CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA)

The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultra-low concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity. This process is termed the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). The proposed method was validated by detecting an inflammatory factor, human interleukin-6 (human IL-6), and a tumor marker, human vascular endothelial growth factor (human VEGF), which achieved limit of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/m (0.81 fM), respectively, demonstrating that CLISA is at least 102-fold more sensitive than conventional ELISA.