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Abstract：
One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3\'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.
摘要：
现代分析和生物分析化学的新兴趋势之一涉及用纳米酶（具有类酶催化活性的纳米颗粒）取代酶标记（例如辣根过氧化物酶）。由于酶和纳米酶通常通过不同的催化机制起作用，预计最佳反应条件也会有所不同。纳米酶底物的优化通常集中在确定理想的pH和温度上。然而,在某些情况下,即使这一步也被忽视了，和为酶设计的商业底物制剂被利用。本文证明，不仅pH值，而且底物缓冲液的组成，包括缓冲物质和添加剂，显着影响纳米酶产生的分析信号。增强剂如咪唑在商业底物中的存在降低了纳米酶的催化活性，这在本文中通过使用3,3'-二氨基联苯胺(DAB)和普鲁士蓝作为模型显色底物和纳米酶来证明。相反，对底物缓冲液的简单修饰大大增强了纳米酶的性能。具体来说,在本文中，证明了诸如柠檬酸盐之类的缓冲液，MES,HEPES,和TRIS,含1.5-2MNaCl或NH4Cl，与商业DAB制剂相比,通过普鲁士蓝显著增加DAB氧化并产生更高的信号。本文的中心信息是，底物组成的优化应该是基于纳米酶的测定法开发中不可或缺的一步。在这里，提出了普鲁士蓝纳米酶的DAB底物组成的逐步优化。优化的基材在效率方面优于商业制剂。通过在几种常用的免疫染色技术中的应用,肯定了优化后的DAB底物的有效性,包括组织染色，免疫球蛋白免疫印迹分析,和抗SARS-CoV-2抗体的斑点印迹分析。
