Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.
Keywords: CRISPR/Cas12a; Cytokines; Interferon-γ; Strand displacement amplification; Tandem reaction.
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