Study On The ELISA-like Method Of Detection For Fibrin Based On Homing Peptide

Cancer is considered one of the most deadly diseases in the world.It is widely believed that early detection of cancer is the most effective way to reduce the mortality rate in solid cancers.Therefore,diagnosing cancers at an operable stage is desirable.Early identification and detection of tumor markers is critical for the treatment of cancer patients and their survival rate.Tumor marker is a protein that is usually found in the blood or urine when the cancer is present.They can be products of the cancer itself or the body's response to cancer or other diseases.Fibrin is the final product of the blood coagulation cascade.Fibrin clots are not formed under normal conditions.They are usually accompanied by several pathological conditions,such as cardiac or cerebral infarction,injuries,acute inflammation,cancer invasion and metastasis.It has been found that fibrin is deposited in the stroma of many tumors.Therefore,the fibrin can be used as an important marker for the diagnosis and monitoring of some tumors.In this work,the tumor marker fibrin was used as the research object,and the homing peptide CREKA has specific affinity towards fibrin.Several homing peptides based on ELISA-like were developed for the detection of fibrin with high sensitivity and selectivity.The paper is divided into the following four parts:1.Selective and sensitive determination of fibrin using functionalized gold nanoparticlesA new ELISA-like method was established for the detection of fibrin by ABTS-H₂O₂system,which realized the rapid and specific detection of fibrin.The streptavidin?SA?coated gold nanoparticles?SA-AuNPs?were used as the basis of functional probes.Biotin-modified G-quadruplex-hemin DNAzyme and CREKA were conjugated with SA-AuNPs with the interaction of biotin and avidin.The above complex was added and interacted with fibrin.Under optimized experimental conditions,the assay showed a linear toward fibrin concentration in the range of 0.056-16.8 nM with a correlation of 0.9971.The regression equation was A=0.0956+0.0056 c?c is the concentration of fibrin,nM?.The limit of detection for fibrin was experimentally determined to be 0.014 nM.2.Homing peptide based ELISA-like chemiluminescence method for selective and sensitive determination of fibrinA new ELISA-like method was established based on CREKA-specific recognition of fibrin.The G-quadruplex-hemin DNAzyme with peroxidase activity was coupled to CREKA to form the DNAzyme-CREKA complex.The above complex as a probe was added into the plates and interacted with fibrin.The chemiluminescent signal is directly used for quantitative analysis of fibrin by luminol-H₂O₂ system.This method has high sensitivity and easy operation.Under optimized experimental conditions,the assay showed a linear toward fibrin concentration in the range of 0.112-5.6 pM with a correlation of0.9975.The regression equation was I_CL=10028.0+1690.9 c?c is the concentration of fibrin,pM?.The limit of detection for fibrin was experimentally determined to be 0.056pM.3.Highly sensitive detection of fibrin based on ELISA-like method of rolling circle amplificationThis work used RCA technology to produce long and repetitive G-quadruplex domain chains based on the characteristic that DNA can be easily replicated.Each G-quadruplex portion was conjugated with hemin to form G-quadruplex-hemin DNAzyme,which achieves a large enrichment of DNAzymes.The above complex as a probe was added to added into the plates and interacted with fibrin.The self-amplification mechanism of this DNAzyme,combined with the signal amplification mechanism of the catalytic reaction luminol-H₂O₂ system,which improves further the sensitivity of detecting fibrin.Under optimized experimental conditions,the assay showed a linear toward fibrin concentration in the range of 1.68-168 fM with a correlation of 0.9975.The regression equation was I_CL=15456.6+93.6 c?c is the concentration of fibrin,fM?.The limit of detection for fibrin was experimentally determined to be 0.56 fM.4.Highly sensitive determination of fibrin using functionalized gold nanoparticles and rolling circle amplificationWe present a robust,sensitive,ELISA-like method that utilizes rolling circle amplification?RCA?and gold nanoparticles?AuNPs?as a signal enhancement method.In this work,the gold nanoparticles were conjugated with CREKA and rolling circle amplification primer DNA.A large number of repetitive G-quadruplex domains can be obtained by RCA technology,which combined with hemin to achieve enrichment of DNAzyme.The above complex as a probe was added into the plates and interacted with fibrin.The content of fibrin can be determined indirectly via luminol-H₂O₂ reaction system.Under optimized experimental conditions,the assay showed a linear toward fibrin concentration in the range of 1.4-112 fM with a correlation of 0.9964.The regression equation was I_CL=52807.5+622.6 c?c is the concentration of fibrin,fM?.The limit of detection for fibrin was experimentally determined to be 0.84 fM.