Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical鈥揅hemical鈥揅hemical Redox Cycling for the Detection of E. coli O157:H7

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• 作者：Md. Rajibul Akanda ; Vellaiappillai Tamilavan ; Seonhwa Park ; Kyungmin Jo ; Myung Ho Hyun ; Haesik Yang
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：February 5, 2013
• 年：2013
• 卷：85
• 期：3
• 页码：1631-1636
• 全文大小：327K
• 年卷期：v.85,no.3(February 5, 2013)
• ISSN：1520-6882

文摘

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鈥揷hemical鈥揷hemical (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(NH₃)₆³⁺ 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 10³ to 10⁸ colony-forming units/mL.