Pallu Justine, Rabin Charlie, Hui Pan, Moreira Thamires S, Creste Geordie, Calvet Corentin, Limoges Benoît, Mavré François, Branca Mathieu
Université de Paris, Laboratoire d'Electrochimie Moléculaire, UMR 7591, CNRS F-75013 Paris France
Chem Sci. 2022 Feb 11;13(9):2764-2777. doi: 10.1039/d1sc06086d. eCollection 2022 Mar 2.
The strength of autocatalytic reactions lies in their ability to provide a powerful means of molecular amplification, which can be very useful for improving the analytical performances of a multitude of analytical and bioanalytical methods. However, one of the major difficulties in designing an efficient autocatalytic amplification system is the requirement for reactants that are both highly reactive and chemically stable in order to avoid limitations imposed by undesirable background amplifications. In the present work, we devised a reaction network based on a redox cross-catalysis principle, in which two catalytic loops activate each other. The first loop, catalyzed by HO, involves the oxidative deprotection of a naphthylboronate ester probe into a redox-active naphthohydroquinone, which in turn catalyzes the production of HO by redox cycling in the presence of a reducing enzyme/substrate couple. We present here a set of new molecular probes with improved reactivity and stability, resulting in particularly steep sigmoidal kinetic traces and enhanced discrimination between specific and nonspecific responses. This translates into the sensitive detection of HO down to a few nM in less than 10 minutes or a redox cycling compound such as the 2-amino-3-chloro-1,4-naphthoquinone down to 50 pM in less than 30 minutes. The critical reason leading to these remarkably good performances is the extended stability stemming from the double masking of the naphthohydroquinone core by two boronate groups, a counterintuitive strategy if we consider the need for two equivalents of HO for full deprotection. An in-depth study of the mechanism and dynamics of this complex reaction network is conducted in order to better understand, predict and optimize its functioning. From this investigation, the time response as well as detection limit are found to be highly dependent on pH, nature of the buffer, and concentration of the reducing enzyme.
自催化反应的优势在于其能够提供一种强大的分子扩增手段，这对于提高众多分析和生物分析方法的分析性能非常有用。然而，设计一个高效的自催化扩增系统的主要困难之一是需要兼具高反应活性和化学稳定性的反应物，以避免不良背景扩增带来的限制。在本研究中，我们设计了一种基于氧化还原交叉催化原理的反应网络，其中两个催化循环相互激活。第一个循环由HO催化，涉及将萘硼酸酯探针氧化脱保护为具有氧化还原活性的萘氢醌，而萘氢醌又在还原酶/底物偶联物存在的情况下通过氧化还原循环催化HO的产生。我们在此展示了一组具有更高反应活性和稳定性的新型分子探针，其产生了特别陡峭的S形动力学曲线，并增强了对特异性和非特异性反应的区分能力。这意味着能够在不到10分钟的时间内灵敏地检测低至几纳摩尔的HO，或者在不到30分钟的时间内检测低至50皮摩尔的氧化还原循环化合物，如2-氨基-3-氯-1,4-萘醌。导致这些出色性能的关键原因是萘氢醌核心被两个硼酸酯基团双重掩蔽而具有的延长稳定性，从需要两当量的HO才能完全脱保护这一点来看，这是一个违反直觉的策略。为了更好地理解、预测和优化其功能，我们对这个复杂反应网络的机制和动力学进行了深入研究。通过这项研究，发现时间响应以及检测限高度依赖于pH值、缓冲液的性质和还原酶的浓度。
