Versatile method of cholinesterase immobilisation via affinity bonds using Concanavalin A applied to the construction of a screen-printed biosensor.
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 micromol l(-1) with a sensitivity of 3.6 mA l 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.
Bucur
Bogdan
B
Centre of Phytopharmacy, University of Perpignan, URA 5054, 52 Avenue Paul Alduy, 66860, Perpignan Cedex, France.
Danet
Andrei Florin
AF
Marty
Jean-Louis
JL
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002802:Cholinesterases / Q000032:analysis / Q000737:chemistry*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003208:Concanavalin A / Q000032:analysis / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D010575:Pesticides / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D010575:Pesticides; D003208:Concanavalin A; D002802:Cholinesterases