Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China.
Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China; School of Chemistry and Materials Science, Guizhou Education University, GaoXin Road 115, Wudang District, Guizhou, 550000, PR China.
Talanta. 2022 Jan 15;237:122949. doi: 10.1016/j.talanta.2021.122949. Epub 2021 Oct 11.
Heterogeneous analysis has great application prospects in the detection of post-translational modification (PTM) enzymes with the advantages of signal enhancement, less sample demand, and high sensitivity and selectivity. Nevertheless, once the substrate was fixed on a solid interface, the steric hindrance might limit the approaching of catalytic center to the substrate, thus reducing the efficiency of PTM. Herein, we suggested that the avidin-modified interface could be used to develop heterogeneous sensing platforms with biotin-labeled substrates as the probes, in which the enzymatic PTM was performed in solution and the heterogeneous assay was conducted on a solid surface. The sensing strategy integrates the advantages but overcomes the defects of both homogeneous and heterogeneous assays. Protein kinase A (PKA) and histone acetyltransferase (HAT) were determined as the examples by using sequence-specific peptide substrates. The signal changes were monitored by HRP-based colorimetric assay and antibody-amplified surface plasmon resonance (SPR). The methods were used for analysis of cell lysates and evaluation of inhibition efficiency with satisfactory results. The strategy can be used for the detection of a variety of biological enzymes and provide a new idea for the design of various heterogeneous biosensors. Thus, this work should be of great significance to the popularization and practical application of biosensors.
异质分析在检测翻译后修饰（PTM）酶方面具有很大的应用前景，具有信号增强、样品需求少、灵敏度和选择性高的优点。然而，一旦底物固定在固体界面上，空间位阻可能会限制催化中心接近底物，从而降低 PTM 的效率。在这里，我们建议可以使用亲和素修饰的界面来开发具有生物素标记底物作为探针的异质感测平台，其中酶 PTM 在溶液中进行，而异质测定在固体表面上进行。该传感策略结合了均相和异相测定的优点，但克服了它们各自的缺陷。我们使用序列特异性肽底物作为实例来确定蛋白激酶 A（PKA）和组蛋白乙酰转移酶（HAT）。通过基于 HRP 的比色测定法和抗体放大表面等离子体共振（SPR）来监测信号变化。该方法用于细胞裂解物的分析和抑制效率的评估，结果令人满意。该策略可用于检测各种生物酶，并为各种异质生物传感器的设计提供新的思路。因此，这项工作对于生物传感器的普及和实际应用具有重要意义。
