original magn@cation ~600.
the size range lo-50pm with no apparent dependence on HTT. The surface morphology of these pellets does, however, exhibit a strong HTT dependence. It is
Fig. 2. SEM of 900 K sulphanilic acid carbon pellets, shown in cross-section. On*ginalmagnification ~60.
327
Fig. 3. (X) Sulphanilic acid carbon pellet densities and (W) electron spin concentrations as a function of HlT
clear that the pellet formed from 900 K SAC has an outer surface which is far smoother and generally less featured and less open than the 700 K SAC pellet. This trend continues as HTT increases. Figure 3 shows the variation in pellet density at different HTTs. It can be seen that the density is at a maximum in the 900 K carbon. The figure also shows our earlier data for the spin concentration of particulate SACS (Dunne & Harker, 1974; Roulston et al., 1990), which also reach a peak in the same carbon. Despite this similarity in shape no direct relationship is apparent. Density variations are unlike the trend described by Kinoshita (1988). However, maximum spin concentrations around this HTT have been previously observed (Harker ec al., 1966; Mrozowski, 1988; Lewis & Singer, 1981 and references therein). We have concluded previously that the free spins observed within SACS are associated with odd alternate n-type radicals which are known to be typically aromatic in nature. These spins have further been shown to be very stable to chemical attack by gases at elevated temperatures. Such stability can be described in terms of reactive centres becoming trapped within the structure of the carbon. The extent to which these centres become trapped would be dependent upon the
328
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extent of convolution of the carbon particle and this would in turn affect the density. This may therefore go some way to explaining the correlation between density and free spin concentration. Figure 4 shows the results of base neutralisation studies, elucidating the nature of the acidic surface oxides present within SAC. Whilst base neutralisation cannot be used to positively identify the nature of surface groups on its own, the interpretations herein are made in conjunction with previously published infrared and electron spectroscopic data for these carbons (Roulston et al., 1990).Using these techniques it was possible to qualitatively conclude the oxygen present to be associated with carboxylic. phenolic, lactonic and quinolic surface functionalities (listed in decreasing order of acidic strength). Positive chemical identification of the groups can thus be undertaken on the basis of their differing acidity and the presence of four distinct acidic responses to the various strength bases employed (Boehm
Enzyme substrate incorporating semiconducting amorphous carbons 17 \ 16 .15 -.
'\
12 -11 -IO .9
--
2
.-
I
--
Fig. 5, Room temperature electrical resistance variation with Hi’T of MC. et al., 1965). It can be seen that although no single
group peaks at 900 K, the overall levels of acidic oxides detected (Fig. 4e) do peak at this HTT. The reason for this is not yet clear. Room temperature electrical resistance measurements on the pelletised carbons falls sharply to a few ohms as HTT is increased (Fig. 5). This reduction in electrical resistance with increasing HTT is typical of many carbons (Kinoshita, 1988). This variation in electrical resistance with HTT also affects the curves recorded by cyclic voltammetry, shown in Fig. 6. The size of the currents recorded in these plots at HTTs below 1000 K, appears to vary with electrical resistance. However, it is not certain whether the distinctive overall shape occurs as a sole result of this high resistance or as a result of the distributive capacitance, resulting from charging phenomena (Gagnon, 1975). Cyclic voltammograms (CV) of SACS prepared above 1000 K clearly exhibit a single oxidation/ reduction peak, indicative of a single redox couple. Of the acidic oxides discussed previously, only the lactonic functionality would seem unlikely to be responsible for this couple as a result of its low levels in the 1100 and 1200 K SAC.
329
The peaks seen in the CVs of pellets formed from these higher H’IT carbons and the overall shape of the CVs, are very similar to those recorded for the chemically oxidised form of Vulcan XC-72 by Kinoshita and Bett (1973). However, unlike these workers, we have noticed a strong dependence of peak potential on ramp rate and are unable to attempt an identification of the redox couple associated with these peaks on this basis. Such a dependence of peak potential on ramp rate is usually indicative of a quasi-reversible or irreversible process. However, a discussion of the kinetics of the electrochemistry of these pellets will not be addressed immediately. Figure 7 shows the response of potential to pH for the 1400 K carbon pellet. Approximately 2 min were required to obtain a stable reading from this HTT carbon. From these data, a gradient of -55 mV per pH unit has been calculated with a potential intercept of 470 mV. Pellets formed from carbons prepared at lower HTTs did not give a sufficiently stable reading to be recorded. The approximately Nemstian behaviour of the 1400 K SAC is obviously similar to that seen by Puri (1970) who recorded a gradient of -59 mV per pH unit with an intercept of 500 mV for a sugar charcoal treated with hydrogen. Similar results are reported in the reviews by Kinoshita (1988) and Midgeley and Mulcahy (1983). Puri concluded that this result was consistent with the quinone/ hydroquinone redox couple acting as a hydrogen electrode and this may also account for the response of the 1400 K SAC to pH and thus imply this couple as the electrochemically active species. Indeed, this couple is generally believed to be the major redox couple on carbons (Kinoshita, 1988). Results of the immobilisation of glucose oxidase to pelletised SAC are presented in Fig. 8. Assay of these apparent activities was necessary via a two-step reaction, due to an interaction between the carbons and the chromogen ABTS. This reaction involved the reduction of the oxidised form of the chromogen and although adsorption may have occurred, this was not obvious. Work will be undertaken to study this interaction further as the reaction appears to be dependent on the HTT. Furthermore, the effect was seen to diminish with time, implying a non-catalytic interaction with a chemical, functional group. This two-step approach to assay of glucose oxidase activity was further permitted by the lack of interaction between SACS and hydrogen peroxide.
i
(A)
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Fig. 6. Cyclic voltammograms of a range of S4C.s in 1 M H$O, at a ramp rate of 100 m Vs-‘. Using an SCE as wference electrode and a platinum foil counter electrode.
Enzyme substrate incorporating semiconducting amorphous carbons
331
Clearly, there is no direct correlation between the levels of glucose oxidase immobilised to each H’lT carbon (Fig. 8) and the levels of carboxylic group found by base neutralisation (Fig. 4a). Although immobilisation of the glucose oxidase is specifically to carboxylic groups, varying surface area of the pellets with HTF (discussed above) and adsorption of the enzyme by groups other than the carboxyl may lead to the apparent independence of immobilisation and carboxylic levels. Further work is needed in this area.
CONCLUSIONS
Fig. 7. Dependence ofpotential of 1400 K SAC electrode on solution pH (V SCE).
We have shown here that SAC pellets possess two key features needed for biosensors. It can be seen from the base neutralisation levels (Fig. 4) that several acidic surface oxides are available across the range of HTTs studied in this investigation for the immobilisation of enzymes. Following from this, it has been shown that the immobilisation of the enzyme glucose oxidase is attainable at reasonably consistent levels across the H’IT range. Electrical resistance of these pellets and CV in sulphuric acid tend to suggest that only the higher HTTs would be of use in the development of a biosensor as a result of high resistance associated with the lower temperature carbons. Electrochemically, the affinity of the 1400 K SAC towards interaction with protons (as seen in Fig. 7) may be of importance in the electrical detection of enzyme activity, particularly involving proton exchanges. Work is currently under way to elucidate the electrochemical processes associated with these pellets and to evaluate their use in biosensors. In addition, we are concentrating on the chemistry and electrochemistry of immobilised enzymes, both in the presence and absence of mediators and are looking at methods of enhancing the activity of immobilised enzyme. REFERENCES Boehm, H. P., Diehl. E. &Heck, W. (1%5). Identification
0
“7m
Em
9m
lmo
iim
iao
I
13110ra
HAT TREATIENT TEUPEMTIBE IK)
Fig. 8. Activities ofglucose oxidase immobilised to pelktised sulphanilic acid carbons.
of functional groups in surface oxides of carbon. Proc 2nd ConJ on Ind. Carbon and Graphite. SCI, London, pp. 369-79. Cho. Y. K. & Bailey. W. M. (1978). Immobilisation of enzymes on activated carbon: Properties of immobilised Glucoamylase, Glucose oxidase and
332 Gluconolactone. Biotech. Bioeng., 20, 165 l-65. Dunne. L. J. & Harker. H. (1974). Structure and magnetic properties of molecular crystalline ‘carbons’ prepared by thermal decomposition of sulphanilic acid in the solid state. Philos. Mag., 30, 1313-18. Gagnon, E. G. (1975). The triangular voltage sweep method for determining double layer capacity of porous electrodes. IV. Porous carbon in potassium hydroxide. 1 Electmchem. Sot., 122, 521-5. Harker, H., Gallagher, J. T. & Parkin, A. (1966). Reaction of carbon with oxidising gases: the role of unpaired electrons. Carbon, 4,401-9. Harker, H. & Sherwood, P. M. A. (1973). X-ray photoelectron studies of sulphur in carbon. Philos. Mag., 27, 1241-4. Kinoshita. K. (1988). Carbon, Electrochemistry and Physicochemical Properties. Wiley, New York. Kinoshita. K & Bet& J. A S. (1973). Potentiodynamic
S. A. Roulston et al. analysis of surface oxides on carbon blacks. Carbon, 11,403-l 1. Lewis. I. C. & Singer, L. S. (1981). Electron spin resonance and the mechanism of carbonisation. Chemistry and Physics of Carbon, 17. l-88. Midgeley, D. & Mulcahy, D. E. (1983). Carbon substrate ion-selective electrodes. Ion-selective Electrode Rev., 5, 162-242. Mrozowski, S. (1988). ESR studies of carbonisation and coalitication processes part II. Biological materials. Carbon, 26, 531-41. Put-i. B. R. (1970). Surface complexes on carbons. In Chemistry and Physics of Carbon, Vol. 6, ed. P. L. Walker. Dekker. New York. pp. 191-282. Roulston, S. A., Dunne, L. J., Clark A D. & Chaplin, M. F. (1990). Electronic and structural properties of amorphous semiconducting carbons prepared by thermal decomposition of sulphanilic acid in the solid state. Philos. Mag.. 62, 243-60.
Development of an enzyme substrate incorporating semiconducting amorphous carbons for use in biosensors S. A. Roulston, M. F. Chaplin, L. J. Dunne & A. D. Clark South Bank Polytechnic,
London.
SE1 OA& UK
(Received 4 May 1990; revised version received 23 July 1990; accepted 7 August 1990)
Abstrack Carbonaceous materials formed by the thermal decomposition of sulphanilic acid in the temperature range 700-1400 K are being studied for their potential as enzyme supports and electron mediators in biosensors. These carbons have been formulated into cylindrical pellets. Chemical analyses for the functional groups have established the presence of carboxylic. phenolic. lactonic and quinolic surface’ acidic oxides. This assignment is supported by infrared and electron spectroscopy. The enzyme glucose oxidase has been immobilised to carboxylic residues in the pellets via the carbodiimide method, with up to 8 X IO-’ units of enzyme immobilised per cm2 of the pellet (geometric area). Electrochemical investigations of the pellets have shown that the potential of the 1400 K electrode varies with pH showing approximately Nemstian behaviour. Cyclic voltammetry in I M sulphuric acid with these carbon pellets, show the existence of a single oxidation/reduction peak on carbons prepared above IOOOK. The nature of the group/redox couple responsible for this peak is not yet certain. Keywords: amorphous carbons. biosensor, cyclic voltammetry. electron spin resonance, glucose oxidase. hydrogen electrode, surface functional groups.
susceptible to thermal decomposition, the control over the decomposition conditions and the potential for post formation modifications all contribute to a rich variety in the characteristics of the carbon and allow desirable features to be selected. We have previously characterised sulphanilic acid carbons (SACS), prepared in the heat treatment temperature (HTQ range 700-l 100 K (Roulston ec al., 1990). Our aim is to incorporate these carbons into a biosensor and thus it is essential that a full understanding of the physics and chemistry of these carbons and electrodes
INTRODUCTION Carbonaceous materials formed by the thermal decomposition of organic molecules possess two significant features which lend themselves for use in biosensors. Functional groups present on the surface allow chemical immobilisation of enzymes and the nature and abundance of these groups can be manipulated by a variety of techniques. Further to this, such carbons have good electrical conductivity, facilitating possible electron transfer with the biological agent of a biosensor. The number of substances which are 325 Biosenson & Bioeltwnnics
095~5663/91/$03.50~
1991
Elsevier Science Publishers Ltd. England. Printed in Great Britain
326 which incorporate them is achieved. In this paper, we give details of the characteristics of SACS and electrodes incorporating them and outline the implications of such features for the development of biosensors.
EXPERIMENTAL The preparation of SACS follows Harker and Sherwood (1973) though we have extended the range of HIT to 700-1400 K The following experimentation on these carbons concentrates on carbons which have been formulated into cylindrical pellets but also includes some work on the particulate carbons. The pellets have a cross sectional area of I.42 cm2 and weigh about 150 mg. Although SAC is not the only constituent of these pellets, it should be noted that it is the major constituent and that it is the only chemically and electrochemically active component. No further details of pellet formation can be given here. Geometric measurements of pellet diameter and height were made at room temperature and used to derive the density of the pellets. Electrical resistance measurements of the pellets were also performed at room temperature. Scanning electron micrographs of these pellets were taken using an Hitachi S-450 SEM. Base neutralisation studies were performed on SACS following the method developed by Boehm and co-workers (Boehm et al., 1965). Here, 100 mg samples of the particulate carbon were mixed constantly for 24 h at room temperature with 25 ml of 50 mM solutions of one of four bases of differing strength. The carbon was filtered and samples of the reaction solution titrated against 50 mM HCI to a set pH and compared to standard base solutions. For electrochemical investigations and immobilisation of glucose oxidase, the pellets were mounted into the form of an electrode assembly. The initial mounting was via attachment of the pellet using paraffin wax to a hollow, polyacetate tube. The edges of the pellet were subsequently sealed with wax such that only one flat face was exposed to the solutions of interest. For electrochemical investigations, mercury was added to the hollow region of the electrode assembly and electrical connection achieved by a platinum wire. Cyclic voltammetry of these mounted pellets
S. A. Roulston et al. was undertaken in outgassed 1 M sulphuric acid at room temperature in a single compartment cell with a saturated calomel reference electrode (SCE) and a platinum foil counter electrode. Outgassing was performed by passing nitrogen through the solution for 1 h and all experiments were performed under a nitrogen atmosphere. Compensation was made for cell resistance drop but not for resistance arising from the pellets. A Princeton Model 362 scanning potentiostat was used for potential generation and interfaced to an Amstrad 1640 equipped with Condecon 300 software supplied by EG&G instruments, Sorbus House, Wokingham, Berks RGI 1 2GY, UK. Immobilisation of the enzyme glucose oxidase has been undertaken on the pelletised form of the carbons following Cho and Bailey (1978). Mounted carbon pellets were immersed in 12 ml of sodium acetate buffer (pH 5.5) containing 10 mM ethyl-dimethylaminopropyl-carbodiimidehydrochloride and allowed to react for 24 h. The pellets were washed three times with buffer to remove unbound carbodiimide and subsequently immersed in 12 ml of buffer containing glucose oxidase at 9 IU ml-’ for 48 h. The pellets were washed repeatedly in buffer, until no enzyme activity was detected in the supematant. Assay of apparent immobilised enzyme activity was achieved via a two step calorimetric assay in acetate buffer. In the first step, reaction of the immobilised glucose oxidase with an aerated solution containing excess glucose was allowed for a set length of time and the reaction stopped by removal of the pellet from the reaction solution. The second step was via a standard calorimetric assay for hydrogen peroxide involving a portion of the supernatant with the enzyme peroxidase and the chromogen azino-bis-ethylbenzthiazoline-sulphonic acid (ABTS). No glucose oxidase enzyme activity was detected in this solution. RESULTS AND DISCUSSION Figures 1 and 2 show scanning electron micrographs of pellets formed from 700 and900 K SAC. They represent cross-sectional views of the pellets with the outer surface of the pellet at the top and the inner structure below. The particulate appearance of the inside of these pellets closely resembles the irregularly shaped particles of unpelletised SACS shown previously (Roulston et al., 1990). The particles within the pellet fall into
Enzyme substrate incorporating semiconducting amorphous carbons
Fig. 1. SEA4 of 700 K sulphanilic acid carbon pellets, shown in cross-section. Original magn@cation ~600.
the size range lo-50pm with no apparent dependence on HTT. The surface morphology of these pellets does, however, exhibit a strong HTT dependence. It is
Fig. 2. SEM of 900 K sulphanilic acid carbon pellets, shown in cross-section. On*ginalmagnification ~60.
327
Fig. 3. (X) Sulphanilic acid carbon pellet densities and (W) electron spin concentrations as a function of HlT
clear that the pellet formed from 900 K SAC has an outer surface which is far smoother and generally less featured and less open than the 700 K SAC pellet. This trend continues as HTT increases. Figure 3 shows the variation in pellet density at different HTTs. It can be seen that the density is at a maximum in the 900 K carbon. The figure also shows our earlier data for the spin concentration of particulate SACS (Dunne & Harker, 1974; Roulston et al., 1990), which also reach a peak in the same carbon. Despite this similarity in shape no direct relationship is apparent. Density variations are unlike the trend described by Kinoshita (1988). However, maximum spin concentrations around this HTT have been previously observed (Harker ec al., 1966; Mrozowski, 1988; Lewis & Singer, 1981 and references therein). We have concluded previously that the free spins observed within SACS are associated with odd alternate n-type radicals which are known to be typically aromatic in nature. These spins have further been shown to be very stable to chemical attack by gases at elevated temperatures. Such stability can be described in terms of reactive centres becoming trapped within the structure of the carbon. The extent to which these centres become trapped would be dependent upon the
328
S. A. Roulston et
(8
al.
(4 /
0 700
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/
.
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lop
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”
1300
140
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Fig. I-contd.
“T
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Fig. 4. HTT dependence of acidic oxides on SAC. as derived by base neutralisation experiments.
extent of convolution of the carbon particle and this would in turn affect the density. This may therefore go some way to explaining the correlation between density and free spin concentration. Figure 4 shows the results of base neutralisation studies, elucidating the nature of the acidic surface oxides present within SAC. Whilst base neutralisation cannot be used to positively identify the nature of surface groups on its own, the interpretations herein are made in conjunction with previously published infrared and electron spectroscopic data for these carbons (Roulston et al., 1990).Using these techniques it was possible to qualitatively conclude the oxygen present to be associated with carboxylic. phenolic, lactonic and quinolic surface functionalities (listed in decreasing order of acidic strength). Positive chemical identification of the groups can thus be undertaken on the basis of their differing acidity and the presence of four distinct acidic responses to the various strength bases employed (Boehm
Enzyme substrate incorporating semiconducting amorphous carbons 17 \ 16 .15 -.
'\
12 -11 -IO .9
--
2
.-
I
--
Fig. 5, Room temperature electrical resistance variation with Hi’T of MC. et al., 1965). It can be seen that although no single
group peaks at 900 K, the overall levels of acidic oxides detected (Fig. 4e) do peak at this HTT. The reason for this is not yet clear. Room temperature electrical resistance measurements on the pelletised carbons falls sharply to a few ohms as HTT is increased (Fig. 5). This reduction in electrical resistance with increasing HTT is typical of many carbons (Kinoshita, 1988). This variation in electrical resistance with HTT also affects the curves recorded by cyclic voltammetry, shown in Fig. 6. The size of the currents recorded in these plots at HTTs below 1000 K, appears to vary with electrical resistance. However, it is not certain whether the distinctive overall shape occurs as a sole result of this high resistance or as a result of the distributive capacitance, resulting from charging phenomena (Gagnon, 1975). Cyclic voltammograms (CV) of SACS prepared above 1000 K clearly exhibit a single oxidation/ reduction peak, indicative of a single redox couple. Of the acidic oxides discussed previously, only the lactonic functionality would seem unlikely to be responsible for this couple as a result of its low levels in the 1100 and 1200 K SAC.
329
The peaks seen in the CVs of pellets formed from these higher H’IT carbons and the overall shape of the CVs, are very similar to those recorded for the chemically oxidised form of Vulcan XC-72 by Kinoshita and Bett (1973). However, unlike these workers, we have noticed a strong dependence of peak potential on ramp rate and are unable to attempt an identification of the redox couple associated with these peaks on this basis. Such a dependence of peak potential on ramp rate is usually indicative of a quasi-reversible or irreversible process. However, a discussion of the kinetics of the electrochemistry of these pellets will not be addressed immediately. Figure 7 shows the response of potential to pH for the 1400 K carbon pellet. Approximately 2 min were required to obtain a stable reading from this HTT carbon. From these data, a gradient of -55 mV per pH unit has been calculated with a potential intercept of 470 mV. Pellets formed from carbons prepared at lower HTTs did not give a sufficiently stable reading to be recorded. The approximately Nemstian behaviour of the 1400 K SAC is obviously similar to that seen by Puri (1970) who recorded a gradient of -59 mV per pH unit with an intercept of 500 mV for a sugar charcoal treated with hydrogen. Similar results are reported in the reviews by Kinoshita (1988) and Midgeley and Mulcahy (1983). Puri concluded that this result was consistent with the quinone/ hydroquinone redox couple acting as a hydrogen electrode and this may also account for the response of the 1400 K SAC to pH and thus imply this couple as the electrochemically active species. Indeed, this couple is generally believed to be the major redox couple on carbons (Kinoshita, 1988). Results of the immobilisation of glucose oxidase to pelletised SAC are presented in Fig. 8. Assay of these apparent activities was necessary via a two-step reaction, due to an interaction between the carbons and the chromogen ABTS. This reaction involved the reduction of the oxidised form of the chromogen and although adsorption may have occurred, this was not obvious. Work will be undertaken to study this interaction further as the reaction appears to be dependent on the HTT. Furthermore, the effect was seen to diminish with time, implying a non-catalytic interaction with a chemical, functional group. This two-step approach to assay of glucose oxidase activity was further permitted by the lack of interaction between SACS and hydrogen peroxide.
i
(A)
-1.2x1; -0.4
E(V)
1 1.0
700K
1lOOK
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I
E(V)
1.0
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800K
1200K
1.1x10-
1.0x10-3
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i(A)
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4
E(V) 1300K
1.0
i (A)
-2JXfLT -0.4
I
E(V)
1OOOK
1.0
1400K
Fig. 6. Cyclic voltammograms of a range of S4C.s in 1 M H$O, at a ramp rate of 100 m Vs-‘. Using an SCE as wference electrode and a platinum foil counter electrode.
Enzyme substrate incorporating semiconducting amorphous carbons
331
Clearly, there is no direct correlation between the levels of glucose oxidase immobilised to each H’lT carbon (Fig. 8) and the levels of carboxylic group found by base neutralisation (Fig. 4a). Although immobilisation of the glucose oxidase is specifically to carboxylic groups, varying surface area of the pellets with HTF (discussed above) and adsorption of the enzyme by groups other than the carboxyl may lead to the apparent independence of immobilisation and carboxylic levels. Further work is needed in this area.
CONCLUSIONS
Fig. 7. Dependence ofpotential of 1400 K SAC electrode on solution pH (V SCE).
We have shown here that SAC pellets possess two key features needed for biosensors. It can be seen from the base neutralisation levels (Fig. 4) that several acidic surface oxides are available across the range of HTTs studied in this investigation for the immobilisation of enzymes. Following from this, it has been shown that the immobilisation of the enzyme glucose oxidase is attainable at reasonably consistent levels across the H’IT range. Electrical resistance of these pellets and CV in sulphuric acid tend to suggest that only the higher HTTs would be of use in the development of a biosensor as a result of high resistance associated with the lower temperature carbons. Electrochemically, the affinity of the 1400 K SAC towards interaction with protons (as seen in Fig. 7) may be of importance in the electrical detection of enzyme activity, particularly involving proton exchanges. Work is currently under way to elucidate the electrochemical processes associated with these pellets and to evaluate their use in biosensors. In addition, we are concentrating on the chemistry and electrochemistry of immobilised enzymes, both in the presence and absence of mediators and are looking at methods of enhancing the activity of immobilised enzyme. REFERENCES Boehm, H. P., Diehl. E. &Heck, W. (1%5). Identification
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Fig. 8. Activities ofglucose oxidase immobilised to pelktised sulphanilic acid carbons.
of functional groups in surface oxides of carbon. Proc 2nd ConJ on Ind. Carbon and Graphite. SCI, London, pp. 369-79. Cho. Y. K. & Bailey. W. M. (1978). Immobilisation of enzymes on activated carbon: Properties of immobilised Glucoamylase, Glucose oxidase and
332 Gluconolactone. Biotech. Bioeng., 20, 165 l-65. Dunne. L. J. & Harker. H. (1974). Structure and magnetic properties of molecular crystalline ‘carbons’ prepared by thermal decomposition of sulphanilic acid in the solid state. Philos. Mag., 30, 1313-18. Gagnon, E. G. (1975). The triangular voltage sweep method for determining double layer capacity of porous electrodes. IV. Porous carbon in potassium hydroxide. 1 Electmchem. Sot., 122, 521-5. Harker, H., Gallagher, J. T. & Parkin, A. (1966). Reaction of carbon with oxidising gases: the role of unpaired electrons. Carbon, 4,401-9. Harker, H. & Sherwood, P. M. A. (1973). X-ray photoelectron studies of sulphur in carbon. Philos. Mag., 27, 1241-4. Kinoshita. K. (1988). Carbon, Electrochemistry and Physicochemical Properties. Wiley, New York. Kinoshita. K & Bet& J. A S. (1973). Potentiodynamic
S. A. Roulston et al. analysis of surface oxides on carbon blacks. Carbon, 11,403-l 1. Lewis. I. C. & Singer, L. S. (1981). Electron spin resonance and the mechanism of carbonisation. Chemistry and Physics of Carbon, 17. l-88. Midgeley, D. & Mulcahy, D. E. (1983). Carbon substrate ion-selective electrodes. Ion-selective Electrode Rev., 5, 162-242. Mrozowski, S. (1988). ESR studies of carbonisation and coalitication processes part II. Biological materials. Carbon, 26, 531-41. Put-i. B. R. (1970). Surface complexes on carbons. In Chemistry and Physics of Carbon, Vol. 6, ed. P. L. Walker. Dekker. New York. pp. 191-282. Roulston, S. A., Dunne, L. J., Clark A D. & Chaplin, M. F. (1990). Electronic and structural properties of amorphous semiconducting carbons prepared by thermal decomposition of sulphanilic acid in the solid state. Philos. Mag.. 62, 243-60.