Versatile method of cholinesterase immobilisation via affinity bonds using Concanavalin A applied to the construction of a screen-printed biosensor

摘要

Development of new and more reliable methods to immobilise biomolecules has emerged rapidly due to a continuous need for more stable, sensitive and reliable biosensors. This paper reports a new method of acetylcholine-esterase (AChE) immobilisation based on the high affinity interaction between the glycoproteic enzyme and Concanavalin A (Con A). In order to establish the nature of the link formed between the glycoenzyme, lectin and support, three different configurations are presented. The optimum immobilisation procedure was further used for biosensor manufacturing. The non-specific adsorption is around 3% and the chemical cross-linking of the proteins is avoided. The optimised method allows loading of the working electrode surface with different amounts of enzyme ranging from 0.3 to 3.3 mIU with a good operational stability. The sensor showed a linear response range to acetylthiocholine substrate between 10 and 110 mumol l(-1) with a sensitivity of 3.6 mA 1 mol(-1). The applicability of the method to the detection of organophosphorus insecticides resulted in a detection limit of 10(-8) mol l(-1) for chlorpyriphos. (C) 2004 Elsevier B.V. All rights reserved.