Study On Rapid Detection Method Of SPD-L1 And AFP In HCC Patient Serum Based On Graphene Oxide Special Fiber Bragg Grating Biosensor

Hepatocellular carcinoma（HCC）accounts for more than 90%of primary hepatic carcinoma,which is the most common type of primary hepatic carcinoma.HCC is the fifth largest cancer worldwide and the third most deadly cancer in the world.HCC has a poor prognosis and a high risk of recurrence,which is particularly important for early diagnosis of HCC.Alpha-fetoprotein（AFP）has been widely used as a marker for early diagnosis of HCC for many years.However,the sensitivity of AFP in the diagnosis of HCC is only 60%to 70%,and its clinical diagnostic value is extremely limited.In recent years,lots of studies have shown that the soluble programmed death factor ligand 1（sPD-L1）is significantly different in the serum of hepatocellular carcinoma patients and healthy people,which indicate that sPD-L1 could be the auxiliay serum marker for the clinical diagnosis of HCC.A large number of studies have confirmed that combined detection of HCC markers can significantly improve the sensitivity and diagnostic accuracy.At present,the detection methods of serum markers mainly include enzyme linked immunosorbent assay（ELISA）,Immune colloidal gold technique,protein chip technique,chemiluminescence immunoassay,etc.ELISA is time-consuming and strenuous,and the results are easily influenced by external factors.Immune colloidal gold technique has low sensitivity,it is qualitative,not quantitative,protein chip technique requires high quality laboratory equipments,and equipments are expensive.Chemiluminescence immunoassay consumes few samples,is easy to operate,and the detection results are relatively accurate.However,it requires patients to bear high detection costs.Therefore,it is of great significance to explore efficient,specific,accurate and low cost detection technology for the rapid diagnosis of HCC.At present,the serological detection of HCC markers mainly depends on the antigen-antibody response,but the quality of domestic HCC serum marker antibody products is uneven,and the price of imported products is expensive.The preparation of high efficiency,high specificity and low cost antibodies to HCC serum markers can provide an effective reagent for the early and rapid diagnosis of HCC.Excessively tilted fiber grating（Ex-TFG）biosensor has advantage of high detection sensitivity,specificity,no tags,low cost,which can realize real-time detection.Ex-TFG biosensor has been successfully applied in the detection of heart failure biomarker NT-proBNP,porcine circovirus type 2（PCV2）,Newcastle disease virus（NDV）,avian influenza virus and procalcitonin（PCT）.We Combined the specificity of antigen-antibody response with the sensitivity of FBG sensor detection,a breakthrough has been made in the sensitivity of detection,and high-level research papers has been published.In this paper,the Ex-TFG biosensor developed earlier was further improved to improve the sensitivity,stability and specificity of the detection.In this study,prokaryotic expression system was used to prepare serum marker proteins sPD-L1 and AFP.BALB/c mice were immunized with sPD-L1 by long-term immune method.Anti-sPD-L1 monoclonal antibody（mAb）was prepared by polyethylene glycol（PEG）cell fusion technology.It has provided an effective reagent for early rapid diagnosis of HCC.Gold nanoshells（AuNS）and graphene oxide（GO）were coated on the surface of the Ex-TFG after hydroxylation and silanization,and activate the carboxyl group（-COOH）on the surface of GO.In order to bind the antibody by a-NH-CO-covalent bond.After the blank spots on the grating surface were closed with skim milk powder,different concentrations of antigens were detected.The repeatability,specificity and clinical sample detection of the sensor were verified.Thus,a rapid detection technique for serum markers based on Ex-TFG biosensor was initially established,which provides a new detection method for the rapid diagnosis of liver cancer.The research contents of this paper are as follows:（1）According to the human PD-L1 gene sequence published by GenBank（GenBank registration number:AY254342.1）and AFP gene sequence（GenBank registration number:NM₀01134.2）,the codon preference of escherichia coli was optimized after sequence analysis to make it suitable for escherichia coli expression system respectively.Full-length target genes sPD-L1 and AFP were synthesized by chemical synthesis respectively.And connect them to plasmid vector pET28a（+）respectively.The recombinant plasmids sPD-L1-pet28a（+）and AFP-pet28a（+）were constructed.The recombinant plasmid was transformed into E.coli competent cell BL21（DE3）for induction and expression respectively,and the induction expression conditions were optimized.Affinity chromatography of the target protein was performed by nickel column,the purity,concentration and specificity of the purified recombinant protein sPD-L1、AFP were identified by SDS-PAGE,BCA and Western blot.（2）The recombinant protein sPD-L1 prepared above was mixed with adjuvant and used as immunogen for long-term immunization of BALB/c mice.Splenic cell suspension was prepared from spleen of mice after 4 times of immunization,mixed them with SP/20cells.Cell fusion was performed by PEG method.Indirect ELISA was used to screen the fused and subcloned cells for more than two rounds.The screened monoclonal positive cell lines were expanded and injected into the abdominal cavity of BALB/c mice to produce ascites monoclonal antibodies.The antibody subtypes of monoclonal cell lines were identified.The ascites monoclonal antibody was purified by Protein A column,and detected by SDS-PAGE,BCA,Western blot and ELISA.（3）The grating was cleaned with 5%dilute HNO₃,anhydrous ethanol and RO water,0.2M NaOH was soaked to make the surface of the optical fiber filled with-OH in order to achieve hydroxylation.The hydroxylated grating region was immersed in an appropriate1%MPTMS to be filled with-SH in order to achieve silanization.The centrifugally treated gold nanoshells（AuNS）were coated with the grating surface overnight.After amination of the gold nanoshell,200uL of GO dispersion was soaked overnight in the grating region.The carboxyl group（-COOH）on the surface of GO was activated by EDC and NHS.sPD-L1monoclonal antibody and AFP monoclonal antibody were modified respectively,and the grating was sealed with 5%skim milk powder.Different concentrations of sPD-L1 antigen and clinical serum were detected,moreover the specificity of the biosensor were evaluated.Results:（1）The recombinant plasmids pET28a（+）-sPD-L1 and pET28a（+）-AFP showed obvious specific bands at 668bp and 1875bp respectively after enzyme-digestion identification,the sequencing results were consistent with the known corresponding sequences,indicating that the recombinant plasmids were successfully constructed.The target protein has the highest expression when the recombinant strain pET28a（+）-sPD-L1/BL21（DE3）was induced at 37℃,0.5mmol/L IPTG,and 9h.the precipitation expression level of recombinant proteins sPD-L1 and AFP were higher than that of supernatant which was identified by SDS-PAGE.After nickel column affinity chromatography,the target proteins sPD-L1 and AFP has above 90%purity and their concentration can reach945.7ug/mL and 191.1ug/mL respectively.Western blot analysis showed that the target proteins sPD-L1 and AFP could bind well with their corresponding market monoclonal antibodies,and showed specific bands of about 28KD and 75KD.（2）Cell fusion trial indicated the fusion rate was 86.7%,and the positive rate was 80.4%.After two rounds of subclonal screening,a total of 4 monoclonal cell lines were obtained,which were named 1-3H,2-2G,2-9G and 1-10C,respectively.The identification results of monoclonal antibody subtypes showed that 1-10C,2-2G were IgG2b however 2-9G was IgG2a.SDS-PAGE showed that the purified ascites monoclonal antibody showed bands at the positions of 50KD and 25KD,which were consistent with the heavy and light chains of IgG antibody.After concentration determination by BCA method,the concentration of purified ascites monoclonal antibody was high,reaching 2.064mg/mL.ELISA showed that the titer of 2-2G and 2-9G ascites monoclonal antibody could reach more than1:2048000.After repeated cryopreservation,resuscitation and multiple passage,all the four hybridoma cell lines could secrete highly effective antibodies,and the obtained monoclonal antibody was proved to be specific to sPD-L1 antigen by western-blot identification.（3）The optical fiber surface of Ex-TFG was modified by hydroxylation,silanization,gold nanoshell coating,amination,go coating,activation of carboxyl group,modification of sPD-L1 antibody,and sealing,the results showed that the wavelength redshifts of each step were 0.235nm,0.255nm,0.866nm,0.006nm,0.64nm,0.076nm,0.3nm,and 0.04nm.The results show that all the steps were ideal.The lowest limit of detection of sPD-L1 antigen by the biosensor is 1pg/mL.The detection range is 1pg/mL-10ng/mL.The Ex-TFG was modified by hydroxylation to activation of carboxyl group,then modified AFP antibody and sealing,the results showed that the wavelength redshifts of each step were0.225nm,0.047nm,0.832nm,0.736nm,1.152nm,1.168nm,1.408nm,1.68nm,the results show that all the steps were relatively ideal.The detection range of AFP antigen by the biosensor is 5pg/mL-10ng/mL.Specific experiments showed that different concentrations of BP26protein,the serological marker of brucellosis,had no specific reaction,the result indicated the good specificity of the sensor.The Immunosensor was developed to detect AFP and sPD-L1 level in the serum of HCC patients and healthy people.the results show that the resonant wavelength offset of HCC patients serum and healthy serum between two markers exist significant differences in the test,and AFP and sPD-L1 level in patients serum appeared certain positive correlation in the same test,which show that the sensor has high specificity in the detection of AFP and sPD-L1.It can meet the requirements of clinical tests.Conclusion:In this study,sPD-L1 and AFP gene engineering bacteria were constructed,and the sPD-L1 and AFP proteins were successfully expressed in the prokaryotic expression system efficiently.Four hybridoma cell lines which can stably secrete anti-sPD-L1monoclonal antibodies were obtained by long-term immunoassay,PEG cell fusion and limited dilution subcloning.Using the prepared antigens and antibodies,a rapid detection method of AFP and sPD-L1 based on the Ex-TFG biosensor was established.The sensitivity,repeatability,specificity,and clinical applications of the sensor were tested.This method has the advantages of high sensitivity,high specificity and good repeatability which can be used to detect AFP and sPD-L1 in serum samples,it has provided a new detection technology and means for the diagnosis of HCC.