Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Jun Xue,Hui Zhou,Jintao Yi

doi:10.3390/molecules26102962

Abstract

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

Highlights

Protein, an important component in human cells and tissues, participates in the physiological activities of the body, catalyzes the cell processes and plays important roles in the research of bioengineering, medical diagnosis, treatment and proteomics [1,2,3]
We explored the use of a molecular beacon (G3MB) based on G-triplex in conjunction with a terminal protection assay for the enzyme amplification detection of protein receptors
These results indicated that the G3MB1-b probe based on G-triplex DNA and thioflavin T could be used for streptavidin detection by the steric hindrance effect from streptavidin-biotin to protect the G3MB1-b probe against exonuclease III (Exo III) and cause the fluorescence change

Summary

Introduction

An important component in human cells and tissues, participates in the physiological activities of the body, catalyzes the cell processes and plays important roles in the research of bioengineering, medical diagnosis, treatment and proteomics [1,2,3]. The oligonucleotides of small-molecule-linked DNA act as coding sequences for identifying the linked organic molecules, but offer immediate signal amplification via polymerase chain reactions. This technology has encouraged many researchers to develop different analytical strategies to apply to the detection of H5N1 antibodies [9], FITC antibodies [10] and folate receptors [11,12]. We developed a simple and label-free biosensor for protein detection based on the thioflavin T and G-triplex molecular beacon (G3MB). Compared with traditional G-quadruplex molecular beacons (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance

Experimental Section

Results and Discussion

Conclusions