A Theoretical Model for Immobilized Enzyme Inhibition Biosensors

The development and experimental verification of a theoretical model for immobilized enzyme inhibition biosensors is reported. The model predicts that the percentage of inhibited enzyme (I %), after exposure to an inhibitor, is linearly related to both the inhibitor concentration ([I]B) and the square root of incubation time (t1/2): I%=Kt1/2[I]B×100 The model is based on diffusion limited inhibitor transport and takes into account the heterogeneous nature of enzyme inhibition that results from immobilization of an enzyme at the surface of a transducer. Experimental verification of the model was achieved using an electrochemical (amperometric) acetylcholine sensor. The immobilized enzyme used in this study was acetylcholinesterase, while the organophosphorus pesticide, paraoxon, was employed as the inhibitor. A second enzyme, choline oxidase, was co-immobilized to facilitate electrochemical detection of the substrate. To satisfy the conditions required by the proposed model a specially designed biosensing device was fabricated employing a microdisk array as the transducer. Acetylcholinesterase (0.00025 units) and choline (1.0 units) were co-immobilized onto the array using a water-based polyurethane/polyethylene oxide colloidal dispersion matrix. A dialysis membrane was used as an active barrier to prevent fouling of the array and to restrict enzyme leaching. The device was used for both batch and flow injection analysis and experimental results verifying the proposed model are presented using both modes.

[1]  Giuseppe Palleschi,et al.  Determination of organophosphorus insecticides with a choline electrochemical biosensor , 1992 .

[2]  Plamen Atanasov,et al.  Potentiometric immunoelectrode for fast assay based on direct electron transfer catalyzed by peroxidase , 1996 .

[3]  I. Kovach.  Competitive irreversible inhibition of enzymes in the presence of a substrate: scope and limitations. , 1991, Journal of enzyme inhibition.

[4]  C. Tran-Minh,et al.  Determination of organophosphorous and carbamate insecticides by flow injection analysis. , 1992, Analytical biochemistry.

[5]  H. Hara,et al.  Analytical application for chemicals using an enzyme sensor based on an ISFET , 1993 .

[6]  G. Palleschi,et al.  Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor , 1995 .

[7]  Gennady Evtugyn,et al.  Sensitivity and selectivity of electrochemical enzyme sensors for inhibitor determination. , 1998, Talanta.

[8]  H. Nam,et al.  Potentiometric biosensors using immobilized enzyme layers mixed with hydrophilic polyurethane , 1998 .

[9]  C. Tran-Minh,et al.  Immobilized Enzyme Probes for Determining Inhibitors , 1985 .

[10]  Petr Skládal,et al.  Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase , 1992 .

[11]  Jean-Louis Marty,et al.  Amperometric flow-through biosensor for the determination of cholinesterase inhibitors , 1999 .

[12]  J. Nicaud,et al.  Detection of organophosphorous pesticides with an immobilized cholinesterase electrode. , 1984, Journal of analytical toxicology.

[13]  D. Barceló,et al.  Sampling of polar pesticides from water matrices , 1997 .

[14]  M. Morita,et al.  Application of a cholinesterase biosensor to screen for organophosphorus pesticides extracted from soil. , 1995, Talanta.

[15]  A. L. Hart,et al.  The response of screen-printed enzyme electrodes containing cholinesterases to organo-phosphates in solution and from commercial formulations , 1997 .

[16]  C Cremisini,et al.  Anticholinesterase activity measurement by a choline biosensor: application in water analysis. , 1993, Biosensors & bioelectronics.

[17]  B. Hoffmann,et al.  Polyurethane Enzyme Membranes for Chip Biosensors , 1989 .

[18]  Richard John,et al.  Development of a generic microelectrode array biosensing system , 2000 .

[19]  W N ALDRIDGE,et al.  Some properties of specific cholinesterase with particular reference to the mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E 605) and analogues. , 1950, The Biochemical journal.

[20]  C. Burstein,et al.  Increased paraoxon detection with solvents using acetylcholinesterase inactivation measured with a choline oxidase biosensor , 1997 .

[21]  Félix Pariente,et al.  Determination of Organophosphorus and Carbamate Pesticides Using a Piezoelectric Biosensor , 1998 .

[22]  R. Kitz,et al.  Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. , 1962, The Journal of biological chemistry.

[23]  Kathrin Stein,et al.  Flow injection analysis of paraoxon with the use of an immobilized acetylcholinesterase reactor , 1996 .

[24]  J. Marty,et al.  Detection of paraoxon by continuous flow system based enzyme sensor , 1998 .