Immunoassay Based On Electroge Nerated Chemiluminescence Amplified By Magnetic Nanobeads And Electrochemiluminescence Resonance Energy Transfer

In chapter one of this thesis, We develop an ultrasensitive electrochemiluminescence (ECL) method for the determination of biomolecules. This method uses magnetic submicrobeads (SMBs) as the carrier of a large number of luminophore molecules to amplify the signal. In this method, ECL of the luminophore molecules on the surface of the SMBs are detected. Using the protocol, an ultrasensitive immunoassay method is provided. First, one streptavidin-coated SMB (SA-SMB) is conjugated to double-antibody sandwich of antigen structure. Next, a huge number of Ru(bpy)32+species are bound to the SA-SMB. The SA-SMBs with Ru(bpy)32+species are immobilized on an Au electrode by means of a magnet. In the presence of tri-n-propylamine, a curve of ECL intensity versus potential with a maximum ECL intensity (Im,ECL) at 1.35 V is obtained.Im,ECL can be used to quantify Ag. Since one target Ag corresponds to one SA-SMBs with a large number of Ru(bpy)32+, as well as the Ru(bpy)32+-loaded SA-SMBs are immobilized on the Au electrode, through the signal amplification,the second ECL wave of Ru(bpy)32+is increased greatly, leading to increased sensitivity. Using this method, a linear relationship between Im,ECL and the CEA concentration is obtained in a range of 1×10-14 to 3×10-13 mol/L.In chapter two, we report a novel resonance energy transfer (RET) technique, electrochemiluminescence resonance energy transfer (ECRET). In ECRET, the emitters of luminol/H2O2 system are generated at an electrode through electrochemical reaction and act as the electrochemiluminescent donors to emit light with a maximum emission of 460 nm. The red fluorescent luminescent semiconductor nanocrystals (quantum dots, QDs), having a maximum emission at 655 nm, are chosen as the acceptors. When a potential was applied to the electrode, the electrochemical donors transferred energy to the QD acceptors, producing efficient ECRET. The ECRET technique could be applied for the investigation of biomolecular interactions of nucleic acids, DNA assay and immunoassay.