Microscale thermophoresis sensor for homogeneous and one-step protein detection based on dual aptamers binding...

Rapid and sensitive detection of proteins is in great demand in disease diagnostics, drug discovery, basic research, and other biomedical applications. The emerging microscale thermophoresis (MST) technique, which enables to monitor fluorescence responses of fluorescent molecules under heating in real time, provides an attractive avenue for sensing transducer, with advantages in no separation and immobilization, ratiometric measurement, rapid analysis, and high throughput. Here, we developed a one-step, selective, and sensitive MST sensor for detection of protein based on a DNA switch assembled by binding a pair of aptamer probes on the same protein. These two aptamer probes share an additional short complementary sequence at terminals, and one is labeled with a fluorophore for signal output. They can bind to protein target, forming a sandwich complex and greatly enhancing local concentrations of aptamer probes, and the complementary sequences hybridize together, assembling a DNA switch with a duplex structure. The DNA switch can be opened upon heating in MST analysis, generating remarkable difference in MST responses over the unbound aptamer probes. Target protein can be detected by measuring the significant MST signal changes. We achieved detection of thrombin and platelet-derived growth factor BB (PDGF-BB) with this DNA switch-based MST sensor. The proposed strategy is promising for sensitive and selective detection of various target proteins.