Spatially Profiling Trace Cytokine Signatures From Microscopically Derived Skin Samples to Probe Skin Disease Inflammation

Inflammatory skin diseases, such as atopic dermatitis, hidradenitis suppurativa and psoriasis, involve chronic aberrations to innate and adaptive immunity, driven by pro-inflammatory cytokines. 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-scale concentrations. Here, a digital surface-enhanced Raman spectroscopy (SERS) immunoassay has been developed for multiplex detection of IL-17A, IL-22, IL-23 and TNF in punch biopsy skin using psoriasis as a model. It is performed using single-molecule counting via analyte compartmentalization on a nanopillar array and single-particle 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 µg to as little as 0.34 µg 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.