Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen. Monitoring sortase activity and screening its inhibitors are of great significance to fundamental understanding of the infection mechanism and pharmaceutical development. Herein, we developed a digital single-particle imaging method to quantify sortase A (SrtA) activity based on transpeptidation-mediated assembly and enumeration of gold nanoparticles (GNPs). The assay utilizes two peptide stands, in which one has the SrtA recognition sequence LPXTG motif while the other carries an oligoglycine nucleophile at the one end and a biotin group at the other. The presence of SrtA enables the ligation of two peptides and allows for the immobilization of streptavidin-functionalized GNPs. Thus, SrtA activity can be quantified by imaging and enumeration of the surface-assembled GNPs at the single-particle level via dark-field microscopy. The single-particle method was highly sensitive to SrtA activity with a low detection limit of 7.9 pM and a wide linear dynamic range from 0.05 to 50 nM. Besides detection of SrtA in complex biological samples such as Gram-positive pathogen lysates, the proposed method was also successfully applied to estimate the half-maximal inhibitory concentration (IC₅₀) values of SrtA inhibitors (curcumin, berberine hydrochloride and quercetin). The present method, combining single-GNP counting and dark-field imaging, provides a facile and novel analytical tool for SrtA activity and its inhibitor screening.

转肽酶分选酶催化的表面蛋白的转肽在革兰氏阳性病原体的感染过程中起关键作用。监测分选酶活性并筛选其抑制剂对于基本了解感染机理和药物开发具有重要意义。在本文中，我们开发了一种数字单颗粒成像方法，可基于转肽介导的金纳米颗粒（GNP）的组装和计数来量化分选酶A（SrtA）的活性。该测定利用两个肽架，其中一个具有SrtA识别序列LPXTG基序，而另一个在一端带有寡糖亲核试剂，在另一端带有生物素基团。SrtA的存在能够连接两个肽，并可以固定链霉亲和素官能化的GNP。从而，SrtA活性可以通过暗场显微镜在单颗粒水平上对表面组装的GNP进行成像和枚举来量化。单颗粒方法对SrtA活性高度敏感，检出限低至7.9 pM，线性动态范围从0.05至50 nM。除了在复杂的生物样品（如革兰氏阳性病原体裂解物中）中检测SrtA之外，该方法还成功地用于估算半数最大抑制浓度（IC）。₅₀）SrtA抑制剂（姜黄素，盐酸小ber碱和槲皮素）的值。本方法结合了单GNP计数和暗场成像，为SrtA活性及其抑制剂筛选提供了一种简便而新颖的分析工具。