| Biomolecules such as biothiols play important roles in the regulation of various normal physiological and pathological processes.Their physiological levels in our body are closely related to many diseases.Therefore,it is of great significance to develop simple,sensitive and low-cost methods for rapid and accurate determination of disease-related biomolecules.In this dissertation,the determination of some important biomolecules in complex systems and the diagnosis of diseases were further studied.With the help of chemometrics moment method who owns the multi-resolution and global feature description ability,the problems of signal overlap between multi-target components and noise interference in complex systems are solved.The main contents of each chapter are as follows:Chapter 1 IntroductionThis section mainly summarizes the development of nano materials for the determination of some important biomolecules and applications of chemometrics in quantitative analysis and disease diagnosis in complex systems.At the same time,the research work and innovation of this dissertation were introduced.Chapter 2 Study on the conjugation of resveratrol to the glutathione thiyl radicalFound in various natural food products,many in vitro evidence indicated that resveratrol（RES）has been linked to neuroprotective and cardioprotective effects and prevent cancer development.However,clinical trials have been conducted with varying results,making the effect of RES controversial.In this paper,we demonstrated that Acrolein（ACR）,an endogenous toxic metabolite,could be conjugated by reduced glutathione（GSH）in the form of glutathione thiyl radical（GS·）.Notably,we discovered that the drug RES could be also conjugated with the high levels of endogenous GS·in cancer cells.A radical trapping agent was employed to capture the GS·and the mechanisms of the two reactions were further studied by UV-Vis spectrometry,mass spectrometry and theoretical calculations.Density Functional Theory（DFT）results showed that the reactions between GS·and RES is easier occur than GS·with ACR.Besides,the formation of the adduct GS-RES in cancer cell was obtained when RES was added during incubation.Further study indicates that over 77.6%of the RES was consumed in cancer cells.High concentrations of GSH were observed in many cancer cells,so it could be inferred that endogenous GS·may be one of the important factors to cause the decreased chemotherapeutic efficacy of anti-tumor drugs,which should be pay special attention in clinical therapeutics and drug development.Chapter 3 Simultaneous quantification of multiple endogenous biothiols in cancer cell based on multi-signal fluorescent probeGlutathione（GSH）,Cysteine（Cys）and Homocysteine（Hcy）are three important biothiols in organisms.As they usually coexist in our bodies,in order to meet the practical demands,the simultaneous quantitative determination of the three thiols is of great necessity.Although large numbers of fluorescent probes（including multi-signal fluorescent probes）have been developed,it is still difficult for the simultaneous quantitative analysis of intracellular thiols due to the spectral overlap and the complexity of the intracellular environment.In this contribution,a multi-signal fluorescent probe was employed for simultaneously quantifying intracellular GSH,Cys and Hcy.DFT calculations were employed to further study the mechanism of interaction between fluorescent probe and the three thiols.The results showed that the reaction between the probe and Hcy needed the lowest excitation energy.The Tchebichef image Moment（TiM）method was employed for the simultaneous quantitative analysis of the three thiols.As the feature variables of target components,TiMs calculated from the grayscale images of fluorescence 3D spectra were used to establish the quantitative linear models by stepwise regression.The precisions of intra-day and inter-day were less than 5.6%and 8.7%,respectively.The limit of detection（LOD）for Cys,GSH and Hcy were 0.11μM,0.35μM and0.18μM,respectively.The recoveries were from 97.0%to 105.9%.In addition,the proposed approach was applied to the simultaneous quantitative determination of Cys,GSH and Hcy in the MCF-10A cell and MDA-MB-231 cancer cell.The obtained results indicated that the concentrations of three thiols in cancer cell were higher than that in the normal cell.This study not only provides an effective approach for quantifying multi-target bio-molecules within complicated intracellular environments,but also further extends the applications of multi-signal fluorescent probes.Chapter 4 Simultaneous determination of monoamine neurotransmitters in human blood serumNeurotransmitters are endogenous chemical messengers that transmit,enhance,and convert specific signals between neurons and other cells.They can be regarded as biomarkers in the diagnosis of many diseases.Herein,an electrochemical method combining chemometrics was developed for simultaneous quantification of monoamine neurotransmitters including Dopamine（DA）,Epinephrine（EP）,Norepinephrine（NE）and serotonin（5-hydroxytryptamine,5-HT）in human blood serum.A reduced graphene oxide（RGO）modified glassy carbon electrode（RGO/GCE）was prepared via electrodeposition method and the modified electrode was characterized by scanning electron microscopy（SEM）and electrochemical techniques.The results of differential pulse voltammetry（DPV）measurement showed that the voltammetric signals of the four neurotransmitters overlapped significantly.To facilitate the simultaneous determination of the neurotransmitters,a chemometric tool of Tchebichef curve moment（TcM）method was employed.The TcMs calculated from the voltammograms were used to establish the models by stepwise regression.The precisions of intra-day and inter-day were less than 3.5%and 8.1%,respectively.The LOD for DA,EP,NE and 5-HT were 0.074μM,0.104μM,0.084μM and 0.097μM,respectively.The recoveries were from 83.5%to111.6%.The results indicated that the proposed approach is simple and reliable for the simultaneous determination of multiple neurotransmitters in human blood serum.Chapter 5 One-pot facile synthesis of CuNCs/RGO nanocomposite for the sensitive detection of heparin in human serum sampleA simple and facile one-pot approach for the synthesis of copper nanoclusters decorated reduced graphene oxide（CuNCs/RGO）nanocomposite was proposed,in which the CuNCs attached to the surface of the reduced glutathione（GSH）functionalized RGO through ligand exchange via their thiol functionalities.The synthesized nanocomposite was verified by structural characterizations,and the further investigation of density functional theory indicated that Cu3R2 cluster(R=C10H16O6N3S)with the lowest energy was the most stable structure in GSH-capped CuNCs.The obtained CuNCs/RGO nanocomposite exhibited rather weak fluorescence due to the photoinduced electron transfer from CuNCs to RGO.Upon the addition of heparin（Hep）,the significant enhancement of fluorescence at 595 nm was achieved,which was developed to detect Hep in human serum samples with high selectivity and sensitivity.The quenching mechanisms of CuNCs/RGO nanocomposite and the sensing of Hep were further studied.Besides,the linear range was 0.1-10μM with the detection limit of 26 n M in buffer solution containing 2%human serum sample,and satisfactory recovery in the range of 96.6%-104.7%was obtained,suggesting that the propose strategy could be applied to the determination of Hep in serum.Chapter 6 An effective approach for the early diagnosis of colorectal cancer based on three-dimensional fluorescence spectra of human blood plasmaColorectal cancer（CRC）is a common malignancy in the gastrointestinal tract,and its screening rates remain relatively low in the general population due to the lack of specific symptoms and effective methods.It is still in urgent need to develop the rapid and reliable approach to the early diagnosis of CRC.Biological molecules related to malignant tissues such as purine nucleotides and porphyrins could produce a wide range of fluorescence emission spectra in the UV-visible region.Herein,based on the 3D fluorescence spectra of human blood plasma,a combination strategy of Tchebichef image moments coupled with partial least squares-discriminate analysis（TiM-PLS-DA）was proposed for robust detection of CRC from three classes（CRC patients,adenoma patients and healthy subjects）.The established model provided an 84%correct classifications for CRC prediction.Venetian blinds with 10-fold cross validation was carried out.The error rate for classification both in cross validation and test set were less than 0.16.Sensitivity and specificity for CRC prediction were 0.95 and 0.88,respectively.In addition,the diagnostic capacity of the method was evaluated by receiver operating characteristics（ROC）analysis with an area under the curve（AUC）for CRC was 0.94.These results demonstrate that the proposed TiM-PLS-DA method based on the 3D fluorescence spectra of human plasma has great advantage for accurate CRC determination,which will provide a promising alternative approach for cancer diagnostics.Chapter 7 The main conclusions and outlook. |
