| Resin microspheres have attracted enormous research interest in biomedical applications, because they are found to be controlled morphology and size, good stable and biocompatible. The surface of resin microspheres can be not only modified with a number of functionalizations, but also directly coupled enzymes, antibody and drugs, due to their reactive groups (hydroxy, aldehyde group, carboxyl and amino group). This dissertation investigates a surfactant-free and template-free method to synthesize biomolecule-based resin microspheres with different sizes by selecting different reaction monomers (dopamine, aminosalicylic acid, serotonin and tryptophan). The nano-Au resin composite microspheres prepared by a convenient in-situ synthesis of Au nanoparticles on resin microsphere surface show excellent electrical conductivity and high surface enhanced Raman scattering (SERS) activity. The molecules with distinguishable electrochemical and SERS signals decorated resin microspheres can be designed to act as electrochemical and SERS immunosensor for one or more lung cancer biomarkers detection. The major contributions are as follows:1. Resin microspheres with different sizes are prepared by selecting different reaction monomers (dopamine, aminosalicylic acid, serotonin and tryptophan). Discuss the relationship between the concentrations of reaction monomer and morphology of resin microspheres; the relationship between chemical structure of biomolecules and particle size of resin microspheres. The nano-Au resin composite microspheres prepared by a convenient in-situ synthesis of Au nanoparticles on resin microsphere surface for the first time. The concentration of HAuCl4 has an important role in the morphology of Au nanoparticles. The interparticle distance of the adjacent gold nanoparticles on the surface of resin microsphere can produce hotspots, which are beneficial for the SERS enhancement effect. Nano-Au resin composite microspheres can be applied in the fields of electrochemical and SERS analysis, due to the conductivity and SERS activity of microspheres.2. An novel electrochemical immunoassay for carcinoembryonic antigen (CEA) determination have been developed based on the immobilization of antibody onto nano-Au resin composite microsphere and thionine decorated glassy carbon electrode. Firstly, nano-Au resin composite microsphere with large specific surface area can effectively increase the surface area of the electrode and the immobilization amount of CEA antibody. Secondly, anti-CEA can covalent adsorb on the working electrode surface by the formation of Au-N bond or condensation reaction with amino on the protein molecules and nano-Au resin composite microspheres. The detection sensitivity of the sensor and electron transfer rate can be improved, due to conductive performance of composite microspheres. The proposed immunoassay also presented good sensitivity, selectivity and storage stability.3. A multiplexed electrochemical immunosensor is developed for simultaneous detection of CEA and neuron specific enolase (NSE) by choosing two redox materials with distinguished peaks. This study used a fluorine doped tin oxide (FTO) conductive glass as working electrode. The redox molecules [toluidine blue (TB) and Prussian blue (PB)] are absorbed on the surface of these microspheres, which is helpful for absorbing antibodies and signal generation. The prepared TB/AuNPs/TCCR and PB/AuNPs/TCCR microspheres are used as substrate to easily construct electrochemical immunosensor. Under the optimized conditions, the linear range of the proposed immunosensor is estimated to be from 0.2 to 25 ng/mL for both CEA and NSE. At a signal to noise rate of 3, the lowest detectable concentration is 0.11 and 0.08 ng/mL for CEA and NSE, respectively.4. In order to avoid the damage of signal molecule and improve the stability of the detection, a multiplexed SERS immune probe is developed for detection of one or two lung cancer biomarkers by choosing one or two Raman signal molecules with distinguished SERS peaks. A sandwich structure contains Raman dye-labeled nano-Au resin composite microsphere with the first antibody, the second antibody immobilized on the electrode modified chitosan stabilized AuNPs, and target antigens. Thus, in the presence of the target antigens, the Raman dye (Nile blue A, NBA)-labeled nano-Au resin composite microsphere could be bonded to the modified electrode surface by antibody-antigen-antibody interactions. The SERS immune probe is applied for detecting the SERS signals of NBA to indirectly detect CEA concentrations. Meanwhile, a multiplexed SERS immune probe is developed for simultaneous detection of CEA and NSE by choosing two Raman signal molecules with distinguished SERS peaks.5. A method has been raised to fabricate a dual mode and multiplexed method to detect lung cancer markers based on gold nanoparticles resin composite microspheres by the signals of electrochemistry and SERS techniques. Two Raman dyes (NBA and thionine) with both electrochemical and SERS characteristic peaks have been selected through a series of screening tests. A simultaneous immunoassay for CEA and cytokeratin-19 (CK-19) based on NBA and thionine (THI) labelled nano-Au resin composite microspheres is developed by using electrochemical and SERS techniques. A sandwich structure contains Raman dye-labeled nano-Au resin composite microspheres with the first antibody, the second antibody immobilized on the electrode modified chitosan stabilized Au nanoparticles, and target antigens. In the presence of the target antigens, the Raman dye-labeled nano-Au resin composite microspheres could be bonded to the modified electrode surface by antibody-antigen-antibody interactions. The immunocomplex can be used for electrochemical and SERS tests. In electrochemical mode, the lowest detectable concentration is 0.01 ng/mL and 0.04 ng/mL for CEA and CK-19. In SERS mode, the detection limit is 0.62 ng/mL and 1.01 ng/mL for CEA and CK-19, respectively. This reliable and effective strategy for CEA and CK-19 determination has potential for application in diagnosis and treatment of lung cancer by using the complementary advantages of the electrochemical and SERS technologies. This method provides a new insights about the early diagnosis of lung cancer. |
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