Research On Chemometric Theory And Application To Fluorescence-based Chem/Bio Sensing Technologies

Due to the advantages of high sensitivity, low consumption, cheap equipment, simple operation, and the capability of real-time non-destructive analysis of complex systems, fluorescence spectroscopy is among the most widely used methods in the field of food science, biological chemistry and clinical analysis, etc. However, direct fluorescence analysis is applicable only to fluorescence substances which are relatively few compared to non-fluorescence substances. The analysis of non-fluorescence substances using fluorescence spectroscopy involves the fluorescent probes, which can be roughly classified into three categories, i.e., intensity-based fluorescent probes, ratiometric fluorescent probes and generalized ratiometric fluorescent probes. Ratiometric fluorescent probes and generalized ratiometric fluorescent probes are more popular than intensity-based fluorescent probes due to their higher robustness to variations in probe loading, bleaching, illumination intensity, and optical path length, etc. conventional univariate ratiometric model based on the ratio between two fluorescence intensities at two emission or excitation wavelengths is generally adopted in quantitative fluorescence analysis using ratiometric fluorescent probes or generalized ratiometric fluorescent probes. However, co nventional univariate ratiometric model is applicable only to the quantitative determination of analytes of interest in simple homogeneous systems without interfering fluorescence substances. When it is applied to complex heterogeneous systems, the accuracy of the quantitative results would be seriously degraded by the presence of scatterers, absorbers and interfering fluorescence substances. This dissertation aims to address this issue by developing novel quantitative model for fluorescence analysis of complex heterogeneous systems using either ratiometric fluorescent probes or generalized ratiometric fluorescent probes. The details are as follows :1. Quantitative determination of free Mg²⁺ in turbid media by the combination of ratiometric probe with a novel quantitative fluorescence model（Chapter 2）Magnesium ion(Mg²⁺) is an essential inorganic mineral nutrient for life and presents in every cell type in almost every organism, it is also a cofactor in more than 300 enzyme systems that participate in diverse biochemical reactions. The quantification of Mg²⁺ is therefore rather important. Fluorescent spectrometry coupled with fluorescent Mg²⁺ indictors is one of the most popular techniques for the quantification of Mg²⁺ in complex systems such as tissues and cells. However, many ratiometric fluorescent indicators designed for the detection of Mg²⁺（such as Mag-Indo-1） can also chelate Ca²⁺ with an even lower dissociation constant, which results in the interference of Ca²⁺ during the quantification of Mg²⁺ in samples containing both Ca²⁺ and Mg²⁺. To address this problem, a novel quantitative fluorescence model was proposed. The proposed quantitative fluorescence model in combination with fluorescence probe Mag-Indo-1 was applied to the quantification of free Mg²⁺ in turbid samples with coexistence of interfering Ca²⁺. Experimental results showed that the proposed quantitative fluorescence model could effectively eliminate the interference of coexisting Ca²⁺. The concentrations of free Mg²⁺ in turbid media can be determined by the proposed quantitative fluorescence model with an average relative predictive error value of about 6.0%. The results achieved in this contribution fully demonstrated that the application of advanced calibration model could realize accurate quantification of the ion of interest in the presence of similar interfering ions in turbid media using a ratiometric fluorescent indicator without a pronounced selectivity for the ion of interest.2. Quantitative determination of analytes of interest in turbid media through combining generalized ratiometric probes with a novel quantitative fluorescence model（Chapter 3 and Chapter 4）Although ratiometric fluorescence probes possess many advantages over intensity-based fluorescence probes, the complex in both design and synthesis mechanism and the complicated synthesis process of ratiometric fluorescence probes hinder their wider applications in practice. In chapter 3, under the guidance of the novel quantitative fluorescence model, a generalized ratiometric fluorescence probe was constructed by combining an intensity-based fluorescence probe（indole） with an inert fluorescence dye（vitamin B6）, and applied to the quantification of NO2- in simulated and real-world turbid media. Experimental results showed that the combination of the generalized ratiometric fluorescence probe and the novel quantitative fluorescence model could realize sensitive and accurate quantitative analysis of NO2- in turbid media with an average relative predictive error of about 4.5%, limit of detection of 1.3 ng/m L, and limit of quantification of 3.9 ng/m L. The proposed method has the advantages of simplicity, fast measurement speed, and no need for sample preprocessing, it is therefore especially suitable for direct analysis of the analytes of interest in turbid media.In chapter 4, the concept of generalized ratiometric fluorescence probes was further developed and applied to direct quantitative determination of epinephrine（EPI） in plasma samples by utilizing the reaction between E PI and o-phenylenediamine and an inert fluorescence dye. Experimental results showed that the proposed method achieved accurate quantitative analysis of EPI in plasma samples without any pretreatment. The average relative predictive error was less than 8%, comparable to the results obtained by LC-MS/MS. The presence of protein and other substances in plasma samples seemed have no significant influence on the quantitative results of the proposed method.3. Quantitative determination of the analytes of interest in turbid media by the combination of a novel quantitative fluorescence model with generalized ratiometric fluorescence nanoprobes based on PEBBLEs（Chapter 5 and Chapter 6）The combination of intensity-based fluorescence probes with inert fluorescence dyes to construct generalized ratiometric fluorescence probes can to some extent expand the application range of intensity-based fluorescence probe. The concept of generalized ratiometric fluorescence probes constructed by simultaneously adding two fluorescence dyes into the samples to be tested is too simple to be applied to complex systems such as cells and biological tissues, because of the difficulty in keeping the ratio between the intensity-based fluorescence probe and the inert fluorescence dye constant in cells and biological tissues. Another type of generalized ratiometric fluorescence probes（i.e. probes encapsulated by biologically localized embedding） is to encapsulate an intensity-based fluorescence indicator and an inert reference fluorescence dye in the pores of stable nano-materials. The generalized ratiometric fluorescence nanoprobes based PEBBLE techniq ue are applicable to complex systems such as cells and biological tissues since the ratios between intensity-based probes and inert reference dyes are constant even if the amounts of generalized ratiometric fluorescence nanoprobes loaded into cells/tissues vary. During quantitative analysis using generalized ratiometric fluorescence nanoprobes based PEBBLE technique, univariate ratiometric models were generally adopted to build the calibration models. However, conventional univariate ratiometric models are not suitable for qu antitative analysis of complex heterogeneous systems. In chapter 5, a generalized ratiometric Ca²⁺ fluorescence nanoprobe based on PEBBLE technique has been developed from fluorescence probe Rhod-2 and inert fluorescence dye eosin B, and combined with a novel quantitative fluorescence model to quantify free Ca²⁺ in simulated and real-world turbid media. Experimental results demonstrated that the method proposed in this chapter obtained accurate quantitative results for free Ca²⁺ in turbid samples, for real-world turbid samples, the average relative predictive error obtained from novel quantitative fluorescence model results is about 2.7%, and recovery rates in the range of 93.1%¹01%, which were on a par with those of atomic absorption spectrometry.The stringent requirements for the selection of encapsulated reference dye in the synthesis and application process of PEBBLEs makes the selection of reference dye very difficult, thus further limits the extensive utilization of PEBBLEs in quantitative analysis, while in subsequent experiments, we found that carbon quantum dots（C-dots） have many advantages such as good water-solubility, stability of fluorescence signals, high fluorescence quantum yields, adjustable excitation and emission wavelength, good dispersibility, low toxicity and good biocompatibility, etc. Thus, in chapter 6, a generalized ratiometric fluorescence nanoprobe for free Ca²⁺ was designed and fabricated by encapsulating Rhod-2 and C-dots inside stable polyacrylamide PEBBLEs nano-materials. In combination with the novel quantitative fluorescence model, the generalized ratiometric fluorescence nanoprobe developed in this chapter was also applied to the quantitative determination of free Ca²⁺ in turbid samples. Experimental results revealed that the quantitative results of the proposed met hod for free Ca²⁺ in turbid samples were rather accurate with an average relative predictive error of less than 4%, comparable to the accuracy and precision of atomic absorption spectrometry.