Novel Spectral Quantitative Models And Their Applications In Complex Multiphase Systems

Because of its high sensitivity, simple implementation as well as relatively cheap equipment compared to the mass spectrometer, polarograph and electron microprobe, fluorescence spectroscopy is a widespread and popular analysis method. Fluorescence spectroscopy has been widely applied and played an important role in fields of pharmaceutical analysis, clinical examination, environmental monitoring, food security, biochemistry, etc. The underlying assumption of using fluorescence spectroscopy to do quantitative analysis is that the relationship between the fluorescence intensity of the sample under test and the concentration of the target fluorophore in the sample follows a simple linear model. Such an assumption only holds for homogeneous aqueous systems. However, when the samples under study are heterogeneous systems such as turbid media, biological tissues and cell suspensions, the presence of practically unavoidable scatterers and background absorbers in such samples causes so-called scattering effects and background absorption effects, which can significantly distort the shape and intensity of fluorescence spectra of fluorophores, invalid the generally assumed linear model, and hence greatly complicate the in-situ quantitative measurement of fluorophores in complex multiphase systems. It is therefore highly desired to develop a simple but effective method to eliminate the effects of scattering and absorption on the fluorescence spectra of fluorophores and realize accurate quantitative analysis of fluorophores in complex multiphase systems.Surface-enhanced Raman spectroscopy（SERS） has many advantages, such as exquisite sensitivity, excellent molecular specificity, less water interference and reduced photo-bleaching. It is a powerful technique for the investigation of molecular structure, interface properties, molecular interaction, surface adsorption behavior of moleculars, etc, and has been widely applied to many areas such as analytical chemistry, surface science as well as biological science. The molecule of interest must be adsorbed onto a suitable roughened metal surface of substrate to achieve SERS. Therefore, the SERS effect relies heavily on the preparation of enhancing substrates. The SERS signal of analyte molecules depends on not only the analyte concentration but also the physical property of enhancing substrate. The difficulty in producing highly stable and reproducible SERS enhancing substrates renders SERS still to be a qualitative or semi-quantitative detection technique at the present stage. It is therefore imperative to develop new strategy/technique to separate the SERS signal contributions induced by changes in analytes’ concentrations from those caused by variations in physical properties of SERS substrate, and hence realize quantitative SERS analysis of complex systems.This dissertation aims to solve the problems mentioned above through developing novel fluorescence and SERS quantitative techniques for complex multiphase systems. The details are as follows.1. Novel fluorescence quantitative technique for complex multiphase systems（Chapter 2）The presence of practically unavoidable scatterers and background absorbers in turbid media such as biological tissue or cell suspensions can significantly distort the shape and intensity of fluorescence spectra of fluorophores, and hence greatly hinder the in-situ quantitative determination of fluorophores in turbid media. In this chapter, a quantitative fluorescence model（QFM） was proposed to explicitly model the effects of the scattering and absorption on fluorescence measurements when the scattering and absorption properties of calibration samples are reasonably close to the sample properties. Based on t he proposed model, a calibration strategy was developed to remove the detrimental effects of scattering and absorption, and hence realize accurate quantitative analysis of fluorophores in turbid media. A proof-of-concept model system—the determination of free Ca²⁺ in turbid media using Fura-2 was utilized to evaluate the performance of the proposed method. Experimental results showed that QFM can provide quite precise concentration predictions for free Ca²⁺ in turbid media, probably the best results ever achieved for turbid media without the use of advanced optical technologies. QFM has not only good performance but also simplicity of implementation. It can be developed and extended in many application areas such as ratiometric fluorescent sensors for quantitative live cell imaging.2. Quantitative SERS technique and its applications in environmental and biomedical fields（Chapter 3 to Chapter 6）The concept of generalized ratiometric indictor based on surface-enhanced Raman spectroscopy was first introduced and successfully implemented in the detection of Cd²⁺ in environmental water samples using Au nanoparticles（Au NPs） modified by trithiocyanuric acid（TMT）（Chapter 3）. Without the use of any internal standard, the proposed method achieved accurate concentration predictions for Cd²⁺ in environmental water samples with recoveries in the ranges of 91.8¹08.1%, comparable to the corresponding values obtained by atomic absorption spectroscopy. The limit of detection and limit of quantification were estimated to be 2.9 and 8.7 n M, respectively. More importantly, other species presented in water samples which can not react with TMT and has weaker binding ability to Au NPs than TMT, do not interfere with the quantification of Cd²⁺. Therefore, it is expected that the combination of the generalized ratiometric indictor based surface-enhanced Raman spectroscopy with the proposed Au NPs-TMT probing system can be a competitive alternative for the primary screening of Cd²⁺ pollution.To overcome the disadvantages of conventionally used dry and wet SERS detection methods, surface-enhanced Raman spectroscopy（SERS） based on conical holed enhancing substrates was proposed（Chapter 4）. Compared with silver colloids deposited plane glass substrates（dry method）, the silver colloids deposited conical holed glass substrate with the optimal diameter and depth specifications exhibited ten-folds of increase in the signal enhancing rate, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of the optimal conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte（s） of interest（wet method）. The optimal conical holed glass substrate in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy(MEM_SERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4.1% and a limit of detection of about 15.3 n M using a portable i-Raman 785 H spectrometer. Other chemical species presented in plasma samples did not seem to interfere with the quantification of 6-mercaptopurine. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEM_SERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc.Ametryn is a selective triazine herbicide belonging to the s-triazine family. Because of its toxicity, persistence and accumulation in the environment as well as its effects on the environment and human health, it is listed as a chemical pollutant that needs to be monitored. Surface-enhanced Raman spectroscopy（SERS） coupled with an advanced chemometric model proposed by some of the present authors—multiplicative effects model(MEM_SERS) was applied to quantitative analysis of ametryn in water samples of the Xiangjiang River（Changsha, China）（Chapter 5）. The adoption of MEM_SERS model was to eliminate the detrimental effects caused by variations in the physical property of enhancing substrate, the intensity and alignment/focusing of laser excitation source. Experimental results showed that the combination of SERS with MEM_SERS can provide quite precise concentration predictions for ametryn in water samples of the Xiangjiang River with an average relative prediction error of about 4.8%. The combination of SERS with MEM_SERS can compete with LC-MS/MS in terms of precision and accuracy of quantitative results. The limit of quantification was about 90 n M. More importantly, no laborious reference methods（e.g. HPLC） were needed to build the MEM_SERS calibration model, since the MEM_SERS calibration model built on the calibration samples prepared with ultrapure water could provide satisfactory quantification results for the test samples prepared with water collected from Xiangjiang River. Therefore, it is reasonable to expect that SERS in combination with MEM_SERS model would become a competitive alternative in routine quantitative analysis of ametryn in environmental water samples.In order to further promote the application of MEM_SERS model, SERS technique in combination with MEM_SERS was applied to the quantification of methimazole in three kinds of plasma and tablet samples（Chapter 6）. Experimental results showed that the combination of SERS technique with MEM_SERS model could effectively mitigate the detrimental multiplicative effects caused by the heterogeneity in the physical properties of SERS enhancing substrates on the SERS signals of complex samples, and hence realized the accurate quantitative determination of methimazole in plasma and tablet samples. The average relative predictive error of MEM_SERS for the concentration of methimazole in three kinds of plasma samples were less than 10%, the limit of detection was 32.1 n M. The recovery rates of MEM_SERS for the concentrations of methimazole in tablet samples were in the range of 93.3¹10.9%, comparable to the corresponding values of LC-MS/MS experiments. The quantification method for methimazole developed in the present contribution has the advantages of simplicity, high sensitivity and accuracy. It may hold great potential to be expected as a promising alternative for quantitative analysis of methimazole in biomedical samples.