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 x 10(-11) to 2 x 10(-9) M with a detection limit of 8 x 10(-12) M.

Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

“…For example, 4',6-diamidino-2-phenylindol (DAPI) [43], SYBR Green I (SG) [44], Malachite green [43,45], crystal violet [46], and OliGreen [47] have all been incorporated into aptasensors due to their ability to exhibit enhanced fluorescence after binding with aptamers. The majority of these biosensing applications exclusively use well-studied aptamers as proof-of-concept studies that bind to targets such as dyes and ATP.…”

G-quadruplexes for luminescent sensing and logic gates

“…The addition of thrombin induces the dissociation of the thrombin aptamer due to the formation of the thrombin-aptamer G-quadruplex ensemble, resulting in a reduced fluorescence signal of EB owing to its weak interaction with G-quadruplex. A switch-on label-free detection platform for thrombin was devised by Jin and co-workers (28) using a single strand-to-quadruplex approach. This assay using CV as a G-quadruplex-selective dye achieved a detection limit of 8 pM for the detection of thrombin with selectivity over IgG and lysozyme.…”

Mesoporous carbon microparticles as a novel fluorescent sensing platform for thrombin detection

“…Up to now, many detection approaches including electrochemistry Radi et al, 2006;Wu et al, 2007;Lee et al, 2008), colorimetry Xu et al, 2009), fluorescence (Jin et al, 2010;Li et al, 2007;Wang and Liu, 2009;Ho and Leclerc, 2004;Lin et al, 2006), and surfaceenhanced Raman scattering (SERS) (Cho et al, 2008;Hu et al, 2009) etc., have been developed. The basic principle in these approaches mainly lies in target-induced conformational change of TB aptamer (TA).…”