| Chemical toxicants derive from a wide variety of resources and varieties.Under the complex international circumstances,various chemical toxicant emergencies occur from time to time,such as paraquat intoxication,ricin intoxication,Nerve agent terrorist assassination,which poses a serious threat to national security,public security and people’s health.In view of such chemical toxicant hazard events,it is urgent to develop the analysis and detection techniques with strong structural confirmation of on-site rapid detection,screening confirmation and traceability,which are of great significance for on-site rescue,clinical diagnosis and treatment,forensic identification and anti-terrorism investigation and evidence collection.The analysis techniques with strong structural confirmation can usually provide rich,clear and sharp fingerprint information,mainly represented by Raman spectroscopy,mass spectrometry and NMR spectroscopy,etc.In recent years,with the rapid development of science and technology,a variety of advanced analytical techniques with simple pretreatment,fast,sensitive and accurate have emerged,for example,surface-enhanced Raman spectroscopy(SERS),matrix-assisted laser desorption ionization mass spectrometry imaging(MALDI-MSI),ambient ionization mass spectrometry and portable miniaturized mass spectrometry technique and related equipment,etc.SERS is a molecular vibration spectroscopy technique that can provide clear,sharp peak information and rich fingerprint information with the advantage of simple,fast and sensitive.It has produced good applications in the on-site rapid detection of toxicants such as paraquat,cyanide,mustard gas,and is known as "the next generation of new techniques for on-site detection of chemical toxicants".However,SERS is mostly used for qualitative or semi-quantitative detection.Due to the influence of unstable factors such as laser intensity fluctuations and differences in physical properties of the substrate,it is urgent to overcome problems such as poor quantitative analysis accuracy.MALDI-MSI is a non-destructive,rapid and high-throughput new molecular imaging technique that can simultaneously perform qualitative,quantitative and localization analysis.It has developed rapidly in the analysis of the spatial distribution of various biomolecules such as drugs,metabolites,peptides and proteins on surface or internal of tissues.It can simultaneously realize the spatial distribution and quantitative analysis of toxicant,drugs and their metabolites in tissues,and identify the distribution and accumulation process of toxicant and drugs in tissues and organs,which has become a powerful tool for pharmacological and toxicological research.In recent years,it has gradually been applied to plant research,showing a good application prospect in the identification of medicinal plants,distribution of metabolites,plant defense,plant seed traceability,etc.Therefore,this thesis is mainly aimed at the urgent need for rapid on-site detection and traceability of chemical toxicants,focusing on the development of sensitive,accurate and simple on-site rapid quantitative SERS method of paraquat in biological samples and toxicokinetic study of paraquat and castor beans tracing techniques based on MALDI-MSI towards the highly toxic pesticides paraquat and Ricinus communis biomarkers.It provides new technical methods for promoting the understanding of toxic mechanism and public safety-related events.The thesis was divided into five chapters.The first chapter is a preface.Firstly,the definition,classification,and threats to national and public security of chemical toxicants were briefly described.The physical and chemical properties,toxicity mechanisms,public safety hazards,and the status and characteristics of various toxicological analysis and detection techniques of paraquat and endogenous toxicants and components in castor beans were summarized one by one.Then,the characteristics and application progress of two representative advanced analytical and detection techniques that have developed rapidly in recent years,including SERS and MALDI-MSI,were described respectively.Finally,based on the above research background and current research status,the basis and main research contents of this thesis were proposed.In response to the urgent need for the rapid and quantitative determination of biological samples from paraquat poisoning,a rapid,simple,sensitive and accurate internal standard-surface enhanced Raman spectroscopy(IS-SERS)quantification method without sample pretreatment was developed to determine the concentration of paraquat in plasma and lung tissue in the second chapter.By using the structural analogue diquat as the internal standard,the problem of uneven "hot spots" of SERS was overcome and the accuracy of SERS quantification was improved.The determination of a single sample was fulfilled within 10 seconds via this method,and paraquat had a good linearity in the range of 1～500 μg/L(plasma sample)and 1～100μg/g(lung sample),with the limit of detection(LOD)and limit of quantitation(LOQ)of 0.5 μg/L and 0.1 μg/g(plasma sample),and 5 μg/L and 1 μg/g(lung sample),respectively.A complete methodological validation according to guidelines for biological sample analysis was perform and the results meet the standard requirements.It was further applied to the toxicokinetics on paraquat in mouse plasma and lung tissue.The results indicated that paraquat was absorbed quickly in mouse plasma and lung tissue,and eliminated slowly.It was prone to accumulate in target organs after entering the blood.The method has been successfully applied to the determination of a clinical intoxication sample.Through the method,it can also be revealed that common biological sample pretreatment methods,such as precipitated protein,may cause an underestimation of the content and toxicity of paraquat,providing new clues for clinical treatment.Currently,there is no specific antidote for paraquat intoxication,it is urgent to deeply understand the toxicity mechanism and toxicokinetics of paraquat,so as to establish an evaluation method for searching for effective antidotes.In the third chapter,the visualization and toxicokinetics study of paraquat in zebrafish were further studied based on MALDI-MSI technique.The results indicated that paraquat had a fast absorption rate in zebrafish.After absorption,it could be widely targeted in brain,gill,kidney,liver and other organs.The concentration of paraquat reached the maximum value in 30 minutes,mainly distributed in kidney and intestine to accelerate the excretion of paraquat.The concentration of paraquat reached the peak value twice in 6hours and mainly accumulated in various toxic metabolism and excretory organs again,Paraquat was excreted in the form of prototype through the kidneys and excreted in the body through feces.After that,the concentration decreased rapidly.However,its half-life was long and the elimination rate in the body was slow,Paraquat could still be detected after 24 hours.Meanwhile,with regard to various potential antidotes that have been discovered so far,taking sodium salicylate as an example,its detoxification effect on paraquat intoxication was preliminarily verified from two dimensions of spatial distribution and quantitative analysis based on MALDI-MSI technique.Compared to SERS,MALDI-MSI provided more dimensional information about spatial distribution and accumulation processes and promoted understanding of toxicokinetic characteristics of paraquat from a multidimensional perspective.It provided more valuable information for the clinical treatment of paraquat intoxication and was an advanced analytical tool for the study of toxicant dose-effect relationship.Finally,in view of the need for forensic and anti-terrorism investigations to trace the source of castor beans in the incidents of intoxication and terrorist caused by ricin and crude extracts of castor beans,in the fourth chapter,a complete and efficient MALDI-MSI method was established based on endogenous peptide biomarkers(Ricinus communis biomarkers,RCBs)for the first time for castor beans which is rich in lipids and fragile when processed.The tissue pretreatment schemes in sample preparation were comprehensively examined,mainly including the preparation of tissue sections,the selection and optimization of matrix,the optimization of key parameters of matrix coating,and the detailed screening and discussion of eight organic solvent washing protocols.Finally,an alternate six-step washing protocol of isopropanol and Carnoy’s solution was developed,which effectively removed a large amount of lipids in castor bean tissue sections and greatly improved the sensitivity of RCBs.This is the first systematic work on organic solvent washing protocol to improve the analysis of peptide of plant tissue in MALDI-MSI,providing a useful reference for the preparation of lipid-rich plant tissue samples.In the fifth chapter,the MALDI-MSI method was applied to visualize the spatial distribution of RCBs in nine castor beans from nine different sources,including domestic and foreign.Meanwhile,a stable isotope internal standard MALDI-MSI quantitative method was established and obtained the distribution and content differences of RCBs in castor bean tissue section from different sources.The results indicated that the content of RCBs in castor beans from nine sources were not significantly different,but the spatial distribution characteristics were significantly different.RCBs were mainly distributed in the whole endosperm of tissue,but the distribution was uneven.In the castor beans from Ethiopia and Pakistan,two foreign germplasm sources,RCBs were mainly concentrated in testa and embryo.Finally,MALDI-MSI data sets of castor bean from nine different sources were effectively classified by multivariate statistical analysis methods,including principal component analysis,deep neural network and t-distributed stochastic neighbor embedding.It indicated that castor beans from Ethiopia,Pakistan and China could be clearly distinguished,while Inner Mongolia autonomous region,Shanxi province and other provinces or autonomous regions in China were significantly different.It revealed valuable classification clues about nationality,altitude and the potential application value of RCBs in traceability analysis,providing a new research perspective and analysis technique for the traceability analysis of castor beans intoxication. |
PDF Full Text Request