Single Nanoparticle Counting Technique And Its Application On Thrombin Assay

Single Nanoparticle Counting (SNC) is a novel single molecule detection method derived from Florescence Correlation Spectroscopy (FCS) or Resonance Light Scattering Correlation Spectroscopy (RLSCS). Determining the concentration of analyte by counting the photon burst during the Brownian motion in a highly focused laser beam, SNC shows certain advantages such as lower detection of limit and capable of detection in complex solution without separation. To date, SNC has only applied to quantum dots or dyes whose signals are florescence. SNC using noble metal nanoparticles (such as gold nanoparticles and silver nanoparticles) as probes, have not yet been reported, mainly due to the fact that the wavelength of resonance light scattering is difficultly distinguished with that of background light.Noble metal nanoparticles have broad applications in biological assays due to their unique optical properties. In order to combine powerful noble metal nanoparticles and sensitive SNC method, we established a SNC system based on the resonance light scattering of noble nanoparticles, including data processing strategy, the selection of data process parameter and detection conditions. We applied this SNC system to the detection of AuNPs as well as AgNPs. Furthermore, as a proof-of-principle we also investigated thrombin assay by SNC system using two assay strategies. One of them utilized the specific binding property of thrombin-binding aptamers and thrombin. The other utilized the reaction that fibrinogen can polymerized under the catalysis of thrombin. The results show that the latter method is more promising with linear range from 0.32 pM-32 pM and very low detection limit (0.44 pM), in the mean while it is also simple and fast in sample preparation and detection, low sample assumption by taking advantage of SNC. We also detected the concentration of prothrombin in human plasma. Above all, we believe that SNC will be an alternative detection method in biological assay based on noble nanoparticles and have broad future prospects in clinical and fundamental research.