Acetylcholinesterase-ISFET based system for the detection of acetylcholine and acetylcholinesterase inhibitors.
A bioelectronic hybrid system for the detection of acetylcholine esterase (AChE) catalytic activity was assembled by way of immobilizing the enzyme to the gate surface of an ion-sensitive field-effect transistor (ISFET). Photometric methods used to characterize bonded enzyme and linker layers on silicon substrates confirm the existence of a stable amino-cyanurate containing AChE monolayer. The transduction of the enzyme-functionalized ISFET, in ionic solutions, is detected in response to application of acetylcholine (ACh). Recorded sensitivity of the modified ISFET to ACh has reached levels of up to 10(-5)M. The Michaelis-Menten constant of the immobilized AChE is only moderately altered. Nevertheless, the maximum reaction velocity is reduced by over an order of magnitude. The ISFET response time to bath or ionophoretic application of ACh from a micropipette was in the range of a second. The catalytic activity of the immobilized AChE is inhibited in a reversible manner by eserine, a competitive inhibitor of AChE. We conclude that the immobilized enzyme maintains its pharmacological properties, and thus the described bioelectronic hybrid can serve as a detector for reagents that inhibit AChE activity.
Hai
Aviad
A
Department of Neurobiology, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Ben-Haim
Deborah
D
Korbakov
Nina
N
Cohen
Ariel
A
Shappir
Joseph
J
Oren
Ruthi
R
Spira
Micha E
ME
Yitzchaik
Shlomo
S
D000109:Acetylcholine / Q000032:analysis* / Q000737:chemistry; D000110:Acetylcholinesterase / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002800:Cholinesterase Inhibitors / Q000032:analysis* / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014173:Transistors, Electronic
D002800:Cholinesterase Inhibitors; D004800:Enzymes, Immobilized; D000110:Acetylcholinesterase; D000109:Acetylcholine