BIOSENSING DOPAMINE USING LIVING AND DEAD PSEUDOMONAS PUTIDA CELLS

Abstract

Two types of microbial sensors for dopamine measurement were successfully developed using living and dead cells of Pseudomonas putida P8, respectively. Dead cells were prepared by short-time exposure of the vacuum dried microbial cells to high temperature. The dead cells were found to retain the viability and activity of their enzyme systems. A newly fabricated sensor has to be preconditioned in a well-stirred and well-aerated 0.1 M, pH 7.0 phosphate buffer containing dopamine for it to fully develop and stabilise its good dopamine sensing efficacy. It took about 9 days to fully activate the living cell sensor and 15 days for the dead cell sensor. At 25°C, the living cell sensor showed a sensitivity of 0.0433 µA/µM dopamine in a 0.1 M, pH 7.0 phosphate buffer over a linear range ofl-200 µM, compared with 0.0384 µA/µM and 1-150 µM for the dead cell sensor. The response of living cell sensor peaked at pH 7.0 and increased monotonically with temperature from l 5°C to 3 5°C. The dead cell sensor showed a better tolerance than the living cell over a wider range of pH. Its response remained substantially the same over the pH range from 5 to 8. Prolonged contact with high concentration of dopamine caused deactivation and reduced sensitivity of the sensor response. Both the sensors showed good response stability and reproducibility with a coefficient of variation of 2.9% for the living cell sensor and 2. 5% for the dead cell sensor over a period of 2 months provided they were kept in a dopamine-free 0.1 M phosphate buffer at room temperature in between measurements and overnight. One or two days reconditioning in the presence of dopamine was found necessary to restore its sensing characteristics when the sensor was not used for more than 5-7 days. A remarkably long service life of 4 to 5 months was found for both the sensors. The extent of interference on dopamine sensing by other chemical solutes including some chemical analogues, metabolic intermediates and those commonly found in a dopamine physiological test sample was examined by calibrating the sensor against these compounds in the same manner and under the same conditions as for dopamine. Among the 26 other organic compounds examined, both sensors showed highest response towards dopamine. For the living cell sensor, tyramine, tyrosine, catechol and arterenol showed a relative sensitivity of over 50% with respect to dopamine whereas no response was observed for homovanillic acid, oxalic acid and riboflavin. Comparable results were obtained for the dead cell sensor. This study showed conclusively that biosensors prepared with both living and dead cells of Pseudomonas putida PS showed good dopamine sensing. The dead cells were prepared according to Tan and Qian's procedure (1996a; 1996b) in which the cells were killed by short-time exposure to high temperature. The enzyme system retained sufficiently high stability and activity for sensing purposes.