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Herein, a hydrogel microneedle\u2010integrated electrochemical biosensor is developed as a complementary ex vivo molecular sensing approach for wash\u2010free and anti\u2010interference monitoring of miRNAs via negative\u2010potential readout. The sensing platform employs a four\u2010way junction (FWJ) nucleic acid nanoreservoir to encapsulate the electroactive indicator doxorubicin (DOX), which is released upon target miRNA\u2010triggered strand displacement. The characteristic oxidation potential of DOX (\u22120.65\u00a0V) enables detection in a negative potential window, thereby reducing co\u2010oxidation interference by endogenous electroactive species. The porous hydrogel microneedle architecture facilitates rapid TIF sampling and directional diffusion of released DOX toward the electrode interface. The biosensor achieved a detection limit of 1.25 pM for miRNA\u201021 with an interassay variability of &lt;1.9%. Preliminary analysis of clinical TIF samples from breast cancer and healthy donors further supported the feasibility of direct miRNA detection, yielding an AUC of 0.92. This work offers a novel strategy by integrating ex vivo in situ TIF sampling with negative\u2010potential readout to achieve wash\u2010free and anti\u2010interference detection of biomarkers in complex physiological matrices.<\/jats:p>","DOI":"10.1002\/adhm.71694","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T06:53:57Z","timestamp":1788850437000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hydrogel Microneedle\u2010Integrated Electrochemical Sensor With Negative\u2010Potential Readout Toward Wash\u2010Free and Anti\u2010Interference Monitoring of miRNAs in Tumor Interstitial Fluid"],"prefix":"10.1002","volume":"15","author":[{"given":"Xiyue","family":"Xie","sequence":"first","affiliation":[{"name":"Key Laboratory of Biorheological Science and Technology College of Bioengineering Ministry of Education Chongqing University  Chongqing 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