Application Of Resonance Rayleigh Scattering Spectra In Detection Of Tumor Markers And Tumor Cells Of Esophageal Carcinoma

According to the National Cancer Center's statistical research,China is currently the country with the highest incidence and mortality of esophageal cancer in the world.The Taihang Mountains in Shanxi,Henan,and Hebei provinces are high-risk areas for esophageal cancer in China,and more than 90%of these cases belong to the esophageal squamous cell carcinoma?ESCC?.Early detection and treatment are effective way to reduce the mortality of esophageal cancer,so the early diagnosis of esophageal cancer is of great significance.Traditional digestive tract endoscopy and pathological examination have been ineffective in detecting early esophageal cancer,and the development of early cancer screening technology based on tumor markers and tumor cells has become a research hotspot in recent years.The study of functionalized gold nanoparticle biosensors offers new opportunities for the diagnosis and treatment of cancer.Owing to its excellent characteristics in the recognition and detection of biological macromolecules,Resonance Rayleigh scattering?RRS?technology combined with functionalized gold nanoprobes has shown good application prospects in the detection of tumor markers and tumor cells.In this paper,ESCC tumor markers epidermal growth factor receptors EGFR and HER2 were used as targets.Gold nanoparticles were modified with antibodies and aptamers to obtain functionalized gold nanoprobes,and the specific recognition of targets by probes was performed.And the resulting RRS spectral signal changes,enabling quantitative detection of ESCC tumor markers and tumor cells.The specific research contents and results are as follows:1.Antibody immobilized cysteamine?Cys?functionalized gold nanoparticles?AuNPs?was proposed as immunosensor,and of resonance Rayleigh scattering?RRS?for detection.First,Cys stabilized AuNPs?Cys-AuNPs?was prepared by the reduction of chloroauric acid with sodium borohydride in the presence of Cys.Further,anti-EGFR antibody?Cetuximab,C225?was covalently linked to the Cys-AuNPs by carbodiimide-mediated amidation protocol to yield the C225-AuNPs immunoprobe.Based on the specific binding of C225 to EGFR,a RRS method was established to determine the concentration of EGFR.Under the optimal conditions,the concentration of EGFR is related to the intensity of RRS in the range from 30.0-130.0 ng·mL^-1with a low detection limit of 4.0 ng·mL^-1.Meanwhile,the proposed immunosensor exhibited excellent selectivity and anti-interference property.The method was applied to the determination of EGFR in human serum and cancer cell lysate samples with satisfactory results.2.Reduction of chloroauric acid with sodium citrate to obtain gold nanoparticles?AuNPs?,thiol-modified anti-EGFR aptamer?Apt?covalently linked to the surface of AuNPs via Au-S bond to obtain functionalized nanoconjugate probe.The binding of EGFR to the Apt-AuNPs probe resulted in a remarkable enhancement of the RRS.The scattering enhancement??I?is linear to the EGFR concentration in the range of 30.0-110.0 ng·mL^-1 with a limit of detection of 0.7 ng·mL^-1.The applicability of the developed method in the detection of EGFR in esophageal cancer cell lysates and human serum samples was experienced.The mechanism of selectivity and the reasons for RRS enhancement have been discussed.3.Gold nanoparticles?AuNPs?were modified by anti-EGFR antibody?Ab?and anti-EGFR aptamer?Apt?to obtain multifunctional Apt-AuNPs-Ab nanoconjugate immunoprobe.The probe is specifically combined with EGFR to form large volume binding products that exhibited a remarkable enhancement of the RRS intensity,and to appear characteristic RRS peak at 312 nm.The scattering enhancement??I?is linear to the EGFR concentration in the range of20.0-100.0 ng·mL^-1 with limit of detection of 0.1 ng·mL^-1.The immunoassay was successfully tested and validated by using ESCC cell Eca109 cell lysates,as well as human serum samples.The experimental results show that the Apt-AuNPs-Ab nanoprobe has higher selectivity and sensitivity,and is more suitable for RRS detection of low concentration EGFR.4.Using the ESCC cell Eca 109 as a model,and the multifunctionalized Apt-AuNPs-Ab probe was used to bind to cells and perform RRS assay.The results indicate that the probe is highly specific for the EGFR protein on the cell surface,and thus binds to cells.The binding system between probe and cell has specific RRS spectral characteristics,and the cell concentration is positively correlated with the RRS signal intensity.The detection range of Eca109 is between 1.0×10²-5.0×10⁵ cell·mL^-1,and the detection limit is up to 20 cell·mL^-1.The method has been successfully applied to the detection of Eca109 cells in human serum samples,and has certain application value in the detection of low concentration cancer cells in early stage of esophageal cancer.5.In this part,we synthesized two aptamer-modified gold nanoparticles?Probe I and Probe II?that separately target two types of esophageal cancer markers:EGFR and HER2.Three different types of ESCC cells:Eca109?EGFR?+??,KYSE510?HER2?+??,and KYSE150?EGFR?+?and HER2?+??were used to interact with the probe,respectively,and the RRS assay was performed.The results showed that Probe I+Probe II can quantitatively detect the above three cells with detection limits of 15 cell·mL^-1?Eca109?,18 cell·mL^-1?KYSE510?and 12 cell·mL^-1?KYSE150?.Based on this,a new quantitative analysis strategy for RRS of low-concentration esophageal cancer cells was proposed.