Fluorescence Quantitative Analysis Technology Based On Probe Technology And Advanced Calibration Theory

Fluorescence spectroscopic technique has feartures of high sensitivity,simple operation,low cost and low reagent consumption,and has been widely applied to various fields.However,the existence of matrix effects and background interference effects will greatly affect the accuracy of the quantitative results of fluorescence spectroscopic technique,and hence greatly hinder the application of fluorescence spectroscopic technique to quantitative analysis of complex real-world samples.In order to achieve accurate quantification of target analytes in complex real-world samples by fluorescence spectroscopic technique and widen its application scope,a novel multivariate calibration strategy based on probe technologies has been proposed in this thesis.Along with the quantitative fluorescence model for generalized ratiometric probes(QFM_GPR)proposed by our research group,the novel multivariate calibration strategy has been successfully applied to the quantification of target analytes in complex real-world samples.The details are as follows:In chapter 2,a probe technique-based generalized standard addition multivariate calibration strategy(GSAM_probe)was designed for the analysis of fluorescence signals with both matrix effects and background interference effects.The performance of the proposed strategy was tested on a proof-of-concept system of quantifying Zn²⁺in real-world samples such as rice and zinc gluconate oral solution using a selective fluorescence probe dansylaminoethyl-cyclen.Experimental results demonstrated that one standard addition sample was enough for the proposed strategy to provide satisfactory quantitative results for Zn²⁺in the real test samples,which were by far better than the corresponding results of the classical univariate standard addition method.The main contributions of this research are 1)to greatly improve the accuracy of quantitative results of optical chem/biosensing techniques,and 2)to open up an avenue for quantitative analysis of small-volume biological samples or some precious samples using invasive techniques in fields such as biomedical sciences and clinical diagnosis,etc.In chapter 3,a ratiometric fluorescent probe was constructed by exploiting the specific recognition of a modified thrombin aptamer?TBA?for thrombin in the presence of Mg²⁺.The quantification information of thrombin in serum can be extracted by GSAM_probe from the relative change in the fluorescence intensities of two fluorephores FAM and TAMRA modified on TBA caused by the presence of thrombin.Experimental results have shown that GSAM_probe is also applicable to quantitative analysis using ratiometric fluorescent probes,and can achieve accurate quantitative analysis of target analytes in complex real-world systems such as serum.In chapter 4,a generalized ratio metric fluorescence probe of"an inert fluorephore plus an intensity-based fluorescent probes"type was constructed based on an ATP aptamer,ThT and butyl rhodamine B.In combination with the QFM_GPR model,the newly designed generalized ratio metric fluorescence probe has been successfully applied to accurate quantitative analysis of ATP in both normal human cells and liver cancer cells.The results have demonstrated that the combination of fluorescence spectroscopic technique and the QFM_GPR model has great potential for ATP quantification in cells.