Research And Application On Fluorescence Biosensor Based On Aptamer

With the development of life science and medical research,people have put forward higher requirements for highly sensitive and highly specific detection of biological samples.As a front-end component for biological detection,biosensor usually include bioreceptors,transducers,and amplification units,which play an irreplaceable role in the entire detection process.At the same time,with the continuous progress of new biotechnology,nanoscience,electronic information and other disciplines,new antigens,antibodies,nucleic acid aptamer,etc.have emerged as recognition units,and new fluorescent dyes,quantum dots,carbon quantum dots,etc.have been used as conversion Labeling materials,new nucleic acid-based enzymes and no enzymes are used as signal enhancement and amplification methods,enabling biosensors to further develop in the direction of small sample consumption,high specificity and sensitivity,fast response speed,short reaction time,and small size.,Promoting biosensors to play an increasingly important role in life science testing,clinical diagnosis,food safety,environmental monitoring,etc.As the most commonly used identification element in biosensors,antibodies play an important role in clinical immunodiagnosis.However,antibodies need to rely on animal extraction and synthesis and purification,so there are large differences between batches,and there are disadvantages such as low detection sensitivity at low abundance,poor thermal stability,and high cost.Aptamer is a kind of oligonucleotide sequence.As a recognition element,it can bind to various types of targets such as proteins,small molecules,cells,microorganisms,etc.with high affinity and specificity.Compared with antibodies,nucleic acid aptamer can be obtained through in vitro screening,so they can be prepared by chemical synthesis.They have many recognition types,easy modification,low cost,good thermal stability,good consistency between batches,and no immunogenicity.Advantages,research has begun in many fields such as disease diagnosis,drug release,targeted therapy,and food safety.In this paper,for the detection of tumor disease markers,the main target is the highly sensitive detection of cells and protein markers in the sample,the nucleic acid aptamer is used as the main recognition unit,and the hybrid chain reaction(HCR)and strand displacement reaction(SDR)and other amplification strategies,combined with new carbon quantum dots and molecular beacons as fluorescent labeling technologies,have designed and developed a variety of new fluorescent biosensing methods based on nucleic acid aptamer.These detection methods have high sensitivity,good specificity,and Simple operation and low cost.To further facilitate the application,this article also designed an automated fluorescence quantitative analysis instrument to quickly detect the corresponding samples.First,a novel cancer cell detection method based on N and S co-doped carbon quantum dots is studied.In this method,a kind of carbon quantum dots with fluorescent properties co-doped with N and S were synthesized,and the characteristic of the nucleic acid aptamer to specifically recognize Hep G2 cells was used to label BHQ on the aptamer sequence.The fluorescence of the carbon quantum dots is quenched by the labeled aptamer sequence.When the Hep G2 target cell exists,the nucleic acid aptamer binds to the target cell,and the carbon quantum dots are released.The aptamer and the carbon quantum dots in the suspension are further separated by centrifugation,and finally the fluorescence intensity of the carbon quantum dots is measured.To assess the concentration of cancer cells.This method realizes the detection of Hep G2 cells in the range of 5-50000 cells/m L,and has good linearity in the range of 5-1000 cells/m L,and the minimum detection limit is 5cells/m L.The aptamer sensor of this method does not have any signal amplification process,has high detection sensitivity,low instrument requirements,and low detection cost,and is a promising clinical analysis tool.Next,a fluorescent method for detecting proteins combined with the reaction of carbon quantum dots and HCR was developed,and a DNA aptamer probe with a hairpin structure was designed to achieve specific recognition of the MUC1 target protein.In the presence of the target protein of MUC1,MUC1 binds to its aptamer to change the conformation of the probe,exposing the single-stranded HCR priming sequence,triggering the interaction of other auxiliary probes to generate a hybridization chain reaction,forming a long double Strand DNA.Because the synthetic carbon quantum dots are modified on the trigger auxiliary probe,the nucleic acid aptamer-carbon quantum dot complex is adsorbed on the surface of graphene oxide due to the ?-?stacking effect when the HCR reaction does not occur,and the fluorescence is quenched.When the HCR reaction occurs,the carbon quantum dots can be far away from the graphene oxide surface,the fluorescence emission resumes,and the peak intensity can be used to indicate the concentration of MUC1.Using this method,the detection of MUC1 protein is realized,the linear range is 10 fg/m L-100 pg/m L,and the lowest detection limit is 2.3 fg/m L.The preparation and combination of carbon quantum dots in this method are very simple.Through the strong fluorescence emission of carbon quantum dots and the HCR-assisted non-enzyme signal amplification process,ultrasensitive detection of target proteins is achieved,and graphene oxide is an excellent The fluorescent quencher can reduce the background fluorescence,which is conducive to the reliable analysis of trace proteins.Then,a sensor method of nucleic acid aptamer molecular beacon combined with target cycle amplification strategy was designed.The fluorescent signal of four hairpin probes was used to construct a DNA star structure to detect TNF-? protein.When the target protein is present,the TNF-? target protein binds to its nucleic acid aptamer,the stem part of the molecular beacon is opened,and it hybridizes with the transition probe,auxiliary probe,and signal probe to form a DNA triangle star structure.Continue to cyclically amplify and release the corresponding fluorescent signal.The fluorescence sensor developed based on this strategy achieves high-sensitivity detection of TNF-?,with a linear range of 10 pg/m L-1000 pg/m L,and the lowest detection limit of 5 pg/m L.The method does not require cleaning and thermal cycling conditions,and only requires10 minutes for one reaction.Since no enzyme is involved in the reaction,it has the advantages of high sensitivity,high selectivity,low cost,simple operation and the like.Finally,the application research of fluorescence quantitative detection instrument based on nucleic acid aptamer is carried out.A set of automated fluorescence detection analyzer is designed.Theoretical calculation of fluorescence model,fluorescence optical path design and selection,fluorescence filtering and calibration algorithm design are also conducted.After the instrument design is completed,according to the characteristics of the fluorescence emission signal,the fluorescence signal is smoothed and filtered and the baseline calibration is processed.The accuracy,precision and linear range of the instrument are tested to verify the performance parameters of the overall system.For the TNF-? target protein,corresponding test strips are prepared and successfully applied with the developed fluorescence detection instrument.