{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T16:11:17Z","timestamp":1787242277770,"version":"build-2736575974"},"reference-count":40,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T00:00:00Z","timestamp":1753315200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["21874082"],"award-info":[{"award-number":["21874082"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["RXRC2022004"],"award-info":[{"award-number":["RXRC2022004"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Fundamental Research Funds for the Central Public Welfare Research Institutes","award":["21874082"],"award-info":[{"award-number":["21874082"]}]},{"name":"Fundamental Research Funds for the Central Public Welfare Research Institutes","award":["RXRC2022004"],"award-info":[{"award-number":["RXRC2022004"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>The CRISPR\/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR\/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR\/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4\u2013103.5%. This dual-amplification strategy\u2014operating without target pre-amplification\u2014featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.<\/jats:p>","DOI":"10.3390\/bios15080479","type":"journal-article","created":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T10:28:09Z","timestamp":1753352889000},"page":"479","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["CRISPR\/Cas12a-Chemiluminescence Cascaded Bioassay for Amplification-Free and Sensitive Detection of Nucleic Acids"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7779-2763","authenticated-orcid":false,"given":"Xiaotian","family":"Guan","sequence":"first","affiliation":[{"name":"Institute of Biopharmaceutical and Healthcare Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peizheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Biopharmaceutical and Healthcare Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Wang","sequence":"additional","affiliation":[{"name":"Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100010, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7906-2885","authenticated-orcid":false,"given":"Shuqing","family":"Sun","sequence":"additional","affiliation":[{"name":"Institute of Biopharmaceutical and Healthcare Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2","DOI":"10.4103\/0973-6247.126679","article-title":"Nucleic acid testing-benefits and constraints","volume":"8","author":"Hans","year":"2014","journal-title":"Asian J. 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