Application Of Chemometric Methods To The Fields Of Spectrometric Analysis And Optical-sensing

Due to its high sensitivity,fast analysis speed,simple operation,spectrometric methods and optical chemo/bio sensors have wide applications in life sciences,drug analysis,food safety,environmental monitoring and other fields.The spectrometric signals are generally affected by many factors other than the concetrations of the analytes of interest such as the phase state of the samples under test（such as solid-liquid coexistence,water and oil coexistence）,background interferences,matrix effects,light scattering,ionization efficiency,and the physical properties of the enhancing substrate,which causes the relationship between concentrations of the analytes of interest and the intensities of spectrometric signals to deviate significantly from a linear or simple non-linear model.Therefore,it is rather difficult to achieve accurate quantification of the analytes of interest in a complex system by the use of simple calibration methods.This thesis attempts to use the spectral shape deformation quantitative theory（SSD）to mitigate the detrimental effects of light scattering,the physical prop erties of the enhancing substrate and ionization efficiency on the quantitative results of spectrometric methods and optical chemo/bio sensors,develop a novel calibration strategy based on background correction to solve the problem of background interferences encountered in quantitative analysis of complex samples using optical chemo/bio sensors,and design a generalized standard addition method through the combination of the calibration strategy based on background correction and standard addition method for accurate quantification of the anlytes of interest in samples with severe matrix effects.The details are as follows:1.The application of SSD to the filed of chemo/bio sensing based on fluorescence and Raman spectrscopies（Chapters 2 to 4）Abnormal levels of ALP are closely related to many diseases.It is therefore is an important biomarker for clinical diagnosis.In Chapter 2,a ratiometric fluorescent sensing method based on Mn O₂ nanosheets was developed for ALP detection.The proposed method in combination with SSD achieved satisfactory quantitative results for ALP in real-world serum samples,with accuracy comparable to the corresponding results obtained by an automatic biochemical analyzer.Its recovery rates for the spiked serum samples were in the range of 98.4～115.0%.The limit of detection and limit of quantification were estimated to be 0.09 and 0.30 UL^-1,respectively.Compared with other fluorescent methods for ALP detection,the proposed method is more robust to interferences.Morevoer,the s ample volume used in the proposed method can be much less than the volume required by the automatic biochemical analyzer.Therefore,it is reasonable to expect that the proposed ratiometric fluorescent sensing method can be further developed to be a compet itive alternative for ALP detection.Cadmium ion(Cd²⁺)is one of the most toxic heavy metal ions.It can cause serious damage to human health even at extremely low daily doses.Rice produced in some areas of Southern China contain levels of Cd exceeding the Chinese food safety limit of 0.2 mg·kg^-1 for rice grain which poses a serious threat to human health.In Chapter 3,a simple but effective SERS method for the quantification of Cd²⁺in rice was developed by integrating trithiocyanuric acid（TMT）functio nalized gold nanoparticles（TMT-Au NPs）,a conical holed substrate,and SSD together.With the aid of potassium ethyl xanthate（PEX）as a masking agent for Pb²⁺,TMT-Au NPs can selectively detect Cd²⁺in rice samples.The extra SERS signal enhancement by the conical holed glass substrate can to some extend ease the sensitivity problem caused by the use of TMT-Au NPs as a ratiometric SERS probe for Cd²⁺.SSD can correct the confounding effects caused by variations in the number and distribution of"hot spots"on or close to the surfaces of Au NPs.The above three parts complemented each other to realize accurate and precise SERS quantification of Cd²⁺in rice.The quantitative results of the proposed method for the rice samples were consistent to the corresponding values determined by the reference method ICP-MS.Its recovery rates for Cd²⁺in the rice samples spiked with different amounts of Cd²⁺were in the range of93.8 to 109.4%.Its limit of detection（LOD）and limit of quantification（LOQ）were estimated to be 8 and 24μg·kg^-1,respectively,completely satisfying the requirements for the detection of Cd²⁺in rice.Therefore,the proposed method has the potential to become a fast screening method for the detection of Cd²⁺in rice.6-thioguanine（6-TG）is a conventional medicine for the treatment of leukemia and other cancers.Its safe dose range is relatively narrow.Thus,the use of 6-TG needs to be strictly controlled.In Chapter 4,a core-shell SERS enhanced substrate was synthesized and combined with SSD for the determination of 6-TG in serum and cell lysate.The quantitative results of the proposed method were consistent with the corresponding results obtained by high performance liquid chromatography（HPLC）.Its average recovery rate was within the range of 95.1～107.0%.The limit of detection and limit of quantification were estimated to be about 1 and 3 n M,respectively,which were better than the corresponding values of the HPLC method.The proposed method has some advatages over other SERS based methods for 6-TG detection.It can achieve accurate and sensitive quantitative analysis of 6-TG in complex biological samples,thanks to the capability of SSD in mitigating the detrimental influence on SERS signals caused by the variations in the physical propert ies of enhancing substrate,and the inherent merit of the internal standard embedded core-shell SERS enhancing substrate in avoiding the competitive adsorption between the internal standard and the target analyte on the surfaces of the SERS enhancing subst rate.2.The application of SSD to quantitative mass spectrometric analysis（Chapter 5）The base excision repair is very important to maintain the integrity of the genome.Uracil-DNA glycosylase（UDG）is one of the essential enzymes involved in this process.Its abnormal expression is closely related to many diseases.So it is necessary to quantify UDG activity in clinical diagnosis.In Chapter 5,a mass spectrometric method for UDG detection was proposed based on strand displacement amplification and SSD.Its working mechanism is as follows:UDG can hydrolyze uracil bases in ds DNA to form a apurinic/apyrimidinic（AP）site which breaks to form sticky ends in the presence of endonuclease IV.A large number of a deliberately designed ss DNA fragment can be produced through strand displacement amplification reaction.Quantification of UDG activity can then be indirectly realized through the detection of the deliberately designed ss DNA fragment using mass spectrometry.SSD can correct the influence of factors other than the concentraitons of the analytes of interest on the intensities of mass spectrometric signals.The strand displacement amplification reaction is employed to increase the detection sensitivity.The proposed method was successfully applied to the quant ification of UDG activity in cell lysates of Hela and MCF-7 cell lines.Its average recovery rate was in the range of94.1～111.3%.The limit of detection and limit of quantification were estimated to be4×10^-4 and 1.3×10^-4U·L^-1,respectively,by far better than other mass spectrometric methods for UDG detection.3.The application of background interference correction strategy and generalized multivariate standard addition to the filed of chemo/bio sensing based on fluorescence and circular dichroic spectroscopies（Chapters 6 and 7）In Chapter 6,a probe-technique based background interference correction strategy was proposed to mitigate the influence of spectroscopically active interferences on the quantitative results of optical chemo/bio-sensors for complex samples.The principle of the proposed method is briefly described as follows:First of all,the background spectrum of a test sample can be mureasured before the addition of the molecular probe for the anlyte of interest.The spectra of both the stan dard samples and the test sample measured after the addition of the molecular probe were then projected onto an orthogonal complement to the space spanned by the background spectrum of the test sample.After projection,the transformed spectra of both the standard samples and the test sample contain no contribution from the background interferences.The calibration model built on the transformed spectra of the standard samples can then be used to accurately predict the concentration of the analyte of interest from the transformed spectrum of the test sample.The proposed method in combination with Pb²⁺probe（K⁺-stabilized PS2.M）was successfully applied to the quantificaiton of Pb²⁺in environmental samples using circular dichroism spectroscopy.Its quantitative results were in good consistence with those of a reference method based on a flame atomic absorption spectrometry,and significantly better than the corresponding results obtained by the calibration model built on the raw spectral data of the standard samples.Though the classical standard addition method can effectively correct the influence of matrix effects on quantitative results of spectrometric analysis,it cannot eliminate the systematic predictive errors caused by the presence of background interferences in test samples.Nevertheless,matrix effects and background interferences are generally coexistent in real-world samples.In Chapter 7,a generalized multivariate standard addition strategy was developed by the integration of the probe technique-based background interference correction strategy and standard addition method,and was applied to quantitative analysis of Al³⁺in samples with significant matrix effects.Differing from convetional standard addition methods,the calibration model of the generalized multivariate standard addition strategy is built on the transformed spectra of the standard samples.The transformed spectra of the standard addition samples are used to determine the optimal value of the model parameter.Theretically,one standard addition sample was enough to make the generalized multivariate standard addition strategy work properly,which is a valuable and desired feature in quantitative analysis of small-volume biological samples or some precious samples using invasive techniques.With the use of only one standard addition sample,the proposed method had provided satisfactory quantitative results for Al³⁺in the digested solutions of food samples.