{"title":"Gold Nanoparticle-Assisted CRISPR-Cas12a-Based Activity Assay for Highly Sensitive Detection of Trypsin.","authors":"Sathishkumar Munusamy, Haiyan Zheng, Jun Chen, Shuo Zhou, Juanhua Kong, Rana Jahani, Yuan Zhao, Xiyun Guan","doi":"10.1021/acsabm.5c00854","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":" ","pages":"6379-6387"},"PeriodicalIF":4.7000,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1021/acsabm.5c00854","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/6/16 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
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.
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.