Akanda Md Rajibul, Joung Hyou-Arm, Tamilavan Vellaiappillai, Park Seonhwa, Kim Sinyoung, Hyun Myung Ho, Kim Min-Gon, Yang Haesik
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Korea.
Analyst. 2014 Mar 21;139(6):1420-5. doi: 10.1039/c3an02328a.
Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical-chemical-chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH3)6(3+), enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH3)6(3+) is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [β-galactosidase, 4-amino-1-naphthyl β-D-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V vs. Ag/AgCl) minimizes the interference effect of electroactive species (L-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL(-1) for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples.
对临床样本中的生物标志物进行即时检测（POCT）对于快速且经济高效的诊断至关重要。然而，开发一种兼具超灵敏度和简便性的电化学POCT技术极具挑战性。我们报道了一种无干扰的电化学侧向流免疫测定法，该方法能够对血清进行一步超灵敏检测。利用电化学 - 化学 - 化学（ECC）氧化还原循环与酶标记物的酶促反应相结合来实现高信号放大。涉及六氨合钌（III）[Ru(NH3)6(3+)]、酶产物和三（3 - 羧乙基）膦（TCEP）的ECC氧化还原循环取决于pH值，因为尽管Ru(NH3)6(3+)的形式电位与pH无关，但酶产物和TCEP的形式电位会随着pH值降低而升高。考虑到ECC氧化还原循环对pH的依赖性，引入了一种理想的酶标记物、底物和产物组合[分别为β - 半乳糖苷酶、4 - 氨基 - 1 - 萘基β - D - 吡喃半乳糖苷和4 - 氨基 - 1 - 萘酚]，以确保酶产物快速且选择性地进行ECC氧化还原循环，同时保持低背景水平。在低施加电位（相对于Ag/AgCl为0.05 V）下进行的选择性ECC氧化还原循环可将血清中电活性物质（L - 抗坏血酸、对乙酰氨基酚和尿酸）的干扰效应降至最低。在滴加血清后仅11分钟，无需使用额外溶液即可获得肌钙蛋白I的检测限为0.1 pg mL(-1)。此外，该侧向流免疫测定法适用于实际临床样本的分析。
