An Ultrasensitive DNA Self-assembly Based Enzyme-free Immunoassay

Objective:The abnormal expression of protein disease markers is closely related to the occurrence and development of cancer.Therefore,the highly sensitive detection of proteins is of great significance in disease diagnosis and postoperative monitoring.Signal amplification technology are now commonly used to improve detection sensitivity.This study aimed to establish an accurate and sensitive fluorescent immunosensor.Combining immunomagnetic beads with DNA self-assembly signal amplification technology,with the help of fluorescence sensing platform and flow cytometry,the target protein can be accurately and quantitatively detected in an enzyme-free and constant temperature environment.By replacing the target sequence,it is expected to realize the detection of different protein disease markers,and provide new methods and ideas for the in vitro diagnosis of diseases.Methods:(1)Characterization of DNA self-assembly and optimization of the parameters.The amplified products were verified and characterized by non-denatured polyacrylamide gel electrophoresis,atomic force microscopy,and fluorescence spectrophotometer;The nonlinear hybridization chain reaction(NHCR)sequence was optimized by designing probes containing different amounts of toeholds to reduce background leakage;The reaction time and temperature of the NHCR process were optimized by fluorescence spectrophotometer;(2)Construction of fluorescent immunosensor.The magnetic beads were activated and coated with captured antibodies,t PSA antigen,and biotin-labeled antibody was added to form sandwich complexes.Then,streptavidin,biotin-labeled DNA trigger,and the NHCR system were introduced to quantitatively detect fluorescence on magnetic beads by flow cytometry.The experimental parameters were optimized and the linear range and selectivity of the method were investigated;the serum samples were tested.(3)Study on the universality of fluorescence immunosensor.The antigen-antibody pair was replaced by f PSA to verify the universality of the sensor and investigate the linear range and detection limit of detection.(4)To investigate the effect of bead size on sensor performance.The effect of magnetic beads at different diameters(2μm,3μm,5μm)on the signal and detection limit of f PSA was compared.Results:(1)During DNA self-assembly,background leakage can be effectively controlled when the number of toeholds of the assistant B probe is reduced by 2 bases;The optimum time of NHCR amplification was 60min and the temperature was 37℃.(2)The optimal reaction conditions of fluorescent immunosensor for t PSA detection were as follows:the amount of antibody captured was 20μg,the concentration of streptavidin was 1μg/m L,and the concentration of Substrate A was 0.15μM.The linear equation is y=5.516 x-6.672,R~2=0.9975,and the detection limit is 1.57pg/m L.(3)Immunofluorescence sensor can be successfully used for quantitative study of f PSA,y=3.2399 x+4.6991,linear correlation coefficient R~2=0.9986(n=3),detection limit calculation is 3.06 pg/m L.(4)The study showed that the smaller the particle size of the magnetic beads,the stronger the generated signal and the lower the detection limit.Conclusions:In this paper,a convenient and accurate universal fluorescence immune-sensing strategy was successfully established.The immunomagnetic bead technology was combined with DNA enzyme-free isothermal amplification,and avidin-biotin was used as a bridge to convert the signal of protein to fluorescence signal for quantitative detection of protein.This biosensor has a good linear range and detection limit,and shows good sensitivity in clinical sample analysis.By changing the antibody pairs,various biomarkers could be detected in the same manner.We hoped that this biosensor will provide new methods and ideas for clinical diagnosis and POCT of cancer in its early stages.