Research On Novel Nanoemitters And Their Electrogenerated Chemiluminescence Bioanalytical Applications

Functional nanomaterials have been widely used for bioanalysis,such as cell image,in vivo observation,and multiplexed immunoassay and detection of nucleic acid.Electrogenerated chemiluminescence or electrochemiluminescence(ECL)as an analytical technique combined with electrochemical and chemiluminescence,has been used in immune analysis,food safety and water quality monitoring.ECL luminophores are usually divided into inorganic systems,organic systems and nanomaterials systems,but only inorganic complex Ru(bpy)32+ system to achieve the commercialization.At present,nanomaterials are mostly based on semiconductor quantum dot system,which has heavy metal toxic elements,leading to serious health and environmental problems and suppress the extended application of bioanalysis.In this dissertation,two kinds of novel ECL nanoemitters,two-dimensional(2D)carbon nitride nanosheets and polymer dots,have been developed to explore the interaction mechanism between the new nanometer emitter and the bioanalyte.By combining nanotechnology,morphology and spectroscopic characteriazation and chemical biology,new analytical and biosensing strategies were designed.This dissertation includes the following five parts:1.Electrochemiluminescent DNA Sensing Using Carbon Nitride Nanosheets as Emitter for Loading of Hemin Labeled Single-Stranded DNAThis work uses this emitter to construct an ECL sensing platform for sensitive DNA detection through its adsorption ability toward single-stranded DNA(ssDNA).The adsorption of hemin-labeled ssDNA on CNNS leads to in situ consumption of dissolved oxygen via hemin-mediated electrocatalytic reduction,thus decreases the formation of coreactant and quenches the ECL emission of CNNS.The ECL sensing platform is designed using hemin-labeled ssDNA to recognize the target DNA,which results in the departure of hemin-labeled hybridization product from the CNNS modified electrode,thus inhibits the annihilation of coreactant and recovers the ECL emission.Under optimized conditions,the proposed sensing strategy shows a wide detection range over 6 orders of magnitude and wondrously high sensitivity with a detection limit down to 2.0 fM.Moreover,the ECL sensor exhibits good performance with excellent selectivity,high reliability,and acceptable fabrication reproducibility.The sensitive sensing strategy provides a new paradigm for the design of ultrasensitive detection method.2.Ratiometric Electrochemiluminescence Detection of Circulating Tumor Cells and Cell-Suxface GlycansThis work designed a dual-potential ratiometric electrochemiluminescence(ECL)system for highly sensitive detection of(circulating tumor cells,CTCs)and simultaneous evaluation of glycan expression on cell surface.Two kinds of ECL nanoemitters,carbon nitride nanosheets and luminol-reduced gold nanoparticles as cathodic and anodic nanoemitters respectively,were prepared to successfully achieve the dual-potential ratiometric ECL.The carbon nitride nanosheets were decorated with gold nanoparticles to conjugate aptamer for specific capture of CTCs,and the anodic nanoemitters were functionalized with lectin for specific recognition of surface glycans.Using MCF-7 CTCs as model target,the proposed ratiometric ECL sensor exhibited excellent analytical performance with a wide linear detection range and a detection limit down to 2 cells.This method could be further used for profiling the variation of glycans such as N-acetylglucosamine,N-acetylneuraminic acid and mannose on cell surface upon treatment.The proposed strategy realized highly sensitive determination of cell concentration,and accurate analysis of glycan expression on cell surface,showing promising application.3.Silole-Containing Polymer Nanodot:An Aqueous Low-Potential Electrochemiiluminescence Emitter for BiosensingA novel D-A conjugated polymer backbone containing silole and 9-octyl-9H-carbazole units was synthesized via Sonogashira reaction.This silole-containing polymer(SCP)was further used to prepare SCP dots with a nanoprecipitation method,which showed an electrochemiluminescence(ECL)emission at relatively low potential in aqueous solution.The strong anodic ECL emission could be observed at +0.4 V(vs Ag/AgCl)with a peak value at +0.78 V in the presence of tri-n-propylamine(TPrA)as a co-reactant,which came from the band gap emission of the excited SCP dots.The ECL emission could be quenched via resonance energy transfer from the excited SCP dots to an acceptor.Thus,a low-potential anodic ECL sensing strategy was proposed for ECL detection of the acceptor-related analytes.Using dopamine as the analyte,whose electro-oxidation product could act as the energy acceptor to quench the ECL emission of SCP dots,the ECL detection method showed a detection limit of 50 nM and high anti-interference ability.This work demonstrates an example of polymer dots as an ECL emitter and its potential application in ECL detection methodology.4.Ru(bpy)32+ Incorporated Luminescent Polymer Dots:Double-Enhanced Electrochemiluminescence for Detection of Single-Nucleotide PolymorphismEncapsulating a large amount of electrochemiluminescence(ECL)active molecules into one nanoparticle for single target recognition can greatly improve the sensitivity of ECL assay.This work used luminescent conjugated polymer as a carrier to synthesize Ru(bpy)32+ doped polymer dots(RuPdots).The RuPdots with an average size of 20 nm and encapsulation of about 1354 Ru(bpy)32+ showed greatly enhanced ECL emission.Using tripropylamine as a co-reactant,the resonance energy transfer from the excited polymer dots to the encapsulated Ru(bpy)32+ was demonstrated,which led to 15.7-fold higher ECL emission than that of Ru(bpy)32+ with the same amount at gold electrode.By coupling the double-enhanced ECL signal of this nanostructure with specific ligase detection reaction,a sensitive ECL biosensor was constructed for specific detection of single-nucleotide polymorphism(SNP).Using mutant KRAS gene as a model target,the ECL SNP detection method showed a linear range from 1 fM to 1 nM and a detection limit of 0.8 fM.The designed RuPdots provided a novel signal amplification strategy and could be used as a universal label for ultrasensitive ECL biodetection.5.Highly Efficient Electrochemiluminescence of Cyanovinylene-contained Polymer Dots in Aqueous Medium and Its Application in Imaging AnalysisLuminescent semiconducting polymer dots(Pdots)have attracted intense attention in the field of electrochemiluminescence(ECL)due to their nontoxic features.To utilize the nontoxic Pdots to achieve sensitive ECL detection and bioimaging,this work studied the ECL behaviors of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)](CN-PPV)Pdots in aqueous solution,which introduced an electron-withdrawing cyano group to p-phenylenevinylene for enhancing the luminescent efficiency.The CN-PPV Pdots could be both electrochemically oxidized to positively charged state and electrochemically reduced to negatively charged state,which led to annihilation ECL emission.The order of oxidation and reduction greatly influenced the annihilation behavior.In the presence of co-reactant,tri-n-propylamine(TPrA)or S2O82-,the CN-PPV Pdots showed strong band-gap ECL emission at 602 nm,which followed two different routes and gave the ECL efficiencies of 11.22%and 1.84%,respectively.The high ECL efficiency allowed CN-PPV Pdots/TPrA system for ECL imaging analysis.As a proof-of-methodology,an ECL imaging method was designed via the chelating interaction of metal ions and Pdots to achieve the high selectivity.The proposed ECL imaging chip-based sensor exhibited excellent analytical performance with a wide linear range from 100 pM to 100 ?M and a detection limit of 67 pM.Compared with the ECL methods based on direct intensity measurement;the developed ECL imaging method possessed the advantages of simplicity,rapid and high-throughput,and had application potential in monitoring water and food quality.