Investigation Of Single-particle Quantitative Analysis And Applications By Darkfield Tracking Microscopy

Due to the unique phenomenon of localized surface plasmon resonance,gold nanoparticles have been widely used as high-performance optical probes in the field of biosensing and chemical analysis.Compared with traditional ensemble methods for bulk measurements,single-particle analysis techniques using gold nanoparticles as probes often exhibited higher resolution and sensitivity for the applications in biochemical detection and diagnostic analysis.These techniques enabled the detection of changes in spectrum(or color),intensity,number as well as optical anisotropy.With these techniques,ultrasensitive detection of gasotransmitter,disease-related molecules such as protein and DNA have been achieved on the glass substrate by simply analyzing target-induced signal responses of individual particles.However,these techniques would suffer from several drawbacks such as unsatisfactory accuracy and precision,because nonspecific adsorption and stochastic clustering of particles on substrate could produce uncontrollable false signals for those paradigms of detecting single particles on the liquid/solid interface.To address this problem,it is necessary to develop singleparticle analysis techniques that enable quantitative detection of free moving particles in homogenous solution without the loss of sensitivity and accuracy.Among the mostcommonly used single-particle technologies,single-particle tracking enables the detection of diffusion behaviors and dynamics of individual particles by analyzing particle movement under optical microscope in complex systems.However,quantitative detection strategy method based on single-particle tracking technology has not been developed till now.In this thesis,we established a quantitative detection method based on single-particle mobility under dark-field tracking microscopy,and applied the application for the detection of cancer markers inhomogeneous solution in a ratiometric manner.The detailed works are as follows:In Chapter 2,by taking advantage of the correlation of hydrodynamic effects with motion behavior of nanoparticles,we developed a quantitative detection strategy based on dark-field tracking microscopy and examined the figure of merits for this technique including the capabilities of resolution and anti-interference.Firstly,we used the single-particle tracking technique for characterizing the hydrodynamic sizes of five types of gold nanoparticles,in order to evaluate their ability to distinguish the sizes of nanoparticles.The results indicated that two types of nanoparticles could be distinguished by the single-particle tracking technology when their size difference is as small as ～ 5 nm.Then we demonstrated the capability of this technique for discriminating the changes in hydrodynamic sizes of gold nanoparticles before and after protein adsorption.Moreover,this method is also competent for detecting the changes in hydrodynamic size caused by the particle aggregation as well as the content of aggregates and monomers in solution from single-particle mobility analysis.Therefore,combining with the strategy of target-induced probes aggregation,the single-particle tracking technology could be used to achieve the quantitative detection of targets,which established the foundation to develop single-particle quantitative detection methods suitable for homogeneous solution systems.In Chapter 3,gold nanoparticles modified with capture antibody and detection antibody of carcinoembryonic antigen(CEA)were used as probes(Anti-CEA@Au NPs),combining with sandwich immunoassay strategy,to explore the quantitative detection capability of dark-field single-particle tracking.Since the addition of CEA with different concentrations would cause different degrees of aggregation of the probes,the change of the aggregate-to-monomer ratio in a system can be obtained by analyzing the average velocity distribution of probes at different CEA concentrations.According to the relationship between the aggregate-to-monomer ratio and the concentration of CEA,the quantitative detection of CEA can be achieved with a linear range from 50 to 750 p M and the limit of detection is 50 p M,which could meet the requirements of clinical detection of CEA.Meantime,the method also displays good selectivity and anti-interference ability,which could be used for the CEA detection in serum samples.Due to the limitations of camera detection performance,the method needs to disperse the reacted probes in a high-viscosity solvent for imaging,which limits its application to some extent.In order to solve this problem,we tried to introduce a restricted liquid film model to explore the restricted movement of gold nanoparticles preliminarily through dark-field imaging technology,which provided a reference for developing the quantitative detection methods based on single-particle mobility analysis.