Fabricating Electrochemiluminescence Biosensors Based On Luminophore Functionalized Graphene Composites For Disease Markers Detection

Disease markers play an important role in the early diagnosis,prevention,treatment and prediction of diseases.It is therefore of great importance to develop highly selective and ultrasensitive detection methods for the disease markers when diagnosing related diseases.Electrochemiluminescence?ECL?biosensors have been extensively applied in environmental monitoring,medical analysis,and food safety.Graphene based composites with outstanding electrical conductivity,large specific surface area,chemical stability,good biocompatibility and other properties own enormous superiorities that can dramatically reduce background signals,shorten response time,and improve the detection sensitivity of ECL biosensors.In particular,the hydrophilic oxygen-functionalities?carbonyl,hydroxyl and carboxyl groups?on the composites surface are conducive to immobilizing ECL luminophores and biomolecules with increased amounts and favorable dispersion,thus showing great potentials in designing efficient ECL sensing strategies for biosensor constructions.In this thesis,a series of luminophores functionalized graphene composites with high ECL efficiency and stability were fabricated and applied to develop biosensors for biomarkers detection,offering some methodological basis for the early clinical diagnostics of diseases.The research contents and main results of this thesis are as follows:1)Herein,a label-free ECL immunosensor was fabricated for prostate specific antigen?PSA?based on luminol functionalized platinum nanoparticles loaded on graphene sheets?Lu-Pt@GS?.In this study,Lu-Pt@GS was utilized as a sensing platform,where for the first time luminol was employed as both ECL luminescence reagent and reductants to in-situ reduce H₂PtCl₆ forming Pt NPs on surface of GS.In the reduction process,lots of Pt NPs could attach on luminol surface,which intensify the ECL signals of luminol and also supply active sites with micro friendly environment for the fruitful immobilaztion of PSA antibodies.Strong and stable ECL signals were obtained by utilizing single-step cycle pulse,which could prevent the consecutive reaction among H₂O₂ and luminol.Under optimized conditions,the fabricated ECL immunosensor acquired a wide linear response range of PSA concentration from 1 pg/mL to 10ng/mL with a pretty low detection limit of 0.3 pg/mL?S/N=3?.Besides,the label-free ECL immunosensor exhibited good stability,repeatability and selectivity.2)A novel label-free ECL immunosensor was designed for PSA based on silver/silver orthophosphate/graphene oxide composites?Ag/Ag₃PO₄/GO?as sensing platform as well as antibody carriers.In this study,Ag₃PO₄ nanoparticles were firstly exploited as novel luminophore for solid-state ECL detection and possible luminescence mechanism was also proposed.GO,offering ultrahigh specific surface area and excellent conductivity,was exploited as qualified substrate for Ag₃PO₄ nanoparticles,which not only contribute towards electrons transfer enhancement for whole ECL system,but also the sensitivity of immunosensor was further improved.Given that the outstanding biocompatibility of silver nanoparticles?Ag NPs?was used to immobilize PSA antibody?anti-PSA?via the covalent coupling among silver and amino group,to improve the sensitivity of fabricated immunosensor.Under optimized conditions,the fabricated ECL immunosensor acquired a wider linear response range of PSA concentration from 1 pg/mL to 80 ng/mL with a pretty low detection limit of 0.3 pg/mL?S/N=3?.Further,the label-free ECL immunosensor exhibited good stability,repeatability and selectivity.3)In this part,an efficient ECL immunosensor was designed for the detection of insulin based on electrochemiluminescence resonance energy transfer?ECL-RET?between Ag₃PO₄and Pd@Au core-shell nanoflower.Ag/Ag₃PO₄/GO composites were used as sensing platform for capture-antibody?Ab₁?incubation.The ECL emission of Ag₃PO₄ could improve extraordinarily through the synergetic assistance of Ag NPs with GO.GO exhibited ultrahigh specific surface area and excellent electrical conductivity to enhance the loading capacity of Ag₃PO₄ and Ab₁,and Ag NPs with spectacular biocompatibility and catalytic property could immobilize Ab₁ through Ag-N bond and further accelerate electron transfer to catalyze the SO₄^·-radicals generation for improving the ECL emission of Ag₃PO₄.Pd@Au core-shell nanoflower was employed to label detection antibody?Ab₂?.The ECL spectrum of Ag₃PO₄,performing as ECL donor,presented fine overlaps with the absorption spectrum of Pd@Au core-shell nanoflower which performed as energy acceptor,triggering obvious reductions in ECL intensity.Under optimized conditions,the fabricated ECL-RET immunosensor acquired a wider linear response range of insulin concentration from 1 pg/mL to 80 ng/mL with a pretty lower detection limit of 0.02 pg/mL?S/N=3?.This ECL-RET strategy will reveal a great prospective applications in the early detection of lower concentrations markers.4)In this chapter,an effective ECL immunosensor was designed for the detection of insulin based on ECL-RET between ZnIn₂S₄ utilized as energy donor and gold decorated silicon dioxide nanoparticles?Au@SiO₂ NPs?as energy acceptor.ZnIn₂S₄ nanosheets?NSs?,which showed strong ECL emission in the presence of potassium persulfate?K₂S₂O₈?with ECL spectral band at 370–720 nm,were in situ grew on the surface of RGO?Zn In₂S₄/RGO?through a facile one-step hydrothermal method.Integrating with the morphological and electrical advantages of RGO and AgNPs,the ECL emission of ZnIn₂S₄ nanosheets at 551 nm was boosted dramatically.Furthermore,Au@SiO₂ NPs whose UV-vis absorption spectra at 450–650 nm were fabricated firstly as a matched ECL acceptor for Ag/ZnIn₂S₄/RGO.Because of well spectral overlap,Au@SiO₂ NPs could quench ECL emission of ZnIn₂S₄ significantly,causing obvious reductions in ECL intension.Under optimized conditions,the fabricated ECL-RET immunosensor acquired a wider linear response range of insulin concentration from 1pg/mL to 80 ng/mL with a pretty lower detection limit of 0.034 pg/mL?S/N=3?,the extremely-efficient ECL-RET immunosensor holds superb potential in detecting the insulin and other biomarkers in human serum for the early diagnostics of severe diseases.