Synthesis And Luminescence Property Study Of Multifunctionalized Nanomaterials And Their Applications In Label-free Bioassays

As the development of nanotechnology, various novel functionalized nanomaterials were explored and used in our daily life. Owing to their superior chemiluminescence (CL) property, stability and repeatability, functionalized nanomaterials with CL property have attracted close attention of science researchers. CL functionalized nanomaterials were applied to clinical disease diagnosis, new drug development, environmental monitoring and biological images. In recent years, multi-functionalized nanomaterials come into our sight because of their better CL signal, detection limit and sensitivity, thus are of great potential in application. Label-free analysis methods are simple, fast and low-cost, thus has being a major trend in analysis method development. This dissertation aims at developing novel multi-functionalized nanomaterials and exploring their properties and applications in label-free bioassays. Firstly, a novel lucigenin and protein enzyme bifunctionalized graphene oxide (GO) nanomaterials were developed. Based on these bifunctionalized GO nanomaterials, three label-free CL biosensors for detection of small biomolecules, including glucose, uric acid and choline, were designed by replacing different enzymes. Then a label-free method for the detection of specific DNA sequences was developed based on gold nanoparticles bifunctionalized with a chemiluminescent reagent and a catalyst. The method for the measurement of the target concentration and binding constants between DNA aptamers and target simultaneously was expanded to the targets that could inhibit chemiluminescence reactions. It was also found that dissociation constants of aptamer-binding targets, antibody-antigen complexes, protein-binding small molecules and double-strand DNA hybrids could be measured by this method. In the end, two kinds of dual-signal nanomaterials with CL property and fluorescence (FL) property were developed and one of them was applied to pesticide sensing-array. The details are as follows:1. A general strategy to prepare GO lucigenin & enzyme composites was developed. The composites have well CL property and enzyme catalytic activity. The assembly of GO lucigenin & GOD composites was studied by high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM) and circular diehroism (CD) and confirmed the successful synthesis of the composites. In this sensing strategy. GO lucigenin composite was first prepared by vigorously stirring GO with lucigenin. Then the functionalization of GOD was achieved by simply storing GOD with GO lucigenin at 4℃ overnight to form GO lucigcnin & GOD composite. When glucose was incubated with GO/lucigcnin & GOD composite for 50 min to generate H2O2, followed by the injection of 0.2 M NaOH. CL signal was detected due to the reaction of lucigenin with H2O2,The present biosensor has been successfully applied for the detection of glucose in human serum samples. Compared with previously reported methods, this sensing strategy is homogeneous and reagentless and avoids complicated assembly procedure and pretrcatment of serum sample, showing good stability, repeatability, high selectivity and simplicity. Moreover, this strategy has been demonstrated to be a general strategy by replacing GOD with other enzymes such as uricase and choline oxidase for the detection of small molecules such as uric acid and choline. The proposed biosensor is a general detection method.2. A label-free method for the detection of specific DNA sequences was developed based on gold nanoparticles bifunctionalized with a chemiluminescent reagent and a catalyst proposed in previous study. In this method, the bifunctionalized gold nanoparticles could be enhanced by DNA sequences and biotinylated single-strand DNA which was complementary with target DNA was taken as capture probe. It was possible to determine the specific DNA sequences related with diseases including mycobacterium tuberculosis (TB), hepatitis B virus (HBV), and myelocytomatosis viral oncogene (v-myc). The detection limits of three specific DNA sequences were 4.8× 10-14 M、5.9×l10-12 M and 8.0×10-12 M, respectively. The sensitivity of this CL method is superior to those of most previously reported label-free methods. Moreover, this label-free CL method is much simpler, faster, and more economic.3. A general method for the determination of the binding constant of specific interactions and binding target concentration simultaneously was proposed based on that target could influence the CL reaction of ABEI functional ized gold colloid with H2O2. The method could be applied on the target that enhance or inhibit the CL reaction. It was also found that dissociation constants of aptamer-binding targets, antibody-antigen complexes, protein-binding small molecules and double-strand DNA hybrids could be measured by this method. Compared with the existing method for determination of binding constant, our method is much easier, cheaper and faster and has better applicability. Meanwhile, the method could determinate the concentration of the target owing to the specificity of specific interactions. Its sensitivity is superior to other label-free method.4. A novel ABEI functionalized graphene quantum dots with CL and FL property was proposed. Firstly, graphite oxide was exfoliated and cut into small 2-dimension graphene oxide. Then small graphene oxide was further cut to obtain GQDs by reduction reaction. At lastly, CL reagent ABEI was functionalized to GQDs by non-covalent interactions including π-π stacking force, electrostatic attraction and hydrogen bonding and AEBI functionalized GQDs was obtained. The CL and FL property of ABEI-GQDs was great thus could be used as dual-signal probe for determination. A pesticide-sensing array was developed for pesticide differentiation by utilizing the effect of various pesticides on the CL and FL signal of ABEI-GQDs. It was found that the sensing strategy could distinguish different pesticides and different pesticide concentrations.5. A straightforward one-pot strategy was developed, without necessity of larger AuNP precursors or dangerous organic solvents for the preparation of high-quantum-yield AuNCs with excellent FL and CL property. In this method,11-mercaptoundecanoic acid was taken as capping agent and ABEI as reductant. The resulting ABEI-AuNCs exhibited a bright orange emission at 580 nm when irradiated with incident light at 280 nm, and weaker emission at 440 nm. In addition, the CL property of ABEI-AuNCs was better than ABEI-AuNPs. Thus it was a potential probe for developing new dual-model detection method and could be applied on biological images, biosensor and catalyst.