Yao Yu , Yi Liu , Ning Xu , Lin Li , Yaming Yang , Xiaolei Liu , Lianjing Zhao , Xue Bai
{"title":"A CRISPR/Cas12a mediated click immunoassay catalyzed by in situ formation of clickase for highly sensitive detection of Trichinella spiralis","authors":"Yao Yu , Yi Liu , Ning Xu , Lin Li , Yaming Yang , Xiaolei Liu , Lianjing Zhao , Xue Bai","doi":"10.1016/j.bios.2025.117521","DOIUrl":null,"url":null,"abstract":"<div><div>The accurate and high-throughput detection of trace protein targets remains an ongoing challenge in the field of food safety testing. This research article presents a highly sensitive CRISPR/Cas12a-mediated click immunoassay for the sensitive detection of <em>Trichinella spiralis</em> (<em>T. spiralis</em>) in meat samples. By simultaneously conjugating activator ssDNA and monoclonal antibodies to gold nanoparticles, the CRISPR/Cas12a system was introduced into the immunoassay. To overcome the challenges associated with the preparation, storage, and transportation of FQ probes in CRISPR/Cas12a systems, the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was employed instead. The designed ssDNA was both the substrate of trans-cleavage activity of Cas12a and the synthetic template of the artificial clicking enzyme copper nanoparticles (CuNPs), which could effectively catalyze the CuAAC reaction to generate the desired signal output. The fluorescence intensity showed a linear relationship with <em>T. spiralis</em> crude protein concentration ranging from 3.125 to 100 ng/mL, and the detection limit was 0.35 ng/mL, which is three orders of magnitude lower than that of ELISA (LOD: 309.75 ng/mL). This method can accurately detect a single <em>T. spiralis</em> larva in 100 g of pork. Collectively, the strategy of combining CRISPR/Cas12a system and CuAAC reaction opens up a novel avenue for developing a highly sensitive, simple and convenient fluorescence assay.</div></div>","PeriodicalId":259,"journal":{"name":"Biosensors and Bioelectronics","volume":"283 ","pages":"Article 117521"},"PeriodicalIF":10.7000,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biosensors and Bioelectronics","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0956566325003951","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
The accurate and high-throughput detection of trace protein targets remains an ongoing challenge in the field of food safety testing. This research article presents a highly sensitive CRISPR/Cas12a-mediated click immunoassay for the sensitive detection of Trichinella spiralis (T. spiralis) in meat samples. By simultaneously conjugating activator ssDNA and monoclonal antibodies to gold nanoparticles, the CRISPR/Cas12a system was introduced into the immunoassay. To overcome the challenges associated with the preparation, storage, and transportation of FQ probes in CRISPR/Cas12a systems, the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was employed instead. The designed ssDNA was both the substrate of trans-cleavage activity of Cas12a and the synthetic template of the artificial clicking enzyme copper nanoparticles (CuNPs), which could effectively catalyze the CuAAC reaction to generate the desired signal output. The fluorescence intensity showed a linear relationship with T. spiralis crude protein concentration ranging from 3.125 to 100 ng/mL, and the detection limit was 0.35 ng/mL, which is three orders of magnitude lower than that of ELISA (LOD: 309.75 ng/mL). This method can accurately detect a single T. spiralis larva in 100 g of pork. Collectively, the strategy of combining CRISPR/Cas12a system and CuAAC reaction opens up a novel avenue for developing a highly sensitive, simple and convenient fluorescence assay.
期刊介绍:
Biosensors & Bioelectronics, along with its open access companion journal Biosensors & Bioelectronics: X, is the leading international publication in the field of biosensors and bioelectronics. It covers research, design, development, and application of biosensors, which are analytical devices incorporating biological materials with physicochemical transducers. These devices, including sensors, DNA chips, electronic noses, and lab-on-a-chip, produce digital signals proportional to specific analytes. Examples include immunosensors and enzyme-based biosensors, applied in various fields such as medicine, environmental monitoring, and food industry. The journal also focuses on molecular and supramolecular structures for enhancing device performance.