Signal Amplification In Electrochemiluminescence Biosensors

Electrochemiluminescence（ECL）is the successful marriage of electrochemistry and chemiluminescence,which has the advantages of high signal-to-noise ratio and good controllability.The high sensitive ECL methods are widely used in the detection of environmental pollutants,bioactive molecules,nucleic acids,and tumor markers.To obtain higher sensitivity,signal amplification plays an important role in ECL methods.A variety of signal amplification strategies have been developed for high sensitive biosensors.In this thesis,anodic stripping voltammetry,biometallization and co-reaction accelerator as signal amplification approaches are combined with ECL to determine tumor markers and dopamine.The main research contents are outlined below:1.Ratiometric ECL immunosensor using single ECL luminophoreRatiometric ECL is helpful to improve the signal-to-noise ratio and signal stability of ECL methods.Usually,two kinds of ECL luminophores as signal and reference are used in the ratiometric ECL measurements.In this work,a ratiometric ECL immunosensor using only single ECL emitter is reported.The feasibility of this method is demonstrated by using carcinoembryonic antigen（CEA） as a model analyte.Porous g-C₃N₄ nanosheets were immobilized on the surface of conductive polymer film of poly（2-Aminoterephthalic acid） doped with carbon nanotubes on glassy carbon electrode（GCE） and used as ECL luminophore.The immune recognition stages are carried out on 96 well plate.The second antibody labeled with Cu S nanoparticles is used as the signal tag.After the immune recognition,the Cu S in sandwich immunocomplex was dissolved.Then the dissolved Cu（Ⅱ）was reduced to Cu on surface of GCE/g-C₃N₄.In the presence of K₂S₂O₈ as the co-reactant,the cathodic ECL of GCE/g-C₃N₄（Cu） is measured and used as the reference signal in the ratiometric ECL measurement.During the subsequent anodic potential scanning process,Cu in GCE/g-C₃N₄（Cu） is oxidized to Cu（Ⅱ）,anodic ECL of GCE/g-C₃N₄ is generated with triethanolamine（TEA）as the co-reactant.The anodic ECL of GCE/g-C₃N₄ is quenched by Cu（Ⅱ）within the Helmholtz layer on the electrode surface and used as the ECL signal for CEA determination.The limit of detection（LOD）for CEA is 0.3 fg/m L,which is one order of magnitude lower than that in signal-on ECL immunosensor with g-C₃N₄ as the signal tag（3 fg/m L）.The combination of biometallization and anodic stripping voltammetry with ratiometric ECL can enhance significantly the sensitivity and reliability in immunosensors.2.Enhancement of ECL FAPbBr₃ in aqueous phase by conductivity and co-reactantTiO₂ nanoparticles and carbon nanotubesPerovskite nanocrystals（NCs）are regarded as potential candidates as ECL emitters due to their excellent photoluminescence ability.Herein,we report the ECL behavior of FAPb Br₃（FA⁺stands for CH（NH₂）₂⁺）NCs in the aqueous media.It was shown that the ECL of FAPb Br₃NCs was obtained by hole-injection to oxidize their reduced states,with the emission peak centered around 534±1 nm and the full width at half maximum of 31±0.5 nm.The annihilation ECL of FAPb Br₃NCs was 4.3±0.4 folds enhanced by adding 8%（w/w）CNTs.In the presence of tripropanolamine as the co-reactant,the anodic ECL intensity of FAPb Br₃ was enhanced by a factor of 29 in compared with its annihilation ECL.Moreover,the intensity of FAPb Br₃NCs was enhanced further 4.8,10.6 and 21.6 folds by using TiO₂ nanoparticles（NPs）,CNTs,TiO₂ NPs and CNTs as the co-reaction and conductivity accelerators,respectively.The anodic ECL of GCE/CNTs@FAPb Br₃NCs@Ti O₂NPs was applied to determination dopamine in the linear range of 0.01-10μM,with the LOD of 2.9 nM.3.Determination of alpha-fetoprotein by dual signal amplification ratiometricECL immunosensorIn the proposed double signal amplification approach,the sandwiched immunocomplex was formed in microplate by using alkaline phosphatase（ALP）labeled secondary antibody as the signal tag.The ALP in the sandwiched immunocomplex was used as the catalyst for the hydrolysis of ascorbate 2-phosphate to produce ascorbic acid.Pd（Ⅱ）in the solution was reduced to Pd nanoparticles（Pd NPs）by as-obtained ascorbic acid.The as-obtained Pd NPs on the surface of the microplate were used as high efficiency catalyst for electroless Ni-P plating,realizing the second signal amplification to further improve the sensitivity.Then Ni-P was dissolved by H₂O₂+HCl,and the concentration of Ni（Ⅱ）was detected by ECL method using g-C₃N₄ as ECL emitter.The Ni（Ⅱ）related to immune reaction was electrodeposited on GCE/CNTs-Ti O₂NPs@g-C₃N₄/Bi.In the presence of K₂S₂O₈ as the co-reactant,the cathode ECL intensity of GCE/CNTs-Ti O₂NPs@g-C₃N₄/Bi（Ni）was measured and used as the reference signal in the ratiometric ECL.During the anodic potential scanning,the Ni（0） on surface of the electrode was oxidized to Ni（Ⅱ）,which quenched the anodic ECL signal of g-C₃N₄.According to the intensity ratio of anodic/cathodic ECL of g-C₃N₄,Ni（Ⅱ）concentration was detected with the detection limit is 0.12 pM.Alpha fetoprotein（AFP）was used as model analyte to verify the feasibility of the above multi-level signal method.The detection limit of AFP was 0.3 fg/m L,improving the sensitivity and stability of ECL immunosensor.