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Here, an enhanced one\u2010pot, helicase\u2010assisted RPA (hRPA)\u2010combined, dual\u2010CRISPR\/uAsCas12a (EOD\u2010CRISPR) assay is described which can be lyophilized on a 3D\u2010printed microfluidic disc to achieve field\u2010deployable multiplex bacteria detection. In EOD\u2010CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D\u2010printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD\u2010CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point\u2010of\u2010need testing, EOD\u2010CRISPR\u2010lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e.,\n                    <jats:italic>Bacillus cereus<\/jats:italic>\n                    ,\n                    <jats:italic>Salmonella enterica<\/jats:italic>\n                    ,\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    , and\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    O157:H7), the onsite detection platform is validated and the sensitivity (80%\u201388.9%) and specificity (92.3%\u2013100%) are comparable to those of standard PCR methods. Therefore, the field\u2010deployable multiplex EOD\u2010CRISPR assays holds great potentials for onsite bacteria detection and beyond.\n                  <\/jats:p>","DOI":"10.1002\/advs.202509355","type":"journal-article","created":{"date-parts":[[2025,9,16]],"date-time":"2025-09-16T14:36:53Z","timestamp":1758033413000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Enhanced One\u2010Pot Dual\u2010CRISPR\u2010Based Assay Lyophilized on a 3D\u2010Printed Disc for Field\u2010Deployable Multiplex Bacteria Detection"],"prefix":"10.1002","volume":"12","author":[{"given":"Yuqing","family":"Shen","sequence":"first","affiliation":[{"name":"Key Laboratory of Environmental Medicine and Engineering Ministry of Education School of Public Health Department of Nutrition and Food Hygiene School of Public Health Southeast University  Nanjing 210009 P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Lu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering School of Biological Science and Medical Engineering Southeast University  Nanjing 210009 P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Biao","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering School of Biological Science and Medical Engineering Southeast University  Nanjing 210009 P. R. 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