| In recent years,the progress of biosensors and biochemical analysis have greatly promoted the development of analytical chemistry and life science.Mass spectrometry is one of the most important biological analysis technologies.It has many advantages in bioactive molecule detection,such as high sensitivity(less than nmol),high speed,high throughput,having the ability to provide accurate molecular weight and structure information at the same time,the possibility of being used for qualitative or quantitative analysis and can be combined with a variety of chromatographic separation technology.All these features make mass spectrometry a promising technique for bioactive molecule detection compared to other technologies.In this thesis,a variety of biosensing approaches have been constructed bas ed on mass spectrometry technology.These methods have the advantages of simplicity,rapidness,low-cost,good stability,high sensitivity,high selectivity and high-throughput for protein and nucleic acid detection.The main contents of the thesis are out lined as follows.In chapter 2,a DNA target fragmentation assay based on mass spectrometry has been developed for DNA methylation level profiling.This method is highly sensitive,allowing direct profiling of DNA methylation using genome DNA without pre-amplification and being able to accurately determine DNA methylation level in specific genes.The selectivity was achieved by combining precise restricted cleavage of genomic DNA with selective capture using magnetic separation.The high sensitivity was obtained through non-enzymatic degradation of target DNA into single bases followed by MS/MS detection.Detection limit of 5-methylcytosine(5m C)reached 0.11 p M.The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples,implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.In chapter 3,DNA target fragmentation assay has been applied to detect trinucleotide repeat length.Trinucleotide repeat length detection is critical for diagnosis,risk assessment,evaluation of aggressiveness of the diseases,and understanding of the molecular mechanisms of repeat size mutation.To demonstrate the potential of our DNA target fragmentation assay,the trinucle otide repeat expansion gene associated with Huntington’s disease was chosen as the case of study.The assay is based on magnetic capture and acidic degradation of target polymerase chain reaction(PCR)amplicons followed by mass spectrometry(MS)detection.This method has advantages in high sensitivity,speed and accuracy.In MS/MS mode,detection limit of cytosine base can reach as low as 18.23 f M.The required minimum amount of genomic DNA is 0.07 μg for a single CAG copy number detection.The developed assay is able to be extended for detecting trinucleotide repeats associated with other diseases.Hence,this method may improve the methodologies for trinucleotide repeat detection and provide a new paradigm for identifying nucleic acid sequence variations.In chapter 4,a highly sensitive mass spectrometry based immunoassay has been developed for protein detection utilizing liposome nanoparticles as mass tag.Protein is an important type of disease biomarker.However,some of the protein content is usually very low,particularly in the blood,urine,and other complex matrices,this brings enormous challenge for its detection.Mass spectrometry can be used for ultrasensitive phosphatidylcholines(PC)detection because PC can be easily ionizedIn our assay,target proteins were captured by magnetic beads,at the same time immunoconjugated with detection antibodies tagged with liposome to form a sandwich immunecomplex structure.Highly sensitive protein detection was achieved based on mass spectrometry measurement of PC.Detection limits of 0.03 ng/m L for prostate-specific antigen(PSA),0.2 ng/m L for carcinoembryonic antigen(CEA)were achieved.Mass spectrometry provides accurate mass information which can help the identification of target proteins and avoid false positive results usually met in enzyme-linked immunosorbent assay(ELISA).In chapter 5,a single molecule detection method has been developed for nucleic acids based on mass spectrometry counting of single liposome particle.Before disease symptoms appear,a very small amount of nucleic acids have already existed that are biomarkers for clinical diagnosis.However,it is difficult to detect ultra-low nucleic acids using existing methods.Hence,establishing ultra-sensitive detection method for nucleic acids is particularly urgent and important.Magnetic beads were used to capture target DNA and liposome particles were used as mass tags for single particle measurement.Liposome was able to be released from magnetic beads via photocatalytic cleavage.Hence,one DNA molecule corresponded to one liposome particle and could be counted by mass spectrometric measurement.Ultrasensitive detection of DNA(10-18 M)could be achieved in this method.Detection of RNA copy number of the hepatitis C viral(HCV)from clinical samples was demonstrated in this thesis. |
