Highly Sensitive And Selective Detection Of Fibrin Based On Bifunctional DNA Nanozyme

With the development of science and technology,cancer,as one of the major diseases threatening human life and health,has gradually became a controllable and curable‘chronic disease'.Then the early detection of cancer plays a vital role in the treatment of cancer.Since fibrin deposits in the tumor cells,which leads to tissue embolism and injury,and even provides a bridge for the growth and metastasis of cancer.Therefore,fibrin can be used as a tumor marker to realize the early diagnosis of cancer.In this work,a new high sensitivity and high selectivity approach for the detection of fibrin was developed using bifunctional DNA nanostructures,which was mainly divided into three parts:1.Highly sensitive detection of fibrin based on double-stranded DNA hemp string reduced background signalIn this paper,due to reducing the function of background signal by double stranded DNA hemp string,a novel,highly sensitive and selective method for the detection of fibrin was developed,combined with the specific recognition of homing peptide CREKA.First,Double-stranded DNA was designed to form a G-quadruplex structure connecting with hemin to obtain G-quadruplex-hemin DNAzyme at the end,which catalyze luminol to generate chemiluminescence signals.And the other end of the double-stranded DNA connects to the homing peptide CREKA,which could recognize fibrin.Because its double-stranded structure hardly generate non-specific adsorption,which reduces the background signal and improves the sensitivity of the system.Under the optimal experimental conditions,the linear regression equation of the system is I = 4071.14 +64.68 c(c is the concentration of fibrin,p M),The assay showed a linear toward fibrin concentration in the range of 1.12 p M to 112 p M with a correlation coefficient of 0.9981.The limit of detection of fibrin was experimentally determined to be 0.085 p M.2.High sensitivity detection of fibrin using double stranded DNA nanowires through hybrid chain reaction.In order to improve the sensitivity of the method,on the basis of the previous work,a new approach of high sensitivity detection of fibrin was developed,combined with hybrid chain reaction(HCR).First,the DNA hairpins H1 and H2 were designed,in which part of the DNA sequence could be hybridized and the terminal sequence can be folded to form G-quadruplex structure.Thus,double-stranded DNA nanowires with multiple G-quadruplex structures were formed by hybridization chain reaction.The probe molecules could recognize fibrin and catalyze the luminol to generate strong chemiluminescence signals.Under the optimal experimental conditions,the linear regression equation of the system is I = 5863.36 + 125.71 c(c is the concentration of fibrin,p M),The assay showed a linear toward fibrin concentration in the range of 0.14 p M to 72.5 p M with a correlation coefficient of 0.9993.The limit of detection of ds DNA was experimentally determined to be 0.012 p M.Compared with the previous work,the amount of reagents was reduced,and the sensitivity was improved.3.High selective detection of fibrin by Sandwich-like enzyme-linked immunoassay assayIn this work,the fibrin antibody was coated on the enzyme-labeled plate to conduct primary selection of fibrin.In the presence of fibrin,the fibrin antibody interactd with fibrin.The remaining protein was eluted through the plate washing process.The double-stranded DNA probe using the homing peptide CREKA as the recognition molecule performs secondary selection of fibrin.Through these two selections,the selectivity of the system for detecting fibrin was greatly improved.Under the optimal experimental conditions,the linear regression equation of the system is I = 3399.39 + 62.33 c(c is the concentration of fibrin,p M),The assay showed a linear toward fibrin concentration in the range of 0.87 p M to 145 p M with a correlation coefficient of 0.9991.The limit of detection of ds DNA was experimentally determined to be 0.061 p M.