Detection Of Carbohydrate Antigen 125 In Serum Of Lung Cancer Patients By Nanomaterial Composite Modified Electrochemical Immunosensor

Objective Lung cancer is one of the malignant tumors with the highest mortality rate.Early diagnosis of cancer is of great significance for improving the quality of life of patients.However,due to the lack of obvious symptoms in the early stage of the disease and the lack of effective physical examination,most cases are not diagnosed until the advanced stage.As important indicators for early diagnosis of cancer,tumor markers have received extensive attention in recent years.Carbohydrate antigen 125(CA125)acts as a cancer-specific biomarker,due to its abnormal expression in patients,it is usually accompanied by an increase of level in serum.At present,the detection methods for CA125 mainly include enzyme-linked immunosorbent assay(ELISA),electrochemiluminescence immunoassay,radioimmunoassay and electrochemical immunosensing technology.Electrochemical immunosensing technology is a research hotspot in the field of immunosensing because of its advantages of low detection cost,short analysis time,high sensitivity and miniaturization of instruments.Based on the above characteristics of electrochemical immunosensing technology,this study aims to construct a new electrochemical immunosensor modified with nanomaterials for the detection of CA125,and to provide a new method for the detection of CA125 in serum.Methods A nanocomposite-nitrogen-doped reduced graphene oxide@carboxylated multi-walled carbon nanotube(N-r GO@CMWCNTs)was modified on the surface of glassy carbon electrode(GCE),and its high specific surface area and excellent conductivity were used to achieve the first signal amplification of the immunosensor.Secondly,the carboxyl groups(-COOH)in the nanocomposite were activated by the combined action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS).Then,the chitosan@gold nanocomposite(CS@Au NPs)was modified on the surface of GCE through the dual effects of electrostatic adsorption and chemical bonding.Because CS@Au NPs also has a high conductivity,it can achieve the second signal amplification of the immunosensor.Finally,through the formation of gold-sulfur bonds(Au-S)between the antibody and the gold nanoparticles,the CA125 antibodies were immobilized on the surface of the modified electrode,which could be used to specifically recognize the CA125 antigens.Scanning electron microscopy,raman spectroscopy,UV-vis spectrophotometer and fourier transform infrared spectroscopy were used to characterize the morphology and structure of the synthesized nanomaterials and modified electrodes.Cyclic voltammetry(CV)and differential pulse voltammetry(DPV)were used to characterize the modification process of the electrodes.In order to achieve the best condition of the immunosensor,the key conditions affecting the experimental results,such as the drop amount of N-r GO@CMWCNTs and CS@Au NPs,the incubation time and concentration of CA125 antibodies,were optimized.In addition,a series of methodological verification were carried out for the constructed immunosensor,including the detection linear range,the detection limit of the method,the precision,stability,selectivity and practicality of the immunosensor.Finally,the constructed immunosensor was used to detect the actual serum samples of lung cancer patients.Results(1)Scanning electron microscopy images,raman spectra,UV-Vis absorption spectra and fourier transform infrared spectra of the nanocomposites and the modified electrodes showed that the nanocomposites were successfully synthesized and the glassy carbon electrode was successfully modified.The SEM characterization results of N-r GO@CMWCNTs showed that N-r GO had a lamellar structure,and CMWCNTs had a tubular structure with a diameter of about 15 nm.CMWCNTs prevented the stacking of N-r GO lamellar,and the lamellar composition of N-r GO and tubular CMWCNTs was uniform.The EDS characterization results of N-r GO@CMWCNTs showed that nitrogen from urea was successfully doped and evenly distributed in the mixed carbon nanomaterials,and the nitrogen content of the nanocomposite was calculated to be 3.87%.The SEM characterization results of CS@Au NPs showed that the synthesized Au NPs were uniformly dispersed in solution with a particle size of about 50 nm.The SEM characterization results of the modified electrode show that N-rGO@CMWCNTs forms a complex three-dimensional structure on the electrode surface,and a large number of gold nanoparticles bind on the surface.The Raman spectra of N-r GO@CMWCNTs demonstrate the successful doping of nitrogen and synthesis of nanocomposites.The ultraviolet-visible absorption spectra of CS@Au NPs demonstrate the presence of gold in the solution in terms of elements.The Fourier transform infrared spectroscopy of CS@Au NPs confirms the existence of free amino groups and hydroxyl groups on the surface of CS in solution.(2)The trend of CV curves and DPV curves indicated the successful construction of the immunosensor.(3)After experimental optimization,the optimal volume of N-r GO@CMWCNTs was 4μL,the optimal volume of CS@Au NPs was 5 μL,the optimal incubation time of CA125 antibody was 12 h,and the optimal incubation concentration of CA125 antibody was 50 μg/m L.(4)Under the optimal experimental conditions,the linear range of CA125 detection by the constructed immunosensor was 1 m U/m L-100 U/m L,and the detection limit was as low as 0.4 m U/m L.The linear regression equation was Y=8.3689 lg X+33.681,and the linear correlation coefficient was 0.998.(5)The relative standard deviation(RSD)of CA125 antigen measured by different modified electrodes was ≤5%.When the same concentration of CA125 antigen was measured continuously with the same electrode,≥90% of the initial signal could still be maintained until the 10 th day.Glucose,ascorbic acid and uric acid were selected as interference substances,and their interference rates were all less than 10%.(6)Low,medium and high concentrations of CA125 antigen standard solution were added to the serum of healthy volunteers for recovery experiments.The recovery rates were 103.9%,107.7% and 94.5% respectively,and the corresponding relative standard deviations were 7.07%,4.17% and 3.88% respectively.(7)The constructed immunosensor was used to detect CA125 in the serum of 10 patients with lung cancer.The result showed that the CA125 concentrations in their serum were between 0.14 U/m L and 259 U/m L,which is consistent with the actual condition of lung cancer patients.Conclusions In this study,a glassy carbon electrode modified with N-r GO@CMWCNTs and CS@Au NPs was constructed.(1)Due to the high conductivity of N-r GO@CMWCNTs and CS@Au NPs,the combined effect of them can realize the double signal amplification of the immunosensor.Therefore,the constructed electrochemical immunosensor can detect CA125 in serum with ultrasensitive sensitivity.(2)After optimizing the key conditions affecting the experimental results,such as the amount of N-r GO@CMWCNTs and CS@Au NPs,the incubation time and concentration of CA125 antibodies,the linear range of CA125 detection in serum by the sensor was 1 m U/m L-100 U/m L,and the detection limit was as low as 0.4 m U/m L.(3)Through a series of methodological verification,the electrochemical immunosensor has good precision,high accuracy and strong anti-interference ability.At the same time,the recovery rate of the newly established method is good.(4)CA125 levels in serum of lung cancer patients were successfully detected by the new method,which provides a new method for the detection of CA125 in serum.