Burestedt E, Nistor C, Schagerlöf U, Emnéus J
Department of Analytical Chemistry, University of Lund, Sweden.
Anal Chem. 2000 Sep 1;72(17):4171-7. doi: 10.1021/ac000128z.
The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for colorimetric and CDH detection, respectively. Matrix effects were less pronounced with the CDH biosensor than with colorimetric detection.
目的是开发一种快速通用的酶流动免疫分析方法（EFIA），该方法使用β-半乳糖苷酶（β-GAL）标记结合比色检测以及一种新型安培生物传感器作为标记检测器。该安培生物传感器是该团队之前为测定地表水样品中的双酚而开发的。抗原（Ag，分析物）、示踪剂（Ag*，用β-GAL标记的抗原）和抗体（Ab）进行离线孵育。达到平衡后，将样品引入流动系统。抗体复合物AgAb和Ag*Ab被捕获在蛋白G柱中，而游离未结合的示踪剂被洗脱，并在底物反应后由下游的安培生物传感器进行检测。酶标记β-GAL将底物4-氨基苯基-β-D-吡喃半乳糖苷（4-APG）转化为4-氨基苯酚（4-AP），随后由纤维二糖脱氢酶（CDH）修饰的固体石墨电极检测4-AP。4-AP首先在相对于Ag/AgCl为+300 mV的电极表面被氧化，形成的4-亚氨基醌（4-IQ）在纤维二糖存在下被CDH还原回4-AP。通过将EFIA与CDH生物传感器相结合，一个示踪剂分子的整体信号在两个阶段被放大，即一个酶标记将底物转化为许多4-AP分子，其次这些分子被CDH生物传感器进一步放大。使用2-硝基苯基-β-D-吡喃半乳糖苷（2-NPG）作为β-GAL底物，通过比色检测研究了EFIA在示踪剂与β-GAL的摩尔比、温度、流速等方面的最佳条件。研究了比色检测和CDH生物传感器检测的性能，并将这两种方法应用于加标地表水样品中模型化合物阿特拉津的测定。比色检测和CDH检测的检测限分别为0.056±0.008和0.038±0.007 μg L⁻¹，IC50值分别为2.04±0.294和0.42±0.08 μg L⁻¹。与比色检测相比，CDH生物传感器的基质效应不太明显。
