The Theoretical Model, Method, and Applications of Scattering Photon Burst Counting Based on an Objective Scanning Technique.

Scattering photon burst counting (SPBC) is a single-particle detection method, which is based on measuring scattering photon bursting of single nanoparticles through a detection volume of <1 fL. Although SPBC has been used for bioassays and analysis of nanoparticles, it is necessary to establish its theoretical model and develop a new detection mode in order to further enhance its sensitivity and enlarge its application fields. In this paper, we proposed a theoretical model for the confocal SPBC method and developed a novel SPBC detection mode using the fast objective scanning technique. The computer simulations and experiments documented that this model well describes the relation between photon counts and experimental parameters (such as nanoparticle concentration and diameter, temperature, and viscosity). Based on this model, we developed a novel SPBC detection mode by using the fast objective scanning technique. Compared to the current confocal SPBC method, the sensitivity of this new method was significantly increased due to the significantly increased photon counts per sampling time, the linear detection range is from 0.9 to 90 pM, and the limit of detection is reduced to 40 fM for 30 nm gold nanoparticles. Furthermore, this new method was successfully applied to determine the enzyme activity of caspase-3 and evaluate the inhibition effectiveness of some inhibitors.