390139 Molecular Bioelectronics: Enzyme-Swnt Conjugates for Bioanalytical Biochips
Generation-3 (Gen-3) biosensors that are based on direct electron transfer are reagentless  and hold the potential for long term in vivo stability to enable fully implantable continual monitoring of various analytes of a wide variety of physiological conditions and pathologies . Among the novel approaches for establishing a Gen-3 biotransduction mechanism, ultrasonic processing  of single walled carbon nanotube-enzyme (e.g. Glucose oxidase, GOx) conjugates  was investigated as a function of sonication time (5 and 60 min), SWNT functionalities (-OH and –COOH) and tube length (<1 µm and 5-10 µm) . Using the blue shift (Δλmax) in the absorbance maximum of the FAD-apoenzyme, the change in the sum of α-helix and β-sheet in the circular dichroism spectra, and the change in the kcat/KM and n of the enzyme conjugate expressed relative to the pristine GOx, were found to show only modest change to the enzyme’s structure during supramolecular conjugation. Short SWNTs appeared to support conjugate formation with no egress of FAD for the enzyme; medium SWNT appeared to support conjugate formation moderately while the long and non-functionalized SWNT caused partial denaturation leading to the egress of FAD.
Microdisc electrode arrays (MDEA 5037 Pt) were microlithographically fabricated on borosilicate glass from 10 nm TiW adhesion layer and 100 nm e-gun deposited Pt. The Pt layer was patterned into working, counter and reference electrodes and all electrodes covered by Si3N4 which was windowed to reveal a pattern of microdiscs (37 discs, each f=50 µm, A= ???cm2) over the working electrode and exposed counter and reference electrodes. Biofabrication  of enzyme amperometric biosensors using the process of electropolymerization (Py to PPy at 0.8 V vs. Ag/AgCl) onto the MDEA 5037 in the presence of glucose oxidase (1 mg/ml, DI water, 100 mC/cm2) resulted in GOx, GOx-ferrocene monocarboxylic acid and GOx-SWNT biotransducers that were responsive to glucose and that allowed parallel evaluation of the relative efficacy of Gen-1, Gen-2 and Gen-3 mechanisms of transduction. The bioanalytical performance of sensitivity (S), linear dynamic range (LDR) and detection limit [3(SD(blank)/S] as well as apparent enzyme kinetic parameters of KM, Imax, and kcat, were obtained from the dose-response curves for mutarotated glucose solutions (0.0 mmol/L to 13.7 mmol/L in PBS 7.2) at RT and compared to solution phase behavior. There was a good linear relationship (R2=0.99) between glucose concentration and response from 2.0 mM – 9.0 mM. The sensitivity was 10-3 mA/mM and the detection limit was 0.002 mA/mM.
Keywords: Biosensors, Bioimmobilization, Nanotubes, Direct Electron Transfer, Glucose-SWNT
 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.
 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.
 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.
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