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Various minimally invasive methods exist that allow for proteomic analysis of affected skin lesions in microscale concentrations. However, analyzing cytokines in small skin samples using traditional immunoassays is challenging due to many cytokines being detectable only at pico\u2010scale concentrations. Here, a digital surface\u2010enhanced Raman spectroscopy (SERS) immunoassay has been developed for multiplex detection of IL\u201017A, IL\u201022, IL\u201023 and TNF in punch biopsy skin using psoriasis as a model. It is performed using single\u2010molecule counting via analyte compartmentalization on a nanopillar array and single\u2010particle active SERS nanotags. This SERS nanopillar assay demonstrated a high degree of specificity and sensitivity, detecting cytokines down to 104 aM. Promisingly, this assay successfully detected cytokines in microscopically derived skin punch biopsy samples containing 13.84\u00a0\u00b5g to as little as 0.34\u00a0\u00b5g of total protein, revealing distinct cytokine profiles. The assay was also able to show the differences in cytokine levels between perilesional and lesional psoriasis skin samples. The high detection sensitivity of the digital SERS nanopillar assay, combined with cytokine profiling in biopsy samples, shows promise for sensitive immunological diagnosis and therapeutic monitoring of inflammatory skin diseases.<\/jats:p>","DOI":"10.1002\/smtd.202501964","type":"journal-article","created":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T04:52:08Z","timestamp":1778647928000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Spatially Profiling Trace Cytokine Signatures From Microscopically Derived Skin Samples to Probe Skin Disease Inflammation"],"prefix":"10.1002","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-0719-549X","authenticated-orcid":false,"given":"Aditya","family":"Krishnakumar","sequence":"first","affiliation":[{"name":"Centre For Personalised Nanomedicine Australian Institute For Bioengineering and Nanotechnology (AIBN) The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5766-3468","authenticated-orcid":false,"given":"Zhen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Centre For Personalised Nanomedicine Australian Institute For Bioengineering and Nanotechnology (AIBN) The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Courtney","family":"Vedelago","sequence":"additional","affiliation":[{"name":"Centre For Personalised Nanomedicine Australian Institute For Bioengineering and Nanotechnology (AIBN) The University of Queensland  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8034-9856","authenticated-orcid":false,"given":"Timothy J.","family":"Liu","sequence":"additional","affiliation":[{"name":"Frazer Institute The University of Queensland Dermatology Research Centre  Brisbane Queensland Australia"},{"name":"Department of Dermatology Princess Alexandra Hospital  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yung\u2010Ching","family":"Kao","sequence":"additional","affiliation":[{"name":"Frazer Institute The University of Queensland Dermatology Research Centre  Brisbane Queensland Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H. 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