Construction Of Sensing Platform Based On Fluorescence Quantum Dots And Its Application In Biological Analysis

As biomarkers for disease diagnosis,biological enzymes and nucleic acids are closely related to human life and health.In order to meet the needs of early diagnosis and accurate treatment of diseases,it is urgent to develop the efficient,sensitive and accurate biological molecular analysis methods.Among various methods for detecting biological enzymes and nucleic acids,fluorescence detection method is widely used because of its advantages of simple operation,high sensitivity and good specificity.To construct highly selective and sensitive fluorescence probes is a key step in the fluorescence detection of biomolecules.Quantum dots have become promising fluorescent nanoprobes due to their good photostability and high biocompatibility.In this paper,we have prepared quantum dots with different fluorescence properties as fluorescent probes and applied them in the detection of biological enzymes and nucleic acids.The experimental results display that the designed sensing platforms have excellent detection ability in complex biological samples.The main research contents are as follows:In the first chapter,we briefly introduced the research background of fluorescent biosensors and the representative new fluorescent nanomaterials——quantum dots.It mainly included the synthesis methods,fluorescence sensing mechanisms and applications.On this basis,we proposed the main content and research significance of this paper.In the second chapter,a fluorometric and colorimetric dual-mode biosensing platform for the specific detection of Kras gene was constructed based on the target recycling amplification strategy.The hairpin DNA（h-DNA）contained the recognition sequence of Kras gene and the segment of G-rich that can form G-quadruplex.The h-DNA specifically hybridized with the Kras gene to form double stranded DNA（ds DNA）,and it was cleaved by the Exo III to trigger the target recycling amplification process.A large amount of G-quadruplex could be produced by the above amplification method.In the presence of hemin,the hemin/G-quadruplex complex was formed.Its peroxidase-like activity could catalyze the oxidation of H₂O₂and further oxidize 2,4-dichlorophenol（2,4-DP）.4-Aminoantipyrine（4-AP）was coupled with the oxidation product of 2,4-DP to form a red complex（quinone imine）.The quinone imine had a characteristic absorption peak at 506 nm and thus quenched the fluorescence of Ag In Zn S QDs（AIZS QDs）.The activity of Kras gene was sensitively detected by the changes of both fluorometric and colorimetric signals.The limits of detection（LOD）were as low as 0.12 p M and 0.85 p M,respectively.Satisfactory results were obtained for measuring the Kras gene in human serum,suggesting that this method showed great potential in diagnosis and analysis.In the third chapter,we designed a fluorescent and colorimetric dual-signal sensing platform for thrombin determination based on the DNA-enhanced peroxidase like activity of iron-cobalt oxide nanosheets（Fe Co-ONSs）.In this work,two different single-stranded DNA:S1 and S2 were involved.S1 was used to enhance the catalytic activity of Fe Co-ONSs,and S2 was modified on the surface of MBs to construct MBs-S2 for subsequent magnetic separation.These two ss DNA contained two different aptamers of thrombin.When thrombin was added to the system,S1 and MBs-S2 quickly combined with thrombin and the amount of S1 decreased in the solution after magnetic separation.The enhancement of peroxidase like activity of S1on Fe Co-ONSs was weakened,resulting in the inhibition of the catalytic oxidation process of substrate TMB.The formation of ox TMB and the fluorescence quenching efficiency of Mo S₂QDs were further affected.Therefore,the quantitative detection of thrombin can be achieved by measuring the absorption signal of ox TMB and the fluorescence signal of Mo S₂QDs.In addition,this sensing strategy can be used to develop various sensing platforms based on biomolecule-aptamers.In the fourth chapter,we designed a ratio fluorescence sensing platform for the determination of Bch E activity based on the adsorption ability of CoOOH NSs to DNA and the target recycling amplification strategy.Initially,single-stranded DNA（ss DNA）were adsorbed on the surface of CoOOH NSs.Bch E could promote the hydrolysis of iodized s-butyrylthiocholine（BTCh）into reducing substance-thiocholine,which can decompose CoOOH NSs into Co²⁺and release ss DNA.In the presence of hairpin DNA（h-DNA）,ss DNA hybridized with h-DNA to form double stranded DNA（ds DNA）with blunt 3′-hydroxylated terminus.Then,a large number of G-quadruplex（in h-DNA）were released through Exo III assisted target recycling amplification strategy.In the presence of thioflavin T（Th T）,the yellow fluorescent substance G-quadruplex/Th T was generated.The addition of organophosphorus pesticides can significantly inhibit the hydrolysis of BTCh by Bch E,and then inhibit the decomposition of CoOOH NSs.Target recycling amplification process could not be triggered due to the lack of ss DNA.Thus,the yellow fluorescent G-quadruplex/Th T could not be produced.During the whole process,the fluorescence emission of Hg-Zn Se QDs at 630 nm as a reference signal remained unchanged.In this method,the LODs of Bch E and OPs were 0.026 m U/L and 0.47 ng/m L,respectively.Our developed biosensing method showed excellent specificity and it has been successfully applied in the analysis of Bch E activity in human serum samples,and OPs in environmental water.In the fifth chapter,we constructed a dual-mode sensing system for ultra-sensitive detection of human HIV DNA based on the 3-D DNA walker signal amplification and target recycling amplification.The locked swing arm and track strand were modified on the surface of MBs to construct 3-D DNA walker.The track chain was a hairpin structure and contained the segment of G-rich that can form G-quadruplex.HIV DNA can hybridize with the blocker DNA on the swing arm to activate the swing arm.After the swing arm was activated,it hybridized with the track strand to form ds DNA and was cleaved by Exo III to release G-quadruplex.When hemin was added to the system,it combined with G-quadruplex to form G-quadruplex/hemin complex with peroxidase-like activity.G-quadruplex/hemin complex can oxidize o-phenylenediamine（OPD）to yellow product 2,3-diaminophenazine（DAP）,resulting in the fluorescence quenching of Mo S₂QDs at420 nm and fluorescence enhancement of DAP at 560 nm.In this process,each swing arm could combine multiple track chains to amplify the signal,and the target recycling amplification also occurred at the same time.The interference of other components in complex samples was eliminated due to the realization of magnetic separation by using MBs.In addition,our designed sensing platform can be used to detect different virus genes by changing the recognition sequence,providing a common strategy for the analysis of various viruses.In the sixth chapter,we systematically summarized the contents of this paper and discussed the future perspectives of quantum dots.