| The photon burst counting method achieves quantitative analysis of sample concentration by collecting photon burst signals generated by excitation of the sample in a very small detection micro-region.The photon burst counting method based on confocal optical configuration has the advantages of less consumption,high sensitivity and low detection limit,and has broad application prospects in the field of homogeneous analysis.The sensitivity of the photon burst counting method mainly depends on parameters such as the number of photon burst counts obtained per unit time,but lacks theoretical model for the photon burst counting method to systematically study these parameters.Based on the excellent resonance light scattering properties of Gold Nanoparticles(GNPs),this paper established a theoretical model for photon burst counting method using GNPs as probes.The effects of parameters such as laser intensity and probe size on the sensitivity of photon burst counting method were systematically studied.The theoretical model and method of photon burst counts based on objective scanning were proposed for high-sensitivity homogeneous analysis.This method can significantly improve the sensitivity of the conventional photon burst counting method.All the reasearch contents and results are shown as follows:1.Establishment of a theoretical model for photon burst counting method.The theoretical model of single-particle photon burst counting method was proposed.GNPs are used as optical probes.The GNPs were used as a model to systematically study the effects of particle size,detection time,viscosity,temperature and light intensity on photon burst counts.The experimental results verified the theoretical model.Guided by the theoretical model,the objective scanning technology was introduced into the photon burst counting method.Then the photon burst counting model based on objective scanning or directional flow was established,which further increased the number of photon burst counts of GNPs per unit time,and significantly improved the sensitivity of this method.We successful established a new method for single-particle photon burst counts with high sensitivity and low detection limit.With the introduction of the objective scanning technique,the sensitivity of this method was increased by 4 times.The detection range was 0.9 to 90 p M,and the detection limit was reduced from 200 f M to 40 f M.This new method has the advantages of high sensitivity,low detection limit,low sample consumption.2.Based on the constructed objective scanning photon burst counting method,a homogeneous analysis method for anti-ds DNA was established.This method firstly modified ds DNA to the surface of GNPs to form GNPs probes.Then the anti-ds DNA antibody was added to the GNPs probe solution.As the concentration of anti-ds DNA antibody increases,antibody induced GNPs probes aggregate to form complex,then the number of particles in sample solution decreased,resulting in a decrease in photon burst counts.By optimizing the experimental conditions,the linear range of this method was 10 to 100 n M and the detection limit was 5.0 n M under optimal conditions.Compared with the existing methods,this new method has the advantages of high sensitivity,specificity,good reproducibility,fast and simple,and has broad application prospects in clinical diagnosis and life sciences. |
