Research On The Extension And Application Of Spectral Shape Deformation Quantitative Theory To The Analysis Of Complex Systems

During quantitative analysis of samples using instrumental techniques,it is generally assumed that the relationship between concentrations of the analytes of interest and corresponding absolute signal intensities follows a linear or simple non-linear model.However,the signal intentities of real-world complex samples depend on not only the concentrations of the analytes of interest but also some uncontrolled samples' physical properties or experimental conditions,which undermines the commonly adopted assumption of linear or simple non-linear relationship.For example,ion suppression,the gradual fouling of the ion source,and aging of the ion multiplier might lead to changes in the overall sensitivity and signal stability of mass spectrometry and compromise the accuracy and precision of quantitative mass spectrometry.In addition,nuclease assisted recycling amplification strategies can be used to enhance the sensitivity of mass spectrometry for the detection of deoxyribonucleic acid(DNA)and ribonucleic acid(RNA).However,the relationship between the mass spectral signal intensity and the concentration of the analyte of interest(i.e.,DNA or RNA)is rather complicated,and cannot be quantitatively modeled by commonly used methods.How to realize sensitive and accurate quantitative analysis of biomolecules such as DNA and RNA in complex biological samples by the combination of nuclease assisted recycling amplification strategies and mass spectrometry is a relatively new topic in the field of mass spectrometric analysis.As another example,the surface enhanced Raman spectroscopic(SERS)signal of a sample is not only related to the concentration of the analyte of interest in the sample but also links to the microstructural properties of SERS enhancing substrates.The difficulty in preparing highly reproducible SERS enhancing substrates in terms of the number and distribution of "hot spots" renders SERS still to be a qualitative or semi-quantitative detection technique at the present stage.How to improve the accuracy and precsion of SERS quantative results is a bottleneck problem in the further development of SERS technique.This thesis attempts to use the spectral shape deformation quantitative theory(SSD)to address the above mentioned issues in quantitative mass spectrometry and quantitatvive SERS assays.The details are as follows.1.The application of SSD to quantitative mass spectrometry(Chapter 2 to Chapter 5)Hormones may be added into cosmetic products to perform specific functions.The addition of hormones in cosmetics has potential hazards on human health.The determination of hormones in cosmetics is therefore of great importance to ensure the quality of cosmetics and safe use of cosmetics.In Chapter 2,tandem mass spectrometry coupled with liquid chromatography(LC-MS/MS)was combined with the spectral shape deformation quantitative theory to quantitatively determine three hormones(i.e.estrone,hydrocortisone and progesterone)in cosmetics(toner and essence emulsion).The adoption of the spectral shape deformation quantitative theory in the analysis of LC-MS/MS data was to address the problem of sensitivity and signal instability of LC-MS/MS over time caused by the gradual fouling of the ion source,vacuum instability,and ion suppression,etc.Experimental results showed that the spectral shape deformation quantitative theory could effectively mitigate the detrimental effects of signal instability of LC-MS/MS over time,and achieved quite accurate quantitative results for estrone,hydrocortisone,and progesterone in both toner and essence emulsion samples with average relative prediction error values no larger than 10.0%.Spectroscopic-based exonuclease III(Exo-III)assisted target recycling amplification techniques have exquisite sensitivity for DNA detection.Unfortunately,they are only capable of equivocally discriminating the perfect complementary target DNA sequences and the base mismatched DNA sequences based on the differences in signal intensities.As a result,they are liable to produce false positive results.Theoretically,the use of mass spectrometry instead of absorption or emission spectroscopy as the tool for signal detection can avoid false positive results to a large extend,since mass spectrometry can detect the DNA fragments produced during the Exo-III assisted target recycling amplification process.However,the distribution pattern of DNA fragments produced by Exo-III assisted target recycling amplification is generally different for samples with different concentrations of the target DNA sequence,which hinders the extraction of both qualitative and quantitative information of the target DNA from mass spectral measurements using traditional univariate or multivariate models.In Chapter 3,an advanced model was derived based on SSD theory for the qualitative and quantitative analysis of the mass spectral measurements of DNA fragments produced by Exo-III assisted target recycling amplification.Experimental results demonstrated that the integration of Exo-III assisted target recycling amplification,mass spectrometry and the advanced model could achieve sensitive and accurate quantitative results for a target short DNA sequence in complex biological medium.More interestingly,the proposed model could unambiguously identify single nucleotide polymorphisms based on the distribution patterns of residual DNA fragments.Therefore,with the aid of the proposed model,mass spectrometry based on Exo-III assisted recycling amplification has great potential for sensitive,selective,and relatively low-cost detection and quantification of short DNA sequences in clinical diagnosis and biomedical research.MicroRNAs(miRNAs)are a group of naturally occurring non-protein-coding endogenous small RNA that can regulate the expression of gene.They are highly tissue-specific biomarkers in clinical diagnosis and prognosis and exhibit abnormal expression levels in cancer cells.Therefore,effective methods for sensitive and reliable detection of miRNAs are of great importance to clinical diagnosis of cancer.In Chapter 4,a mass spectrometric platform for label free and sensitive detection of miRNAs was developed by integrating reverse transcription reactions of miRNAs and Exo-III assisted signal amplification strategy with the advanced calibration model designed for the analyisis of complex mass spectral data produced in Exo-III assisted signal amplification in Chapter 3.Experimental results demonstrated that the proposed platform could achieve accurate quantitative analysis of miRNA-141 in total RNA extracts from Hela,MDA-MB231 and 22RV1 cells.Its quantitative results were in good agreement with the corresponding results obtained by the conventional fluorescence real-time polymerase chain reaction(PCR)assays.The mean recovery rates of its quantitative results were within the range of 95.0~108.6%.The limit of detection(LOD)and the limit of quantification(LOQ)were estimated to be 1.1 and 3.4 pM,respectively.Since the reverse transcription reactions of miRNAs and hydrolysis mechanism of Exo-III for duplex DNA are all sequence-independent,the proposed mass spectrometric platform can be applied to the quantification of an arbitrary miRNAs.More importantly,the proposed mass spectrometric platform can unambiguously discriminate the target miRNAs from the non-target miRNAs with single nucleotide polymorphisms(SNPs)based on the differences in distribution patterns of residual DNA fragments produced in Exo-III assisted signal amplification.The proposed mass spectrometric platform has the potential to become a routine tool for analyizing miRNAs in complex biological samples.As important biomarker candidates for cancer screening and early detection research,miRNAs generally undergo synergistic adjustment in tumor and cancer cells.The occurrence of cancers is often associated with aberrant expression of multiple miRNAs.Therefore,methods capable of simultaneously,sensitively and selectively detecting multiple miRNAs are of great significance for early diagnosis and clinical research of cancers.In Chapter 5,the hydrolysis patterns of the duplex-specific nuclease(DSN)on different types of DNA/miRNA heteroduplex were explicitly investigated.Based on the information obtained during the experiments and SSD theory,a mass spectrometric method was proposed for label free and multiplexed detection of multiple miRNAs(i.e.,miRNA-141,miRNA-21 and let-7a).The proposed method effectively mitigated the ion suppression effects among multiply charged negative ions of multiple DNA fragments and possible background interferences in complex biological samples,and realized accurate and reliable simutaneous quantifcation of miRNA-141,miRNA-21 and let-7a in samples of Hela and MDA-MB231 cell extracts with recovery rates within the range of 89.2~111.6%.It is reasonable to expect that proposed mass spectrometric method can be a competitive alternative for label free and multiplexed detection of multiple miRNAs.2.The application of SSD to quantitative SERS spectroscopy(Chapter 6)Dopamine(DA)is one of the most important neurotransmitters for its role in modulating and regulating of the central neural and cardiovascular system.In Chapter 6,silver nanoparticles modified with iron-nitrilotriacetic acid were prepared and combined with SSD to quantify DA in complex biological samples by SERS technique.Iron-nitrilotriacetic acid was served as a selective SERS proble for DA.And an advanced calibration model based on SSD was derived to effectively mitigate the detrimental effects on the quantitative results of SERS technique caused by the variations in the physical properties of SERS enhancing substrate s.Experimental results demonstrated that the proposed method achieved quite accurate and precise quantification for DA in complex biological samples such as plasma and urine with recovery rates varying within the range of 91.9~112.0%,surprisingly better than the corresponding values of the quantitative results obtained by LC-MS/MS.