Gao Fengli, Liu Lin, He Cancan, Chang Yong, Wang Weiqiang
Henan Province Key Laboratory of New Opto-electronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang 455000, China.
Shiyan Key Laboratory of Biological Resources and Eco-Environmental Protection, Department of Chemistry and Environmental Engineering, Hanjiang Normal University, Shiyan 442000, China.
Biosensors (Basel). 2025 Jun 4;15(6):354. doi: 10.3390/bios15060354.
Nanoquenchers with a single quenching cofactor exhibit limited fluorescence quenching efficiency. In this work, a metal-organic hybrid with dual quenching cofactors (Cu and pyrroloquinoline quinone or PQQ) was prepared by metal-coordinated assembly and used as a nanoquencher for a protease assay with enhanced quenching efficiency. The peptide substrate with an oligohistidine (His) tag was labeled with a fluorophore. Caspase-3 was determined as a protease example. The substrate was attached onto the surface of the Cu-PQQ nanoquencher by a metal coordination interaction between the unsaturated Cu on the nanoparticle surface and the His tag in the peptide. The cleavage of the peptide substrate by enzymatic hydrolysis led to the release of a fluorophore-conjugated segment from the nanoquencher surface, thus turning on the fluorescence. The nanoprobe was used to determine caspase-3 with a linear range of 0.01-5 ng/mL and a detection limit of 7 pg/mL. Furthermore, the method was used to evaluate inhibition efficiency and monitor drug-induced cell apoptosis. In contrast to other means of peptide immobilization, such as physical adsorption and covalent coupling, the strategy based on the metal coordination interaction is simple and powerful, thereby achieving assays of caspase-3 activity in lysates with a satisfactory result. The work should be valuable for the design of nanoquenchers with multiple quenching cofactors and the development of novel biosensors.
具有单一猝灭辅因子的纳米猝灭剂表现出有限的荧光猝灭效率。在本工作中，通过金属配位组装制备了一种具有双猝灭辅因子（铜和吡咯喹啉醌或PQQ）的金属有机杂化物，并将其用作纳米猝灭剂用于蛋白酶分析，以提高猝灭效率。带有寡聚组氨酸（His）标签的肽底物用荧光团标记。以半胱天冬酶-3作为蛋白酶实例进行测定。底物通过纳米颗粒表面的不饱和铜与肽中的His标签之间的金属配位相互作用附着在Cu-PQQ纳米猝灭剂的表面。肽底物通过酶促水解裂解导致荧光团共轭片段从纳米猝灭剂表面释放，从而开启荧光。该纳米探针用于测定半胱天冬酶-3，线性范围为0.01-5 ng/mL，检测限为7 pg/mL。此外，该方法用于评估抑制效率并监测药物诱导的细胞凋亡。与其他肽固定化方法（如物理吸附和共价偶联）相比，基于金属配位相互作用的策略简单且有效，从而实现了对裂解物中半胱天冬酶-3活性的测定，结果令人满意。这项工作对于设计具有多个猝灭辅因子的纳米猝灭剂和开发新型生物传感器具有重要价值。
