The Application Of Spectrometric Techniques To The Detection Of Biomarkers And Authentication Of Chinese Medicinal Materials

Biomarkers are enzymes,nucleic acids,metabolic small molecules and other substances existing in body fluids such as blood and urine.Changes in the concentration of biomarkers generally reflect the occurrence and development of diseases.Chinese medicinal materials play very import roles in the treatment of many acute and chronic diseases.For instance,the effectiveness and safety of traditional Chinese medicine for the treatment of patients with COVID-19 by the World Health Organization.Many biomarkers mainly exist in complex systems such as blood at very low concentration level,which makes their quantification rather difficult.Chinese medicinal materials are also highly complicated systems.Authentication of Chinese medicinal materials is therefore not an easy task.Hence,it is still quite necessary to develop methods for accurate and fast detection of biomarkers and authentication of Chinese medicinal materials.In this thesis,efforts have been made to investigate the application of mass spectrometry,surface-enhanced Raman spectroscopy,excitation-emission matrix fluorescence spectroscopy and their combinations with chemometric methods to the accurate and fast quantification of biomarkers and authentication of Chinese medicinal materials.The main research contents are as follows:In the second chapter of this thesis,a novel platform for the detection of ribonuclease H（RNase H）was developed by taking advantage of the hydrolyzation ability of RNase H against RNA strand in RNA/DNA hybrid duplexes and integrating hybridization chain reaction amplification and inductively coupled plasma mass spectrometry.Experimental results showed that the proposed platform had a wide linear detection range and sensitivity for RNase H,and successfully realized accurate quantification of RNase H in lysate samples of Hep G2 and He La cell lines.In chapter 3,a simple but effective method for the quantification of glucose was developed by the combination of surface-enhanced Raman spectroscopy with the spectral shape deformation quantitative theory.The working mechanism of the proposed method for the detection of glucose is as follows.Under the catalyzation of glucose oxidase,glucose can be decomposed into H₂O₂,which can catalyze the oxidization of o-phenylenediamine（OPD）into 2,3-diaminophenazine（DAP）with the presence of silver nanoparticles,and cause significant changes in SERS measurements.The quantitative information of glucose can then be extracted from the resulting changes in SERS measurements by the spectral shape deformation quantitative theory.The proposed method was successfully applied to the detection of glucose in serum samples.Its quantitative results with recovery rates varying from 82.9 to 115.6%were basically consistent with those of a commercial glucose meter.In chapter 4,excitation-emission matrix fluorescence spectroscopy was combined with chemometric method,and applied to the identification of adulteration of atractylodes macrocephala koidz（AMK）samples and quantification of adulteration levels in these samples.Experimental results revealed that partial least squares discriminant analysis（PLS-DA）,k-nearest neighbors（k NN）and random forest（RF）could accurately identify adulteration of AMK samples based on their fluorescence measurements with correct classification rates no less than 93%.In particular,PLS-DA achieved a correct classification rate of 100%,and performed best among the three chemometric methods.Moreover,the adulteration levels of five types of substances adulterated in AMK samples could be accurately determined by the corresponding partial least squares regression models with root mean square error values no larger than2.34%.