Electrochemiluminescence Biosensing And Imaging Methods Based On Functionalized Magnetic Particles

Electrogenerated chemiluminescence(electrochemiluminescence,ECL)is one kind of luminescence occurred at/near the surface of electrode as a result of electrochemical reactions and the chemiluminescence(CL)reactions.ECL biosensing method generally uses biomolecular probes and ECL reagents/materials to measure the concentration of target molecules by translating a biochemical interaction into a quantifiable ECL signal.ECL biosensing has the advantages such as,strong controllability,high sensitivity,simple instrument,widely linear range,fast speed,easily automated.It is a very important issue to design biointerfaces in ECL biosensing for a given target because the accuracy,sensitivity,and reproducibility of the biointerface-based methods depend on the performance of the biointerface.In general,for the construction of biointerfaces in most of the ECL biosensors,the molecular recognition probes/ECL probes(ECL reagent-labeled molecular recognition probes)are fixed on the surface of the solid electrode through the approaches such as,physical adsorption,embedding,self-assembly,or covalent bonding methods.This type of construction biointerface methods of the ECL biosensors has played an important role in basic theoretical research.However,because this kind method for construction of the ECL biosensors was usually only once measurement,it is subject to certain restrictions in practical applications.Immuno-magnetic beads-based ECL automated analysis,such as Roche automated ECL immunoassay,has become more and more widely used in clinical analysis.However,Roche automated ECL immunoassay is based on flow system and Pt electrode taken as working electrode,which makes the commercial instruments to be very expensive,in addition,few application of multi-component assay.ECL imaging,a kind method that combines ECL and imaging technology,has become more and more favored by researchers in multi-biomarker assay and cell imaging because it has the advantages such as,high sensitivity and large amount of information.However,the ECL mechanism of large-sized microspheres/cells on the electrode surface is still unclear.In view of the problems of the above-mentioned ECL biosensing methods,this dissertation devotes the research issue "Functional magnetic particles-based electrogenerated chemiluminescence biosensing and imaging",aims to develop new ECL biosensing methods and imaging strategies based on functionalized magnetic particles.This work was financially supported from National Natural Science Foundation of China,"Point of Care Testing Based on Electrogenerated Chemiluminescence Biosensing"(No.21775098)and "Magnetically Guided Electrochemiluminescent Biochips and Their Clinical Application"(No.21974081).There are 6 chapters in the thesis.Chapter 1 is the general introduction.The principle of ECL,ECL luminophores,advantages and disadvantages of ECL biosensing are briefly introduced,and then the recent progress of metal complexes-based ECL biosensing,magnetic beads-based ECL biosensing,quenching-based ECL biosensing,and ECL imaging,are reviewed.Finally,the research purpose and main contents are presented.Chapter 2 presents one work on "Electrogenerated chemiluminescence immune-sensing on magnetic bead/gold nanoelectrode ensembles".Based on the preliminary research work,a gold nanoelectrode ensembles(Au-NEE)platform taken as a disposable electrogenerated chemiluminescence(ECL)platform with immunomagnetic microbeads for ECL immunoassays was developed.It was found that the peak-shaped voltammograms were obtained at the Au-NEE,attributed to the active and inactive total diffusional overlap.The ECL intensity/active area at Au-NEE was 12.9 folds in the Ru(bpy)32+-tri-n-propylamine(TPA)system and 19.6 folds in the luminol-H2O2 system,compared with that at the Au macroelectrode,mainly attributed to the diffusion overlap of the Au-NEE and the edge effect of the individual gold nanodisks of the Au-NEE.The ECL immunoassay based on the Au-NEE platform with magnetic microbeads for the determination of cancer biomarkers was developed.Carbohydrate antigen 19-9(CA 19-9)was chosen as a model analyte while biotinylated CA 19-9 antibody combined with the streptavidin coated magnetic microbeads was taken as the capture probe,and ruthenium complex-labeled CA 19-9 antibody was used as the signal probe.A "sandwich" bioconjugates on the magnetic beads were transferred onto the ECL platform,and then the ECL measurements were performed in TPA solution.The developed method showed that the ECL peak intensity was directly in proportion to the concentration of CA 19-9 in the range from 0.5 to 20 U/mL with a limit of detection of 0.4 U/mL.This work demonstrates that the Au-NEE can be employed as a useful disposable ECL platform with the merits of cheapness,low nonspecific adsorption and practical application.The work provided a promising approach for the point-of-care test for the determination of protein biomarkers.Chapter 3 presents one work on "Synthesis and electrogenerated chemiluminescence properties of 8 kinds of hydrophobic cyclometalated iridium(?)complexes".On the design of cyclometalated iridium(?)complexes,it is to be adopted that in the main ligands,increasing the ?-electron conjugated structure and introducing a thienyl group can reduce the ECL potential and tune the emission wavelength of the complexes,while in the ancillary ligand,introducing the electron-withdrawing group can tune the emission wavelength and increasing hydrophobic alkyl chains can improve the hydrophobicity of cyclometalated iridium(?)complexes.In this work,8 kinds of hydrophobic cyclometalated iridium(?)complexes were designed and synthesized,and mainly characterized on the UV-Vis absorption spectra,fluorescence spectra,fluorescence lifetime,fluorescence quantum yield,cyclic voltammetry curve,annihilation and co-reactant ECL behaviors in acetonitrile solution.The relationship between complexes structure and its properties,combined with density functional theory calculations also were discussed.This work demonstrates that the increasing the ?-electron conjugated structure on the main ligands could reduce the ECL potential and tune the emission wavelength in red-shift,and increasing the ?-electron conjugated structure on the main ligands could improve the ECL quantum efficiency of the complexes.However,compared with thienyl group,phenyl group does not improve the ECL quantum efficiency.The introduction of electron-withdrawing group on the ancillary ligands could tune the emission wavelength in red-shift while increasing hydrophobic alkyl chains on the ancillary ligands can improves the hydrophobicity of the complex and has little effect on the ECL properties.This work provided some important information for the design and synthesis of ECL complexes and the selection of ECL complexes in liposomes.Chapter 4 presents one work on "Magnetic nanoparticle@lipid film ferrocene/ruthenium bipyridine complex intermolecular electrogenerated chemiluminescence quenching and its application in biosensing,,aim at to develop a new ECL biosensing method based on bio-cleavage on the magnetic nanoparticles.One hydrophobic ruthenium complex[Ru(bpy)2dcbpy]Cl2 was synthesized and its ECL properties were investigated.The lipid film-coated magnetic nanoparticles(Fe3O4 NP@lipid)were synthesized.When Fe3O4 NP@lipid were taken as an important carrier and[Ru(bpy)2dcbpy]Cl2 was taken as an ECL signal probe and cholesterol-(CH2)6-HS SKLQK-F c(Fc=ferrocene)was taken as a recognition probe-quencher,the functionalized magnetic nanoparticles,named as all-probe magnetic nanoparticles(Fe3O4 NP@lipid/signal probe/recognition probe-quencher),were synthesized and their ECL properties were investigated.It was found that after the recognition probe-quencher(cholesterol-Ahx-HSSKLQK-Fc)was inserted into the surface of magnetic nanoparticle signal probe/lipid film(Fe3O4 NP@lipid/[Ru(bpy)2dcbpy]Cl2),the ECL quenching constants of the ECL signal probe by quencher Fc was 137 folds than one that the signal probe and the recognition probe-quencher were inserted into the surface of the lipid film-coated indium tin oxide(ITO)electrode,and 391 folds than one that the ECL quenching constants of Fc-COOH and[Ru(bpy)2dcbpy]2+in the homogeneous solution.Importantly,in this work,a new ECL biosensing method based on bio-cleavage on the magnetic nanoparticles for the determination of prostate specific antigen(PSA)has been developed,in which PSA was chosen as a model analyte and the all-probe magnetic nanoparticles were drop-coated on the surface of the GC electrode,which was taken as the biosensing interface.The liner range from 0.1 to 2.0 ng/mL PSA and the limit of detection of 0.01 ng/mL PSA(S/N=3)were achieved for the developed method.The proposed approach provided a new idea for the bio-cleavage ECL biosensing using Fe3O4 NP@lipid.Chapter 5 presents one work on "Electrogenerated chemiluminescence imaging of single partial on side-view via single particle approaching",aim at to develop a new strategy for the single magnetic bead-based ECL imaging analysis.Considering that the light shielding effect of the ECL from the micron magnetic beads on the surface of the electrode led to the weak luminescence signal on the "top-view" model using CCD,this work proposed a new ECL imaging strategy on the "side-view" model using CCD.In this work,ECL bio-conjugates(biotin-peptide-Rul)were loaded on the surface of one micron magnetic bead(MB@SA)to form one ECL particle(MB@SA/biotin-peptide-Rul).This ECL particle was taken as a model analyte.The electrode system was designed by using a GC electrode as working electrode and using the robotic arm of the three-dimensional electric platform as approaching element,in which the ECL particle was adhered to the tip of one capillary glass tube or carbon fiber,which was fixed on the platform.It was found that the diffusion current of Fc-OH in the solution gradually decreased with increasing the size of the magnetic bead and also with decreasing the distance between the magnetic beads and the electrode surface,indicating that the magnetic bead hinders the diffusion of electroactive substance.The ECL intensity of the ECL particle at the ECL imaging increased with increasing the diameter of the magnetic beads(1.0-2.8 ?m)while that decreased with the increasing of the magnetic beads(2.8-15 ?m),indicating that the large-diameter magnetic beads have a serious light shielding effect.For the ECL particle with a diameter of 15 ?m,the ECL imaging intensity was observed using"side-view" model,while could not be observed using "top-view" model.Additionally,PMT was also taken as a detector for recording the ECL intensity-potential profiles of the ECL particle,which was drop-coated on the surface of the GC electrode.It was found that two ECL peaks occurred and the ratio of the two peak ECL intensities was related to the diameters of the ECL particles(0.30-10 ?m),and in the ECL mechanism,the ECL emission of the ECL particles in the small sizes was dominated by directly electrochemical oxidation of Ru(bpy)32+pathway,while that in the large sizes was dominated by the chemical reaction between Ru(bpy)3+and TPA' generated by electrochemical oxidation of TPA.This work provided a new approach for the research of single-particle catalysis and single cell ECL imaging.Chapter 6 presents the conclusion and outlook.The main conclusion of this thesis was summarized.The shortcomings of this thesis were analyzed.The aspects of this work that needs to be further improved and supplemented were discussed.Additionally,the prospect for ECL biosensing and imaging methods based on the functionalized magnetic particles were presented.