Autocatalysis-Integrated Bioorthogonal (Poly)Catalyst-Linked Immunosorbent Assay for Living Cell Membrane Antigens.

Abstract

Immunoassay methods, notably enzyme-linked immunosorbent assays (ELISAs), renowned for their signal amplification capabilities, are extensively employed in scientific research and clinical diagnostics. However, the instability of enzymes and their sensitivity to cellular environments present significant challenges for the broad application of ELISA in living cells. In this work, we present a bioorthogonal (poly)catalysis-linked immunosorbent assay (BCLISA) designed for the detection of cell membrane antigens, which involves coupling bioorthogonal catalysts based on small molecules or polymers to antibodies. After screening, we opted for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) as the core reaction system. The polymer-based catalysts exhibit enhanced reactivity at the same molecular concentration due to their multiple catalytic sites. Polytriazoles formed during the CuAAC reaction have the ability to chelate Cu(I), therebypromoting faster catalysis. By harnessing this autocatalytic feature, we successfully increased the signal amplification potential of BCLISA. Ultimately, this autocatalysis-integrated BCLISA technique was employed for antigen detection and imaging on both in vitro and living cell membranes. This approach offers a new method for the detection and imaging of low-abundance antigens on living cells.