Novel Biosensors Study For The Detection Of Nucleic Acid And Protein

Nucleic acid and protein are fundamental substances that lives by them. There won't be life without nucleic acid and protein. When a gene gets expressed, that means its protein is produced. Proteins exist inside each one of us to help carry out our daily functions. Nucleic acid serves as information-carrying molecules in biological systems and is the founder of life. Proteins are the performers of life action. With the development of life science and biomedicine, study of disease and nucleic acid has become the hot research. Gene mutation will result in disease. False transcription will lead to protein dysfunction and disease. Therefore, sickness prevention and disease diagnosis are very important. DNA mutation detection and protein quantification could be helpful for disease monitoring. And then developing novel detection assay for nucleic acid and protein is the main task for researchers.In this dissertation, we aimed to study new biosensor which applied in DNA and protein quantitative analysis. A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component. The sensitive biological elements contain nucleic acid, protein, and enzyme. The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into a measurable effect, such as an electrical signal and fluorescence signal. Bioreceptors are the key to specificity and selectivity for biosensor. They are responsible for binding the analyte of interest to the sensor for the measurement. The general bioreceptors are antibody-antigen, enzymes and nucleic acids aptamer-protein. Biosensor can be classified into many types, such as immunosensor, enzyme biosensor and glucose biosensor. Nowadays as advanced detection means, biosensor methods have wide perspective and practicability in further. The project focus on developing biosensor methods based on the fluorescent probe and DNA aptamer which can be used in detection and analysis for nucleic acid and protein, as follow: 1. Through studying the interaction between pyrene probe and nucleic acid, it was found that DNA induce the aggregation of the positively charged pyrene probe and gave out strong pyrene excimer emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for a biosensor based on label-free fluorescence probe for nucleic acid sensing.2. Through studying the interaction between pyrene probe and normal/mismatched nucleic acid, mismatched ssDNA cleaved with nuclease S1 gave no induced pyrene probe aggregation, but the DNA containing the complementary sequences could have duplex DNA and show strong excimer fluorescence. So the DNA containing full-match and containing one-mismatch showed different chain length after nuclease digestion, which resulted in diversity excimer fluorescence. Based on the phenomenon, we developed a biosensor based on label-free fluorescence probe and enzymatic reaction for detection of one-base mutation on DNA and analysis of single nucleotide polymorphism (SNP).3. The method relies on the specific binding between the nucleic acid aptamer and its target protein, and the binding between the single-stranded DNA binding (SSB) protein and the single-stranded nucleic acid (aptamer or molecular beacon). Lysozyme binding to its aptamer prevents SSB protein binding, and the subsequent binding of the free SSB protein to molecular beacon (MB) results in a turn-on fluorescence signal, which can be used for lysozyme quantification. By using the molecular beacon, we have developed a molecular beacon-biosensor based on the aptamer-protein recognition and protein displacement for lysozyme assay in salive.4. We have study the interaction of MB-SSB and the SSB protein cycles. Microscale of SSB protein could not open the MB and give out turn-on fluorescence. However, with the addition of LNA primers. DNA polymerase and dNTP, after the DNA polymerization. SSB protein was displaced by the new strand and went on to bound the next MB until open all the MB. and then gave out the strong fluorescence signal. We are aimed to show fluorescence biosensor based on the molecular beacon and SSB-induced strand displacement amplification for SSB protein analysis. In the process of nucleic acid recognizition and detection, we chose pyrene as a fluorescence probe to produce signal. Negatively charged nucleic acid could induce positively charged pyrene probe aggregation, which generated excimer emission. We demonstrate that the induced excimer fluorescence is related to the concentration, length, sequence. According to conclusion, we designed this fluorescence biosensor which used to detect one-point mutation.In the process of proteins recognizition and detection, we take advantage of specific bind between DNA-aptamer and protein. Meanwhile, molecular beacon "molecular light swich" was chosen as a reporter probe to give out fluorescence signal. What's more, a kind of cycled single-stranded binding protein was introduced to recover the fluorescence of MB. On the basis of the principles, we worked out a project which combined lysozyme-SSB displacement with SSB cycling-MB signal amplification. It turned out that our method could apply to SSB protein detection.After scientific and all-around investigation, the four biosensors have been used in aspects of nucleic acid recognition, one-base mutation and protein qunantitive. Label-free fluorescence probe based biosensor is low-cost and rapid. Nuclei acid aptamer based biosensor is high specific and selective...