A CRISPR/Cas12a mediated click immunoassay catalyzed by in situ formation of clickase for highly sensitive detection of Trichinella spiralis

IF 10.7 1区 生物学 Q1 BIOPHYSICS
Yao Yu , Yi Liu , Ning Xu , Lin Li , Yaming Yang , Xiaolei Liu , Lianjing Zhao , Xue Bai
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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.
CRISPR/Cas12a介导的点击酶原位形成催化的点击免疫分析法用于高灵敏度检测旋毛虫螺旋体
微量蛋白靶点的准确、高通量检测一直是食品安全检测领域面临的挑战。本研究提出了一种高灵敏度的CRISPR/ cas12a介导的click免疫分析法,用于肉类样品中旋毛虫(T. spiralis)的灵敏检测。通过同时偶联激活剂ssDNA和单克隆抗体到金纳米颗粒,将CRISPR/Cas12a系统引入免疫分析。为了克服CRISPR/Cas12a系统中FQ探针的制备、储存和运输相关的挑战,我们采用了铜(I)催化叠氮化物-炔环加成(CuAAC)反应。所设计的ssDNA既是Cas12a反式裂解活性的底物,又是人工点击酶铜纳米颗粒(cups)的合成模板,可以有效催化CuAAC反应产生所需的信号输出。荧光强度与螺旋体粗蛋白浓度在3.125 ~ 100 ng/mL范围内呈线性关系,检出限为0.35 ng/mL,比ELISA法(LOD: 309.75 ng/mL)低3个数量级。该方法可准确检测出100克猪肉中单个螺旋体幼虫。综上所述,CRISPR/Cas12a系统与CuAAC反应相结合的策略为开发高灵敏度、简单方便的荧光检测开辟了一条新的途径。
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来源期刊
Biosensors and Bioelectronics
Biosensors and Bioelectronics 工程技术-电化学
CiteScore
20.80
自引率
7.10%
发文量
1006
审稿时长
29 days
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.
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