Sensitive and enzyme-free detection for single nucleotide polymorphism using microbead-assisted toehold-mediated strand displacement reaction

This report described a free-enzyme, convenient and inexpensive genotyping biosensor capable of detecting single nucleotide polymorphism at normal temperature based on the combination of toehold-mediated strand displacement reaction (toehold-SDR) and microbead-capture technique. The biosensor consists of a pre-hybridized strand formed by a reporter probe and a capture probe. In the presence of a mutant sequence, there is no toehold-mediated strand displacement and the reporter probe cannot be released from the pre-hybridized strand. Microbeads capture the fluorescent pre-hybridized strand through biotin–streptavidin interaction, so microbeads give out significant fluorescence signal, while there is no fluorescence in the solution. However, in the presence of a matched target, the strand displacement is effectively initiated and the reporter probe is released from pre-hybridized strand. After adding microbeads, the solution produces bright fluorescence, while microbeads have no obvious signal. Genotypes are identified conveniently according to the fluorescence intensity of the solution. The method provides a simple and inexpensive strategy to detect point mutation. Moreover, this biosensor shows the linear relationship in the range of 1–40 nmol/L and reaches a detection limit of 0.3 nmol/L.

Graphical abstractThis report described a free-enzyme, convenient and inexpensive genotyping biosensor capable of detecting point mutation at normal temperature based on the combination of toehold-SDR and microbead-capture technique.Figure optionsDownload full-size imageDownload as PowerPoint slide