Label-free protein recognition using aptamer-based fluorescence assay

Abstract

Monitoring proteins in real time and in homogeneous solution without using external labels has always been a difficult task. In this paper, we have developed a label-free method for the ultrasensitive detection of thrombin in homogeneous solutions. High-affinity thrombin-binding aptamer (TBA) used as molecular recognition probe, and fluorophore, crystal violet (CV), was chose as fluorescence signal probe. The fluorescence of CV enhanced significantly when the free CV solution was mixed with single-stranded TBA. In the presence of human thrombin, the fluorescence of CV decreased after the specific interaction between TBA and thrombin. Using the fluorescence change, we are able to selectively detect the thrombin in homogeneous solutions. The conformation transformation was investigated by circular dichroism (CD) spectra measurements. Our method has been shown to be simple and effective without any labelling of the probe or of the target, and this procedure poses minimum effects on the binding properties of the proteins. This assay is highly selective and ultrasensitive. Under the optimum conditions, the method exhibits a dynamic response range from 2 × 10⁻¹¹ to 2 × 10⁻⁹ M with a detection limit of 8 × 10⁻¹² M.