Anal. Chem. 1993, 65,
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Enzyme Switch Responsive to Glucose P. N. Bartlett' and P. R. Birkin School of Chemistry, Uniuersity of Bath, Bath BA2 7AY, U.K.
INTRODUCTION Inthispaperwedescribeforthefirsttimeanenzymeswitch responsive toglucose. Theideaofprducingaswitchsensitive to,and selectivefor, a chemical species is extremely appealing. Thefirstexamplesofsuchdevices weredescribedby Wrighton et al.,'.* who demonstrated switches based on conducting polymer films electropolymerized across a gap of micron dimensions between two gold electrodes. These devices were responsive to redoxspecies including hydrogen,' oxygen,l and complex metal ions2 and made use of the well-documented changes in conductivity which accompany oxidation and reduction of these This idea was extended hy Matsue et al.8 by the incorporation of an enzyme into the polymer to produce a device responsive to the substrate for that enzyme. In their device they immobilized the enzyme diaphorase (NADH dehydrogenase, EC 1.6.99) in an electropolymerized poly(pyrro1e) film grown across a gap. In the presence of a suitable mediator (anthraquinone-2-sulfonic acid, sodium salt), they were able to link the oxidation of NADH, catalyzed by the enzyme,to the reduction of the poly(pyrrole) film. Thus, starting with the film in its oxidized, conducting, state they showed that on addition of NADH the resistance of their device increased as the polymer was converted to its reduced, insulating, state. This switching process took -15-20 min. In the present work, the switch is constructed by immobilizingglucose oxidase (EC 1.1.3.4) in anelectropolymerized film of poly(l,2-diaminohenzene)grown on top of a poly(aniline) base film, Figure 1. The switch employs tetrathiafulvalene (TTF) as a redox mediator7 capable of shuttling charge between the enzyme and the conducting polymer. In the oxidized state, at +0.5 V vs SCE and at pH 5.0, poly(aniline) isinsulating. Onadditionofglucosethepoly(aniline) is reduced through the following set of reactions
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+ GOx(ox) GOx(red) + gluconolactone GOx(red) + 2TTF* GOx(ox) + 2TTF TTF + PANI(ox) TTF' + PANI(red)
&D-ghCOSe
where GOx(ox) and GOx(red) represent the oxidized and reducedformsoftheenzyme,TTF+andTTFare theoxidized and reduced forms of the mediator, and PANI(ox) and PANl(red) represent the oxidized, insulating, and reduced, conducting, forms of the poly(ani1ine) film. Present addreas: Depanmmr of Chemistry. Universihl of Southamp ton, Southampton. SO9 5". U.K. ( I , Tharkeray. .I.W.: Wnghmn, M.S . J . Phjd. Chem. 19%. 90,fifi74. (2) Paul. E. W.:Rirco.A.d.; Wrighton, M.S.J.Phyr. C h m 19R5,R9, 1441. 13) Ofer.. D.:. Crooks. R. M.:.Wriehton. I .M. S.J. Am. Chem. Soe. 1990. 112,1869. (4) Olmedo, L.; Chanteloube, I.; Gennain, A,; Petit, M.Synth. Met. 1989, 30, 159. (5) Schiavon, G.; Sitran, S.: Zotti, G . Synth. Met. 1989,32,209. ( 6 ) Matsue, T.: Nishizaura, M.: Sawaguchi, T.: Uchida, I. J. Chem. Sac., Chem. Commun. 1991,1029. (7) Bartlett, P. N.; Bradford, V. Q. J . Chem. Soe., Chem. Commun. 1990,1135.
/ po1y(1.2-diarninobenzene) poly(ani1ine)
. carbon electrodes
Figure 1. Enzynmswnch responslvetoglucose. The enzyme glucose ox&se Is Immobilized in a thin insulating film of poly(l,2dlaminobenzene) deposned on top of ihe poiy(aniline)film
,
..
.
..
- 0 . 5 V "8 SCE
:,......
.,.
,. ....
,WO
Figure 2. Operation of the swnch. Upon exposure to glucose the switch is turned from " o f f to '"on''. The swnch is reset by holding ihe film at + O S V vs SCE in a background buffer solution.
EXPERIMENTAL SECTION The switches were fabricated on screen-printed carbon microband electrodes. These were fabricated on a PVC base by screen printing successivelayersof carbon followed by twolayers of dielectric, followed by another layer of carbon, and finally by two layers of dielectric. The dielectric provides insulation between the two carbon microhand electrodes. The microbands are typically 0.5 em long and l(t15 pm wide with the gap 2W25 pm wide. The surface of the electrodes was exposed by cooling the printed PVC in liquid nitrogen and then fracturing across the electrode. Poly(ani1ine)was grownacross the gap by holding the potential of the two carbon electrodes for 20 s at +0.9 V vs SCE in a solution containing 200 pL of aniline (Aldrich)and 5 cm3 of NaHSOI (Aldrich) acidified to pH -0 with 0.5 cm3 of concentrated HzSOd (95-98% AR grade, Aldrich). The typical total charge passed during this deposition was 3 mC, corresponding to a film thickness of -20 pm. Glucose oxidase is irreversiblydamaged if placed in solutions belowpH58.9andsotheenzymeisincompatible withthestrongly acidic conditions required to grow the poly(ani1ine) film. We therefore chose to entrap the enzyme in an insulating poly(1,P diaminohenzene) film electrochemicallydeposited on top of the poly(ani1ine) bridge. This film wa8 chosen because it can he electrochemicallypolymerized at a potential where poly(aniline) is conducting and it produces a highly active enzyme film.'0 This (8)Chen, T. L.;Weng, H. S. Biotechnol. Bioeng. 1986,28. 107. (9) Bauin, J. C.; Hultin, H. 0.Bioteehml. Bioeng. 1982,.?.4,1225. (10) Malitesta, C.; Palmisem, F.; Torsi, L.:Zambonin, P. G . Anal. Chem. 1990,62,2735. F, 1993 American Chemical SocleC,
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8,APRIL 15, 1993 111B experiments the buffer was purged with argon for 20 min to remove dissolved oxygen. Subsequentexperiments showed that the same results were obtained in air-saturated buffer. In some experiments the Triton XlOO and TTF were omitted from the buffer solution. At the start of each measurement the device was held at +0.5 V vs SCE for 30 s to oxidize the poly(ani1ine). The device was then disconnected from the potentiostat, and a bias of 20 mV was applied between the two electrodes (see Figure 2). The drain current, i b , flowing through the poly(ani1ine) between the two carbon electrodes was recorded as a function of time with and without glucose present.
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Increasing glucaac concentration
c 8 C
3 C
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2 C
E
4
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40
RESULTS AND DISCUSSION
60 80 100
[glucoae]/mmol
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dm-'
No glucose 0 0
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Tirne/a
recorded for a 20-mV bias Flgure 3. Plots of the drain current, as a function of time following addition of an allquot of glucose to a cibatdphosphate pH 5 buffer solutlon containing 0.5 mol dm-3 Na2S04/ 0.5 vol 96 Triton XlOO and saturated with lTF. Between each measurement the device was washed and reoxidized at +0.5 V vs SCE. The inset shows a calibration plot of S, as a function of the glucose concentration. was done by transferring the poly(ani1ine)-coatedelectrodes to a pH 5 citric acid (Fisons)/NazHPO~(Fisons) buffer" solution containing 0.5 mol dm3 NazS04,5 mmol dm3 1,a-diaminobenzene, and 167 pmol dm3 glucose oxidase. The electrodes were left in solution at open circuit for 6 min to allow adsorption of ~ J ~ poly(l,2the enzyme to the poly(ani1ine) s u r f a ~ e . ~The diaminobenzene)f i i was then deposited by holding the potential at +0.4 V vs SCE for 15 min. Finally, the device was placed in a stirred solution containing 3 cm3pH 5.0 buffer, 50 p L of DMSO (Aldrich),12pLofTritonXlOO(Aldrich),andsolidTTF (Aldrich) for at least 2 h to allow incorporation of TTF in the device. The final working devices were stored at room temperature in this TTF-containing solution. Responses to glucose were recorded in a pH 5 citric acid/ NazHPO4 buffer solution containing 0.5 mol dm3 NazS04/0.5 vol % Triton X100, saturated with TTF. For our initial (11)Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. Data For Biochemical Research, 3rd ed, Oxford University Press: Oxford, England, 1986. (12) Szucs, A.; Hitchens, G. D.; Bockris, J. 0. M. J.Electrochem. Soe. 1989,136, 107.
(13) Wahlg-ren, M.; Arnebrant, T. Trends Biotechnol 1991,9,201. (14) Followingsubmissionof this manuscript two papers have appeared describing the use of conducting polymers to make biosensor devices: Nishizawa, M.; Matsue, T.; Uhida, I. Anal. Chem., 1992,64,2642. Hoa, D. T.; Suresh Kumar,T. N.; Punekar, N. S.; Srinivasa, R. S.; Lal, R.; Contractor, A. Q. Anal. Chem. 1992,64,2645. In both cases these devices work by responding to localized pH changes in the polymer film. This approach suffers from the well-documentedproblems of all potentiometric pH responsive enzyme devices: Eddowes, M. J.; Pedley, D. G.; Webb, B. C. Sens. Actuators, 1985, 7,233. In the device described in this paper the change in conductivity is brought about by the mediated reduction of the conducting polymer and not as the result of local pH changes.
Before the addition of glucose idrain 0 because, at +0.5 V vs SCE a t this pH, the poly(aniline1 is in its insulating state. On addition of glucose there is an initial lag followed by a rapid increase in the drain current, which then levels off to a constant value. Figure 3 shows a typical set of results for one device for arange of glucoseconcentrations. Between each measurement the device was washed in buffer and then the poly(aniline) was reoxidized to +0.5 V vs SCE. The speed of response of the device, as defined by the time taken for i & h to reach half of its final value, tllz, and the slope of the steepest part of the curve, , S both depend on the glucose concentration. The inset in Figure 3 shows a plot of S, as a function of the glucose concentration. In the absence of glucosethe device turns on slowly, tl/z 360 s due to reduction of the poly(ani1ine) film by TTF present in solution. In blank experiments, where either glucose oxidase or TTF was omitted, no responses to glucose were obtained.14 When the TTF was omitted from the buffer solution, the speed of response to glucose decreased with each successive measurement as the device was washed, reoxidized, and reused. The speed of response can be returned to its initial value if the device is placed back into the modificationsolution for a repeat modification period. We attribute the decrease in response time to loss of TTF from the enzyme film during the measurements. No such decrease is observed when the TTF is present in the buffer. Our results show that poly(ani1ine)can be used as the basis for a enzyme switch and that by operating this so that the switch turns on, as opposed to off, in the presence of the enzyme substrate we can obtain responses which are significantlyfaster than those obtained by Matsue et al. for a switch based on a copolymer of poly(pyrro1e).
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ACKNOWLEDGMENT We thank MediSense (UK)Inc. for a CASE studentship for P.R.B. and for assistance with screen printing. RECEIVEDfor review October 20, 1992. Accepted January 22, 1993.
