Electrochemical Characterization of Silicon-Based Gold Microband Electrode Array and its Application for Labelless T-2/HT-2 Toxin Immunosensing

Abstract

Microelectrode arrays (MEAs) are gaining interest in electroanalysis owing to its distinctive voltammetry properties compared to its macro counterparts. Among the MEAs widely fabricated and studied are microdisc array and microband array. We report here the microfabrication of 10 μm microband array (number of band in an array, N=17) and its application in labelless impedimetric sensing of T-2/HT-2 toxin. The microband array has recess depth (i.e. Si3N4 passivation thickness) of 200 nm. Upon fabrication, the device was first characterized via visual inspection and electrochemical analysis. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies were performed in 1 mM ferrocenecarboxylic acid (FCA) in 0.01 M PBS, pH 7.4. At scan rate of 100 mv s-1, cyclic voltammogram for the microband array exhibited a slight peak-shaped CV; and was found to be scan-rate dependent. Experimental limiting current of the microband array (529±7 nA) was slightly lower compared to the calculated theoretical current (632 nA) indicating mixed diffusion profile of the microband array. The device was then employed in immunosensor construction for T-2/HT-2 toxins detection. T-2 mycotoxin and its metabolite (HT-2), are target of concern in the biosensing application due to its lethal toxicity and prominent presence in EU grains industry. Surface functionalization for anti-T-2 monoclonal antibody (mAb) immobilization was first achieved via surface hydroxylation with plasma cleaning and piranha solution treatment, followed by (3-Aminopropyl) triethoxysilane (APTES) silanization and 15 minutes pre-incubation with various concentrations of anti-T-2 toxin mAb in EDC/NHS mixture. The optimal concentrations for anti-T-2 toxin mAb immobilization on the microband array surface was determined at 0.75 mg mL-1. Based on the calibration curve developed in buffer solution system, the functionalized microband array was proven sensitive as it was able to detect T-2/HT-2 toxin at low dynamic working range (0-25 ppb) and limit of quantitation (LOQ) achieved at 4.89 ppb.