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Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes
Biosensors & Bioelectronics 17 (2002) 35 – 43 www.elsevier.com/locate/bios
Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes S. Myler, S.D. Collyer, K.A. Bridge, S.P.J. Higson * Manchester Materials Science Centre, Uni6ersity of Manchester and UMIST, Gros6enor Street, Manchester, M1 7HS, UK Received 12 October 2000; received in revised form 1 June 2001; accepted 6 August 2001
Abstract An outer ultra-thin-polydimethyldichlorosiloxane film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode biosensor. The composite membrane was formed via the condensation polymerisation of dimethyldichlorosilane at the surface of a host porous alumina membrane. Homogeneous polydimethyldichlorosiloxane films of B100 nm thickness acted as effective substrate diffusional barriers and were supported by the underlying porous alumina surface. Glucose and oxygen permeability coefficients were determined using diffusion chamber apparatus. Polysiloxane composite membranes were found to offer some screening functionality towards anionic biological interferents such as ascorbate. On exposure to blood an approximate 25% signal drift was observed during the first 2 h exposure to blood; after this time responses remained almost stable. Whole blood glucose determinations showed a close correlation (r 2 = 0.98) to analyses performed via standard hospital analyses. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Ultra-thin film composite membranes; Blood glucose determinations; Polysiloxane; Permselectivity; Biocompatibility
1. Introduction Enzyme electrodes have continued to attract considerable interest over the past few years due to the simplified alternative analytical regimes they potentially offer in comparison to many more complicated approaches (Cosnier, 1999). The relatively slow acceptance of biosensor technology has, however, unquestionably been hindered, by a number of generic problems associated with lack of robustness (Cosnier, 1999), poor longevity (Kress-Rodgers, 1997) and the frequent inability to effectively screen against interferents that can give rise to erroneous responses (KressRodgers, 1997). Amperometric/oxidase-H2O2 membrane based sensors continue to represent one of the most studied classes of biosensor, due to the ease by which H2O2 levels may be both quantified and related to substrate levels in addition to the commercial availability and * Corresponding author. Tel.: + 44-1612008871; fax: 1612003586. E-mail address: [email protected] (S.P.J. Higson).
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relative robustness of many oxidase enzymes (Bourdillon et al., 1982). Of these, glucose oxidase enzyme electrodes continue to enjoy the greatest interest, and this may largely be explained in terms of the clinical and economic significance of glucose as an analyte (Scheller and Schubert, 1992). It can be argued that in order for biosensors to gain wider acceptance and indeed practical application, they have generically to more adequately meet a number of requirements in terms of specificity, robustness and sensitivity (Higson and Vadgama, 1994). For many clinical applications, for example, the sensor must resist the detrimental effects of both outer surface biofouling and working electrode passivation (Higson et al., 1993a) in addition to being able to exclude interferents which might give rise to erroneous results (Christie et al., 1992). A common approach used within many electrochemical sensors to overcome these problems has been to employ two or more tailored functionalised membranes to (a) minimise biofouling effects (Higson et al., 1993a,b) screen interferents and/or low molecular weight electrode passivating solutes from reaching the working electrode (Higson et al., 1993a).
0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 6 5 - 2
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S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43
Each membrane acts as an additional diffusional barrier which lowers the magnitude of sensor responses (Iwuoha et al., 1999) as well as extending response times (Mullen et al., 1986). One approach adopted by ourselves (Higson and Vadgama, 1995a) and others (Palmisano et al., 1997) has been to couple two or more functionalities into one membrane thereby minimising the total diffusional barrier offered towards solutes (Higson and Vadgama, 1994). One membrane developed within our laboratory, for example, was based upon anodically electrochemically fabricating ultrathin-polymer-film-composite membranes for use within amperometric enzyme based biosensors (Myler et al., 1997). Ultra-thin-polymer-film-composite membranes consist of host porous mechanical support barriers, onto which are bonded a homogeneous ultra-thin-polymerfilms (e.g. B100 nm thickness), that act as the effective separational barrier (Higson and Vadgama, 1994). High solute fluxes may be achieved across such membranes due to the extreme thinness of the separational polymer film (Higson and Vadgama, 1994). In this way, response times may often be minimised in comparison to those obtainable if two or more conventional membranes are used within a sensor (Higson and Vadgama, 1994). This paper describes the use of a polysiloxane thinfilm-composite membrane to provide the outer surface biocompatibility, permselective screening of interferent solutes and linearisation of sensor responses of a model glucose oxidase based enzyme electrode. The thickness of the diffusional barrier is greatly reduced in comparison to conventional membranes and in this way the magnitude of sensor responses are enhanced while response times can also be minimised. The applicability of the polysiloxane thin-film-composite membrane sensor is demonstrated via inclusion and exploitation within a glucose sensor for whole blood glucose determinations, the performance of which is compared with standard analytical techniques used within a hospital clinical biochemistry laboratory.
trolube Ltd, Berkshire) was purchased from Maplin Electronics. Silver DAG paint was purchased from Merck BDH. Porous alumina Anopore membranes were purchased from Whatman International Ltd (Maidstone, Kent, UK). Microporous polycarbonate membranes were purchased from the Poretic Corporation, Livermore, CA.
2.2. Buffers and solutions A phosphate buffer (pH 7.4) comprising 5.27×10 − 2 M Na2HPO4, 1.3× 10 − 2 M NaH2PO4, 5.1× 10 − 3 M NaCl, was prepared in distilled water and was used for the preparation of all aqueous solutions. Glucose solutions of 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 mM concentration were prepared in phosphate buffer for the calibration of enzyme electrodes.
2.3. Preparation of polysiloxane thin-film composite membranes Alumina membranes were sandwiched between two pieces of laboratory film cut so as to leave an exposed circular area (diameter approximately 50 mm) on the uppermost face of the membrane surface, Fig. 1(a). The membrane in this way was then secured and sealed between two stainless steel plates by means of a screwthreaded clamp, and tightened so as to provide an even pressure, (custom designed by the mechanical workshops of the Manchester Materials Science Centre, UMIST, Manchester). Fig. 1(b). The entire assembly was then placed into a glass vessel (1 litre volume), stored in a Gallenkamp 4A 1715 Humidity Oven, at a constant humidity level of 50% and room temperature (25 °C). 2 ml of monomer were added to a glass petri dish within the vessel, and the membrane assembly placed face down and suspended 1 cm above the liquid monomer, Fig. 1(c). The cell was then sealed and left for a given time duration, after which the assembly was removed from the vessel and stored in the humidity oven for a further 60 min to ensure polymerisation of any excess monomer.
2. Experimental
2.4. Electrochemical cell for enzyme electrodes
2.1. Reagents and membranes
A custom designed glass cell was fabricated by E. Boote Glass Blowing Services (Macclesfield, Cheshire, UK) for GOD-based enzyme electrode fabrication, Fig. 2. The cell was designed so that a working platinum electrode was mounted within a glass platform with a flat circular surface, flush with the surrounding glass. In this way alumina membranes, (or enzyme laminates employing alumina membranes), could be clamped between the underlying flat surface of the combined sample chamber and the cell lid, to prevent cracking of the brittle membrane.
Glucose oxidase (GOD) from Asperigillus niger (75% protein, 150 000 U/g solid), and bovine serum albumin (BSA), fraction V, were obtained from Sigma (Poole, Dorset, UK). D-glucose, L-ascorbic acid, sodium hydrogen phosphate, dihydrogen phosphate, sodium chloride, 1,1,1-trichloromethane and dimethyldichlorosilane (all ‘AnalaR’ grade), were purchased from BDH (Poole, Dorset, UK). All chemicals were used without further purification. Electrolube silver conductive paint (Elec-
S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43
The Pt working electrode was polarised at + 650 mV (vs. Ag/AgCl) for the oxidation of H2O2. The cell comprised a central 2 mm diameter platinum disc which served as the working electrode. A platinum counter electrode with a surface area in excess of 2 cm2, together with an Ag/AgCl reference electrode, were suspended within the sample chamber and thus immersed within the analyte sample. A Bank Electrotronik Voltage Scan generator, Wenking model VG72 was used in conjunction with a Ministat potentiostat (H.B. Thompson and Associates, Newcastle-upon-Tyne, UK) for all electrochemical studies; charge current/charge transients were recorded using an IBM compatible PC and Pico software in conjunction with a ‘Picoscope’ analogue to digital converter, purchased from Maplins Electronics, UK.
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electrode, (again with the ultra-thin-polymer-film orientated uppermost), prior to final assembly and fixation by an O-ring.
2.6. Electron microscopy Electron micrographs were taken using Phillips XL 30 FEG electron microscope, using an acceleration voltage of 2.0 kV. For surface examination, ultra-thinpolymer-film-composite membranes were mounted flat onto an aluminium electron microscopy stage, using silver DAG conductive paint, and left to dry in air. For cross-sectional examination, an aluminium stage was cut in half and a section of membrane mounted vertically using silver DAG paint.
2.5. Enzyme laminate fabrication
3. Results and discussion
An enzyme/albumin stock solution was prepared by dissolving glucose oxidase (2560 U ml − 1) and bovine serum albumin (BSA, 0.1 mg ml − 1) in buffer. 6 ml GOD/BSA solution and 3 ml glutaraldehyde (5% v/v in buffer) were mixed rapidly and placed on a 1 cm2 portion of 0.015 mm pore diameter underlying polycarbonate membrane. A 1 cm2 portion of the ultra-thinpolymer-polymer-film composite membrane was placed on top of the enzyme/albumin/gluteraldehyde mixture so that the polymer film remained uppermost, and glass slides were used to compress the enzyme laminate under gentle finger pressure for 5 min. The final crosslinked enzyme laminate was then placed over the working
Siloxanes, such as dimethyldichlorosiloxane may be polymerised via a condensation reaction in the presence of water (Clarson and Semlyen 1993). Dimethyldichlorosiloxane for this study was polymerised at the surface of 0.2 mm pore diameter alumina membranes, a scanning electron micrograph of the surface of which is shown in Fig. 3(a). Membranes were prepared via dimethyldichlorosiloxane vapour deposition at the surface of an alumina membrane, in accordance with the protocol described within the Section 2, and subsequently stored within a dessicator prior to use or characterisation. Membranes were prepared via 30 s, 60 s and 2 mins exposure to dimethyldichlorosiloxane
Fig. 1. Schematic of polysiloxane vapour coating apparatus for the preparation of silicone coated thin-film composite membranes.
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Fig. 2. Custom designed glass cell for the construction of enzyme laminates employing polysiloxane thin-film composite membranes.
vapour within a humid atmosphere. Scanning electron microscopy (SEM) of membranes coated for 30, and 60 s showed an incomplete coverage of the membrane with polymer. Membranes coated via deposition times of \2 mins prevented the passage of all solutes and was, therefore, not studied further. Electron micrographs of alumina membranes coated via 2 mins exposure to dimethydichlorosiloxane vapour in the presence of H2O vapour show, Fig. 3(b), that following this coating duration, the membrane becomes homogeneously coated with a polydimethyldichlorosiloxane film, thereby covering all of the membrane pores. Cross-sectional SEMs show mean thickness of this film to be 80910 nm. It should be remembered in this context, that the microporous membrane serves only as a support for the film which would otherwise be mechanically too fragile to sustain its own integrity. Permeability coefficients, P, for glucose and oxygen were determined for membranes coated via 30 s, 60 s and 2 min polysiloxane vapour deposition exposures, using diffusion chamber apparatus as previously described (Higson et al., 1993b). On each occasion, three membranes of each type were independently tested within the diffusion chamber apparatus and a mean value for each P coefficient value calculated. For clarity only this value is shown in subsequent figures describing permeability coefficients. The permeability coefficients for glucose, Fig. 4, show that as membranes are exposed to longer coating times, the permeability to glucose decreases as the polymer film is progressively laid down at the host membrane surface suggesting that the effective pore area is diminished with further deposition of polymer. It should be noted that the most significant decrease in permeability towards glucose is caused by the first (30 s) deposition of polymer at the host membrane surface. A decrease in O2 permeability is also seen, Fig. 5, as the membrane is progressively
coated via increasing vapour exposure times. The permeability to oxygen continues to decrease as further polydimethyldichlorosiloxane is deposited at alumina membrane surfaces. It should be noted that in accordance with previous studies of thin-film composite membranes, the permeability of all of the membranes towards oxygen is always significantly greater than the corresponding permeability coefficient exhibited towards glucose and this can be explained by the relative molecular weights of the two solutes dictating the rates of solute partitioning across the separatory polymer film. Again the first coating of polymer gives rise to the greatest decrease in permeability for oxygen. Fig. 6 depicts the corresponding glucose/O2 P ratios for the solute permeability data of Figs. 4 and 5, respectively. The first coating of polydimethyldichlorosiloxane gives rise to the greatest lowering of the glucose/O2 P ratio. Extended vapour exposure times result in a continued lowering of this ratio, although the effect becomes less prominent as further polysiloxane is laid down. The membranes showing the smallest glucose/O2 P ratios are those exposed to the longest vapour coating time and therefore, those which posses the greatest deposition of polymer at the alumina surface. It has previously been shown that sensors utilising substrate diffusion limiting membranes with the smallest glucose/O2 P ratios impart the greatest linearity ranges (Myler et al., 1997). Glucose oxidase enzyme electrodes were calibrated for sensors with outer covering membranes coated via 0, 30 s, 60 s and 2 mins exposure to polydimethyldichlorosiloxane vapour, Fig. 7. Reproducible calibration graphs were obtained irrespective of whether aliquots were sampled in order of increasing or decreasing concentrations. Membranes coated via the longest vapour deposition times exhibit the greatest linearity but at the expense of lowered signal magnitudes, Fig. 7. The greatest increase in
S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43
linearity occurs as the first deposition of polysiloxane occurs, which it should be noted correlates with the greatest drop in the glucose/O2 P ratio, Fig. 6. The enzyme electrode curvi-linear range continues to be progressively extended as further polysiloxane coating is applied to the host membrane surface. It is clear, therefore, that all of the coated membranes may act as effective substrate diffusion limiting membranes for the linearisation of sensor responses. It should again be
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noted that although the rate of change of glucose/O2 permeability is greatest for the first polymer depositions (viz the initial 30 s exposure to polysiloxane vapour), membranes with extended coatings continue to impart extended linearity ranges. It follows that the absolute permeability of each solute may also be important in determining final linearity ranges for sensor response. The response times for all of the sensors employing polysiloxane-coated membranes were found to be B1
Fig. 3. SEM surface image of (a) a bare Anopore alumina membrane ( × 4000 magnification) and (b) a polysiloxane thin-film composite Anopore alumina membrane coated via 2 mins exposure to dimethyldichlorosiloxane in the presence of water vapour ( ×4000 magnification).
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Fig. 4. Permeability coefficients (P) for glucose across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
Fig. 6. Glucose/O2 P ratios across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
min which again compares favourably to many contemporary sensors with similar linearity and calibration profiles. One problem that has continued to plague the development and indeed potential commercialisation of many sensors for whole blood determinations, has been the intractable problem of signal drift caused by biofouling effects (Higson et al., 1993a). Silicones have been used as biomaterials (Canham, 1995) and biocompatible coatings (Schmidt et al., 1993) for a range of differing applications for many years and were, therefore, chosen as suitable candidates for possibly helping address this issue. We have previously demonstrated that enzyme electrodes with highly substrate outer covering diffusion limiting membranes may sometimes permit the effective exclusion of anionic biological interferents such as urate and/or ascorbate without the use of conventional permselective membranes (Higson and Vadgama, 1995b). It is thought that this behaviour is related to the anionic nature of the enzyme and albumin (protein) in which it is immobilised and that an interferent ion
may only traverse this layer under conditions of high substrate solute flux and thus enzyme substrate saturation in the case of the oxidases (Higson and Vadgama, 1995b). It is believed that the gluconic acid generated via the enzyme catalysed reaction effectively neutralises the anionic protein layer, thereby allowing anions to traverse this barrier and thus gain access to the working electrode where they may either be oxidised and thus contribute to erroneous signal responses and/or passivate the electrode surface. Enzyme electrodes employing outer covering membranes coated via 2 mins exposure to polydimethyldichlorosiloxane vapour were exposed to glucose in both the presence and absence of 1 mM ascorbate. Ascorbate at this concentration, in the presence of glucose across a range of concentrations, only gave rise to increases of B 1 nA for concentrations of glucose B 30 mM. At glucose responses exceeding 30 mM, the enzyme electrode responses were found to be elevated by \ 15 nA on addition of 1 mM ascorbate, which clearly demonstrates that this enzyme electrode
Fig. 5. Permeability coefficients (P) for oxygen across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
Fig. 7. Glucose enzyme electrode calibrations employing thin-film composite membranes coated via differing vapour deposition times. Uncoated membrane; 30 s coating; 60 s coating; " 120 s coating.
S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43
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Fig. 8. Scanning electron micrographs following 2 h exposure to whole blood for (a) a 0.1 mm pore size anopore microporous alumina membrane and (b) a 0.1 mm pore size anopore microporous alumina membrane coated via 2 mins exposure to dimethyldichlorosiloxane vapour in the presence of H2O (both ×4000 magnification).
behaves in a very similar manner to those already reported, that employ highly substrate limiting coatings (Higson and Vadgama, 1995b). A glucose oxidase enzyme electrode employing ultrathin-film composite membranes fabricated via 2 min exposure to polysiloxane vapour, was fabricated with an underlying 0.015 mm pore size polycarbonate membrane in place of a conventional permselective membrane, (as described in the Section 2). The sensor was first exposed to a pooled human whole blood sample for 2 h. Comparisons to the response of the electrode to 5 mM glucose samples within pH 7.4 phosphate buffer showed a loss
of response of approximately 25% during this time period, although longer exposures to blood did not lead to any further loss of response. For this reason the initial 2 h exposure to blood was treated as a pre-conditioning period prior to any attempted determinations of glucose concentrations. An enzyme electrode that exhibits a stable baseline clearly benefits the analysis of multiple blood samples over a period of time, even if in practice a pre-conditioning exposure to blood period must be introduced in order to obtain stable sensor responses. Scanning electron micrographs of uncoated bare alumina, Fig. 8(a) and polydimethyldichlorosiloxane coated
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alumina membranes, Fig. 8(b) following 5 mins exposure to pooled whole human blood, clearly demonstrate that the degree of biofouling is reduced by the introduction of the polymer coating. Enzyme electrode coatings pre-exposed to blood for 2 h were accordingly re-calibrated prior to the determination of blood glucose calibrations. Patient blood samples were supplied by a local hospital (Manchester Royal Infirmary —UK), within fluoride oxalate tubes to prevent glucose metabolism by blood cells prior to glucose determinations. Blood glucose analyses performed via a polysiloxane ultra-thin film composite enzyme electrode are compared in Fig. 9 to standard hospital biochemistry laboratory analyses performed by the same hospital that supplied the patient samples. The findings show a close correlation (r 2 =0.98) between the two methods.
glucose determinations across a clinically significant concentration range. The composite membrane has also been shown to facilitate the screening of biological anionic interferents due to charge repulsion effects within the underlying enzyme and protein matrix; this behaviour is similar in manner to that observed with other highly substrate diffusion limiting thin-film composite membranes previously reported. Polydimethyldichlorosiloxane was selected, in part, as a candidate for study as a functional polymer coating, due to its well known biocompatibility and use as a biomaterial for many applications. Polysiloxane ultrathin-film composite membranes were found to exhibit enhanced biocompatibility with respect to alumina membranes as demonstrated by reduced surface biofouling effects on exposure to whole blood. Acknowledgements
4. Conclusions Ultra-thin-film polydimethyldichlorosiloxanc composite membranes have permitted the development of a glucose oxidase enzyme electrode for the determination of glucose concentrations within whole blood patient samples. Glucose/O2 P ratios were found to progressively decrease as the pores of the underlying porous alumina membrane became blocked. It is believed that this behaviour can be explained in terms of the relative molecular masses of these two solutes determining the rate of partitioning across the polymer film and thus the permeability of the composite membrane as a whole. It has been demonstrated that ultra-thin-film composite membranes may act as diffusion limiting membranes for the linearisation of sensor responses to allow
Fig. 9. Whole blood glucose determinations via enzyme electrodes employing (Y-axis) 2 mins vapour exposure polysiloxane coating and (X-axis) standard hospital determinations.
The authors would like to thank the EPSRC for a PhD studentship for S. Myler and the Nuffield foundation for financial support. We would also like to thank Rod Hinchcliffe of the Clinical Biochemistry Laboratories— Manchester Royal Infirmary, UK for supplying patient blood samples and for comparative testing. References Bourdillon, C., Thomas, V., Thomas, D., 1982. Electrochemical study of D-Glucose Oxidase auto-inactivation. Enzyme Microb. Technol. 4, 175 – 180. Canham, L.T., 1995. Bioactive silicon structure fabrication through nanoetching techniques. Advanced Mater. 7, 1033 – 1037. Christie, I., Treloar, P.H., Vadgama, P.M., 1992. Plasticized poly(vinyl chloride) as a perinselective barrier membrane for high-selectivity amperometric sensors and biosensors. Anal. Chim. Acta 269, 65 – 73. Clarson, S.J., Semlyen, J.A., 1993. Siloxane Polymers. Prentice Hall, New Jersey, p. 4. Cosnier, S., 1999. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films: A review. Biosens. Bioelect. 14, 443 – 456. Higson, S.P.J., Vadgama, P., 1994. Biosensors: a viable monitoring technology? Med. Biol. Eng. Comput. 32, 601 – 609. Higson, S.P.J., Vadgama, P., 1995a. Diamond like carbon coated films for enzyme electrodes; characterization of biocompatibility and substrate diffusion limiting properties. Anal. Chim. Acta 300, 77 – 83. Higson, S.P.J., Vadgama, P., 1995b. Diamond like carbon films for enzyme electrodes; characterisation of novel overlying permselective barriers. Anal. Chim. Acta 300, 85 – 90. Higson, S.P.J., Desai, M.A., Ghosh, S., Christie, I., Vadgama, P., 1993a. Amperometric enzyme electrode biofouling and passivation in blood. Characterisation of working electrode polarization and inner membrane effects. J. Chem. Soc. Faraday Trans. 89, 2847 – 2851. Higson, S.P.J., Desai, M.A., Koochaki, Z., Vadgama, P., 1993b. Glucose oxidase enzyme electrode: relation between inner membrane permeability and substrate response. Anal. Chim. Acta 276, 335 – 340.
S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43 Iwuoha, E.I., Rock, A., Smyth, M.R., 1999. Amperometric L-lactate biosensors: 1. Lactic acid sensing electrode containing lactate oxidase in a composite poly-L-lysine matrix. Electroanalysis 111, 367 –373. Kress-Rodgers, E. (Ed.), 1997. Handbook of Biosensors and Electronic Noses. CRC Press, London. Mullen, W.H., Keedy, F.H., Churchouse, S.J., Vadgama, P., 1986. Glucose enzyme electrode with extended linearity: application to undiluted blood measurements. Anal. Chim. Acta 183, 59 – 66. Myler, S., Eaton, S., Higson, S.P.J., 1997. Poly(o-phenylenediamine)
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ultra-thin polymer-film composite membranes for enzyme electrodes. Anal. Chim. Acta 357, 55 – 61. Palmisano, F., DeBenedetto, G.E., Zambonin, C.G., 1997. Lactate amperometric biosensor based on an electrosynthesized bilayer film with covalently immobilized enzyme. Analyst 122, 365 –369. Scheller, F., Schubert, F., 1992. Biosensors. Elsevier, London, pp. 85 – 252. Schmidt, S., Horch, K., Normann, R., 1993. Biocompatibility of silicon-based electrode arrrays implanted in feline cortical tissue. J. Biomed. Mat. Res. 27, 1393 – 1399.
Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes S. Myler, S.D. Collyer, K.A. Bridge, S.P.J. Higson * Manchester Materials Science Centre, Uni6ersity of Manchester and UMIST, Gros6enor Street, Manchester, M1 7HS, UK Received 12 October 2000; received in revised form 1 June 2001; accepted 6 August 2001
Abstract An outer ultra-thin-polydimethyldichlorosiloxane film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode biosensor. The composite membrane was formed via the condensation polymerisation of dimethyldichlorosilane at the surface of a host porous alumina membrane. Homogeneous polydimethyldichlorosiloxane films of B100 nm thickness acted as effective substrate diffusional barriers and were supported by the underlying porous alumina surface. Glucose and oxygen permeability coefficients were determined using diffusion chamber apparatus. Polysiloxane composite membranes were found to offer some screening functionality towards anionic biological interferents such as ascorbate. On exposure to blood an approximate 25% signal drift was observed during the first 2 h exposure to blood; after this time responses remained almost stable. Whole blood glucose determinations showed a close correlation (r 2 = 0.98) to analyses performed via standard hospital analyses. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Ultra-thin film composite membranes; Blood glucose determinations; Polysiloxane; Permselectivity; Biocompatibility
1. Introduction Enzyme electrodes have continued to attract considerable interest over the past few years due to the simplified alternative analytical regimes they potentially offer in comparison to many more complicated approaches (Cosnier, 1999). The relatively slow acceptance of biosensor technology has, however, unquestionably been hindered, by a number of generic problems associated with lack of robustness (Cosnier, 1999), poor longevity (Kress-Rodgers, 1997) and the frequent inability to effectively screen against interferents that can give rise to erroneous responses (KressRodgers, 1997). Amperometric/oxidase-H2O2 membrane based sensors continue to represent one of the most studied classes of biosensor, due to the ease by which H2O2 levels may be both quantified and related to substrate levels in addition to the commercial availability and * Corresponding author. Tel.: + 44-1612008871; fax: 1612003586. E-mail address: [email protected] (S.P.J. Higson).
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relative robustness of many oxidase enzymes (Bourdillon et al., 1982). Of these, glucose oxidase enzyme electrodes continue to enjoy the greatest interest, and this may largely be explained in terms of the clinical and economic significance of glucose as an analyte (Scheller and Schubert, 1992). It can be argued that in order for biosensors to gain wider acceptance and indeed practical application, they have generically to more adequately meet a number of requirements in terms of specificity, robustness and sensitivity (Higson and Vadgama, 1994). For many clinical applications, for example, the sensor must resist the detrimental effects of both outer surface biofouling and working electrode passivation (Higson et al., 1993a) in addition to being able to exclude interferents which might give rise to erroneous results (Christie et al., 1992). A common approach used within many electrochemical sensors to overcome these problems has been to employ two or more tailored functionalised membranes to (a) minimise biofouling effects (Higson et al., 1993a,b) screen interferents and/or low molecular weight electrode passivating solutes from reaching the working electrode (Higson et al., 1993a).
0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 6 5 - 2
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S. Myler et al. / Biosensors & Bioelectronics 17 (2002) 35–43
Each membrane acts as an additional diffusional barrier which lowers the magnitude of sensor responses (Iwuoha et al., 1999) as well as extending response times (Mullen et al., 1986). One approach adopted by ourselves (Higson and Vadgama, 1995a) and others (Palmisano et al., 1997) has been to couple two or more functionalities into one membrane thereby minimising the total diffusional barrier offered towards solutes (Higson and Vadgama, 1994). One membrane developed within our laboratory, for example, was based upon anodically electrochemically fabricating ultrathin-polymer-film-composite membranes for use within amperometric enzyme based biosensors (Myler et al., 1997). Ultra-thin-polymer-film-composite membranes consist of host porous mechanical support barriers, onto which are bonded a homogeneous ultra-thin-polymerfilms (e.g. B100 nm thickness), that act as the effective separational barrier (Higson and Vadgama, 1994). High solute fluxes may be achieved across such membranes due to the extreme thinness of the separational polymer film (Higson and Vadgama, 1994). In this way, response times may often be minimised in comparison to those obtainable if two or more conventional membranes are used within a sensor (Higson and Vadgama, 1994). This paper describes the use of a polysiloxane thinfilm-composite membrane to provide the outer surface biocompatibility, permselective screening of interferent solutes and linearisation of sensor responses of a model glucose oxidase based enzyme electrode. The thickness of the diffusional barrier is greatly reduced in comparison to conventional membranes and in this way the magnitude of sensor responses are enhanced while response times can also be minimised. The applicability of the polysiloxane thin-film-composite membrane sensor is demonstrated via inclusion and exploitation within a glucose sensor for whole blood glucose determinations, the performance of which is compared with standard analytical techniques used within a hospital clinical biochemistry laboratory.
trolube Ltd, Berkshire) was purchased from Maplin Electronics. Silver DAG paint was purchased from Merck BDH. Porous alumina Anopore membranes were purchased from Whatman International Ltd (Maidstone, Kent, UK). Microporous polycarbonate membranes were purchased from the Poretic Corporation, Livermore, CA.
2.2. Buffers and solutions A phosphate buffer (pH 7.4) comprising 5.27×10 − 2 M Na2HPO4, 1.3× 10 − 2 M NaH2PO4, 5.1× 10 − 3 M NaCl, was prepared in distilled water and was used for the preparation of all aqueous solutions. Glucose solutions of 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 mM concentration were prepared in phosphate buffer for the calibration of enzyme electrodes.
2.3. Preparation of polysiloxane thin-film composite membranes Alumina membranes were sandwiched between two pieces of laboratory film cut so as to leave an exposed circular area (diameter approximately 50 mm) on the uppermost face of the membrane surface, Fig. 1(a). The membrane in this way was then secured and sealed between two stainless steel plates by means of a screwthreaded clamp, and tightened so as to provide an even pressure, (custom designed by the mechanical workshops of the Manchester Materials Science Centre, UMIST, Manchester). Fig. 1(b). The entire assembly was then placed into a glass vessel (1 litre volume), stored in a Gallenkamp 4A 1715 Humidity Oven, at a constant humidity level of 50% and room temperature (25 °C). 2 ml of monomer were added to a glass petri dish within the vessel, and the membrane assembly placed face down and suspended 1 cm above the liquid monomer, Fig. 1(c). The cell was then sealed and left for a given time duration, after which the assembly was removed from the vessel and stored in the humidity oven for a further 60 min to ensure polymerisation of any excess monomer.
2. Experimental
2.4. Electrochemical cell for enzyme electrodes
2.1. Reagents and membranes
A custom designed glass cell was fabricated by E. Boote Glass Blowing Services (Macclesfield, Cheshire, UK) for GOD-based enzyme electrode fabrication, Fig. 2. The cell was designed so that a working platinum electrode was mounted within a glass platform with a flat circular surface, flush with the surrounding glass. In this way alumina membranes, (or enzyme laminates employing alumina membranes), could be clamped between the underlying flat surface of the combined sample chamber and the cell lid, to prevent cracking of the brittle membrane.
Glucose oxidase (GOD) from Asperigillus niger (75% protein, 150 000 U/g solid), and bovine serum albumin (BSA), fraction V, were obtained from Sigma (Poole, Dorset, UK). D-glucose, L-ascorbic acid, sodium hydrogen phosphate, dihydrogen phosphate, sodium chloride, 1,1,1-trichloromethane and dimethyldichlorosilane (all ‘AnalaR’ grade), were purchased from BDH (Poole, Dorset, UK). All chemicals were used without further purification. Electrolube silver conductive paint (Elec-
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The Pt working electrode was polarised at + 650 mV (vs. Ag/AgCl) for the oxidation of H2O2. The cell comprised a central 2 mm diameter platinum disc which served as the working electrode. A platinum counter electrode with a surface area in excess of 2 cm2, together with an Ag/AgCl reference electrode, were suspended within the sample chamber and thus immersed within the analyte sample. A Bank Electrotronik Voltage Scan generator, Wenking model VG72 was used in conjunction with a Ministat potentiostat (H.B. Thompson and Associates, Newcastle-upon-Tyne, UK) for all electrochemical studies; charge current/charge transients were recorded using an IBM compatible PC and Pico software in conjunction with a ‘Picoscope’ analogue to digital converter, purchased from Maplins Electronics, UK.
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electrode, (again with the ultra-thin-polymer-film orientated uppermost), prior to final assembly and fixation by an O-ring.
2.6. Electron microscopy Electron micrographs were taken using Phillips XL 30 FEG electron microscope, using an acceleration voltage of 2.0 kV. For surface examination, ultra-thinpolymer-film-composite membranes were mounted flat onto an aluminium electron microscopy stage, using silver DAG conductive paint, and left to dry in air. For cross-sectional examination, an aluminium stage was cut in half and a section of membrane mounted vertically using silver DAG paint.
2.5. Enzyme laminate fabrication
3. Results and discussion
An enzyme/albumin stock solution was prepared by dissolving glucose oxidase (2560 U ml − 1) and bovine serum albumin (BSA, 0.1 mg ml − 1) in buffer. 6 ml GOD/BSA solution and 3 ml glutaraldehyde (5% v/v in buffer) were mixed rapidly and placed on a 1 cm2 portion of 0.015 mm pore diameter underlying polycarbonate membrane. A 1 cm2 portion of the ultra-thinpolymer-polymer-film composite membrane was placed on top of the enzyme/albumin/gluteraldehyde mixture so that the polymer film remained uppermost, and glass slides were used to compress the enzyme laminate under gentle finger pressure for 5 min. The final crosslinked enzyme laminate was then placed over the working
Siloxanes, such as dimethyldichlorosiloxane may be polymerised via a condensation reaction in the presence of water (Clarson and Semlyen 1993). Dimethyldichlorosiloxane for this study was polymerised at the surface of 0.2 mm pore diameter alumina membranes, a scanning electron micrograph of the surface of which is shown in Fig. 3(a). Membranes were prepared via dimethyldichlorosiloxane vapour deposition at the surface of an alumina membrane, in accordance with the protocol described within the Section 2, and subsequently stored within a dessicator prior to use or characterisation. Membranes were prepared via 30 s, 60 s and 2 mins exposure to dimethyldichlorosiloxane
Fig. 1. Schematic of polysiloxane vapour coating apparatus for the preparation of silicone coated thin-film composite membranes.
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Fig. 2. Custom designed glass cell for the construction of enzyme laminates employing polysiloxane thin-film composite membranes.
vapour within a humid atmosphere. Scanning electron microscopy (SEM) of membranes coated for 30, and 60 s showed an incomplete coverage of the membrane with polymer. Membranes coated via deposition times of \2 mins prevented the passage of all solutes and was, therefore, not studied further. Electron micrographs of alumina membranes coated via 2 mins exposure to dimethydichlorosiloxane vapour in the presence of H2O vapour show, Fig. 3(b), that following this coating duration, the membrane becomes homogeneously coated with a polydimethyldichlorosiloxane film, thereby covering all of the membrane pores. Cross-sectional SEMs show mean thickness of this film to be 80910 nm. It should be remembered in this context, that the microporous membrane serves only as a support for the film which would otherwise be mechanically too fragile to sustain its own integrity. Permeability coefficients, P, for glucose and oxygen were determined for membranes coated via 30 s, 60 s and 2 min polysiloxane vapour deposition exposures, using diffusion chamber apparatus as previously described (Higson et al., 1993b). On each occasion, three membranes of each type were independently tested within the diffusion chamber apparatus and a mean value for each P coefficient value calculated. For clarity only this value is shown in subsequent figures describing permeability coefficients. The permeability coefficients for glucose, Fig. 4, show that as membranes are exposed to longer coating times, the permeability to glucose decreases as the polymer film is progressively laid down at the host membrane surface suggesting that the effective pore area is diminished with further deposition of polymer. It should be noted that the most significant decrease in permeability towards glucose is caused by the first (30 s) deposition of polymer at the host membrane surface. A decrease in O2 permeability is also seen, Fig. 5, as the membrane is progressively
coated via increasing vapour exposure times. The permeability to oxygen continues to decrease as further polydimethyldichlorosiloxane is deposited at alumina membrane surfaces. It should be noted that in accordance with previous studies of thin-film composite membranes, the permeability of all of the membranes towards oxygen is always significantly greater than the corresponding permeability coefficient exhibited towards glucose and this can be explained by the relative molecular weights of the two solutes dictating the rates of solute partitioning across the separatory polymer film. Again the first coating of polymer gives rise to the greatest decrease in permeability for oxygen. Fig. 6 depicts the corresponding glucose/O2 P ratios for the solute permeability data of Figs. 4 and 5, respectively. The first coating of polydimethyldichlorosiloxane gives rise to the greatest lowering of the glucose/O2 P ratio. Extended vapour exposure times result in a continued lowering of this ratio, although the effect becomes less prominent as further polysiloxane is laid down. The membranes showing the smallest glucose/O2 P ratios are those exposed to the longest vapour coating time and therefore, those which posses the greatest deposition of polymer at the alumina surface. It has previously been shown that sensors utilising substrate diffusion limiting membranes with the smallest glucose/O2 P ratios impart the greatest linearity ranges (Myler et al., 1997). Glucose oxidase enzyme electrodes were calibrated for sensors with outer covering membranes coated via 0, 30 s, 60 s and 2 mins exposure to polydimethyldichlorosiloxane vapour, Fig. 7. Reproducible calibration graphs were obtained irrespective of whether aliquots were sampled in order of increasing or decreasing concentrations. Membranes coated via the longest vapour deposition times exhibit the greatest linearity but at the expense of lowered signal magnitudes, Fig. 7. The greatest increase in
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linearity occurs as the first deposition of polysiloxane occurs, which it should be noted correlates with the greatest drop in the glucose/O2 P ratio, Fig. 6. The enzyme electrode curvi-linear range continues to be progressively extended as further polysiloxane coating is applied to the host membrane surface. It is clear, therefore, that all of the coated membranes may act as effective substrate diffusion limiting membranes for the linearisation of sensor responses. It should again be
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noted that although the rate of change of glucose/O2 permeability is greatest for the first polymer depositions (viz the initial 30 s exposure to polysiloxane vapour), membranes with extended coatings continue to impart extended linearity ranges. It follows that the absolute permeability of each solute may also be important in determining final linearity ranges for sensor response. The response times for all of the sensors employing polysiloxane-coated membranes were found to be B1
Fig. 3. SEM surface image of (a) a bare Anopore alumina membrane ( × 4000 magnification) and (b) a polysiloxane thin-film composite Anopore alumina membrane coated via 2 mins exposure to dimethyldichlorosiloxane in the presence of water vapour ( ×4000 magnification).
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Fig. 4. Permeability coefficients (P) for glucose across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
Fig. 6. Glucose/O2 P ratios across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
min which again compares favourably to many contemporary sensors with similar linearity and calibration profiles. One problem that has continued to plague the development and indeed potential commercialisation of many sensors for whole blood determinations, has been the intractable problem of signal drift caused by biofouling effects (Higson et al., 1993a). Silicones have been used as biomaterials (Canham, 1995) and biocompatible coatings (Schmidt et al., 1993) for a range of differing applications for many years and were, therefore, chosen as suitable candidates for possibly helping address this issue. We have previously demonstrated that enzyme electrodes with highly substrate outer covering diffusion limiting membranes may sometimes permit the effective exclusion of anionic biological interferents such as urate and/or ascorbate without the use of conventional permselective membranes (Higson and Vadgama, 1995b). It is thought that this behaviour is related to the anionic nature of the enzyme and albumin (protein) in which it is immobilised and that an interferent ion
may only traverse this layer under conditions of high substrate solute flux and thus enzyme substrate saturation in the case of the oxidases (Higson and Vadgama, 1995b). It is believed that the gluconic acid generated via the enzyme catalysed reaction effectively neutralises the anionic protein layer, thereby allowing anions to traverse this barrier and thus gain access to the working electrode where they may either be oxidised and thus contribute to erroneous signal responses and/or passivate the electrode surface. Enzyme electrodes employing outer covering membranes coated via 2 mins exposure to polydimethyldichlorosiloxane vapour were exposed to glucose in both the presence and absence of 1 mM ascorbate. Ascorbate at this concentration, in the presence of glucose across a range of concentrations, only gave rise to increases of B 1 nA for concentrations of glucose B 30 mM. At glucose responses exceeding 30 mM, the enzyme electrode responses were found to be elevated by \ 15 nA on addition of 1 mM ascorbate, which clearly demonstrates that this enzyme electrode
Fig. 5. Permeability coefficients (P) for oxygen across polysiloxane thin-film composite Anopore alumina membranes fabricated via a range of differing vapour deposition times.
Fig. 7. Glucose enzyme electrode calibrations employing thin-film composite membranes coated via differing vapour deposition times. Uncoated membrane; 30 s coating; 60 s coating; " 120 s coating.
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Fig. 8. Scanning electron micrographs following 2 h exposure to whole blood for (a) a 0.1 mm pore size anopore microporous alumina membrane and (b) a 0.1 mm pore size anopore microporous alumina membrane coated via 2 mins exposure to dimethyldichlorosiloxane vapour in the presence of H2O (both ×4000 magnification).
behaves in a very similar manner to those already reported, that employ highly substrate limiting coatings (Higson and Vadgama, 1995b). A glucose oxidase enzyme electrode employing ultrathin-film composite membranes fabricated via 2 min exposure to polysiloxane vapour, was fabricated with an underlying 0.015 mm pore size polycarbonate membrane in place of a conventional permselective membrane, (as described in the Section 2). The sensor was first exposed to a pooled human whole blood sample for 2 h. Comparisons to the response of the electrode to 5 mM glucose samples within pH 7.4 phosphate buffer showed a loss
of response of approximately 25% during this time period, although longer exposures to blood did not lead to any further loss of response. For this reason the initial 2 h exposure to blood was treated as a pre-conditioning period prior to any attempted determinations of glucose concentrations. An enzyme electrode that exhibits a stable baseline clearly benefits the analysis of multiple blood samples over a period of time, even if in practice a pre-conditioning exposure to blood period must be introduced in order to obtain stable sensor responses. Scanning electron micrographs of uncoated bare alumina, Fig. 8(a) and polydimethyldichlorosiloxane coated
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alumina membranes, Fig. 8(b) following 5 mins exposure to pooled whole human blood, clearly demonstrate that the degree of biofouling is reduced by the introduction of the polymer coating. Enzyme electrode coatings pre-exposed to blood for 2 h were accordingly re-calibrated prior to the determination of blood glucose calibrations. Patient blood samples were supplied by a local hospital (Manchester Royal Infirmary —UK), within fluoride oxalate tubes to prevent glucose metabolism by blood cells prior to glucose determinations. Blood glucose analyses performed via a polysiloxane ultra-thin film composite enzyme electrode are compared in Fig. 9 to standard hospital biochemistry laboratory analyses performed by the same hospital that supplied the patient samples. The findings show a close correlation (r 2 =0.98) between the two methods.
glucose determinations across a clinically significant concentration range. The composite membrane has also been shown to facilitate the screening of biological anionic interferents due to charge repulsion effects within the underlying enzyme and protein matrix; this behaviour is similar in manner to that observed with other highly substrate diffusion limiting thin-film composite membranes previously reported. Polydimethyldichlorosiloxane was selected, in part, as a candidate for study as a functional polymer coating, due to its well known biocompatibility and use as a biomaterial for many applications. Polysiloxane ultrathin-film composite membranes were found to exhibit enhanced biocompatibility with respect to alumina membranes as demonstrated by reduced surface biofouling effects on exposure to whole blood. Acknowledgements
4. Conclusions Ultra-thin-film polydimethyldichlorosiloxanc composite membranes have permitted the development of a glucose oxidase enzyme electrode for the determination of glucose concentrations within whole blood patient samples. Glucose/O2 P ratios were found to progressively decrease as the pores of the underlying porous alumina membrane became blocked. It is believed that this behaviour can be explained in terms of the relative molecular masses of these two solutes determining the rate of partitioning across the polymer film and thus the permeability of the composite membrane as a whole. It has been demonstrated that ultra-thin-film composite membranes may act as diffusion limiting membranes for the linearisation of sensor responses to allow
Fig. 9. Whole blood glucose determinations via enzyme electrodes employing (Y-axis) 2 mins vapour exposure polysiloxane coating and (X-axis) standard hospital determinations.
The authors would like to thank the EPSRC for a PhD studentship for S. Myler and the Nuffield foundation for financial support. We would also like to thank Rod Hinchcliffe of the Clinical Biochemistry Laboratories— Manchester Royal Infirmary, UK for supplying patient blood samples and for comparative testing. References Bourdillon, C., Thomas, V., Thomas, D., 1982. Electrochemical study of D-Glucose Oxidase auto-inactivation. Enzyme Microb. Technol. 4, 175 – 180. Canham, L.T., 1995. Bioactive silicon structure fabrication through nanoetching techniques. Advanced Mater. 7, 1033 – 1037. Christie, I., Treloar, P.H., Vadgama, P.M., 1992. Plasticized poly(vinyl chloride) as a perinselective barrier membrane for high-selectivity amperometric sensors and biosensors. Anal. Chim. Acta 269, 65 – 73. Clarson, S.J., Semlyen, J.A., 1993. Siloxane Polymers. Prentice Hall, New Jersey, p. 4. Cosnier, S., 1999. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films: A review. Biosens. Bioelect. 14, 443 – 456. Higson, S.P.J., Vadgama, P., 1994. Biosensors: a viable monitoring technology? Med. Biol. Eng. Comput. 32, 601 – 609. Higson, S.P.J., Vadgama, P., 1995a. Diamond like carbon coated films for enzyme electrodes; characterization of biocompatibility and substrate diffusion limiting properties. Anal. Chim. Acta 300, 77 – 83. Higson, S.P.J., Vadgama, P., 1995b. Diamond like carbon films for enzyme electrodes; characterisation of novel overlying permselective barriers. Anal. Chim. Acta 300, 85 – 90. Higson, S.P.J., Desai, M.A., Ghosh, S., Christie, I., Vadgama, P., 1993a. Amperometric enzyme electrode biofouling and passivation in blood. Characterisation of working electrode polarization and inner membrane effects. J. Chem. Soc. Faraday Trans. 89, 2847 – 2851. Higson, S.P.J., Desai, M.A., Koochaki, Z., Vadgama, P., 1993b. Glucose oxidase enzyme electrode: relation between inner membrane permeability and substrate response. Anal. Chim. Acta 276, 335 – 340.
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