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 toeholdmediated 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 addingmicrobeads, 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.