Anal. Chem. 1995, 67,1332-1338
Polyacrylamide=BasedRedox Polymer for Connecting Redox Centers of Enzymes to Electrodes T h i e y de Lumley-Woodyear,t Patrick Rocca,ts* Jamie Lindsay,t Yael h r , l Amlhay Freeman+ and Adam Heller**t Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712-1062, and Department of Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, 69978, Israel
Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C11+/2+(dmebpy = 4,4-dimethyl-2,a’-bipyridine) and cross-linkingwith oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linkingis (1.3 f 0.1) x cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel “wired” horseradish peroxidase (HRP), lactate oxidase (Lox)or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking,and their enzyme content. At the wired HRP electrodes, H202 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalyticallyoxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 pg/ cm2 thick polymer Eilms, were selective for glucose in the presence of physiological concentrations of urate and ascorbate. Polyacrylamide gels are advantageously used in biochemistry as separation matrices for biological macromolecules, as they do not strongly or irreversiblybind proteins or nucleic acids. Enzyme electrodes made with polyacrylamide-containing films have been reported.’ In these, the redox polymer was a copolymer of polyacrylamide and polyacrylate,to which alkylamine-derivatized ferrocene redox centers were covalently bound. The polymer and either glucose oxidase or horseradish peroxidase were crosslinked with glutaraldehyde to form the electrode-modifyingfilm. In these electrodes, only the immediate enzyme-containinglayer communicated electrically with the electrodes. With the polyacrylamide-derivedelectronconducting redox hydrogels that we describe here, higher HzOz electroreduction currents are observed, because multiple layers of the enzyme horseradish peroxidase are connected electrically to electrodes. Specilically the steady-state HzOz electroreduction current density was increased 5@fold. Electrical communication between redox centers of enzymes and metal electrodes is of the essence in amperometric University of Texas at Austin. Present address: Institut National des Sciences Appliquees, Place Emile Blondel, B.P. 08, 76131 Mont-Saint-Aignan’Cedex,France. 5 Tel-Aviv University. (1) Calvo E. J.; Danilowicz C.; Diaz L. /. Chem. Soc., Faraday Trans. 1993,89, 377-84. +
4
1332 Analytical Chemistry, Vol. 67,No. 8,April 75,7995
biosensor~.~-~ One way to realize such communication is through three-dimensional redox p~lymefl-’~ networks that electrically connect enzyme redox centers to electrodes. Different systems of enzyme wiring hydrophilic epoxy cement-based hydrogels are example^.^-^ Redox hydrogel films are unique in both having adequate electron diffusion coefficients (Le., not rate limiting) and being permeable to water-soluble substrates and products of enzymatic reactions. When a cross-linkedredox polymer network electrically “wires”an enzyme that is covalently bound to it, the gel and the electroncollecting metal form the enzyme electrodes. Such electrodes are useful in applications where release of diffusional mediators from the electrodes is to be avoided and where small size is imporkmt.5J4J5 Redox polymer wires based on osmium(bpy)somplexed poly(4vinylpyridine) (PVP) or poly(1-vinylimidazole) (PW) (bpy = bipyridine or its derivative) have been reported.16 These wires formed, upon cross-linking, adequate electron conductors and transferred electrons, originating in the redox sites, to the electrodes. For example, hydrogels were made by cross-linking WI,-Os (n = 3,5, or 10) and glucose oxidase with poly(ethy1ene glycol) diglycidyl ether (PEGDGE). Effective “wiring”of glucose oxidase depended on interaction of segments of the redox polymer with deeply buried redox centers of the enzyme and required strong electrostaticinteraction between the enzyme and the wiring redox polymer. Here we show that, in the absence of such strong interaction with the polyacrylamide-based redox polymer, glucose (2) (a) Degani, Y.; Heller, A/. Am. Chem. SOC.1987,91, 1285-9. @) Degani, Y.; Heller, A J. Am. Chem. SOC.1988,110,2615-20. (3) Schuhmann, W.; Ohara, T. J.; Schmidt, H.-L.; Heller, A J. Am. Chem. Soc. 1991,113,1394-7. (4) Degani, Y.; Heller, A J. Am. Chem. SOC.1989,Ill, 2357-8. (5) F’ishko, M. V.; Katakis, I.; Lindquist, S.-E.;Ye, L.; Gregg, B. A; Heller, A Angew. Chem., Int. Ed. Engl. 1990,29,82-4. (6) Gregg, B. A; Heller, A Anal. Chem. 1990,62, 258-63. (7) Gregg, B. A; Heller, A /. Phys. Chem. 1991,95,5970-5. (8) Gregg, B. A; Heller, A J. Phys. Chem. 1991,95,5976-80. (9) Katakis, I.; Heller, A Anal. Chem. 1992,64, 1008-13. (10) Hale, P. D.; Inagaki, T.; Ikamoto, Y.: Skotheim, T. A J Am. Chem. SOC. 1989,Ill, 3482-3. (11) Hale, P. D.; Boguslavski, L. I.; Inagaki,T.; Lee, H. S.; Skotheim, T. A; Karan, H. I.; Okamoto, Y. Mol. Ctyst. Lis. Cryst. 1990,190,251-8. (12) Hale, P. D.; Boguslavski, L. I.; Inagaki,T.; Karan,H. I.; Lee, H. S.; Skotheim, T. A; Okamoto, Y. Anal. Chem. 1991,63, 677-82. (13) Foulds, N. C.; Lowe, C. R Anal. Chem. 1988,60,2473-8. (14) Wang, D. L.; Heller, A Anal. Chem. 1993,65,1069-73. (15) Linke, B.; Kemer, W.; Kiwit, M.; F’ishko, M.; Heller, A Biosens. Bioelectron. 1994,9,151-8. (16) Ohara, T.; Rajagopalan, R; Heller, A Anal. Chem. 1994,66,2451-7.
0003-2700/95/0367-1332$9.00/0 0 1995 American Chemical Society
oxidase is poorly wired, but horseradish peroxidase, the redox functions of which are superlicial, is well wired. We form the hydrogel from the 7:1copolymer of acrylamide and l-vinylimidazole (PAA-PVI), which we complex with [O~(dmebpy)zClI+/~+. The resulting redox polymer PAA-PVI-Os is highly water soluble. We form sensors by cross-linking PAA-PVI-Os with GOx, LOX, or HRP using glutaraldehyde, the N-hydroxysuccinimide diester of suberic acid, dimethyl suberimidate, or PEGDGE. The resulting redox hydrogels adhered to vitreous carbon electrodes. The best electrodes were made using dimethyl suberimidate as cross-linker. The redox potential of dimethyl suberimidatecross-linkedPAA-PVI-Os was $55 mV (SCE), and a plateau in the electrooxidation current was reached at +150 mV (SCE). When poised at +160 mV (SCE) the interferant (e.g., Figum I. (A) Structure of the poly(acty1amide)-poly(vinylimidazo1e) ascorbate or urate) electrooxidation currents were low. EXPERIMENTAL SECTION Chemicals. Acrylamide (Merck, Catalog No. 800830), vinylimidazole (Aldrich, Catalog No. 23,5466), N,N,N',W-tetramethylethylenediamine (TEMED; Fluka Catalog No. 87690), ammonium persulfate (Merck, Catalog No. 1201) hydrazine hydrate (Aldrich, Catalog No. 22,581-9), 4,4'-dimethyl-2,2'-bipyridine (dmebpy; Aldrich, Catalog No. 24,573-9), suberic acid bis(N-hydroxysuccinimide ester) (Sigma, Catalog No. S 1885), dimethyl suberimidate dihydrochloride (Sigma, Catalog No. D 7636), poly(ethy1ene glycol) diglycidyl ether (PEGDGE 400; Polysciences, Catalog No. 08210), glutaraldehyde (Sigma grade I, 25%aqueous solution, Catalog No. G5882), sodium borohydride (Aldrich, powder, 98% purity, Catalog No. 19,807-2), glucose oxidase (Sigma, Catalog No. G7141) from Aspergillus niger (type X-S, 75% protein), lactate oxidase (Sigma, Catalog No. L 0638) from Pediococcus species, and peroxidase (Sigma, Type XI1 from horseradish, Catalog No. P 8415) were used as received. Os(dmebpy)zClz was prepared as reported.17 Copolymer of Acrylamide and Vinylimidazole. Into a 1 L round-bottomed flask, equipped with magnetic stirrer and thermostated at 40 "C, were added 24 g of acrylamide and 7.0 mL of vinylimidazole dissolved in 150 mL of water, followed by a solution of 0.69 mL N,N,W,N-tetramethylethylenediaminein 50 mL of water and a freshly prepared solution of a well-dried ammonium persulfate (0.6 g dissolved in 150 mL of water). The polymerization reaction was allowed to proceed in a tightly closed vessel with stirring for 30 min. The polymer thus obtained was separated by dropwise addition of the reaction mixture into 2 L of methanol. The precipitate was separated, redissolved in 300 niL of water and reprecipitated in 2 L of methanol. The polymer was separated, incubated under methanol at 4 "C overnight, and dried in an evaporator. Figure lA shows the copolymer of acrylamide and vinylimidazole thus obtained. PAA-FW-dmeOs. The osmium-derivatized polymer was prepared by refluxing Os(dmebpy)zClz (624 mg) with the copolymer of acrylamide and vinylimidazole (430 mg) in 50 mL of ethylene glycol for 3 days under argon. The polymer solution was added dropwise under stirring to an ether-acetone mixture (800 mL/200 mL) . The osmiumderivatized polymer was precipitated in ether, filtered through a low-porosity glass frit, and dried (17) Buckingham, D. A; Dwyer, F.P., Goodwin, H.A; Sargeson,A M.Aust. J Chem. 1964,17, 325.
(PAA-PVI polymer). (B) Structure of the osmium-complexed redox polymer after partial substitution of its amide by hydrazide groups (PAA- PVI-dmeOs).
0
0
POlymCr(0S)
A w
HZO
+NH,NH*
polymcr(os)&""1
(wh)
p-1
(PAA-PM-dmcOs)
Figure 2. Synthesis of PAA-PVI-dmeOs.
under vacuum. The yield was 88%. In order to introduce reactive groups for cross-linking, part of the amide groups of the polymer were substituted by hydrazide groups. This was done by reacting 200 mg of the redox polymer, dissolved in 4.15 mL of water, with 1.56 mL of hydrazine hydrate for 6 h at 40 "C. The solution was added to absolute ethanol (400 mL), and 90% of the liquid was evaporated in order to remove most of the water as its azeotrope with alcohol. The mixture was then centrifuged at 2200 rpm for 1h to separate the insoluble cross-linked polymer. The nonaosslinked PAA-PVI-dmeOs in ethanol was added dropwise with stirring to diethyl ether (500 mL) , reprecipitated, and filtered. The dark purple solid (Le., PAA-PVI-dmeOs, Figure 1B) was dried under vacuum ovemight at room temperature. The yield was 40% ( F i i e 2). Calcd for C52.6H93.zClzN1*8019.30s (M 1502.9): C, 42.04, H, 6.25; C1, 4.72; N, 13.79; Os, 12.66. Found: C, 41.66; H, 5.97; C1, 4.43; N, 13.68; Os, 12.55. Electrodes. Rotating disk electrodes were prepared by embedding vitreous carbon rods (3 mM diameter, V10 Atomergic) in a Teflon shroud using a low-viscosity epoxy resin (Polyscience, Catalog No. 01916). All enzyme electrodes were prepared by syringing the polymer (5 mg/mL in water), the enzyme (0.5, 1, or 2.5 mg/mL in phosphate buffer, pH 7.4), and then the crosslinker (0.1 or 0.2 mg/mL in phosphate buffer, pH 7.4) onto the electrode surface (0.071cm? and by mixing on the surface Figure 3a). The electrodes were allowed to cure for at least 24 h in air at room temperature before use. Measurements. Electrochemical measurements were performed with a Princeton Applied Research 175 universal programmer, and a Model 173 potentiostat or with a Princeton Applied Research Model 400 bipotentiostat. The signal was recorded on a Kipp and Zonen X-Y-E" recorder. Rotating disk electrode Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
1333
A
f NH/NH-
Polymer
-
m'
"H
On the surface
of the electrode (RT)
NH
Polymer
Crosslinker
D:
X
HC-CH II 0
~
X
-HN-Ywuvb%Y-NH-
It
0
Figure 3. (a) The cross-linking reactions investigated: (b) Structures of the cross-linkers (left), (A) suberic acid bis(Khydroxysuccinimide ester), (B) dimethyl suberimidate, (C) poly(ethy1ene glycol) diglycidyl ether, and (D) glutaraldehyde; and their reaction products with amines (right).
experiments were performed with a Pine Instruments AFMSRX rotator with an MSRS speed controller. All chemical measurements were performed using a 20 mM phosphate buffer (PH 7.4) containing 0.14 M NaCl, in a three-electrode cell with a rotating (1000 rpm) glassy carbon working electrode, a saturated calomel reference electrode (SCE), and a platinum counter electrode, isolated from the bulk solution with a Vycor frit (EG&G, Princeton Applied Research). The cells were Nz purged. RESULTS AND DISCUSSION
Cyclic Voltametry of Variously Cross-IinkedPAA-PVIdmeOs-GOx Films. Four cross-linkerswere used: glutaralde 1334 Analytical Chemistry, Vol. 67, No. 8, April 75,7995
hyde, suberic acid bis(N-hydroxysuccinide ester), dimethyl suberimidate dihydrochloride, and PEGDGE. Their structures are shown in Figure 3b. All cross-linked films adhered well to the vitreous carbon electrodes and all, except for the glutaraldehyde cross-linked films, retained -90% of their electroactive centers when soaked in a stirred phosphate buffer at room temperature for 48 h. The cyclic voltammograms of the filmcoated rotating disk electrodes had the classical symmetrical shape, characteristic of reversible oxidation and reduction of a surface-bound species. Their half-wave potential was +55 mV (SCE). This potential was independent (variation A/cmz) and the sensitivity (> 1A cm-2 M-l) are high and HzOz electroreduction is seen already at M HzOz concentration. Apparently the coiled PAA-PVI-dmeOs polymer effectively connects superficial HRP redox centers to electrodes but, unlike PVI-Os, does not form an electrostatic complex with GOx and, as a result, does not effectively wire its redox centers.
1338 Analytical Chemistry, Vol. 67, No. 8, Apd 75, 7995
ACKNOWLEDGMENT We acknowledge support of this work by the Office of Naval Research, the National Science Foundation,the National hstitutes of Health (No. lROlDK42015OlA), and the Robert A Welch Foundation. Received for review November 11, 1994. January 29, 1995.@
Accepted
AC9411031 ~
Abstract published in Advance ACS Abstracts, March 1, 1995.
