Sensitive and homogeneous protein detection based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification

Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.