Assembly of DNA-Templated Bioluminescent Modules for Amplified Detection of Protein Biomarkers

By virtue of its self-illuminating mechanism, the bioluminescence resonance energy transfer (BRET) technique has recently emerged as a promising platform for point-of-care (POC) diagnostics. However, due to the difficulty of incorporating generic affinity elements, such as aptamers and antibodies, current BRET-based methods are still not applicable to most clinically important biomarkers. Furthermore, the inability of these methods to amplify BRET signals leads to limited sensitivity in some applications. Here, we present a modular strategy for amplified BRET detection of protein biomarkers in human peripheral blood samples. In this strategy, a DNA-templated bioluminescent module was constructed by simultaneously binding luciferase and green fluorescent protein to one DNA template in a site-specific manner. The proposed modules showed high energy transfer efficiency and could be assembled into long self-illuminating polymers. Owing to this modular design, aptamers and antibodies were rationally incorporated, enabling specific assembly of multiple bioluminescent modules on one target. This strategy realized amplified BRET assays for human alpha-thrombin and prostate specific antigen (PSA) with the detection limit in the picomolar range using either a spectrophotometer or a smartphone. The modularity of our strategy allowed detection of different biomarkers by simple exchange of affinity elements. Furthermore, the self-illumination and isothermal amplification performance of this strategy make it an attractive tool for POC diagnostics.