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Despite DNA assembly\u2010based barcoding and amplification strategies across the breadth of molecular in situ analysis, an easy\u2010to\u2010design, nonenzymatic, highly efficient, background leakage\u2010avoided, highly specific, and sensitive system is highly required yet is still in its infancy. Spatial confinement nano\u2010assembly can increase the reaction efficiency in a localized isothermal autonomous manner. Here in this work, the DNA assembly that originally relies on random collisions between freely diffusing probes is constructed between two extended linear confined probes, by which a novel confined reaction model named as extended linear confined zipper hybridization chain reaction <jats:italic>(ZHCR<\/jats:italic>) is proposed. ZHCR can significantly improve the efficiency of probe assembly and enable stable assembly within live cells, providing precise in situ target information. ZHCR system is employed to analyze two thyroid cancer\u2010specific miRNAs, achieving in situ tracing and serum content detection. By integrating machine learning algorithms, ZHCR demonstrates significant potential in thyroid cancer auxiliary diagnosis, establishing a versatile platform that enables both highly sensitive homogeneous detection and in situ analysis of low\u2010abundance nucleic acid fragments.<\/jats:p>","DOI":"10.1002\/smll.202500202","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T03:06:28Z","timestamp":1747969588000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Extended Linear Confined Zipper Cascaded Reaction for Highly Efficient Intracellular Imaging and Assisting Diagnosis of Thyroid Cancer"],"prefix":"10.1002","volume":"21","author":[{"given":"Lu","family":"Liu","sequence":"first","affiliation":[{"name":"Medical Examination Centre The First Affiliated Hospital of Chongqing Medical University  Chongqing 400016 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