Detection of inducible nitric oxide synthase using a suite of electrochemical, fluorescence, and surface plasmon resonance biosensors

Abstract

A suite of biosensors for rapid detection of inducible nitric oxide synthase (iNOS) is described. First, a metal-enhanced electrochemical detection (MED) sensor, which relied on the redox properties of a silver monolayer, was developed. The linear detection range was between 8.64 × 10 −2 and 5.4 × 10 1 ng/ml with a detection limit of 1.69 × 10 −4 ng/ml. This method was compared with surface plasmon resonance (SPR) biosensors in which polyclonal mouse anti-iNOS was covalently immobilized onto a gold surface using an iNOS antigen. The linear detection range recorded was between 3.37 × 10 1 and 5.4 × 10 −2 ng/ml with a detection limit of 2 × 10 −3 ng/ml. Finally, an ultrasensitive portable capillary (UPAC) fluorescence immunosensor, in which a mouse anti-iNOS antibody was covalently immobilized onto the inner surface of a capillary and a rabbit anti-iNOS antibody was employed as the secondary antibody, was developed. The resulting signals were found to be directly proportional to iNOS concentrations between 1.52 × 10 −1 and 1.52 × 10 −2 ng/ml with a detection limit of 1.05 × 10 −3 ng/ml. These immunosensors exhibit low cross-reactivity toward potential interferents such as human serum albumin and ovalbumin. The SPR and UPAC biosensors were validated using simulated blood spiked with recombinant iNOS, resulting in recoveries of 85% and 88.5%, respectively. The research presented in this article could potentially provide new ways of detecting NO for diagnostic and biomarker purposes in medical research.