Anal. Chem. 2005, 77, 729-732
Detection of Glucose at 2 fM Concentration Nicolas Mano* and Adam Heller
Department of Chemical Engineering and Texas Materials Institute, The University of Texas at Austin, Austin, Texas 78712
We report the amperometric detection of glucose at 2 fM concentration in a physiological buffer solution at 1 atm O2 pressure. The sensitive assay is based on the close to absolute electroreductive stripping of O2 from the solution near the glucose electrooxidizing anode. The glucose was detected by its electrooxidation on a stationary glassy carbon disk surrounded by an also stationary platinum ring. The disk was coated with a film of glucose oxidase (GOx), electrically “wired” with PVP-[Os(N,N′-dimethyl2,2′-biimidazole)3]2+/3+ (polymer I), having a redox potential of -0.19 V versus Ag/AgCl. The ring was coated with bilirubin oxidase (BOD) “wired” with PAA-PVI[Os(4,4′-dichloro-2,2′-bipyridine)2Cl]+/2+ (polymer II), having a redox potential of + 0.36 V versus Ag/AgCl. The ring-disk electrode was held facing up, and a 30-µL drop was placed on it for the assay, with the ring poised at -0.3 V/ AgAgCl and the disk poised at -0.1 V/ Ag/AgCl. Even though the atmosphere over the drop was O2 at 1 atm pressure, the wired BOD disk scavenged the O2 so effectively that the glucose-reduced FADH2 of GOx was not oxidized by O2, the natural cosubstrate of the enzyme. The lower limit of the glucose concentration that can be detected in a biological sample through a redox couple-mediated electrooxidation reaction is often defined by the dissolved O2 concentration. For selective glucose electrooxidation, the glucoseelectrooxidizing anode must be poised at a potential where electrooxidizable solutes, other than glucose, are not electrooxidized. Even if O2 is not a cosubstrate of the enzyme applied to catalyze the electrooxidation, as is the case for the widely used PQQ-glucose dehydrogenase (GDH), O2 slowly oxidizes the electron-shuttling enzyme-reduced redox mediator and is slowly electroreduced at the potential at which even a poorly O2 electroreduction-catalyzing electrode, such as a vitreous carbon electrode, is poised. In the case of flavoenzymes, such as glucose oxidase, O2 directly competes for FADH2 electrons of the glucosereduced enzyme. The dynamic range of the enzyme electrodes is extended to lower glucose concentrations usually by using fast electron acceptors, such as diffusing ferrocene/ferrocinium couples1 or redox polymers,2,3 which capture the FADH2 electrons more rapidly than O2. * To whom correspondence should be addressed. E-mail: mano@ mail.utexas.edu. (1) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem 1984, 56, 667-661. 10.1021/ac0486746 CCC: $30.25 Published on Web 12/03/2004
© 2005 American Chemical Society
The monitoring of very low glucose concentrations requires, however, reductive scavenging of O2 from the proximity of the glucose anode.4,5 To this end, Ikeda and his colleagues introduced the use of a gold minigrid cathode in front of the redox couplemediated glucose electrooxidizing anode on which the O2 was electroreduced4 and Yarnitzky and co-workers shielded their “wired” glucose oxidase-coated anode, which was poised at +0.4 V versus AgAgCl, by a glucose-permeable, gold-coated membrane cathode, poised at -0.4 V/AgAgCl, on which the O2 was electroreduced.6,7 Both succeeded in detecting accurately 1 mM glucose concentration under 1 atm O2. An alternative, very successful, approach has been to employ instead of glucose oxidase, GDH. Although O2 is not a cosubstrate for GDH, and much lower glucose concentrations can be detected with electrodes coated with this enzyme, some O2 is still reduced at the potentials at which the GDH electrodes are poised, interfering with the assay of glucose at very low concentrations. We recently described a glucose electrooxidizing anode made by “wiring” glucose oxidase (GOx) with poly(4-vinylpyridine)[Os(N,N′-dimethyl-2,2′-biimidazole)3]2+/3+ (polymer I).2 The unique features of this “wire” were its 13-atom-long flexible tethers, binding the redox centers to the backbone, and the reducing redox potential of the dialkylated biimidazole complex of Os2+/3+. The long tethers increased the frequency of effective electrontransferring collisions between reduced and oxidized osmium centers and thereby the apparent electron diffusion coefficient, Dapp, which reached in the cross-linked redox hydrogel 5.8 × 10-6 cm2 s-1, an order of magnitude higher than in earlier redox hydrogels. The effective collection of the electrons from the glucose-reduced GOx allowed poising of the anode at a potential as reducing as - 0.10 V versus Ag/AgCl. We also described a bioelectrocatalyst that was superior to platinum in the four-electron electroreduction of oxygen to water,8 made by wiring Tt-bilirubin oxidase (BOD) with a wire, also having long and flexible tethers between its backbone and its redox centers. Here we apply the efficiently O2-electroreducing “wired” BOD in the scavenging of the interfering O2, and describe the detection of glucose at femtomolar concentrations under 1 atm O2 pressure, even when the glucose electrooxidizing anode is poised at the highly reducing (2) (3) (4) (5)
Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2002, 125, 4951-4957. Calvo, E. J.; Wolosiuk, A. Chem. Phys. Chem. 2004, 5, 235-239. Ikeda, T.; Katasho, I.; Senda, M. Anal. Sci. 1985, 1, 455-457. Ikeda, T.; Hirokazu, H.; Kojiro, M.; Mitsugi, S. Agric. Biol. Chem. 1985, 49, 541-543. (6) Yarnitzky, C. N. J. Electroanal. Chem. 2000, 491, 154-159. (7) Yarnitzky, C. N.; Campbell, C. N.; Caranua, A. M.; Georgiou, G.; Heller, A.; Vreeke, S. M. J. Electroanal. Chem. 2000, 491, 160-165. (8) Mano, N.; Fernandez, J. L.; Kim, Y.; Shin, W.; Bard, A. J.; Heller, A. J. Am. Chem. Soc. 2003, 125, 15290-15291.
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Scheme 1. Schematic Diagram of the Wired GOx on the Glassy Carbon Electrodea
a The glassy carbon electrode is coated with a cross-linked adduct of GOx from A. niger and a redox polymer (PVP[Os(N,N′-alkylanated 2,2′biimidazole)3]2+/3+). The electrons are transferred from glucose to GOx, from GOx to redox polymer I, and from redox polymer I, to the electrode.
potential of -0.1 V versus Ag/AgCl, where any residual O2 would have been electroreduced. EXPERIMENTAL SECTION Chemicals and Materials. GOx from Aspergillus niger (EC 1.1.3.4, 191 units‚mg-1) was purchased from Fluka (Milwaukee, WI) and bilirubin oxidase (EC 1.3.3.5, 1.3 units‚mg-1) from Trachyderma tsunodae was purchased from Amano (Lombard, IL) and purified as previously described.8 Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) from Polysciences, Inc. (Warrington, PA). All solutions were made with deionized water passed through a purification train (Sybron Chemicals Inc, Pittsburgh, PA). Ultrapure O2 and argon were purchased from Matheson (Austin, TX). The synthesis of the GOx-wiring redox polymer PVP-[Os(N,N′-alkylanated-2,2′bi-imidazole)3]2+/3+ (polymer I) and of PVI[Os(4,4′-diamino-2,2′-bipyridine)2Cl]+/2+ (polymer II) was previously reported.9,10 Instrumentation and Electrodes. The measurements were performed using either a CHI 660 or a CHI 832 bipotentiostat (CH-Instruments, Austin, TX) and a homemade Faraday cage. The RRDE electrodes (disk diameter 4.57 mm, ring inner diameter 4.93 mm, outer ring diameter 5.38 mm, collection efficiency 22%) were purchased from Pine Instrument Co. (Grove City, PA). The potentials were measured versus a miniature Ag/AgCl (3 M KCl) reference electrode (Cypress, Lawrence, KS). The counter electrode was a 0.5-mm-diameter platinum wire (BAS, West Lafayette, IN). The electrode preparation and measurement of the films thickness were fully described in a previous paper.2,11,12 The bioelectrocatalyst solutions were deposited under a magnifying loupe while avoiding their spreading to the second electrode. The electrochemical measurements were performed in PBS (pH 7.4 20 mM phosphate-buffered 0.15 M NaCl). To minimize thermal (9) Mano, N.; Kim, H.-H.; Heller, A. J. Phys. Chem. B 2002, 106, 8842-8848. (10) Mano, N.; Kim, H.-H.; Zhang, Y.; Heller, A. J. Am. Chem. Soc. 2002, 124, 6480-6486. (11) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2002, 124, 12962-12963. (12) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 6588-6594.
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Figure 1. Steady-state current of glucose electrooxidation when the glassy carbon of the RRDE electrode is poised at -0.1 V versus Ag/AgCl and the ring is not connected to the potentiostat. (A) t ) 250 s, bubbled with air. t ) 160 s, bubbled with O2; 15 mM glucose, 20 mM phosphate, pH 7.4, 0.15 M NaCl, 37 °C. (B) Steady-state glucose electrooxidation current densities at 2, 4, and 6 fM glucose. 1 atm O2. Other conditions as in (A).
convection in the 30-µL drop on the electrode’s surface, both the shaft of the RRDE and the glucose solution were maintained at 37.5 °C, and to reduce evaporative cooling of the drop’s surface, the atmosphere was maintained near 100% humidity. To avoid spreading of the 30-µL droplet, a hydrophobic circle was drawn around the outer Pt ring with a felt-tip pen containing a hydrophobic ink (DAKO Pen, S 2002, DAKO Corp., Carpinteria, CA). The cathodic catalyst deposited on the Pt ring was made of 44.4 wt % Tt-BOD and 49 wt % polycationic redox copolymer PAAPVI-[Os(4,4′-dichloro-2,2′-bipyridine)2Cl]+/2+, cross-linked with 6.6 wt % PEGDGE, and the anodic catalyst deposited on the glassy carbon electrode consisted of the cross-linked adduct of 39.6 wt % GOx, 48.4 wt % PVP-[Os(N,N′-alkylanated 2,2′biimidazole)3]2+/3+, and 12 wt % PEGDGE.
Scheme 2. Schematic Diagram of the Coated Rotated Ring-Disk Electrodea
a The glassy carbon electrode is coated with a cross-linked adduct of GOx and a redox polymer (see Scheme 1). The Pt ring is coated with a cross-linked adduct of BOD from T. tsunodae and a second redox polymer (PAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)2Cl]+/2+). At the anode, electrons are transferred from glucose to GOx, from GOx to polymer I, and from polymer I to the electrode. At the cathode, electrons are transferred from the cathode to redox polymer II, from redox polymer II to BOD, and from BOD to O2.
RESULTS AND DISCUSSION Because O2 is the natural cosubstrate of glucose oxidase, it competes for the FADH2 electrons of the enzyme. Many publications describe the resulting oxygen interference in glucose assays and Risinger et al. even used glucose and GOx to deoxygenate solutions.13 Oxygen interferes also in the analysis of glucose, and as shown by Ikeda4 and by Yarnitzky7 and their colleagues, its concentration defines the threshold for glucose detection and the dynamic range in mediator-involving of glucose assays. This is also the case when the bioelectrocatalyst is wired GOx14,15 and electron cascade along the path glucose f glucose oxidase f polymer I f anode (eq 1 and Scheme 1). As the O2 partial GOx
β-D-glucose + 2[polymer I]3+ 98 δ-glucono-1,5-lactone + 2[polymer I]2+ + 2H+ (1) GOx
β-D-glucose + O2 98 δ-glucono-1,5-lactone + H2O2 (2)
pressure is increased, the anodic glucose electrooxidation current decreases, because O2 competes with the wire for GOx-FADH2 electrons (eq 2). As seen in Figure 1A, as much as half the current is lost upon switching the bubbled gas from argon to oxygen at 37 °C in a physiological buffer and at 15 mM glucose concentration when the electrode is poised at -100 mV/ AgAgCl, and ∼1/8th of the current is lost upon switching from argon to air. The current loss increases for thicker films and is greater for lower glucose concentrations,12,16 because at high glucose concentration, the O2 flux is consumed at the outer layer of the redox polymer, while a (13) Risinger, L.; Yang, X.; Johansson, G. Anal. Chim. Acta 1987, 200, 313318. (14) Heller, A. Acc. Chem. Res. 1990, 23, 128-134. (15) Heller, A. J Phys. Chem. B 1992, 96, 3579-3587. (16) Mano, N.; Mao, F.; Heller, A. J. Electroanal. Chem. In press.
Figure 2. Rotating ring-disk voltamogramms, under argon, of the wired GOx disk (A, left axis) and wired BOD ring (B, right axis). Disk electrocatalyst: 48.4 wt % PVP-[Os(N,N′-dialkylated 2,2′biimidazole)3]2+/3+, 39.6 wt % GOx, and 12 wt % PEGDGE; total loading of the electrocatalyst 130 µg.cm-2. Ring electrocatalyst: 44.4 wt % Tt-BOD, 49 wt % PAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)2Cl]+/2+, and 6.6 wt % PEGDGE; total loading of the electrocatalyst 60 µg‚cm-2. 20 mM phosphate buffer, pH 7.4, 0.15 M NaCl, 37 °C, 10 mV/s.
substantial part of the inbound glucose flux survives reaction with O2 and penetrates the film. Even though the rapid diffusion of electrons (Dapp ) 5.8 × 10-6 cm2 s-1)2 in the GOx wiring redox polymer I reduces the loss by making the collection of electrons rapid, at low glucose concentration (2 fM), nearly all (17) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals and Applications, 2nd ed.; John Wiley and Sons: New York, 2001. (18) Soukharev, V. S.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2004, 126, 83688369.
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Figure 3. (Trace A) Time dependence of the disk glucose electrooxidation current upon incremental increase of the glucose concentration from 0 to 2 fM, from 2 to 4 fM, and from 4 to 6 fM. (Trace B) Time dependence of the simultaneous ring O2 electroreduction current. Disk poised at -0.1 V versus AgAgCl (right axis); ring poised at -0.3 V versus AgAgCl (left axis). (The reviewer of this paper pointed out that the spikes of the ring current originate in the RC coupling of the ring and the disk through the solution, described by Mani, S.; Bruckenstein, S. J. Electrochem. Soc. 1974, 121, 1439. Every time the current flows at the disk, the ring double layer charges/ discharges. Since the uncompensated resistances from the reference electrode to the disk and ring are different, the ring current should result in the first derivative of the disk current transients as seen in this figure. Ring current spikes result from positive and negative responses at the disk upon glucose addition.)
the FADH2 electrons of GOx are captured by dissolved O2, rather than by polymer I when the atmosphere is 1 atm O2. (Figure 1B). In the stationary ring-disk electrode, the glucose electrooxidizing disk is, however, well shielded by the ring (Scheme 2) on which O2 is electroreduced. (eq 3) Figure 2 shows the cyclic BOD
O2 + 4H+ + 4[polymer II]+ 98 4[polymer II]2+ + 2H2O (3) voltammogram of the modified RRDE under argon at 37 °C in a pH 7 20 mM phosphate and 0.15 M NaCl buffer solution. The
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glassy carbon electrode was modified with wired GOx (bold line, left axis)2 and the Pt ring was modified with wired BOD (thin line, right axis).8,9,12 Their respective voltammograms were characteristics of a reversible surface bound couple with an apparent redox potential of +350 mV versus AgAgCl for the wired BOD and -200 mV versus AgAgCl for the wired GOx. In both, the width of the peaks at half-height, Ewhm, was 90 mV, close to the theoretical value of 90.6 mV for an ideal Nernstian one-electrontransfer reaction,17 indicative of a fast electron transport through the two wires. Figure 3 shows the current of glucose electrooxidation after increasing the glucose concentration from 0 to 2 fM, from 2 to 4 fM, and from 4 to 6 fM with the disk poised at -0.1 V versus Ag/AgCl and the ring at -0.3 V versus AgAgCl. The ring current (left axis) decreased as O2 was consumed and reached its steadystate value in 60 s. After the oxygen was scavenged by the ring, the adding of glucose to 2 fM concentration produced a glucose electrooxidation current, which reached its steady-state value after 80 s. CONCLUSION Glucose concentrations as low as 2 fM can be amperometrically detected in oxygen-depleted solutions. The half-cell potential for the reaction O2 + 4H+ + 4e - T 2H2O at pH 7 and at 37°C is ∼0.57 V versus Ag/AgCl. At the - 0.3 V versus Ag/AgCl potential of rapidly O2 reducing cathode, shielding the glucose electrooxidizing anode, the calculated equilibrium concentration of O2 is well below 1 aM. The wired bilirubin oxidase electrocatalyst is so superior to platinum18 that all O2 reaching the cathode is reduced on arrival. Hence, the residual reducible O2 no longer defines the detection limit. Similar wired oxidase disk electrodes are likely to allow the detection of lactate, L-R-glycerol phosphate, glutamate, or acetlylcholine at low concentrations. ACKNOWLEDGMENT The study was supported by the Office of Naval Research (N00014-02-1-0144) and by the Welch Foundation. N.M. thanks The Oronzio de Nora Industrial Electrochemistry Fellowship of The Electrochemical Society. Received for review November 16, 2004. AC0486746
September
7,
2004.
Accepted
