New principle of direct real-time monitoring of the interaction of cholinesterase and its inhibitors by piezolectric biosensor

被引:37
作者
Makower, A
Halámek, J
Skládal, P
Kernchen, F
Scheller, FW
机构
[1] Univ Potsdam, Inst Biochem & Biol, Dept Analyt Biochem, D-14476 Golm, Germany
[2] Masaryk Univ, Dept Biochem, CS-61137 Brno, Czech Republic
[3] Biosyntan, D-13125 Berlin, Germany
关键词
cholinesterase; inhibitor; piezoelectric; biosensor; real-time; detection;
D O I
10.1016/S0956-5663(03)00089-7
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
This paper describes a new method for the sensitive detection of cholinesterase inhibitors based on real-time monitoring using a piezoelectric biosensor. The cholinesterase inhibitor paraoxon was immobilized on the sensing surface via a chelate complex as the recognition element. At first, the conjugate of N-mercaptoundecanoic acid (MUA) with Nalpha,Nalpha-bis (carboxymethyl)-L-lysine (NTA-Lys) was chemisorbed to form a self-assembled monolayer on the surface of the gold electrode of the piczosensor. In the next step, paraoxon-spacer-hexahistidine conjugate was linked to the MUA-Lys-NTA layer via the chelate complex with Ni2+. The paraoxon-modified surface thus obtained was applied for the binding of human butyrylcholinesterase (BChE). Regeneration of the sensing surface was achieved by splitting the chelate complex with EDTA and depositing a fresh layer of Ni2+ followed by addition of the paraoxon-spacer-hexahistidine. In the presence of free inhibitors like diisopropylfluorophosphate (DFP), binding of BChE to the surface-bound paraoxon was decreased. In this way, a competitive affinity assay for organophosphorus compounds was developed. The limit of detection for DFP as a model compound was 10 nmol/l (ca. 2 mug/l). This new concept seems suitable for constructing biosensors for the group-specific detection of cholinesterase-inhibiting substances like insecticides in the field. (C) 2003 Elsevier Science B.V. All rights reserved.
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页码:1329 / 1337
页数:9
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