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Langmuir 2007, 23, 364-366
Diazonium-Functionalized Horseradish Peroxidase Immobilized via Addressable Electrodeposition: Direct Electron Transfer and Electrochemical Detection Ronen Polsky, Jason C. Harper, Shawn M. Dirk, Dulce C. Arango, David R. Wheeler, and Susan M. Brozik* Sandia National Laboratories, P.O. Box 5800, MS-0892, Albuquerque, New Mexico 87185 ReceiVed October 4, 2006. In Final Form: NoVember 14, 2006 A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 ( 0.7 s-1 and a ∆Ep of 8 mV (V ) 150 mV/s). The large ks and low ∆Ep are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.
The direct electron transfer of enzymes has been the subject of much interest for the development of reagentless amperometric biosensors, biofuel cell elements, bioreactors, and the study of electron-transfer processes.1 The direct electrical communication between the enzyme and an electrode transducer is normally hindered by the insulation of the redox-active center by the protein matrix. Direct probing of the redox protein active site is obtained when a protein-electrode interaction exists such that electrons can transfer directly between the enzyme and electrode. Because redox mediators or electron shuttle molecules are not required, this method is well suited for electron-transfer mechanistic studies and greatly simplifies device fabrication. The method used to entrap or immobilize redox-active proteins onto an electrode can have a profound effect on the electrontransfer properties and overall stability of the protein. Factors that influence these qualities include protein orientation, distance from redox cofactor to the electrode, local protein environment, and film conductivity and electrostatic properties.2 Direct electrical contacting of redox proteins to electrodes has been accomplished by tethering to the redox group of the protein or immobilization via self-assembling monolayers, polycation layering, hydrogel and conducting polymers, clay colloids, or carbon paste, all of which provide electron-conducting pathways between the protein and the electrode.3 Recently, Corgier et al. introduced the use of diazoniummodified antibodies for the direct electrically addressable immobilization of proteins.4 The main advantages of such an immobilization strategy are the ease of preparation, the stability * Corresponding author. E-mail:
[email protected]. (1) (a) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615. (b) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (c) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443. (d) Prodromidi, M. I.; Karayannis, M. I.; Electroanalysis 2002, 14, 241-261. (2) (a) Habermuller, K.; Mosbach, M.; Schuhmann, W. Fresenius J. Anal. Chem. 2000, 366, 560. (b) Joseph, S.; Rusling, J. F.; Lvov, Y. M.; Friedberg, T.; Fuhr, U. Biochem. Pharmacol. 2003, 65, 1817. (3) (a) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877. (b) Gooding, J. J.; Mearns, F.; Yang, W. R.; Lin, J. Q. Electroanalysis 2003, 15, 81. (c) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969. (d) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451. (e) Schuhmann, W. Mikrochim. Acta 1995, 121, 1. (f) Zhou, Y.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 211. (g) Gorton, L. Electroanalysis 1995, 7, 23. (4) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127, 18328.
Figure 1. Preparation of diazonium-modified enzyme electrode: (1) Carboxyl diazonium is covalently attached to HRP by EDC/ NHS cross linking in 0.1 M carbonate buffer, pH 11. (2) DiazoniumHRP is deposited onto an electrode by cyclic voltammetry in 10 mM HCl by sweeping from 0 to -1000 mV at 100 mV/s. Subsequent linear sweep voltammetry of the modified electrode yielded direct electron transfer to the heme cofactor. Addition of peroxide produced catalytic currents from substrate turnover.
of the covalent surface chemistry bond, and the ability to address the protein spatially onto microelectrode arrays. Herein we show the utility of this technique to electrically address the redoxactive enzyme, horseradish peroxidase (HRP). A simple one step process to produce diazonium-protein adducts is introduced. The novelty of immobilizing the enzyme by an addressable electrical deposition is demonstrated. Direct electron transfer of HRP to the electrode is observed. The diazonium-HRPfunctionalized electrode exhibits a high heterogenenous electrontransfer rate constant and a low ∆Ep as well as great efficacy toward catalytic, mediator-free H2O2 detection. The preparation of diazonium-functionalized enzyme electrodes is presented in Figure 1. Carboxyl diazonium molecules are coupled to surface available primary amine groups on HRP via carbodiimide chemistry under basic conditions (1). Unreacted carboxyl diazonium molecules were removed via centrifugation (Supporting Information). The diazonium-functionalized HRP is then electrodeposited onto a glassy carbon electrode via cyclic voltammetry (2). An electrode prepared by depositing the diazonium-modified HRP yielded direct electron transfer between the redox-active site in the enzymes and the electrode surface.
10.1021/la062916a CCC: $37.00 © 2007 American Chemical Society Published on Web 12/14/2006
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Figure 2. Cyclic voltammograms of a diazonium-HRP electrode in 0.1 M PB, pH 7.4, under argon at 150 mV/s. Electrodes prepared via five-cycle deposition from diazonium-functionalized HRP solutions aged for (A) 1 week and (B) 4 weeks at 4 °C. Figure 3. Cyclic voltammograms of a diazonium-HRP electrode in 0.1 M PB, pH 7.4, under argon at 20, 50, 100, 200, 300, 400, 500, 600, 700, and 800 mV/s. (Inset) Plot of Ip,a and Ip,c vs scan rate.
The cyclic voltammogram in Figure 2A shows an anodic peak at -361 mV and the corresponding cathodic peak at -369 mV (V ) 150 mV/s), which are attributed to the FeIII/FeII redox couple of the electroactive heme center of HRP. A control electrode prepared under the same conditions but using a HRP solution without diazonium coupling showed no redox currents. The formal potential, E0′, of the couple was -365 ( 3 mV, which is similar to that of -377 mV reported for HRP entrapped within a solid polymer matrix on a GCE5 and -346 mV for HRP immobilized on a colloidal gold-modified carbon paste electrode (HRP-AuCPE).6 This formal potential is approximately 95 mV more negative than that reported for native HRP in solution.7 This may be due to partial protein unfolding because either portions of the HRP may be in direct contact with the GCE surface or structural stress induced via the multiple diazoniums on a single HRP molecule that may be covalently linked to the surface. Diazoniumfunctionalized HRP stored in solution at 4 °C was stable for more than 1 week, retaining electrodeposition and catalytic properties (Figure 2A). An electrode prepared from a diazoniumHRP solution stored for 4 weeks, presented in Figure 2B, showed a significant decrease in the currents observed at the original couple at -365 mV. An additional redox couple was also observed centered near -150 mV. This change is likely due to partial denaturation of a population of the diazonium-HRP conjugates in which the heme cofactor local environment is altered, leading to a positive shift in the redox potential. Diazonium molecules may form triazine bonds with free amines on the protein surface,8 leading to potential cross linking, denaturation, and hindering of electrodeposition. However, because HRP functionalization occurs under excess carboxyl diazonium all free amines should be conjugated. Also, as the modified HRP readily assembles onto an electrode, it is apparent that amide bonds were formed, providing free diazoniums for surface reaction. The effect of increasing scan rates is shown in Figure 3, and the corresponding plots of cathodic and anodic peak currents versus scan rate (inset) show a linear relationship indicating a surface-confined species. A heterogeneous reaction rate constant, ks, of 10.3 ( 0.7 s-1, and the dependence of potential peak location on scan rate categorize this reaction as quasi-reversible. The relatively high ks obtained for the immobilized diazoniummodified HRP is larger than the 1.13 s-1 reported for HRP immobilized on a DNA film,9 6.04 s-1 for the HRP-Au-CPE,7
and is similar to that obtained from HRP immobilized onto oligo(phenylethynylene) molecular wires.10 The peak-to-peak separation, ∆Ep, is a low 8 mV at V ) 150 mV/s, near the ideal separation of 0 mV for strongly adsorbed systems. This ∆Ep is lower than that reported for the HRP-Au-CPE of 33 mV at 150 mV/s and much lower than the 279 mV at V ) 20 mV/s reported for a HRP/Au colloid/cystamine electrode.11 The high ks and low ∆Ep obtained in this work is attributed to the intimate contact resulting from the close proximity between enzyme and electrode, attached by only one to three phenyl groups. Integration of the reduction peak indicates an electroactive surface coverage of 1.6 × 10-11 mol/cm2. This is equivalent to 65% of a hexagonally closepacked monolayer with an average diameter of 35 Å for globular HRP.12 The peak width at half peak height, ∆Ep1/2, is 166 mV, which is larger than the ideal 90.6 mV. Because HRP contains five to six lysine residues available for conjugation13 to the carboxyl diazonium molecule, it is likely that the electrical deposition results in a distribution of orientations on the electrode surface. This distribution of orientations provides a larger distribution of energy barriers for electron transfer to occur. This may account for the broader reaction peaks obtained from the immobilized HRP.14
(5) Ferri, T.; Poscia, A.; Santucci, R. Bioelectrochem. Bioenerg. 1998, 44, 177. (6) Liu, S.-Q.; Ju, H.-X. Anal. Biochem. 2002, 307, 110. (7) Harbury, H. A. J. Biol. Chem. 1957, 225, 1009. (8) (a) Chen, B.; Flatt, A. K.; Jian, H.; Hudson, J. L.; Tour, J. M. Chem. Mater. 2005, 17, 4832. (b) Hudson, J. L.; Jian, H.; Leonard, A. D.; Stephenson, J. J.; Tour, J. M. Chem. Mater. 2006, 18, 2766. (9) Chen, X.; Ruan, C.; Kong, J.; Deng, J. Anal. Chim. Acta 2000, 412, 89.
(10) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421. (11) Xiao, Y.; Ju, H. X.; Chen, H. Y. Anal. Biochem. 2000, 278, 22. (12) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163. (13) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szoke, H.; Henriksen, A.; Hajdu, J. Nature 2002, 417, 463. (14) Zhang, Z.; Nassar, A.-E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769.
Figure 4. Cyclic voltammograms of a diazonium-HRP electrode in 0.1 M PB, pH 7.4, under argon at 150 mV/s upon addition of peroxide: (A) 0, (B) 0.25, (C) 0.50, and (D) 1.0 mM H2O2.
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Figure 5. Chronoamperometric plot of a diazonium-HRP electrode at a potential of -400 mV in 0.1 M PB, pH 7.4, under argon, with stirring, and three subsequent additions each of 8, 50, and 100 µM H2O2.
The diazonium-modified HRP electrode also shows catalytic activity toward H2O2 reduction as presented in Figure 4. The cyclic voltammetric response shows a large catalytic current upon addition of 0, 0.25, 0.50, and 1.0 mM H2O2 (a-d, respectively) at 150 mV/s. Chronoamperometric experiments at a fixed potential of -400 mV upon three sequential additions each of 8, 50, and 100 µM H2O2, presented in Figure 5, show that such an electrode is highly suitable as a H2O2 detector. The
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steady-state current indicates a fast response time of ∼2 s. The sensitivity compares favorably with existing reports on H2O2 sensors by electrodes based on the direct electron transfer of HRP.15 In conclusion, we show that the direct electrically addressable deposition of diazonium-modified enzymes is highly suitable for electrochemical sensing formats. A simple one-step procedure is presented for diazonium-enzyme conjugation. We also demonstrate for the first time the direct electron transfer of horseradish peroxidase using a direct deposition procedure. The utility of such an electrode for the nonmediated catalytic detection of H2O2 shows great promise as an H2O2 sensor. We believe that future generations of arrayed electrochemical sensors for multianalyte detection and studies into enzyme “wiring” can benefit from protein electrodes prepared by this method. Acknowledgment. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin company, for the United States Department of Energy under contract DEAC04-94AL85000. Supporting Information Available: Related instrumentation, reagents, immobilization schemes, and procedures. This material is available free of charge via the Internet at http://pubs.acs.org. LA062916A (15) Ferapontova, E.; Gorton, L. Electroanalysis 2003, 15, 484.
