Zhou Jinyan, Chen Xueshan, Wei Yingnan, Lu Ruixuan, Wei Zeliang, Huang Ke, Luo Hong, Zhang Jinyi, Zheng Chengbin
College of Chemistry and Material Science, Sichuan Normal University, Chengdu, Sichuan 610068, China.
Key Laboratory of Green Chemistry & Technology of MOE, College of Chemistry, Sichuan University, Chengdu, Sichuan 610065, China.
Anal Chem. 2023 Dec 12;95(49):18303-18308. doi: 10.1021/acs.analchem.3c04723. Epub 2023 Nov 29.
Salivary pepsin has been proposed as a promising diagnostic marker for gastroesophageal reflux disease (GERD). However, the activity of human pepsin is strongly influenced by pH, and the acidic condition (pH ∼ 2) is optimal, which is a contradiction for the pepsin detection kit based on its catalytic activity. Thus, its accurate quantification in saliva (neutral pH) by readily rapid tools with simplicity and low cost is still challenging. Herein, a convenient fluorescence assay has been developed for the rapid detection of pepsin at neutral pH based on its electrostatic interaction with SYBR Green (SG) rather than the bioactivity. At neutral pH, the positively charged SG fluorophore can be effectively adsorbed by the negatively charged pepsin due to the low isoelectric point (pI) and large molecular size of pepsin. Thus, the molecular rotation of SG is limited, and its fluorescence intensity is significantly increased. The strategy was further confirmed to have the same fluorescence response as that of normally active and inactivated pepsin. Due to the unique pI of pepsin, the fluorescence strategy is highly selective for pepsin compared to other proteins. On the basis of this strategy, a smartphone-based fluorescence capture device integrated with a programmed Python program was fabricated for both color recognition and the accurate detection of pepsin within 3 min. Under the optimal conditions, this turn-on sensor allowed for the on-site analysis of pepsin with a detection limit of 0.2 μg/mL. Moreover, this strategy was successfully used to assess salivary pepsin to aid in the noninvasive diagnosis of GERD.
唾液胃蛋白酶已被提议作为胃食管反流病（GERD）一种很有前景的诊断标志物。然而，人胃蛋白酶的活性受pH值影响很大，酸性条件（pH约为2）是最佳条件，这对于基于其催化活性的胃蛋白酶检测试剂盒来说是一个矛盾。因此，用简单且低成本的快速工具在唾液（中性pH）中对其进行准确定量仍然具有挑战性。在此，基于胃蛋白酶与SYBR Green（SG）的静电相互作用而非生物活性，开发了一种方便的荧光分析法，用于在中性pH条件下快速检测胃蛋白酶。在中性pH值下，由于胃蛋白酶的低等电点（pI）和大分子尺寸，带正电荷的SG荧光团可被带负电荷的胃蛋白酶有效吸附。因此，SG的分子旋转受到限制，其荧光强度显著增加。该策略进一步被证实对正常活性和失活的胃蛋白酶具有相同的荧光响应。由于胃蛋白酶独特的pI，与其他蛋白质相比，该荧光策略对胃蛋白酶具有高度选择性。基于此策略，制造了一种集成有编程Python程序的基于智能手机的荧光捕获装置，用于颜色识别和在3分钟内准确检测胃蛋白酶。在最佳条件下，这种开启式传感器能够对胃蛋白酶进行现场分析，检测限为0.2μg/mL。此外，该策略已成功用于评估唾液胃蛋白酶，以辅助GERD的无创诊断。
