Mulchandani A, Kaneva I, Chen W
Department of Chemical and Environmental Engineering, University of California, Riverside 92521, USA.
Anal Chem. 1998 Dec 1;70(23):5042-6. doi: 10.1021/ac980643l.
A fiber-optic microbial biosensor suitable for direct measurement of organophosphate nerve agents was developed. The unique features of this novel microbial biosensor were the recombinant Escherichia coli cells expressing the enzyme organophosphorus hydrolase on the cell surface and the optical detection of the products of enzyme-catalyzed organophosphate hydrolysis. The use of cells with the metabolic enzyme expressed on the cell surface as a biological sensing element provides advantages of no resistance to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively. The use of an optical transducer allows the detection of different organophosphates in a mixture, presently not feasible with acetylcholinesterase-based biosensors. E. coli cells expressing organophosphorus hydrolase (OPH) on the cell surface were immobilized in low melting temperature agarose on a nylon membrane and attached to the common end of a bifurcated fiber-optic bundle. OPH-expressing E. coli cells catalyzed the hydrolysis of organophosphorus pesticides to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The backscattered radiation of the specific wavelength incident light was measured using a photomultiplier detector and correlated to the organophosphate concentration. The best sensitivity and response time were obtained using a sensor constructed with 1.5 mg of cells operating in pH 9, 50 mM HEPES buffer with 100 mM NaCl and 0.05 mM CoCl2 at 30 degrees C. At optimized conditions, the biosensor measured paraoxon, parathion, and coumaphos pesticides with high selectivity against triazine and carbamate pesticides in approximately 10 min. The lower detection limits were 3 microM for paraoxon and parathion and 5 microM for coumaphos. When stored in the buffer at 22 degrees C, the biosensor was stable for over a 1-month period and showed no decline in the response for over 75 repeated usages. The new fiber-optic microbial biosensor is an ideal tool for on-line monitoring of the detoxification process for organophosphate pesticides-contaminated wastewaters but may not be suitable for environmental monitoring.
开发了一种适用于直接测量有机磷神经毒剂的光纤微生物生物传感器。这种新型微生物生物传感器的独特之处在于重组大肠杆菌细胞在细胞表面表达有机磷水解酶，以及对酶催化有机磷水解产物进行光学检测。与分别基于细胞内表达酶的传统微生物生物传感器和基于酶的传感器相比，使用在细胞表面表达代谢酶的细胞作为生物传感元件具有以下优势：对分析物和产物跨细胞膜的传质无阻力，且由于无需酶纯化而成本较低。使用光学换能器可以检测混合物中的不同有机磷，这在目前基于乙酰胆碱酯酶的生物传感器中是不可行的。将在细胞表面表达有机磷水解酶（OPH）的大肠杆菌细胞固定在尼龙膜上的低熔点琼脂糖中，并连接到分叉光纤束的公共端。表达OPH的大肠杆菌细胞催化有机磷农药水解，形成化学计量的发色产物，这些产物在特定波长下吸收光。使用光电倍增管探测器测量特定波长入射光的背向散射辐射，并将其与有机磷浓度相关联。使用在30℃下于pH 9、含有100 mM NaCl和0.05 mM CoCl2的50 mM HEPES缓冲液中运行的1.5 mg细胞构建的传感器，可获得最佳灵敏度和响应时间。在优化条件下，该生物传感器在大约10分钟内对氧磷、对硫磷和蝇毒磷农药具有高选择性，对三嗪和氨基甲酸酯农药具有高选择性。氧磷和对硫磷的检测下限为3 microM，蝇毒磷的检测下限为5 microM。当在22℃的缓冲液中储存时，该生物传感器在1个月以上的时间内保持稳定，并且在超过75次重复使用中响应没有下降。这种新型光纤微生物生物传感器是在线监测有机磷农药污染废水解毒过程的理想工具，但可能不适用于环境监测。
