New Methods For Spectral Analysis Of Proteins And Nucleic Acids

In recent years,the discovery of valuable biomarkers and the sensitive detection of known biomarkers have become an important hotspot in biomedical research.Biomarkers are biological substances,ranging from nucleic acids to proteins,which are present or perturbed in aberrant conditions.When organs,tissues,cells and sub-cells are damaged by disease or external environment,the normal structure and function are changed to produce the substances with the biochemical index.It plays an important role in the identification,early treatment and prevention of diseases to discovery and sensitive detection of such specific biomarkers.The objective of this work was to develop sensitive and specific analytical methods for detection of biomarkers by using Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)and DNA motor-mediated fluorescence signal amplification.MALDI-MS is one of the most soft ionization techniques developed in recent years.It has many characteristics such as high throughput,high resolution.DNA motors have also been widely and successfully used for signal amplification.My research focused on development of new analytical methods for detection of different biomarkers by taking advantages of MALDI and DNA motors.Two specific methods were developed:(1)A single cell analysis method was developed by using MALDI-MS.We developed a MALDI-MS based single cell analysis method and used to biomarkers that could be used for early detection of the RSV virus infection.Briefly,we infected Hep-2 cells with RSV virus and then used the optical microscope to examine whether the cells are infected.To identify whether any new biomarkers are produced,we then used MALDI-MS to analyze the infected and normal cells.In the range of 5000-10000 Da,there were three peaks significantly different between the normal and infected cells,one peak(m/z=6154.25)showing up-regulation and other two peaks(m/z=7658.47 and m/z=9259.82)showing expression regulation upon RSV injected.(2)A toehold-exchange molecular translator was developed and further applied to construction of DNAzyme motors and DNA logic gates for amplified detection of microRNA(miRNA).We first developed a toehold-exchanged reaction-mediated molecular DNA translator that enables translating different input nucleic acid targets into a specific DNA output.We further applied the translator to construction of DNAzyme motors and DNA logic gates that allows homogeneous and amplified detection of nucleic acids under room temperature.Two miRNA targets,miRNA 10 b and miRNA 21 were used to test the proof of concept.A linear range from 0 to 800 pM was obtained for miRNA 10 b and a linear range from 0 to 250 pM for miRNA 21.A limit of detection(LOD)of 3.5 pM was achieved for both miRNA targets.We also demonstrated AND gates,OR gates,and NOT gates by using miRNA 10 b and miRNA 21 as input targets.In conclusion,I developed two different analytical methods: a MALDI-MS-based single cell analysis method and a DNA translator-based fluorescence signal amplification method.The former was used to identify potential biomarker for RVS infection,and the latter one was used for sensitive detection of two miRNA targets that are associated with breast cancer.The identification and sensitive detection of disease biomarkers are of great significance for molecular diagnosis and disease treatment.