Discriminative Detection of Neurotoxins by a Layer-by-Layer Based Carbon Nanotube/bi-enzyme Biosensor

Multi-enzyme systems have attracted considerable interests due to the versatility for multiplexed detection of different analytes. In this study, a novel layer-by-layer (LbL) assembled multi-enzyme biosensor incorporating acetylcholinesterase (AChE) and organophosphate hydrolase (OPH) was proposed for discriminative detection between organophosphorus (OP) and non-organophosphorus (non-OP) neurotoxins. This was achieved through LbL assembly of electrostatically interacted enzyme armored carbon nanotubes (CNT-OPH and CNT-AChE) with a set of cushioning bilayers consisting of CNT-polyethyleneimine (PEI) and CNT-DNA on glassy carbon electrode with electrochemical detection. OPH catalyzes the hydrolysis of OPs (e.g. paraoxon) and AChE can be used for detection of both OPs and non-OPs (e.g. carbamate) through inhibition assays, and thus makes it possible for detection of OP neurotoxins from multi-analytes samples, and determination of the non-OP neurotoxins to their true concentration with AChE inhibition. Optimization of the LbL architecture was initially conducted with penetration analyses including position of active layer, number of total layers and impedance analyses with respect to different concentration of CNTs. Surface morphology and chemical analyses were characterized with Scanning Electron Microscopy and Raman spectroscopy to have a better understanding of the layered structure. The simple and inexpensive LbL approach can be further extended to a wide variety of enzymes in a multi-detection biosensing system, wherein each analyte could be “fingerprinted” with respect to its catalytic response to different enzymes.