New Cross-correlation Spectroscopy Methods And Its Applications Based On Nano Optical Probes

Fluorescence cross correlation spectroscopy(FCCS)is a single molecule optical technology based on fluorescence correlation spectroscopy(FCS).It is a highly sensitive and selective detection technique by the introduction of dual-labelled molecules.FCCS has been applied to study the molecule interaction and homogeneous bioassay.Generally,FCCS needs two different colour lasers,and the alignment of optical configuration is more difficult when compared to conventional FCS with one laser.And two lasers increase the cost of research.In this dissertation,we built the single wavelength excitation fluorescence cross-correlation spectroscopy(SW-FCCS)setup by using quantum dots(QDs)and fluorescent dyes as labelling probes.Then,we for the first time established scattering light and fluorescence cross-correlation spectroscopy(SFCCS)based on FCCS theoretical model and setup by using fluorescent probes and resonance light scattering probes.On the basis of the development of the FCCS setup,the selection and optimization of optical probes,we develop highly sensitive homogeneous immunoassays for cancer biomarkers.The main work is included the following parts.1.We systematically investigated the conjugation of QDs with certain biomolecules using capillary electrophoresis(CE)and FCS methods,and also investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography(SEC)technique,fluorescence spectroscopy and FCS.In bioconjugation procedures,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-hydroxysulfo-succinimide(Sulfo-NHS)were used as linking reagents.We studied the effects of certain factors such as the isoelectric points(pI)of bio-macromolecules and buffer pH on the bioconjugation of QDs,and found that the pIs of bio-macromolecules played an important role in the conjugation reaction.By the optimization of the buffer pH,some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo-NHS as linking agents.Furthermore,we also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer.In purification procedures,we systematically studied the effects of certain factors such as the scales of column,loading volume,elution buffer and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device.And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions.It laid a foundation for the further research.2.We developed a sensitive and rapid method for homogeneous immunoassay of biomarker in human sera by using SW-FCCS.In homogeneous immunoassays,competitive and sandwich immunoreaction modes were used and liver cancer biomarker alpha-fetoprotein(AFP)was used as an assay model.Two colour fluorescent probes,QDs and fluorescent dye,were chosen to label two different antibodies in sandwich mode,and antibodies and antigens in competitive mode.The effects of the incubation temperature,the immune reaction time,the stability of immunocomplexes,the concentration of fluorescent labelled bioconjugates and serum autofluorescence on the homogeneous immunoassay were systematically investigated.Furthermore,an aggregate exclusion method was used to exclude bright signals from large aggregates,and the credible and steady data can be obtained.In optimal conditions,the linear ranges of this method are from 20 pM to 5.0 nM in sandwich mode and from 180 pM to 15.0 nM in competitive mode;and the detection limits are 20 pM and 180 pM in sandwich mode and competitive mode,while ELISA method,with the same pair of antibodies used in our study,has a detection limit of 72 pM.This new method was successfully applied to directly determine AFP level from clinical samples,and our results were basically in agreement with ELISA.3.We for the first time established SFCCS based on the principle of FCCS,using fluorescent probes and resonance light scattering probes.We investigated the influence of separation distance and silver dimers on fluorescence enhancement of fluorescent dyes by sliver nanoparticles(SNPs),and developed SFCCS-based homogeneous immunoassays for the liver cancer biomarker AFP.In optimal conditions,the linear range of competitive immunoassay was from 116 pM to 3.0 nM,and the detection limit was 116 pM;the linear range of sandwich competitive immunoassay was from 23 pM to 15.0 nM,and the detection limit was 23 pM;the linear range of sandwich immunoassay was from 930 fM to 580 pM,and the detection limit was 930 fM.While ELISA method,with the same company used in our study,has a detection limit of 26 pM.This new method was successfully applied to directly determine AFP level from clinical samples,and our results were basically in agreement with ELISA.This method had high sensitivity,good reproducibility and short analysis time.SFCCS could simultaneously detect the resonance light scattering signals and fluorescence signals and analyze their cross-correlation information in hybrid systems,such as cell membranes and live cells.Therefore,SFCCS pave the way to achieve higher resolution and to detect higher order molecular interactions in biological systems.