title: Microplate based optical biosensor for L-Dopa using tyrosinase from Amorphophallus campanulatus.
abstract: Developing a biosensor which is capable of simultaneously monitoring l-Dopa levels in multiple samples besides requiring small reaction volume is of great value. The present study describes the detection of l-Dopa using tyrosinase enzyme extracted from Amorphophallus campanulatus and immobilized on the surface of the microplate wells. Among the different approaches used for immobilizing tyrosinase onto the microplate wells, glutaraldehyde treatment was found to be most effective. Besides enzyme activity, ESEM-EDS (environmental scanning electron microscope-energy dispersive system) and Atomic Force Microscopy (AFM) were also carried out to confirm the immobilization of tyrosinase enzyme onto the microplate well surface. This immobilized biocomponent was then integrated with an optical transducer for l-Dopa detection and it showed good reproducibility. The sensing property of the system was studied by measuring the initial rate of dopachrome formation at 475 nm. The calibration plot gave a linear range of detection from 10-1000 μM and the detection limit was calculated to be 3 μM. The immobilized biocomponent was stable for 41 days and was reused up to nine times. Spiked samples (blood plasma) were also analyzed using this biocomponent. This microplate based biosensor thus provides a convenient system for detection of multiple samples in a single run.
authors.affiliation: Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Electronic address: saini_amardeepsingh@yahoo.co.in.
authors.forename: Amardeep Singh
authors.initials: AS
authors.lastname: Saini
authors.affiliation: Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Electronic address: jkumar@barc.gov.in.
authors.forename: Jitendra
authors.initials: J
authors.lastname: Kumar
authors.affiliation: Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Electronic address: jsmelo@barc.gov.in.
authors.forename: Jose Savio
authors.initials: JS
authors.lastname: Melo
mesh_terms: D031056:Amorphophallus / Q000201:enzymology*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D006801:Humans; D007980:Levodopa / Q000097:blood* / Q000378:metabolism; D057230:Limit of Detection; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D013053:Spectrophotometry; D014159:Transducers
chemical_list: D004800:Enzymes, Immobilized; D007980:Levodopa; D014442:Monophenol Monooxygenase
keywords: Amorphophallus campanulatus; Immobilization; Microplate; Optical biosensor; Tyrosinase; l-Dopa