Setting Up A Novel Protein Biochip For Concurrent Detecting Serum Biomarkers Of Liver Cancer And Evaluating The Clinical Diagnostic Values

Background: Hepatocellular carcinoma(HCC)is a primary liver cancer with high morbidity and mortality,and is one of the most common malignant tumors in the world,which is the main histological subtype of liver cancer.Although histopathological diagnosis and imaging diagnosis of liver cancer are the gold standard and sensitive detection methods,they are not only expensive,complex and harmful to patients,but also unable to be applied to large-scale population screening.Alpha-fetoprotein(AFP)is the most commonly used serum biomarker for HCC screening,but its sensitivity and specificity are not ideal.In recent years,numerous evidences have shown that the combination of multiple biomarkers for HCC can effectively improve the diagnostic value of HCC,and five serum biomarkers including AFP,AFP-L3(Lens culinaris-agglutinin-reactive fraction of AFP),GPC3(Glypican 3),GP73(Golgi protein73)and OPN(Osteopontin)have shown promising diagnostic performance.Objective: Constructing a novel biochip platform using the coupling agent N,N-carbonyldiimidazole(CDI)and mixed sulphur acid for the evaluation of multiple biomarkers and high-throughput screening of HCC.The advantage of this modification is that it could construct a suitable cross-linking environment between the modified surface and the protein and reduce steric hindrance to the greatest extent.Methods: Firstly,16-mercaptohexadecanoic acid and 6-mercaptohexanoic acid were used to form self-assembled monolayer(SAM)on the surface of the gold chip,then zero length coupling agent CDI was used to modify and activate the carboxyl in the end of sulphur acid.Thus,reactive intermediates were formed,which connected with the aminos of antibodies and formed stable amide bond.To optimize the immobilization of anti-biomarker antibodies on the chip and the detection efficiency,we conducted quality control explorative experiments on the temperature of reaction environment,the optimal concentration of captured antibodies and detected antibodies on the chip.Finally,we used the optimal conditions obtained from the above exploratory experiments to carry out the simultaneous detection of all sera with the novel biochip.We determined the standard workflow of protein microarray for simultaneous detection of serum biomarkers in HCC patients and control population based on the optimal conditions obtained from quality control trials.Results: The chip modified with mixture sulphur acids and CDI was successfully constructed,and the ambient temperature suitable for simultaneous detection of HCC biomarkers on the chip was 25℃,and the optimal concentration for immobilization of capture antibodies and detection antibodies were all 50μg/m L.In addition,we found that the serum dilution of 1:8-1:20 could reach the optimal standard,and 1:10 was finally selected.The results of sera detection showed that GPC3 has the highest diagnostic value within the five biomarkers.The Area under ROC curve(AUC)of GPC3 was 0.903,which was higher than AFP(0.826),AFP-L3(0.74),GP73(0.759)and OPN(0.633),and the sensitivity and specificity were 87.5% and 87.5% respectively.Among the double biomarkers panels,AFP+AFP-L3 showed the best performance with AUC 0.980,Youden index 0.83,sensitivity 89.60% and specificity 93.75%.Among the thiple biomarkers panels,AFP+AFP-L3+GPC3 had the highest Youden index(0.987),sensitivity(93.80%)and specificity(93.75%),which was the best triple biomarker panel.AFP+AFP-L3+GPC3+GP73 revealed best value in all four-biomarker panels,with Youden index,sensitivity and specificity of 0.91,94.80% and 96.25%,respectively.When all five biomarkers were combined,the Youden index,sensitivity and specificity were 0.93,96.90% and 96.25%,respectively.Conclusion: The gold biochip based on CDI and mixed sulphur acid has good protein coupling ability and high detection sensitivity,which can be successfully applied to the synchronous detection of five biomarkers for HCC.Simultaneously,among the five HCC biomarkers AFP,AFP-L3,GPC3,GP73 and OPN,GPC3 revealed the highest diagnostic value.When in consideration with combined biomarkers,the panel of AFP+AFP-L3+GPC3 conferred the most appropriate combination mode with sensitivity and specificity of more than 90%.When more than three biomarkers were combined,the increase in diagnostic value would reach a plateau.The novel biochip could become a potential tool for large-scale screening of people at high risk of liver cancer.