CRISPR/dCas9-assisted SERS sensing for transgenic crops based on two-factor verification of 35S promoter and Nos terminator

IF 8 1区化学 Q1 CHEMISTRY, ANALYTICAL

Sensors and Actuators B: Chemical Pub Date : 2025-01-27 DOI:10.1016/j.snb.2025.137293

Ailing Su , Ziqi Chen , Weiqing Xu , Chongyang Liang , Xiangguo Liu , Shuping Xu

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引用次数: 0

Abstract

A nucleic acid analysis platform was built based on the combination of the recombinase polymerase amplification (RPA)-aided CRISPR/dCas9 and surface-enhanced Raman scattering (SERS) technologies, which enables the simultaneous detection of dual nucleic acid fragments, for the purpose of identifying transgenic crops. Two SERS-active nanoprobes (AgNP@Ra@dCas9/sgRNA) were fabricated by grafting the single RNA (sgRNA)-activated dCas9 protein to silver nanoparticles that have been decorated with two different Raman reporters (Ra). RPA primers endow target genes with biotin end that can be captured by the streptavidin-modified magnetic bead (MB@SA), followed by the conjugation of SERS nanoprobes. Thus, strong SERS signals would be detected above the MBs. This SERS platform has been applied for identifying transgenic crops based on the sequence designs specifically for the 35S promoter and Nos terminator simultaneously. Double confirmation of target genes can ensure the accuracy of detection. The whole sensing process, including RPA, dCas9 activation, and target gene identification, can be completed in one tube, within 80 min. This CRISPR/dCas9-SERS analytical platform has been proved to be feasible in detecting plant samples, indicating one successful attempt of the CRISPR/dCas9-SERS systems in multi-target detection.

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基于重组酶聚合酶扩增（RPA）辅助的CRISPR/dCas9和表面增强拉曼散射（SERS）技术的结合，建立了一个核酸分析平台，可同时检测双重核酸片段，用于鉴定转基因作物。通过将单核糖核酸（sgRNA）激活的 dCas9 蛋白嫁接到用两种不同的拉曼报告物（Ra）装饰的银纳米粒子上，制备了两种 SERS 活性纳米探针（AgNP@Ra@dCas9/sgRNA）。RPA 引物赋予目标基因生物素末端，而生物素末端可被链霉亲和素修饰的磁珠（MB@SA）捕获，然后再连接 SERS 纳米探针。这样，磁珠上方就能检测到强烈的 SERS 信号。这一 SERS 平台已被应用于根据 35S 启动子和 Nos 终止子的序列设计同时鉴定转基因作物。目标基因的双重确认可确保检测的准确性。整个传感过程，包括 RPA、dCas9 激活和目标基因鉴定，可在一个试管中完成，耗时 80 分钟。该 CRISPR/dCas9-SERS 分析平台在植物样品检测中被证明是可行的，表明 CRISPR/dCas9-SERS 系统在多靶点检测中的一次成功尝试。

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

Sensors and Actuators B: Chemical 工程技术-电化学

CiteScore

14.60

自引率

11.90%

发文量

1776

审稿时长

3.2 months

期刊介绍： Sensors & Actuators, B: Chemical is an international journal focused on the research and development of chemical transducers. It covers chemical sensors and biosensors, chemical actuators, and analytical microsystems. The journal is interdisciplinary, aiming to publish original works showcasing substantial advancements beyond the current state of the art in these fields, with practical applicability to solving meaningful analytical problems. Review articles are accepted by invitation from an Editor of the journal.