CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection

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 ultralow 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/mL (0.81 fM), respectively, demonstrating that CLISA is at least 10²-fold more sensitive than conventional ELISA.