Homogeneous label-free protein binding assay using small-molecule-labeled DNA nanomachine with DNAzyme-Based chemiluminescence detection.

Detecting the interactions between small molecules and proteins was critical for disease theranostics and drug development. Here we propose a novel universal assay strategy for monitoring small molecule-protein interactions in solution using strand displacement amplification (SDA) mediated by protein binding to small molecule with DNAzyme-based chemiluminescence detection. The DNA polymerase and nicking enzyme assisted SDA could yield a great amount of peroxidase-mimicking DNAzyme sequences which cause significantly chemiluminescence signals, while protein binding to the small molecule label would prevent DNA polymerase from extending nick site and DNAzyme sequence, and thus the chemiluminescence signals would obviously decrease. This strategy was demonstrated using folate and its binding protein (folate receptor), and the results revealed that the developed strategy enable offer a label-free, homogeneous, and highly sensitive chemiluminescence detection of folate receptor with a detection limit of 1pM. At the same time, it has been successfully used for folate receptor detection in human serum. The proposed chemiluminescence sensing method might provide a generic, robust, and high-throughput platform for detecting various small molecule-protein interactions for biological applications.