(559g) Prospects for Direct Electron Transfer in Gen-3 Biosensors and Advanced Biofuel Cells With Swnt-Gox Conjugates

There is pressing need for the design, development and understanding of abio-bio interfaces that permit direct electron transfer (DET) between oxidoreductase enzymes and metallic, carbonaceous or semiconductor electrodes [1]. Generation-3 (Gen-3) biotransduction systems used in biosensors and advanced enzyme biofuel cells may exploit DET enabled by single walled carbon nanotube (SWNT)-enzyme conjugates [2]. In this work we explore the use of diazonium salts to covalently immobilize pyrrole monomer onto glassy carbon electrodes (GCE) and the electropolymerization of Py as a method of biofabrication to immobilize SWNT-GOx conjugates onto platinum and glassy carbon electrodes (GCE). Glucose oxidase (GOx) – single walled carbon nanotube supramolecular conjugates (GOx-SWNT) were produced using ultrasonication and ultracentrifugation [3] and were shown to suffer very little loss of bioactivity regardless of nanotube fiunctionalization [4]. The GOx-SWNT conjugates were immobilized on Φ=100μm Pt microelectrodes (μE) via electropolymerization of pyrrole (Pt|PPy-(GOx-SWNT)) and compared to a typical Gen-1 system wherein GOx was immobilized onto Pt mE via electropolymerization of pyrrole (Pt|PPy-GOx). Electropolymerization times required to achieve 100 mC/cm² were 45 min and 26 min for GOx and GOx-SWNT, respectively. The electrodeposited thin films were then overoxidized (OOPPy) (40 cycles, 100 mV/s, -0.2- +1.3 V vs. Ag/AgCl, PBS 7.2) to eliminate the role of the conductive PPy layer and allow the effect of the presence of SWNTs to be readily observed.  Electrical and electrochemical impedance spectroscopy (EIS) was used to characterize the newly fabricated biotransducers in freshly prepared phosphate buffered saline (1X PBS pH = 7.2) at room temperature in two or three electrode mode, respectively. EIS used non-perturbing 10 mV amplitude over the frequency range 10^-1 – 10⁶ Hz. Multiple scan rate cyclic voltammetry (MSRCV) was performed using 1.0 mM FcCOOH in 1X PBS buffer (pH=7.2). EIS of the Gen-1 (Pt|PPy-GOx) and Gen-3 (PT|PPy-(GOx-SWNT)) biotransducer constructs prepared with increasing electropolymerization charge density caused a dispersion shift towards higher frequencies as well as an increase in the overall impedance magnitude. Both Gen-1 and Gen-3 constructs showed similar behavior for charge densities up to 1 mC/cm² while the Gen-3 constructs exhibited a lower impedance magnitude for charge densities > 1 mC/cm². MSRCV produced the effective areas of the electrodes from an application of the Randles-Sevcik equation and the literature value of D = 4.51 x 10^-6 cm²/s (FcCOOH). There was a ca. 80% reduction of the original electrode area in the case of Pt|OOPPy-GOx compared to a 25% reduction in the case of the Pt|OOPPy-(GOx-SWNT), confirming electrochemical communication between the FcCOOH probe and the underlying Pt electrode. The heterogeneous rate constant k⁰ for the electron transfer (ET) reaction for each biotransduction system was obtained from an analysis of the cyclic voltammograms using the methodology described by Nicholson [5] and a linearized version of the Nicholson approach developed by Leddy et al [6]. The presence of the SWNT within the Pt|OOPPy-(GOx-SWNT) system acts as a conducting medium through the overoxidized ultra-thin film for enhanced electron transfer between the probe molecule in solution and the underlying metallic electrode.

Bioanalytical performance of sensitivity (S), linear dynamic range (LDR) and detection limit [3(SD(blank)/S] as well as apparent enzyme kinetic parameters of K_M, I_max, and k_cat, were obtained from the dose-response curves for glucose and compared to solution phase behavior.

References:

[1] Karunwi O, Wilson AN, Kotanen C, Guiseppi-Elie A. Engineering the Abio-Bio Interface to Enable More than Moore in Functional Bioelectronics. Journal of The Electrochemical Society. 2013;160:B60-B5.

[2] Guiseppi-Elie A, Lei C, Baughman RH. Direct electron transfer of glucose oxidase on carbon nanotubes. Nanotechnology. 2002;13:559.

[3] Guiseppi-Elie A, Choi S-H, Geckeler K, Sivaraman B, Latour R. Ultrasonic Processing of Single-Walled Carbon Nanotube–Glucose Oxidase Conjugates: Interrelation of Bioactivity and Structure. NanoBiotechnology. 2008;4:9-17.

[4] Karunwi O, Guiseppi-Elie A. Supramolecular glucose oxidase-SWNT conjugates formed by ultrasonication: effect of tube length, functionalization and processing time. Journal of Nanobiotechnology. 2013;11:6.

[5] Nicholson RS. Theory and Application of Cyclic Voltammetry for Measurement of Electrode Reaction Kinetics. Analytical Chemistry. 1965;37:1351-5.

[6] Paul HJ, Leddy J. Direct determination of the transfer coefficient from cyclic voltammetry: isopoints as diagnostics. Analytical Chemistry. 1995;67:1661-8.