Gold Nanoparticle-Assisted CRISPR-Cas12a-Based Activity Assay for Highly Sensitive Detection of Trypsin.

IF 4.7 Q2 MATERIALS SCIENCE, BIOMATERIALS
ACS Applied Bio Materials Pub Date : 2025-07-21 Epub Date: 2025-06-16 DOI:10.1021/acsabm.5c00854
Sathishkumar Munusamy, Haiyan Zheng, Jun Chen, Shuo Zhou, Juanhua Kong, Rana Jahani, Yuan Zhao, Xiyun Guan
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引用次数: 0

Abstract

Proteases play important roles in diverse physiological processes, and their malfunction has been implicated in various conditions and diseases. Therefore, development of sensitive methods for protease detection in clinical samples is highly desired for disease diagnosis. Herein, we report an ultrasensitive and selective CRISPR-Cas12a based fluorescent assay for trypsin activity measurement. By taking advantage of the signal amplification brought by a unique magnetic bead-gold nanoparticle assembly, which carries the peptide substrate and tens to hundreds of DNA molecules per peptide molecule, trypsin can be detected with a limit of detection reaching as low as 0.13 ng/mL. Furthermore, the sensor selectivity study was performed by examining several biomolecules commonly present in biological samples, including bovine serum albumin (BSA), human serum albumin (HSA), DNase, RNase, chymotrypsin, elastase, and thrombin. Moreover, trypsin inhibition and serum sample analysis were successfully carried out. Given the ultrahigh sensitivity, the CRISPR-based trypsin activity assay developed in this work can be used as a generic platform for developing sensors for other proteases, offering the potential as a noninvasive/minimally invasive tool for clinical diagnosis.

基于金纳米颗粒辅助crispr - cas12的高灵敏度胰蛋白酶活性检测
蛋白酶在多种生理过程中发挥重要作用,其功能障碍与多种疾病有关。因此,在临床样品中开发敏感的蛋白酶检测方法是疾病诊断的迫切需要。在此,我们报告了一种基于CRISPR-Cas12a的超灵敏和选择性胰蛋白酶活性测定的荧光检测方法。利用独特的磁珠-金纳米粒子组件带来的信号放大,胰蛋白酶可以被检测到,检测限低至0.13 ng/mL。该组件携带肽底物和每个肽分子数十到数百个DNA分子。此外,通过检测生物样品中常见的几种生物分子,包括牛血清白蛋白(BSA)、人血清白蛋白(HSA)、dna酶、RNase、凝乳胰蛋白酶、弹性酶和凝血酶,进行了传感器选择性研究。胰蛋白酶抑制和血清样品分析均成功。鉴于超高灵敏度,本研究中开发的基于crispr的胰蛋白酶活性测定可作为开发其他蛋白酶传感器的通用平台,具有作为临床诊断的无创/微创工具的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
期刊介绍: ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.
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