Studies Of Novel Methods For Protein Detection Based On The Nucleic Acid Amplification Techniques

As the basic material of life, proteins paly important roles in the life and variousforms of life activities. Therefore, the detection of protein has been the focus of therearch. The detection of target using biosensors is sensitive and accurate, even incomplex systems. So biosensing is the new development tendency of protein analysis.Researchers designed many novel biosensors for the rapid and sensitive detection ofprotein.Aptamer is one kind of functional nucleic acids, namely the short DNA or RNA,which can be isolated via vitro selection known as systematic evolution of ligands byexponential enrichment (SELEX). Aptamer can bind to target sepecially, such as smallmolecules, proteins, cells, metal ions, etc. In addition, aptamer can be synthesized andmodifyed conveniently. With more and more aptamers of the proteins being isolated,the aptamers were widely used in the biosensor for the protein analysis.However, the change of signal caused by the binding of aptamer and target proteinwasn’t high enough for sensitive detection of the target. Signal amplificationtechnology has an important effect on improving the detection sensitivity. Numerousbiosensors based on the isothermal nucleic acid amplification technologies wereconstructed for the target sensentive detection, such as, rolling circle amplification,polymerase chain reaction, nicking enzyme signaling amplification, etc.According to the above considerations and the reported literatures, thisdissertation is focused on developing a series of novel biosensors for the proteindetection based on the the isothermal nucleic acid amplification. The detailed methodsare described as follows:In chapter2, an electrochemical sensor based on hybridization chainreaction(HCR) and enzyme-signal amplification was constructed for interferon-gammadetection. Hybridization chain reaction contains two harpin probes that can hybridizewith each other. With the trigger of intiator probe, the two harpin probes bound one byone through hybridization and form many long duplex DNAs with nicks. The captureprobes which can hybridize with the IFN-γ aptamers were immobilized on theelectrode. Upon the addition of target protein, the protein bind with the aptamer, thenthe recognition probe that contain aptamer and intiator probe can not be captured onthe electrode. Therfore the intiator probe can not trigger the HCR on the electrode. The two harpin probes modifyed with biotin and the streptavidin–alkaline phosphatase(SA–ALP) can be captured on the electrode by the specific interaction of biotin/avidin.The SA–ALP as the catalyst converted the electroinactive substrate1-naphthylphosphate into the electroactive derivative1-naphthol, ultimately the target can bedetected accurately.In chapter3, The thrombin was detected based on nicking enzyme assisted signalamplification. The harpin structure of RP was deformed for the binding of thrombinand its aptamer, then released the stem sequence that can hybridize with the loop ofanother harpin probe HP. Therefore the harpin probe HP was open. In this situation,the single strand DNA involving the nicking enzyme recognition sites can hybridizewith the harpin probe HP. With the polymerase extension and the nicking enzymeactivities, an abundance of complementary DNAs that can hybridize with thefluorescent molecular beacons were generated, providing amplified fluorescence signal.At the same time, the complex of probe RP and thrombin was displace d and hybridizedwith the new harpin probe HP. Thereby the circle amplification can improve thedetection sensitivity.In chapter4, exonuclease III-aided amplification technology and graphene oxidewere used for lysozyme detection. Exonuclease III loses activity on3’-protrudingtermini (≥4bases) of double-stranded DNA and single-stranded DNA.10nucleotides3’-overhang of the hairpin DNA probe (HP) was designed, and HP consists of thelysozyme aptamer sequence. Upon the addition of target protein, the HP exhibited aconformational change. Under this circumstances, the HP can hybridize with signalprobe (SP) and showed a blunt3’-terminus of SP. The signal pobe was digested byexonuclease III, facilitating the hybridization of HP with another signal probe and thecycling of digestion of multiple signal probes. The signal probes were digested byexonuclease III into residuals and nucleotides could not be adsorbed on the grapheneoxide, so no fluorescence quenching occurred. The qualitative and quantitativeanalysis of the target can be realized by the detected fluorescence signals. What ismore important, this strategy can potentially be used for the dection of another kind ofsmall molecule and protein.