| Liver cancer is one of the most common cancers in the world and is a common malignant tumor of the digestive system.It is difficult to detect in the early stage.Once it is found,it is mostly in the late stage and the mortality rate is extremely high.Therefore,early diagnosis and early treatment are particularly important.Tumor marker and genetic detection can be used for early screening of cancer.Gene CYP1A1 is closely related to the occurrence of cancer.Tumor marker alpha-fetoprotein(AFP)is also one of the important indicators of liver cancer screening.Therefore,detection of these two substances is of great significance.The traditional SNP detection method relies on gel electrophoresis or gene sequencing technology,which has high requirements on instrument and limits its application.The widely used AFP detection methods include enzyme-linked immunosorbent assay,fluorescent immunoassay,mass spectrum etc.These methods are highly sensitive,can achieve rapid,accurate and semi-quantitative detection of tumor markers in serum.However,the instrument is expensive,requires professional personnel to operate,and the detection cost is high and real-time detection cannot be realized.Therefore,exploring the real-time detection method has research value.Colloidal gold chromatographic strip is a colorimetric-based detection method that utilizes the color depth of the colloidal gold to achieve semi-quantitative detection with naked eyes.Colloidal gold chromatographic strip is widely used in medical diagnosis,environmental monitoring and food safety detection due to their simple operation,rapid detection,low application cost and and can be used for real-time detection.Enzyme signal amplification technology is an excellent signal amplification method,which can improve detection sensitivity by utilizing the catalytic effect of enzyme.Horseradish peroxidase(HRP)can catalyze the substrate 3-amino-9-ethylcarbazole(AEC)to generate red insoluble substances.HRP was labeled on the surface of colloidal gold,and the color signal could be amplified by its catalytic reaction.In view of the shortcomings of traditional SNP and AFP detection methods,such as expensive instruments,complicated operation and inability to meet the requirements of real-time detection,and the absence of joint detection of susceptible gene and tumor marker,a strip biosensor for simultaneous detection of SNP and AFP was established by combining HRP with colloidal gold chromatographic strip.And the discrimination of SNP locus and AFP content in serum were realized.The contents mainly include:(1)A test strip biosensor capable of simultaneously detecting cancer susceptibility gene CYP1A1 and AFP has been established.There were two detection lines in the strip biosensor,one was used for qualitative detection of susceptible gene by base complementary pairing,and the other was used for semi-quantitative detection of AFP by antigen-antibody reaction.Finally,signal amplification was achieved by HRP catalyzing substrate AEC.(2)The feasibility of visual detection of SNP locus and AFP content in colloidal gold test strips was verified,reaction conditions were optimized,and linear range of the detection method was determined.The method was successfully applied to the detection of SNP and AFP in real sample..(3)Results showed that under the optimized conditions,the strip biosensor had a good linear relationship(R~2=0.998)when AFP concentration was in the range of 20-200ng/mL,and the other hundred times of proteins had no obvious interference to the detection,indicating that the method presented good selectivity.The detection of susceptibility gene CYP1A1 and AFP in real blood sample by the strip was in accordance the standard method,and the spiked recovery was between 90%and 110%,which showed that the method had good accuracy and could be used for qualitative judgment of SNP locus base type of susceptibility gene and semi-quantitative detection of tumor marker in actual sample,so it had a good application potential. |
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