Joint Detection Of Early Hepatocellular Carcinoma Markers Based On Surface Plasmon Resonance

Hepatocellular Carcinoma(HCC)is now the second leading cause of cancer death in China,of which mortality as well as morbidity have always been showing an ascending trend.Early diagnosis of HCC can improve the thorough therapeutic effects and the prognosis of this disease and consequently,the overall welfare of HCC patients.However,the efficiency,accuracy and sensitivity of the traditional detection method are not high enough for the early diagnosis of HCC.In this thesis,a joint detection method of early HCC markers based on surface plasmon resonance(SPR)is presented.Alpha-fetoprotein(AFP)and miRNA-125 b were jointly used as the markers,thus the sensitivity and specificity of early diagnosis of HCC were improved explicitly.For the high sensitive and specific joint detection of AFP and miRNA-125 b,the following work has been completed.Firstly,an SPR-based Biacore 3000 system was implemented.Through the combination of its single-channel modification and four-channel serial measurement,only a single trace amount of human serum is required for the quantitative detection of both of the two markers.At the same time,the Flow cell(FC)1 was selected as the reference to eliminate the interference of background light and other environmental factors.Secondly,a CM5 chip attached by a matrix of carboxymethylated dextran was implemented as the sensor.And the biological probes of AFP and miRNA-125 b were covalently modified on its gold surface to eliminate the interference from the complex components(e.g.proteins,glucose,hormones)in human serum and realize the specific detection of AFP and miRNA-125 b.The DNA probe complementary to miRNA-125 b base pairing was fixed in the sensing area of FC2.And the monoclonal antibody against AFP was fixed in FC3’s sensing area.Experimental results show that both surface modifications provided specificity for the two markers detection.But for the detection of the two markers in the human serum,the sensitivity of this direct detection method was required to be enhanced.Thirdly,to achieve a low detection limit of AFP and miRNA-125 b,Double Antibody Sandwich Method(DASM)and S9.6 antibody enhanced method were implemented to enhance the SPR signal and detection sensitivity of the two markers respectively.Experimental results show that AFP(0-400ng/mL)can be accurately detected by DASM and the lowest concentration of miRNA-125 b detected by the S9.6 antibody enhanced method is 200 pM.Lastly,the joint detection method of AFP(0-400ng/mL)and miRNA-125b(0-1000pM)was tested using modified SPR sensor and verified for the early diagnosis of HCC.