CRISPR-based ultrasensitive multiplexed array for the detection of multiple plasma biomarkers for early Alzheimer’s disease diagnosis

CRISPR-based ultrasensitive multiplexed array for the detection of multiple plasma biomarkers for early Alzheimer’s disease diagnosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CRISPR-based ultrasensitive multiplexed array for the detection of multiple plasma biomarkers for early Alzheimer’s disease diagnosis Haiming Luo, Liding Zhang, Changwen Yang, Xuewei Du, Hui Gong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5132416/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. We anticipate that this inexpensive, ultrasensitive, and multi-detection platform can also be widely used to detect other protein-related biomarkers besides AD. Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays Health sciences/Neurology/Neurological disorders/Dementia/Alzheimer's disease Full Text Additional Declarations Yes there is potential Competing Interest. L.H. and Z.L. have a filed patent application on UCMDA through Hainan University (Application No. 2024107318409, filing date 6 June 2024). The other authors declare no competing interests. Supplementary Files SupplementaryInformation.pdf Supplementary information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5132416","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444420968,"identity":"44bf6ec4-57ca-40ef-a1fa-ad36e5bb5ff6","order_by":0,"name":"Haiming Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIie3OMQuCQBTA8ScHtSiuTn2G2yKKPovHQU22BOLY5JI0G/Uhrm9wckOLeuuBQbYXNLYEKU0NpW4N94c3PHg/eAA63R+GAQwOMHHfa68dgYrMKoK6EdGBDPtrV5iBXGApOdx9AfZu9ZuMoowJMy2WWFEw4lyAc+INjymPCSssCFMIkBUKwI7bQM63muSESQHo2YooqyacME4BGa1I6rFkn1KyVRQnUT43HdVEjtmhvAZTspHJpXz444EdN5CPeDVmh3udTqfTfesFQzhL1bvYp/AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6226-5800","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Haiming","middleName":"","lastName":"Luo","suffix":""},{"id":444420969,"identity":"3a86aa7b-95dc-4f46-9e9f-6153bb01c0a7","order_by":1,"name":"Liding Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Liding","middleName":"","lastName":"Zhang","suffix":""},{"id":444420970,"identity":"c84c3153-7eb6-4bb0-89c5-faee36fa4e38","order_by":2,"name":"Changwen Yang","email":"","orcid":"https://orcid.org/0009-0008-3917-0046","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Changwen","middleName":"","lastName":"Yang","suffix":""},{"id":444420971,"identity":"24b250cc-a34a-4900-8d9a-2945d1485d33","order_by":3,"name":"Xuewei Du","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xuewei","middleName":"","lastName":"Du","suffix":""},{"id":444420972,"identity":"96eacd65-f7cc-4c64-b362-ef06c0c90995","order_by":4,"name":"Hui Gong","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Gong","suffix":""},{"id":444420973,"identity":"6b76e4b7-3764-4f43-98dc-8adef453e8d4","order_by":5,"name":"Ying Han","email":"","orcid":"","institution":"Department of Neurology, Xuanwu Hospital of Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Han","suffix":""},{"id":444420974,"identity":"af344dd5-8bbd-4a2a-8e02-c787d5016a63","order_by":6,"name":"Qingming Luo","email":"","orcid":"https://orcid.org/0000-0002-6725-9311","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Qingming","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2024-09-22 12:35:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5132416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5132416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83926905,"identity":"9d34b62e-c3ff-4c4a-ba61-525685ddf350","added_by":"auto","created_at":"2025-06-04 14:40:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1621214,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscriptof6RPACRISPRNC20250417.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5132416/v1_covered_c15ad7ff-5874-4b15-81ef-2e8b1cd9fee8.pdf"},{"id":80866052,"identity":"132647d0-3b77-4f1e-bb1a-06d501422ddd","added_by":"auto","created_at":"2025-04-18 03:34:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1844983,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5132416/v1/e564bbed7362531d0d0ca6d0.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nL.H. and Z.L. have a filed patent application on UCMDA through Hainan University (Application No. 2024107318409, filing date 6 June 2024). The other authors declare no competing interests.","formattedTitle":"CRISPR-based ultrasensitive multiplexed array for the detection of multiple plasma biomarkers for early Alzheimer’s disease diagnosis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5132416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5132416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. 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