Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea.
Anal Chem. 2013 Feb 5;85(3):1631-6. doi: 10.1021/ac3028855. Epub 2013 Jan 17.
Signal amplification by enzyme labels in enzyme-linked immunosorbent assays (ELISAs) is not sufficient for detecting a low number of bacterial pathogens. It is useful to employ approaches that involve multiple signal amplification such as enzymatic amplification plus redox cycling. An advantageous combination of an enzyme product [for fast electrochemical-chemical-chemical (ECC) redox cycling that involves the product] and an enzyme substrate (for slow side reactions and ECC redox cycling that involve the substrate) has been developed to obtain a low detection limit for E. coli O157:H7 in an electrochemical ELISA that employs redox cycling. In our search for an alkaline phosphatase substrate/product couple that is better than the most common couple of 4-aminophenyl phosphate (APP)/4-aminophenol (AP), we compared five couples: APP/AP, hydroquinone diphosphate (HQDP)/hydroquinone (HQ), L-ascorbic acid 2-phosphate/L-ascorbic acid, 4-amino-1-naphthyl phosphate/4-amino-1-naphthol, and 1-naphthyl phosphate/1-naphthol. In particular, we examined signal-to-background ratios in ECC redox cycling using Ru(NH(3))(6)(3+) and tris(2-carboxyethyl)phosphine as an oxidant and a reductant, respectively. The ECC redox cycling that involves HQ is faster than the cycling that involves AP, whereas the side reactions and ECC redox cycling that involve HQDP are negligible compared to the APP case. These results seem to be due to the fact that the formal potential of HQ is lower than that of AP and that the formal potential of HQDP is higher than that of APP. Enzymatic amplification plus ECC redox cycling based on a HQDP/HQ couple allows us to detect E. coli O157:H7 in a wide range of concentrations from 10(3) to 10(8) colony-forming units/mL.
酶标记在酶联免疫吸附测定（ELISA）中的信号放大对于检测少量细菌病原体是不够的。采用涉及多种信号放大的方法是有用的，例如酶放大加氧化还原循环。已经开发出一种酶产物的有利组合[用于涉及产物的快速电化学-化学-化学（ECC）氧化还原循环]和酶底物（用于涉及底物的缓慢副反应和 ECC 氧化还原循环），以在采用氧化还原循环的电化学 ELISA 中获得对大肠杆菌 O157：H7 的低检测限。在寻找比最常见的 4-氨基苯磷酸盐（APP）/4-氨基酚（AP）对更好的碱性磷酸酶底物/产物对的过程中，我们比较了五种对：APP/AP、氢醌二磷酸盐（HQDP）/氢醌（HQ）、L-抗坏血酸 2-磷酸盐/L-抗坏血酸、4-氨基-1-萘基磷酸盐/4-氨基-1-萘酚和 1-萘基磷酸盐/1-萘酚。特别是，我们检查了使用 Ru(NH(3))(6)(3+)和三（2-羧乙基）膦分别作为氧化剂和还原剂的 ECC 氧化还原循环中的信号背景比。涉及 HQ 的 ECC 氧化还原循环比涉及 AP 的循环更快，而与 APP 情况相比，HQDP 涉及的副反应和 ECC 氧化还原循环可以忽略不计。这些结果似乎是由于 HQ 的形式电位低于 AP，而 HQDP 的形式电位高于 APP。基于 HQDP/HQ 对的酶放大加 ECC 氧化还原循环使我们能够在 10(3)到 10(8)个菌落形成单位/mL 的广泛浓度范围内检测到大肠杆菌 O157：H7。
