磁珠辅助一锅rca激活CRISPR/Cas12a电化学发光生物传感器检测柑橘黄龙冰病原菌

IF 10.5 1区生物学 Q1 BIOPHYSICS

Biosensors and Bioelectronics Pub Date : 2025-09-10 DOI:10.1016/j.bios.2025.117986

Yingsi He , Yutian Zhang , Hongzhang Xiang , Kejing Ren , Yashi Yin , Yanru Gao , Yingjie Yang , Weidong Zhang , Lingzhi Liu , Heyou Han , Wenjing Wang

{"title":"磁珠辅助一锅rca激活CRISPR/Cas12a电化学发光生物传感器检测柑橘黄龙冰病原菌","authors":"Yingsi He , Yutian Zhang , Hongzhang Xiang , Kejing Ren , Yashi Yin , Yanru Gao , Yingjie Yang , Weidong Zhang , Lingzhi Liu , Heyou Han , Wenjing Wang","doi":"10.1016/j.bios.2025.117986","DOIUrl":null,"url":null,"abstract":"<div><div>Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging <em>cis</em>-cleavage for template recycling and <em>trans</em>-cleavage to degrade single stranded DNA attached on Ru(bpy)<sub>3</sub><sup>2+</sup>-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of <em>Candidatus Liberibacter asiaticus</em> (<em>C</em>Las) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.</div></div>","PeriodicalId":259,"journal":{"name":"Biosensors and Bioelectronics","volume":"290 ","pages":"Article 117986"},"PeriodicalIF":10.5000,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen\",\"authors\":\"Yingsi He , Yutian Zhang , Hongzhang Xiang , Kejing Ren , Yashi Yin , Yanru Gao , Yingjie Yang , Weidong Zhang , Lingzhi Liu , Heyou Han , Wenjing Wang\",\"doi\":\"10.1016/j.bios.2025.117986\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging <em>cis</em>-cleavage for template recycling and <em>trans</em>-cleavage to degrade single stranded DNA attached on Ru(bpy)<sub>3</sub><sup>2+</sup>-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of <em>Candidatus Liberibacter asiaticus</em> (<em>C</em>Las) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.</div></div>\",\"PeriodicalId\":259,\"journal\":{\"name\":\"Biosensors and Bioelectronics\",\"volume\":\"290 \",\"pages\":\"Article 117986\"},\"PeriodicalIF\":10.5000,\"publicationDate\":\"2025-09-10\",\"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/S0956566325008620\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOPHYSICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biosensors and Bioelectronics","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0956566325008620","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

摘要

黄龙冰（HLB）对全球柑橘产业构成灾难性威胁，必须及早发现病原体进行疾病控制，最大限度地减少经济损失。在此，我们报道了一种单锅电化学发光（ECL）生物传感器，用于整合滚动圆扩增（RCA）激活的CRISPR/Cas12a双裂解活性，以及基于工程磁珠的淬灭ECL发射器。靶标启动的RCA产生激活Cas12a的扩增子，同时利用顺式切割进行模板回收和反式切割来降解附着在Ru(bpy)32+负载磁珠上的单链DNA。这种双扩增策略恢复了ECL信号，实现了对asiaticcandidatus Liberibacter （CLas）核糖核苷酸二磷酸还原酶亚基β基因片段的高特异性超灵敏检测。检测限为2 fM，线性范围为10 fM-1 nM。该集成平台消除了多步骤孵育，在柑橘叶样品中表现出令人满意的性能，为HLB诊断提供了强大的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen

Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging cis-cleavage for template recycling and trans-cleavage to degrade single stranded DNA attached on Ru(bpy)₃²⁺-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of Candidatus Liberibacter asiaticus (CLas) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

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.