Studies Of Fluorescence Methods For Protein Detection Based On Nucleic Acid Amplification Technologies

Proteins are important biological macromolecules,playing important roles in life functions.Recently,accumulative evidences indicated that protein expression or function alteration is related to the disease and even cancer.The protein expression assay is crucial for illustrating the physiological and pathological conditions,illustrating the effect of durgs or environmental factors,understanding the physiological and pathological phenomena,and realizing the occurrence,development and therapeutic effct of disease.Sensitive detection of some proteins in biological sample is also important for the diagnosis,treatment and drug screening.The content of proteins in body fluids is extremely low.Moreover,the complexity of protein metabolism and the interference of endogenous substances also improve the difficulty of protein detection in biological samples.Recently,the commonly used methods for protein detection are Enzyme-linked immunosorbent assay(ELISA),electrophoresis,mass spectrography,electrochemical method and fluorescence method.Among them,the fluorescence method possesses the advantages of simplicity,high sensitivity and excellent specificity.In recent years,sensitive methods based on the nanoparticle amplification approach and nucleic acid amplification approach provided valuable tools for protein detection in biological samples.Based on rolling circle amplification(RCA),hybridization chain reaction(HCR)and catalytic hairpin assembly(CHA),some fluorescence biosensors for protein assay were fabricated in this paper.The analytical model were platelet derived growth factor BB(PDGF-BB),tumor necrosis factor a(TNF-a),interferon y(IFN-?)and tyrosine protein kinase 7(PTK7).There are six parts in this paper:Chapter one is the introduction section,and it summarizes the concept,the detection significance and sensitive methods for protein.In addition,the existing problems for protein assay at the present stage are also summarized.In chapter two,a novel self-locked aptmer probe was constructed.The aptamer probe contained two functional parts:one was the aptamer sequence in the 3'-terminal with the function of molecular recognition,and the other one was the signal transduction sequence in the 5'-terminal with the function of signal transduction.Moreover,the aptamer sequence and the signal transduction sequence could partially hybridize with each other,forming a stem-loop structure.Thus,the aptamer probe could be locked by itself in the absence of target.Due to the intramolecular hybridization,this self-locked aptamer probe was more stable than that locked by another short DNA strand.Thus,it is more conducive to reduce the interference signals generated by the uncontrollable folding of aptamer.By combining the strand displacement amplification(SDA)and dual exponential rolling circle amplification(DE-RCA),a self-locked aptamer probe mediated cascade amplification strategy was fabricated.The ultrasensitive detection of PDGF-BB was achieved,and the detection limit was 0.38 fM with a dynamic range of more than 6 orders of magnitude,which was superior to the same amplification approach that mediated by a blocker DNA locked aptamer probe.In addition,by changing the aptamer sequence,sensitive and selective detection of small molecule adenosine was also achieved,suggesting that the design flexibility of the proposed self-locked aptamer probe and the proposed strategy.In chapter three,an enzyme-free 3-D DNA walker on DNA-coated MNBs was constructed.The walker was powered by the catalytic assembly of DNA hairpins.Triggered by the nucleic acid or protein,the nanoscale motions of the walking device could lead to the generation of a double-stranded DNA with fluorescent output molecule on the surface of MNBs.Compared with the walkers triggered by some protein enzyme reaction,the proposed walker was more simple and general.Taking the Smallpox gene as a model analyst,the walking system revealed good performance with a detection limit of 4.1 fM and an admirable specificity toward the mismatched target.In addition,by changing the target recognition elements,sensitive and selective analysis of proteins was also achieved,suggesting the application flexibility of the proposed walking device.Therefore,this 3-D DNA walker provided a valuable tool for biosensing analysis and could be extended to other specific targets.In chapter four,a multiplexed detection method for cytokines detection was construction based on a dual bar-code strategy and single-molecule counting.In the proposed method,IFN-? and TNF-a were chosen as model analytes,which were correlated with a variety of physiological and pathological processes including latent tuberculosis,HIV infection and melanoma.First,the primary bar-code strands were introduced by the formation of antibody-antigen-magnetic nanoprobe complex.Second,the second bar-code strands were introduced by the progress of multibranched HCR.Third,fluorescence dots could be acquired through the hybridization of the second bar-code strands and the fluorescence probes.Finally,the numbers of fluorescence dots were counted one by one for quantification.Senstive detection of IFN-y and TNF-a were achieved through the dual bar-code strategy,and the detection limit were both 5 fM.Unlike the typical bar-code assay that the bar-code strands should be released and refixed,the proposed method was more direct.Importantly,simultaneous and quantitative detection of IFN--? and TNF-a in human serum was also achieved,suggesting that this strategy was reliable and had a great potential application in biomedical research and early clinical diagnosis.In chapter five,a binding-induced nicking site reconstruction strategy was constructed for quantitative detection of membrane protein on living cell.In this method,first,an aptamer probe for membrane protine PTK7 was designed with an aptamer sequence,a trigger sequence and a nicking site.In the absence of PTK7,the aptamer sequence and the trigger sequence were hybridized with each other,forming a stem-loop structure.At this time,the two sequence of the nicking site were separated.When the binding of the aptamer probe and PTK7 occurred on living cell,the probe conformation would be changed and the nicking sites could be reconstructed,leading to the expose of the nicking site and the trigger sequence.Next,the nicking site could be identified by nicking enzyme,yielding the release of the trigger sequence,which could act as the trigger to initiat the homogeneous cascade RCA and HCR amplification.The proposed strategy revealed excellent sensitivity for PTK7 detection with a detection limit of 0.3 fM.Quantitative assay of PTK7 on living cancer cells was performed,and as low as 10 cells could be detected.Therefore,this propoese strategy provided a novel approach for reliable quantification of membrane protein expression on living cell.Chapter six is a conclusion and expectation section.It mainly summarizes the research result of the paper.