Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: Application for neurotoxins detection

• Published in: Biosensors & bioelectronics Vol. 22; no. 12; pp. 3001 - 3007
• Main Authors: Ramanathan, M., Simonian, A.L.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15-06-2007; Elsevier Science
• Subjects: Array biosensor; Biological and medical sciences; Biosensing Techniques - methods; Biotechnology; DFP; Enzyme Stability; Enzymes, Immobilized - metabolism; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Isoflurophate - analysis; Kinetics; Organophosphorus hydrolase; Paraoxon; Paraoxon - analysis; Parathion; Parathion - analysis; Sensitivity and Specificity; Spectrometry, Fluorescence - methods; TiO2; Titanium; Parathion; Array biosensor; Organophosphorus hydrolase; Paraoxon; DFP; TiO2; Insecticide; Enzyme; Pesticides; Esterases; Fluorescence spectrometry; Phosphoric triester hydrolases; Toxin; Biosensor; Neurotoxin; Hydrolases; Organophosphorus compounds; Aryldialkylphosphatase; Kinetics; Detection; Sensor array; Application; Monitoring
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2006.12.029

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO2 layer to increase the surface area available for immobilization.
The biosensor enabled the detection of 2.5μM paraoxon, and 10μM DFP and parathion, respectively. Very short response time of 30s can be achieved with a total analysis time of less than 2min. When operated at room temperature and stored at 4°C, the waveguide retained reasonable activity for greater than 45 days.