Studies On Electrochemiluminescence Immunoassay Methods Based On Graphitic Carbon Nitride For Detection Of Tumor Markers

Electrochemiluminescence?ECL?immune analysis technology,combines the technique of electrochemical luminescence and immunoassay technology,which can improve the biological protein molecule detection sensitivity and selectivity significantly.This paper around the rapid detection of tumor markers and major requirements,building new type ECL sensor based on carbon nitride materials to realized the sensitive analysis of tumor markers.This thesis research work mainly includes the following sections:1.One-step preparation of disposable multi-functionalized g-C₃N₄ based electrochemiluminescence immunosensor for the detection of CA125A novel,disposable and label-free electrochemiluminescence?ECL?immunosensor,based on multi-functionalized graphitic carbon nitride?g-C₃N₄?coated on the one-off screen-printed carbon electrodes?SPCEs?,for the detection of tumor marker carbohydrate antigen 125?CA125?was developed.The multi-functionalized gC₃N₄ was prepared with amino-coated Fe₃O₄ nanoparticles and CA125 antibody?antiCA125?chemically bound to the surface of carboxylated g-C₃N₄ simultaneously.The Fe₃O₄ nanoparticles ensured the presence of multi-functionalized g-C₃N₄ in the outer Helmholtz plane?OHP?of the ECL immunosensor and even becoming part of the electrode surface.This assembly promoted the electron transfer between g-C₃N₄ and the electrode resulting in greatly enhanced ECL intensity.The CA125 was selectively captured by anti-CA125 leading to effective inhibition of the ECL signal.Under optimal conditions,this disposable ECL immunosensor exhibited a wide dynamic linear detection range for CA125?0.001 –5 U/m L?with a lower detection limit of 0.4 m U/m L,providing high sensitivity.In addition,the immunosensor also exhibited high specificity,good reproducibility and long-term stability.All these properties offer great promise for rapid,simple,selective and effective detection of CA125 in clinical applications.2.An “in-electrode”-type immunosensing strategy for the detection of squamous cell carcinoma antigen based on electrochemiluminescent Au NPs/g-C₃N₄ nanocompositesA novel “in-electrode”-type electrochemiluminescence?ECL?immunosensor for the sensitive detection of squamous cell carcinoma antigen?SCCA?was constructed using magnetic graphene oxide?nano Fe₃O₄@GO?and Au nanoparticles/graphiticphase carbon nitride?Au NPs/g-C₃N₄?.The capture probe was prepared by immobilizing the primary antibody of SCCA?Ab 1?on the nano Fe₃O₄@GO,while the Au NPs/g-C₃N₄ nanocomposites labelled the secondary antibody of SCCA?Ab 2?,which acted as a signal tag.The recognition scaffold was the following: the capture probe was immobilized onto the magnetic electrode surface that caught the target SCCA and finally allowed the immobilization of the signal tag via the interaction between antigen and antibody.Importantly,a high ECL signal could be obtained due to the unique immunocomplex,which ensured all of the g-C₃N₄ on the outmost plane were directly fixed onto the electrode surface and became part of the electrode surface.This resulted in an enhanced efficiency of the g-C₃N₄ for electrochemical luminescence,thus extending the outer Helmholtz plane?OHP?of the proposed electrode and leading to high sensitivity.Taking advantage of both nano Fe₃O₄@GO and Au NPs/g-C₃N₄,the ECL intensity was found to increase logarithmically with SCCA concentration in a wide linear range from 0.001 to 10 ng/m L and with a detection limit of 0.4 pg/m L.The proposed “in-electrode”-type ECL immunosensor was used to analyse SCCA in human serum,and satisfactory recoveries were obtained,indicating that the proposed method was promising for practical applications in the clinical diagnosis of SCCA.3.Potential-resolution “in-electrode” type electrochemilunescence immunoassay based on Au nanoparticles functionalized g-C₃N₄ nanosheet and Ru?bpy?32+ for simultaneous detection dual-targetsHere,a novel potential-resolved “in-electrode” type electrochemiluminescence?ECL?immunosensor was fabricated based on two different types of luminant Ru-NH₂ and Au NPs/g-C₃N₄ to realize simultaneous detection of dual targets.In this strategy,anti-CA1251 and anti-SCCA1 were immobilized on bare gold electrode as capture probes,which could catch the two corresponding target CA125 and SCCA,and the immobilization of the signal tags was all owed via the interaction between antigen and antibody.In this process,?Ru&anti-CA125₂?@GO and anti-SCCA₂-Au NPs/g-C₃N₄ could exhibit two strong and stable ECL emissions at 1.25 V and-1.3 V respectively,which could be used as effective signal tags.Taking advantage of “in-electrode” type ECL immunosensor,all the electrochemiluminophores near the outer Helmholtz plane are “effective” in participating in the electrochemical reactions and emitting ECL signals.Therefore,the dual targets CA125 and SCCA could be detected within the linear ranges of 0.001-100 U/m L and 0.001-100 ng/m L,with detection limits of 0.4 m U/m L and 0.33 pg/m L,respectively.All these results demonstrated that the present potential-resolved “in-electrode” type electrochemiluminescence approach provided a promising analytical method for dual targets analysis with the advantages of simple analytical procedure,small sample volume and lower cost,which made the proposed method potential for clinical detection.