Saini Amardeep Singh, Kumar Jitendra, Melo Jose Savio
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Anal Chim Acta. 2014 Nov 7;849:50-6. doi: 10.1016/j.aca.2014.08.016. Epub 2014 Aug 13.
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.
开发一种能够同时监测多个样品中左旋多巴水平且所需反应体积小的生物传感器具有重要价值。本研究描述了使用从魔芋中提取并固定在微孔板孔表面的酪氨酸酶来检测左旋多巴。在将酪氨酸酶固定到微孔板孔上的不同方法中，发现戊二醛处理最为有效。除了酶活性检测外，还进行了环境扫描电子显微镜-能谱仪（ESEM-EDS）和原子力显微镜（AFM）检测，以确认酪氨酸酶已固定在微孔板孔表面。然后将这种固定化生物组分与用于检测左旋多巴的光学换能器集成，结果显示出良好的重现性。通过测量475nm处多巴色素形成的初始速率来研究该系统的传感特性。校准曲线给出的检测线性范围为10 - 1000μM，计算得出的检测限为3μM。固定化生物组分可稳定41天，且可重复使用多达9次。还使用该生物组分对加标样品（血浆）进行了分析。因此，这种基于微孔板的生物传感器提供了一种在单次运行中检测多个样品的便捷系统。
