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However, detecting biomarkers directly in complex biological environments, particularly whole blood, remains challenging because of nonspecific protein adsorption and nanopore\/nanochannel clogging. Here, a DNA aptamer functionalized nanochannel biosensor is developed with excellent antifouling properties, achieved by coating the nanochannel surface with agarose gel. This gel coating effectively mitigates fouling in diverse biological environments while maintaining comparable sensitivity to uncoated nanochannels for detecting prostate\u2010specific antigen (PSA) in buffer solutions within 20 min. The biosensor exhibits a detection limit of 1 ng\u00a0mL<jats:sup>\u22121<\/jats:sup> for PSA in human serum, matching the performance of commercial enzyme\u2010linked immunosorbent assay (ELISA) kits. Importantly, it successfully differentiates whole blood samples from prostate cancer patients and healthy individuals. The superior antifouling behavior is attributed to the electrically neutral, highly hydrophilic nature, and porous structure of the agarose gel, which prevents the adsorption of large biomolecules while facilitating the diffusion of PSA for aptamer\u2010based capture. This DNA aptamer functionalized nanochannel biosensor with agarose gel coating offers reliable protein detection in complex biological environments, showing great promise in biomedical applications.<\/jats:p>","DOI":"10.1002\/smll.202409966","type":"journal-article","created":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T22:47:13Z","timestamp":1741733233000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Agarose Gel\u2010Coated Nanochannel Biosensor for Detection of Prostate\u2010Specific Antigen in Unprocessed Whole Blood Samples"],"prefix":"10.1002","volume":"21","author":[{"given":"Jing","family":"Zhao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Geomicrobiology and Environmental Changes Engineering Research Center of Nano\u2010geomaterials of Ministry of Education Faculty of Materials Science and Chemistry China University of Geosciences  Wuhan 430074 P. R. 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