Fluorescence Sensing Study Of Proteins Based On DNA Self-assembly And Strand Displacement Signal Amplification Strategy

Living standards of people have increasing with the rapid development of encnomic and science,and thus people tend to put more and more attention to their body health,and some molecular biomarkers play crucial roles in in real biological analyses,especially in early diagnosis of diseases,drug monitoring and other aspects.Therefore,it is of high demand to detect specific trace or even ultra-trace molecular biomarkers,exploiting effective and convenient methods for different targets detection is of central importance for disease diagnosis,prediction and treatment.Fluorescent biosensor is given to be an efficient detection machine of several targets.Fluorescent biosensor has been widly applied in disease diagnosis,environmental monitoring,food and drug safety and scientific research because of its simple operation,fast signal response,strong specificityand homogeneous system.For further improvement of detection level of biomarkers,different kinds of signal amplification methods have combined with fluorescent detetion strategy.Three fluorescent biosensors were constructed based on enzyme-assisted DNA strand displacement reaction and DNA self-assembly signal amplification methods to obtain highly senseitive and selectivity detection of mucin 1(MUC1),nuclear factor-kappa B(NF-kB p50)and interferon-?(IFN-?).The specific research work and results are summarized as follows:Part 1.Cascaded multiple recycling amplifications for aptamer-based ultrasensitive fluorescence detection of protein biomarkersHighly sensitive detection of molecular biomarkers plays a significant role in diagnosing various types of diseases at the early stage.According to previously reported studies,the expression levels of MUC1 in tumor cells were found up-regulated when compared with that in the majority of normal human epithelia of various origins,indicating the potential of MUC1 as a serological or histochemical diagnostic marker to evaluate tumor treatments.Hence,exploiting effective strategies for MUC1 detection is of central clinical importance.We demonstrated in this paper an ultrasensitive aptamer-based fluorescence method for detecting mucin 1(MUC1)in human serum via a cascaded multiple recycling signal amplification strategy.The MUC1 target molecules present in the samples cause structure switching of the hairpin aptamer probes,which initiates three cascaded recycling cycles for the cleavage of the fluorescently quenched signal probes to recover significant fluorescence for highly sensitive detection of MUC1.The developed method has a linear range from100 fM to 1 nM for MUC1 detection.Besides,owing to the substantial signal amplification by the integrated and cascaded recycling cycles,a low detection limit of 35 fM is achieved with high selectivity.Moreover,the monitoring of trace MUC1 in human serum can also be realized with such a method,indicating its great potential for highly sensitive detection of different disease biomarkers.Part 2.Silver ion-stabilized DNA triplexes for completely enzyme-free and sensitive fluorescence detection of transcription factors via catalytic hairpin assembly amplificationTranscription factors are several kinds of functional DNA binding proteins to perform regulatory functions in gene transcription,replication and expression,which plays important roles in gene regulation and have been identified as promising biomarkers for disease diagnosis.On the basis of a new Ag~+-stabilized DNA triplex probe and catalytic hairpin assembly(CHA)signal amplification,we have established a completely enzyme-free and sensitive method for simple fluorescence detection of NF-kB p50(nuclear factor-kappa B),a transcription factor.We found that the employment of Ag~+to stabilize the DNA triplex structure could effectively reduce the background noise.The association of the target NF-kB p50 with the recognition hairpin in the Ag~+-stabilized DNA triplex leads to the release of a single stranded DNA,which is used as the trigger to initiate subsequent CHA between a fluorescently quenched signal hairpin and the recognition hairpin in the triplex DNA structure,thereby resulting in drastically amplified fluorescence recovery for sensitive detection of NF-kB p50.This assay method shows a dynamic concentration range of 5 to 150 pM and a detection limit of 1.5 pM for the detection of NF-kB p50.Besides,the presence of the target molecules can also be selectively discriminated from other non-specific proteins.Moreover,the presence of low concentrations of NF-kB p50 in human serum samples could be monitored with this approach.With the successful demonstration for NF-kB p50,such a method can be potentially extended to detecting other transcription factors in a convenient and sensitive manner without using any enzymes for signal amplification samples.Part 3.Netlike hybridization chain reaction assembly of DNA nanostructures enables exceptional signal amplification for sensing trace cytokinesThe monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment.For example,interferon-gamma(IFN-?)is a crucial inflammatory cytokine,which is produced by several types of immune cells including cytotoxic T-lymphocytes,T-helper cells and other lymphocytes during the immune response.These cells are related to the regulations of immunity,proliferation and differentiation.Therefore,various disease progressions are closely associated with the expression levels of IFN-?,and sensitive detection of IFN-?is thus of great importance.In this work,we proposed a target-responsive netlike hybridization chain reaction(nHCR)DNA nanostructure construction method,which can offer an exceptional signal enhancement,for highly sensitive fluorescence detection of cytokine,interferon-gamma(IFN-?).The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands,which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures.The formation of the DNA nanostructures brings the dyes into close proximity,resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-?.The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-?detection.Besides,with the high specificity of the aptamer probe and the significant signal amplification of the nHCR,such an IFN-?detection strategy shows excellent selectivity and high sensitivity,which can be potentially applied to detect IFN-?in human serums.With such a demonstration of the detection of IFN-?,this proposed method can be extended for detecting different types of biomolecules.