A fluorometric assay of thrombin using magnetic nanoparticles and enzyme-free hybridization chain reaction

A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5-103.3%. Graphical abstract A fluorometry method for thrombin detection using magnetic nanoparticles and enzyme-free hybridization chain reaction.