Novel Polymer Dots Based Electrochemiluminescence And Imaging Applications

Nowadays,electrochemiluminescence(ECL)has played important roles in clinical diagnosis and life analysis because of its advantages of low background,high sensitivity,wide linear range and electrochemical controllability.As a new visualization technology,ECL imaging has made great progress in the fields of single particle analysis,multitarget detection and cell imaging due to its spatial-temporal resolution and high throughput.ECL imaging can more conveniently reveal the relevant information about the electrochemical activity of single particle and single cell.However,most ECL imaging systems still rely on luminol/H2O2 and[Ru(bpy)3]2+/TPrA systems,which usually show the following disadvantages:(1)It is difficult to label the biomolecules with small molecular ECL emitters,and complex reaction processes are needed to functionalize these emitters or their derivatives.(2)When imaging single particle or single cell,high concentration of coreactant,such as amine coreactants,is necessary,which will cause cytotoxicity and environmental pollution and is unfavorable for realtime or in situ detection of living cells.(3)In the detection of low abundance targets,local signal enhancement is necessary.But it is not easy to combine small molecule ECL emitters with a variety of signal amplification strategies.Therefore,developing more suitable luminescent materials is always one of the hot research topics for ECL imaging.Thus,this thesis designs and synthesizes a series of polymer dots(Pdots)as the ECL emitter to develop several ECL imaging systems.A variety of strategies are introduced to these systems for improving the ECL performance of Pdots.Combined with abundant DNA amplification technology,novel ECL visual sensing platforms are developed to achieve high-throughput,high sensitivity and in situ detection of biomarkers.It includs the following four parts:1.Electrochemiluminescent Imaging for Multi-immunoassay Sensitized by Dual DNA Amplification of Polymer Dots SignalA true-color ECL imaging strategy was designed for multi-immunoassay of proteins by coupling highly efficient Pdots with dual DNA amplification.The Pdots functionalized with DNA1 that hybridized with black hole quencher-labelled DNA2 to self-quench the ECL emission of Pdots.After the capture antibody immobilized on Au/ITO slide,it recognized the target protein and then reacted with biotin-labeled detection antibody,streptavidin and biotin-labeled oligonucleotide,respectively.After that,a large number of DNA1 functionalized Pdots could be introduced onto the slide surface by rolling circle amplification of the oligonucleotide to trigger the enzymatically cyclic release of the Pdots from the self-quenched probes to solution in the presence of Exo III.The dual DNA amplification produced greatly amplified ECL signal for true-color ECL imaging.Using carcinoembryonic antigen,cytokeratin-19fragment and neuron-specific enolase as lung cancer-specific biomarkers,the ECL imaging-based multi-immunoassay exhibited excellent performance and could accurately detect these biomarkers in clinical human serum samples for lung cancer screening.2.Dual Resonance Energy Transfer in Triple-Component Polymer Dots to Enhance Electrochemiluminescence for Highly Sensitive BioanalysisA dual intramolecular resonance energy transfer(RET)mechanism with a newly synthesized triple-component Pdots was designed to achieve great ECL enhancement.This mechanism efficiently shortens the path of energy transmission in Pdots,thus greatly promotes the ECL amplification by 380 and 31 times compared to no and single RET systems,and results in a relative ECL efficiency of 23.1%(vs.1 mM[Ru(bpy)3]2+).Using metal-organic frameworks to carry the Pdots,a highly luminescent probe is proposed.By integrating the probe with target-mediated enzymatical circulation amplification and DNA array,a highly sensitive ECL imaging method is designed for simultaneous visual analysis of two kinds of proteins.3.Dual Intramolecular Electron Transfer for In Situ Coreactant-Embedded Electrochemiluminescence Microimaging of Membrane ProteinA dual intramolecular electron transfer strategy and tertiary amine conjugated polymer dots(TEA-Pdots)were designed to develop a coreactant-embedded ECL mechanism and microimaging system.The TEA-Pdots could produce ECL emission at+1.2 V without need of coreactant in test solution.The superstructure and intramolecular electron transfer led to unprecedented ECL strength,which was 132 times stronger than that from the mixture of Pdots with TEA at equivalent.The ECL efficiency was even higher than that of typical[Ru(bpy)3]2+system.Therefore,this coreactant-embedded ECL system could be used for in situ ECL microimaging of membrane protein on single living cells without additional permeable treatment for transporting coreactant.The feasibility and validity were demonstrated by evaluating the specific protein expression on cell surface.4.Potential and Color Resolved Electrochemiluminescence of Polymer Dots for Array Imaging of Multiplex MicroRNAsA potential and color resolved ECL array imaging strategy was designed for highthroughput detection of two microRNAs(miRNAs)based on luminol doped L-Pdots and diethylamine coupled N-Pdots which can generate blue and red ECL emission,respectively.The array was prepared with the mixture of Pdots which were covalently modified with their quencher labelled DNAs to recognize corresponding miRNAs.Upon addition of duplex-specific nuclease,the DNAs hybridized with miRNAs were digested to release the quenchers and miRNAs,which led to the ECL recovery of Pdots and target-cyclic signal amplification.By imaging the array at+0.6 V and+1.0 V and using miRNA-21 and miRNA-205 as the analytes,the blue and red channel images could be extracted to realize the simultaneous and high-throughput sensing of these miRNAs.5.ECL Imaging for Detection of Dopamine Secreted by PC12 Cells based on Single Cell MicroarrayAn ECL imaging platform for quantitative detection of dopamine secretion by single cells was constructed.The platform based on microfluidic single cell array filmed TEAPdots which modified with dopamine aptamer as probes.When the oxygen concentration in the environment decreased,the dopamine secreted by PC 12 cells was captured by the aptamer on TEA-Pdots.After applying the potential of+1.4 V,dopamine was oxidized to quinones,which quenched the ECL of TEA-Pdots.The platform realized the detection of dopamine secreted by single cells and successfully revealed the single cell heterogeneity.