Development of aptamer-based sensors for the real-time detection of proteins

摘要

In this work, the selective molecular recognition capability and high binding affinity of nucleic acid aptamers is integrated with the signal transduction methodology of molecular beacons for real-time monitoring of a protein target. An aptamer recognition element was modified to exist in a stem-loop configuration in the absence of a protein target, and in the presence of the protein target thrombin, the probe changes conformation. Upon binding to the target, this separation causes a physical separation of the attached fluorophore and quencher molecules, thereby allowing for an engineered, single-step recognition and signaling systems. The aptamer signaling probe was found to exhibit a maximum 12-fold change in signal when hybridized with a complement control, and a 3-fold change in signal with an excess of thrombin protein target. The fluorescence increased with increasing concentration of thrombin, until probe saturation where the fluorescence signal did not increase further, but leveled off in intensity. The signaling probe produced a rapid response, with 70% of the maximum signal achieved within a 15 second response time.