| Electrochemiluminescence (ECL) immunosensors combined the merits of ECL technology and immunoassay, are valuable tools for monitoring the antibody or antigen due to the advantages of high sensitivity, high selectivity and low background. The biological recognition system on an immunosensor is pivotal for the trancduction between the biorecognition event and detected signal. In the system, using the material and biological amplificatory technology to specifically transduce and amplify the signal is an effective approach for improving the sensitivity. This research focuses on the preparation of the multi-functionalized nanomaterials, the construction of the immunoreaction interface, the strategy of sensitivity enhancement and the multiplexed assay by Ru(bpy)32+ -based ECL immunosensors. The detail contents are as follows:1. Multi-walled carbon nanotubes and Ru(bpy)32+/nano-Au nano-sphere as efficient matrixes for a novel solid-state electrochemiluminescence sensor.As the large surface area and conductivity of gold nanoparticles, it was employed to prepare the Ru(bpy)32+/nano-Au nano-sphere (abbreviate as Ru-AuNPs) via the electrostatic interactions. And an effective method for immobilization of Ru(bpy)32+ is developed by using the Ru-AuNPs and MWCNTs-Nafion composite nanoparticles for a novel ECL sensor. The experiment confirms that the enhancement of the ECL intensity on the sensor is attributed to following reasons. One hand, the employment of MWCNTs in the Nafion film enlarged the electro-active surface areas to benefit the contact between the signal probe on the composite film and co-reactant used as reinforcing agent. On the other hand, the nano-materials of MWCNTs and nano-Au also improve the conductivity of the self-assembled film so as to increase the quantity of excited state of Ru(bpy)32+ in the unit time and finally cause better properties in luminescence. The ECL sensor was used to detect a kind of alkaloid medicine, Matrine. It showed the good response to the concentration of the Matrine from 2.0×10-6 mol·L-1 to 6.0×10-3 mol·L-1. Furthermore, this work innovatively employs the quantificational count of electro-active surface area to explain the contribution of nanomaterial for ECL response, which brings the new approach into the ECL research region of nanomaterial.2. A new electrochemiluminescence immunosensor based on Ru(bpy)32+ -doped TiO2 nanoparticles labeling for ultrasensitive detection of human chorionic gonadotrophin.As the large surface area and low protons excited energy of TiO2 nanoparticles, a sensitive ECL immunosensor was constructed on the basis of sandwich type immunoreaction with Nafion functionalized TiO2 composite nanoparticles as label. The composite nanoparticles labeled antibody were synthesized by successively loading the antibody and large amounts of Ru(bpy)32+ molecules onto the Nafion coated TiO2 nanoparticles, which is named as Ru-Nafion@TiO2 labeled secondary antibody. They together with the electro-deposited gold nanoparticles (DpAu) were employed to assemble the ECL immunosensor. The capability of the immunosensor was evaluated by detecting the ECL responses of immunosensor on different concentrations of human chorionic gonadotrophin (HCG). Comparing with other immunoassays, the sandwich-type ECL immunosensor assembled with DpAu and Ru-Nafion@TiO2 nanoparticles can perform the ultrasensitive detection to HCG with a low detection limit 0.007 mIU·mL-1.3. Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@Ru and multi-sites biotin/streptavidin affinity.The potential controlling silver catalysis for Ru(bpy)32+ ECL signal at a special potential-0.4-1.25 V was newly developed as the ECL signal amplification strategy for ultrasensitive protein detection. In the proposed Ru(bpy)32+ ECL immune system without any co-reactant, the detected ECL signal was amplified due to the following multiple amplification strategies:(1) the ECL catalysis for Ru(bpy)32+ were performed by electro-inducing the DpAg particles to generate Ag+ ion and controlled by the special potential. The catalyzer Ag+ produced near the electrode surface and reproduced by cyclic potential scan, which improved the catalytical efficiency. (2) The amount of the ECL signal probes linked to secondary antibodies were amplified by the adsorption of Pt nanoparticles and the multiple sites bridge linkage of biotin/SA. These new multiple signal amplification strategies made the proposed ECL immunosensor achieve ultrasensitive detection for model protein human IgG with the detection limit down to 3 pg·mL-1.4. Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay.Based on the associated signal amplificatory technology, an innovatory ECL immunoassay strategy was proposed to detect the newly-developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)32+ The quantificational calculation proved the -5.3x103 cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng-mL-1 range, and a detection limit down to 0.77 pg·mL-1.5. The study on the electrochemiluminescence immunosensor based on the simultaneous multiplexed immunoassay.A new discovery that the cathodal ECL of S2O82- anion appears at -1.4 V promotes the multiplexed assay by ECL immunosensor. The K2S2O8 and Ru(bpy)32+ were chosen as the ECL signal probe and respectively encapsulated in liposome as immune label. Relative to the Ru(bpy)32+ -encapsulated liposome as the label of anti-CEA, the K2S2O8-encapsulated liposome was used to label the anti-AFP. Then they were orderly subjected the sandwich immunoassay on one electrode surface. Finally, these ECL probes were released by the lysing and adsorbed on the electrode suface for the detection. The cathodal and anodic ECL scans were executed synchronously at the potential scope of -1.6 V-1.25 V. And the ECL reponses appeared at -1.4 V for the AFP antigen and at 1.2 V for the CEA. Due to the enhancement of the dissolved oxygen, the ECL response with the S2O82- anion label is larger than that with the Ru(bpy)32+ label. The immunosensor showed linear ranges of 20-250 ng·mL-1 for AFP and 5-150 ng·mL-1 for CEA. This result testifies that the multiplexed assay can be realized in the ECL.
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