| Objective Lung cancer is the main cause of cancer death in China.Non-small cell lung cancer(NSCLC)is the most common form of lung cancer,accounting for about85% of lung cancer cases.Abnormal expression of cytokeratin 19 fragment antigen 21-1is closely related to the occurrence of NSCLC.In malignant lung cancer tissues,a large amount of cytokeratin is released into the blood,which leads to the increase of CYFRA21-1 serum content.Therefore,the early detection of tumor markers is of great significance for the early prevention of cancer.The purpose of this study is to construct an electrochemical immunosensor based on nanocomposite modification to detect CYFRA21-1,a lung cancer marker in serum,providing technical support for early diagnosis of NSCLC.Methods(1)Ordered mesoporous carbon(CMK-3)was combined with carboxylated multi-walled carbon nanotubes(CMWCNTs)to improve the electron transfer rate and realize electrochemical signal amplification.Combined with gold nanoparticles(Au NPs),an electrochemical immunosensor was prepared on the glassy carbon electrode(GCE).The Anti-CYFRA21-1 was incubated on the surface of the electrode,and the antibody CYFRA21-1(Anti-CYFRA21-1)and Au NPs were bound by Au-S bond.(2)The incubation time,concentration and dropping amount of antibody were optimized.(3)Under the optimal experimental conditions,the methodology indexes such as linear range,detection limit,accuracy,precision,stability and selectivity were studied and discussed.(4)The electrochemical immunosensor was applied to the determination of CYFRA21-1 in human serum.Results(1)Scanning electron microscope(SEM)images showed that CMK-3 with good ordered structure was compounded with CMWCNTs through π-π interaction.It could be observed that the Au NPs particles were packed on the CMK-3@CMWCNTs on the surface and in the voids.The results showed that Au NPs@CMK-3@CMWCNTs nanocomposites were successfully synthesized.According to the CV curve,compared with GCE,Au NPs/GCE,CMK-3/GCE,CMWCNTs/GCE and CMK-3@CMWCNTs/GCE,the CV peak current value of Au NPs@CMK-3@CMWCNTs/GCE increased significantly.The results indicated that the nanocomposite was successfully modified on the electrode surface.Compared with Anti-CYFRA21-1/Au NPs@CMK-3@CMWCNTs/GCE,the peak current value of CYFRA21-1/ Anti-CYFRA21-1/Au NPs@CMK-3@CMWCNTs/GCE was significantly higher.It was showed that,the sensor could detected CYFRA21-1 specifically by using specific antibody to capture antigen on the basis of signal amplification of various nanomaterials.(2)As a result of optimized detection conditions,When the incubation time of Anti-CYFRA21-1 increased from 4h to 12 h,the current value difference Δ I(the difference between IP measured by CYFRA21-1 and I0 measured by blank sample)gradually increased,and then decreased after 12 h.The results indicated that the incubation process of Anti-CYFRA21-1 reached the optimal state at 12 h,so the incubation time of antibody was selected at 12 h.The DPV detection of Anti-CYFRA21-1 with the concentration between 10 μg/m L and 100 μg/m L showed that ΔI firstly increased and then decreased with the increase of the concentration and reached the peak at 50 μg/m L,so the antibody concentration was selected to be 50μg/m L.The volume of different nanocomposites was recorded by adding different nanocomposites,Δ I first gradually increased and then decreased,and reached the maximum at 5 μL,so Au NPs@ CMK-3@CMWCNTs composite was selected to be 5μL.(3)Under the optimal conditions,the constructed immunosensor had a linear range of 0.5 pg/m L to 105 pg/m L for the detection of CYFRA21-1 in serum.The logarithmic linear correlation of CYFRA21-1 standard series concentration was good.The linear regression equation was y = 5.2782x+13.012,and the correlation coefficient(r)was 0.998,with a detection limit of 0.2 pg/m L.Under optimal conditions,three concentrations of CYFRA21-1(0.1 ng/m L,1 ng/m L and 10 ng/m L)were added to 2.08 ng/m L of healthy human serum for spike recovery experiment.At the same time,it can remain stable for 13 consecutive days.Ascorbic acid(AA),uric acid(UA),and glucose(GLU),which were common components in serum,were selected as interferers to investigate their interference to the detection results.The interference experiment of 1:100 diluted serum was carried out,and the selectivity was better.In this study,CYFRA21-1 with 0.1ng/m L,1ng/m L and 10ng/m L was selected as the second method.The average values were taken for 6 times in each experiment.The recoveries ranged from 92.3% to 104.5%.The relative standard deviation(RSD)was less than 6.08%(n = 6),and the recoveries were more than 90%,showing the good reliability of the electrochemical immunosensor.(4)The constructed method was applied to the detection of 15 human serum samples(10 lung cancer patients and 5 healthy people).The concentration range of CYFRA21-1 in lung cancer patients was 4.75~44.60 ng/m L,which was higher than that in healthy people.The results showed that the electrochemical immunosensor can be used to detect the lung cancer biomarker CYFRA21-1 in the serum of patients with lung cancer.Conclusion In this study,a new method based on nano-composite modified electrochemical immunosensor was established to detect CYFRA21-1,a lung cancer marker in serum.The method has good selectivity,high sensitivity and reliable detection results. |
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