New principle of direct real-time monitoring of the interaction of cholinesterase and its inhibitors by piezolectric biosensor.
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 piezosensor. 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 microg/l). This new concept seems suitable for constructing biosensors for the group-specific detection of cholinesterase-inhibiting substances like insecticides in the field.
Makower
Alexander
A
Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Golm, Germany. makower@rz.uni-potsdam.de
Halámek
Jan
J
Skládal
Petr
P
Kernchen
Frank
F
Scheller
Frieder W
FW
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002091:Butyrylcholinesterase / Q000737:chemistry*; D002800:Cholinesterase Inhibitors / Q000032:analysis* / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques / Q000295:instrumentation / Q000379:methods*; D007306:Insecticides / Q000032:analysis; D007531:Isoflurophate / Q000032:analysis*; D009862:Online Systems; D010261:Paraoxon / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002800:Cholinesterase Inhibitors; D004800:Enzymes, Immobilized; D007306:Insecticides; D007531:Isoflurophate; D002091:Butyrylcholinesterase; D010261:Paraoxon