Research On Novel Systems Of Quantum Dot-Based Electrogenerated Chemiluminescence And Their Bioanalytical Applications

Quantum dots (QDs), as one kind of functional nanocrystals, due to their advantages in optical and electronic aspects, have been widely used for bioanalysis in cell image, in vivo observation, and multiplexed immunoassay and analysis of DNA hybridization. With various QDs as ECL nanoemitters, electrogenerated chemiluminescence or electrochemiluminescence (ECL) as a widely utilized analytical technology has been developed for designing novel analytical methodologies. However, the reported QDs-based ECL systems normally suffer from highly applied potential as well as the introduction of strong oxidants as the exogenous coreactants, which suppress the extended application of QDs-based ECL bioanalyses. In this dissertation, by combining nanotechnology, morphology and spectroscopic characteriazation and chemical biology, two novel carbon nanomaterials-modified interfaces were at first fabricated to promote the electron transfer, then a series of newly-designed bioanalytical methods based on the solid-state ECL of QDs were developed, finally, a QDs-labeled "signal-on" strategy for ECL immunoassay was proposed based on the electro-driven and low-background nature of ECL. This dissertation includes the following eight parts:1. A Glucose Biosensor Based on Direct Electrochemistry of Glucose Oxidase Immobilized on Nitrogen-Doped Carbon NanotubesA novel biosensor for glucose was prepared by immobilizing glucose oxidase (GOx) on nitrogen-doped carbon nanotubes (CNx-MWNTs) modified electrode. The CNx-MWNTs membrane showed an excellent electrocatalytic activity toward the reduction of O2due to its diatomic side-on adsorption on CNx-MWNTs. The nitrogen doping accelerated the electron transfer from electrode surface to the immobilized GOx, leading to the direct electrochemistry of GOx. The biofunctional surface showed good biocompatibility, excellent electron-conductive network and large surface-to-volume ratio, which were characterized by scanning electron microscopy, contact angle and electrochemical impedance technique. The direct electron transfer of immobilized GOx led to stable amperometric biosensing for glucose with a linear range from0.02to1.02mM and a detection limit of0.01mM (S/N=3). These results indicated that CNx-MWNTs are good candidate material for construction of the third-generation enzyme biosensors based on the direct electrochemistry of immobilized enzymes.2. Amplified Electrochemiluminescence of Quantum Dots by Electrochemically Reduced Graphene Oxide for Nanobiosensing of AcetylcholineA signal amplification system for electrochemiluminescence (ECL) of quantum dots (QDs) was developed by using electrochemically reduced graphene oxide (ERGO) to construct a nanobiosensing platform. Due to the structural defects of GO, the ECL emission of QDs coated on GO modified electrode was significantly quenched. After the electrochemical reduction of GO, the restoration of structural conjugation was observed with spectroscopic, morphologic and impedance techniques. Thus in the presence of dissolved O2as coreactant, the QDs/ERGO modified electrode showed ECL intensity increase by4.2and178.9times as compared with intrinsic QDs and QDs/GO modified electrodes due to the adsorption of dissolved O2on ERGO and the facilitated electron transfer. After choline oxidase (ChO) or ChO-acetylcholinesterase was further covalently cross-linked on the QDs/ERGO modified electrode, two ECL biosensors for choline and acetylcholine were fabricated, which showed the linear response ranges and detection limits of10-210uM and8.8μM for choline, and10～250μM and4.7μM for acetylcholine, respectively. This green and facile approach to prepare graphene-QDs system could be of potential applications in electronic device and bioanalysis. 3. Signal Amplification by Adsorption-Induced Catalytic Reduction of Dissolved Oxygen on Nitrogen-Doped Carbon Nanotubes for Electrochemiluminescent ImmunoassayA signal amplification strategy for electrochemiluminescence (ECL) of quantum dots (QDs) by an adsorption-induced catalytic reduction of dissolved oxygen at the sidewall of nitrogen-doped carbon nanotubes (NCNTs) was designed for ’signal-on’sandwich immunoassay. The NCNTs possessed strong ability toward the adsorption of dissolved oxygen, which induced its electrochemical reduction accompanying with the catalysis of NCNTs. The reduction produced superoxide radical to act as a coreactant of ECL of QDs, leading to an enhanced ECL emission and a new ECL mechanism. The NCNTs were functionalized with polystyrene sulfonate for labeling the signal antibody. With a sandwich immunoreaction, the labeled signal antibody introduced the NCNTs onto QDs-based immunosensor surface to produce an amplification platform for ECL immunoassay. The proposed immunoassay method showed excellent performance with a linear range of6orders of magnitude. The NCNTs-based strategy expanded the ECL application of QDs in biosensing and bioanalysis.4. Electrocatalytic Reduction of Coreactant by Highly Loaded Dendrimer-Encapsulated Palladium Nanoparticles for Sensitive Electrochemiluminescent ImmunoassayA nonenzymatic tracing tag was designed by anchoring polyamidoamine (PAMAM) dendrimer-encapsulated palladium nanoparticles (PdNPs) to single-walled carbon nanohorn (SWNH). The tag was further linked with signal antibody for sensitive electrochemiluminescent (ECL) immunoassay of tumor marker utilizing the electrocatalytic activity of the PdNPs toward reduction of dissolved oxygen, the coreactant of ECL reaction of quantum dots. The PdNPs@[PAMAM-G5]/SWNH nanohybrids were synthesized through the in situ reduction of PdCl42-adsorbed inside SWNH-supported PAMAM, and characterized with morphological, spectroscopic and electrochemical techniques. Both the PAMAM and the SWNH greatly improved the loading capacity of PdNPs, thus enhanced the electrocatalytic ability. Using carcinoembryonic antigen as a model analyte, upon a sandwich-type immunoreaction, the electro-reduction of coreactant catalyzed by the tracing tag and the steric hindrance of formed sandwich immunocomplex decreased the ECL emission, leading to a sensitive ECL immunoassay strategy. This method exhibited acceptable precision and a wide linear range over6orders of magnitude with detection limit down to0.47pg mL-1. The newly designed tracing tag and strategy are promising for applications of the functional nonenzymatic nanohybrids in determination of low-abundant biomarkers.5. Label-Free Electrochemiluminescent Detection of DNA by Hybridization with Molecular Beacon to Form Hemin/G-Quadruplex Architecture for Signal InhibitionA facile label-free electrochemiluminescent (ECL) DNA sensor was designed by using molecular beacon with guanine-rich stem as recognition probe. The ECL emission was produced from surface unpassivated CdTe quantum dots (QDs) co-immobilized with colloidal gold nanoparticles (AuNPs) on chitosan modified electrode surface. The molecular beacon was adsorbed onto the AuNPs by the thiolated stem. Upon the hybridization of molecular beacon with target DNA to open the cycle in the presence of hemin, the dissociated guanine-rich sequence could conjugate hemin to form a G-quadruplex architecture. The formed DNAzyme then catalyzed the reduction of dissolved oxygen, the endogenous coreactant for ECL emission of QDs, leading to a decrease of ECL signal. The variations in surface morphology during the fabrication and recognition processes of ECL sensor were characterized with atomic force microscopy and electrochemical impedance spectroscopy. The ECL signal inhibition linearly depended on the logarithmic value of DNA concentration ranging from5.0fM to0.1nM with a detection limit of0.9fM. This proposed label-free method provided a promising application of QDs-based ECL emission in ultrasensitive DNA assay.6. Electrochemiluminescent Quenching of Quantum Dots for Ultrasensitive Immunoassay through Oxygen Reduction Catalyzed by Nitrogen-Doped Graphene-Supported Hemin A novel peroxidase-like mimic for oxygen reduction was designed by the noncovalent assembly of hemin on nitrogen-doped graphene via axial ligation. The mimic led to sensitive electrochemiluminescent (ECL) quenching of quantum dots (QDs) due to the annihilation of dissolved oxygen, the ECL coreactant, by its enzymatic reduction. Using the mimic with high loading of hemin as a signal tag of the secondary antibody, a novel ultrasensitive immunoassay method for biomarker detection was proposed. In air-saturated pH8.0buffer, the immunosensor constructed by a stepwise immobilization of bidentate-chelated CdTe QDs and capture antibody showed an intensive cathodic ECL irradiation, which could be annihilated upon the formation of the mimic labeled sandwich immunocomplex. Using carcinoembryonic antigen as a model analyte, the immunoassay method showed a linear range from0.1pg mL-1to10ng mL-H and a detection limit of24.0fg mL-’. The immunosensor exhibited good stability, acceptable fabrication reproducibility and practicability. The mimetic enzyme-based ECL quenching strategy provided a powerful avenue for design of ultrasensitive detection method, showing great promise for clinical application.7. Ferrocenyl-Terminated Dendrimer as Efficient Quencher via Electron and Energy Transfer for Cathodic Electrochemiluminscent ImmunoassayA dual quenching effect on the ECL emission of QDs via both electron and energy transfer was demonstrated using an organometaliic framework as quencher. The ECL quencher was designed by covalently linking Fc-CHO to primary amine group of PAMAM dendrimer. The assembled Fc moieties showed electron delocalization among the cyclopentadiene groups, which accelerated the electron transfer from Fc to OH and electron injected QDs to the oxidation product of Fc. The energy transfer from excited QDs to the cyclopentadiene of Fc further quenched the ECL emission. The residual terminal amines endowed Fc@PAMAM with aqueous dispersion and favoured for further biofunctionalization with proteins. Thus a novel immunosensing strategy was proposed for ECL immunoassay with Ab2-Fc@PAMAM as a tracing tag. This method showed a low detection limit, wide calibration range striding over the threshold for clinic diagnosis, excellent stability, good precision, acceptable reproducibility and reliability, indicating promising practicability of the dual quenching effect. 8. Synthesis and Low-Potential Electrogenerated Chemiluminescence of Surface Passivated Phenol Formaldehyde Resin@CdS Quantum DotsA novel method for synthesis of polymer-stabilized quantum dots (QDs) was reported through one-pot hydrothermal reaction. Using this method, phenol formaldehyde resin (PFR) polymer was in situ formed to passivate the surface of CdS QDs with hexamethylenetetramine as the source of both monomer and crosslinking agent, HCHO. The PFR-capped QDs possessed uniform size distribution and controllable surface states and showed a unique electrogenerated chemiluminescence (ECL) spectrum with two peaks at537and573nm, which corresponded to the core and surface states of QDs, respectively. The partially unpassivated surface state produced a low-potential ECL emission with relatively long lifetime at-0.78V. A new signal tag was thus designed for "signal-on" ECL immunoassay by assembling the QDs on poly(diallyldimethylammonium chloride) coated carbon nanospheres to label the signal antibody. With carcinoembryonic antigen as a model, the developed sandwich-typed immunoassay showed a linear range over5orders of magnitude with a limit of detection down to0.83pg mL1. The excellent analytical performance indicated the promising application of the surface passivated PFR-capped QDs in biosensing.