CRISPR-enhanced ELISA based on ssDNA-triggered Cas13a-Csm6 cascade for sensitive profiling of inflammatory biomarkers in sports-related osteoarthritis among martial arts athletes.

Sports-related osteoarthritis (OA), which is common among martial arts athletes exposed to repetitive high-impact loading, is closely associated with complex inflammatory responses. Sensitive detection of inflammatory biomarkers is therefore highly desirable for early diagnosis and disease monitoring. However, conventional enzyme-linked immunosorbent assays (ELISA) are often limited by insufficient sensitivity due to the lack of intrinsic signal amplification. Herein, we report a CRISPR-enhanced ELISA platform based on an ssDNA-triggered Cas13a-Csm6 cascade for ultrasensitive detection of OA-related inflammatory biomarkers. In this strategy, detection antibodies are conjugated with trigger ssDNA, which is retained in the sandwich immunocomplex after target binding and serves as an activator for Cas13a. Activated Cas13a subsequently cleaves the Csm6 activator precursor to generate secondary messengers, thereby initiating a Csm6-mediated amplification process and converting protein recognition into amplified nucleic acid fluorescence output under isothermal and amplification-free conditions. The developed platform enabled sensitive detection of multiple inflammatory biomarkers with low-picogram-per-milliliter limits of detection, including IL-6, IFN-γ, and MMP-3, while maintaining high specificity and good tolerance in simulated biological matrices. Well-resolved multi-analyte analysis was achieved through a spatially resolved ELISA format, allowing parallel detection of different targets in individual wells of the same microplate. The proposed method also showed good agreement with conventional ELISA, demonstrating its analytical reliability. This work provides a versatile and sensitive strategy for spatially resolved parallel protein analysis and offers promising potential for inflammatory biomarker monitoring and OA-related bioanalysis in martial arts athletes.