Development And Applications Of High Performance Immunoassay Microarray Based On Nanopolymer Brush Modified Glass Substrate

Protein microarray is fabricated by immobilization of different protein probes on a piece of solid substrate (glass, plastic, metal et al.) at separate locations in an ordered manner. It has emerged as a powerful tool for screening of protein expression, protein-nucleotide interaction, and protein-small molecule interaction in a parallel and high-throughput manner. In past decades, it has been employed for widely applications in fundamental bioscience, clinical diagnosis, drug discovery, and toxicological research. In comparison with the traditional two-dimensional electrophoresis, mass spectrometry and enzyme linked immunosorbent assay (ELISA), miniaturized protein microarrays provide many advantages, such as high-throughput screening, less sample consumption, short detection time, low cost, and high reliability and so on.Antibody microarray, a main type of protein microarrays, is fabricated with antibodies as capture molecules. Thus proper substrate surface and antibody immobilization strategy are of very importance to achieve high array performance. Ideally, antibodies should be immobilized on the microarray substrates with high density, good stability, expected orientation and maximum bioactivity. Although many strategies including such as glutaraldehyde modification method, thiol modification method, nanomaterials coating method have been developed in past years to modify various substrates for antibody immobilization, they always suffer from some challenges such as random orientation, low attachment efficiency, severe nonspecific adsorption, and poor bioactivity.To overcome these challenges and further improve performance of the antibody microarrays, this thesis work focuses on development of two universal and efficient surface modification strategies based on nanopolymer brush. At the same time, key physical and chemical factors involved in antibody microarray immunoassay were further investigated to explore how the developed strategies improve the performance of antibody microarrays. Research works and results are as follows.1. Development of PGMA-APBA polymer brush-modified glass slide for oriented antibody immobilizationMicroarray substrates were first silanized by APTES which successfully introduced amino group on glass surface; Initiator was then covalently conjugated on APTES-modified slide; the PGMA polymer brush was synthesized via surface initiated atom transfer radical polymerization on side surface; phenylboronic acid functional groups were introduced into the PGMA brush through covalent conjugation between epoxy group on GMA and amino group on APBA, resulting in PGMA-APBA brush. This method combines the advantages of 3D flexible polymer brushes and the PBA-enabled oriented antibody attachment via carbohydrate at Fc portion, resulting in covalent, high density and directional antibody immobilization.2. Performance evaluation of the developed PGMA-APBA-slide in microarray immunoassayPerformance of PGMA-APBA slides was evaluated by study of antibody loading capacity, antibody-antigen (Ab-Ag) interaction efficiency and signal-to-noise ratio. PGMA-slide was also investigated for comparison. Although the antibody loading capacity on PGMA-slides is higher than that on PGMA-APBA-slide, antibody-antigen (Ab-Ag) binding efficiency and signal-to-noise ratio obtained from PGMA-APBA-slides are significantly higher than those from PGMA-slides. It probably results from that PBA-enabled oriented antibody attachment via carbohydrate at Fc portion keeps antigen-binding sites fully expose to their corresponding antigens.3. Phenylboronic acid polymer brush-enabled oriented and high density antibody immobilization for sensitive microarray immunoassayMicroarray immunoassay was performed on PGMA-APBA-slide with rabbit IgG as model analyte, from the experimental results, the detection range obtained from PGMA-APBA-slides is from 10 pg mL-1 to 1 μg mL-1, and LOD is determined to be 1pg mL-1. For PGMA-slide, the detection range is from 100 pg mL/1 to 1μg mL-1, and LOD is determined to be 10 pg mL-1. Therefore, both LOD and dynamic range from PGMA-APBA-slides is better than PGMA- slide.4. Fabrication of 3-D microarray and its microfabrication-free fluidic immunoassay device to improve the array density and enhance the assay performanceThe microfluidic device is assembled with a rectangular glass cuboid and a circular capillary glass tube. The former serves as a 3-D microarray substrate, while the latter is employed as a tubular channel to guide a fluid during the assays and by simple integration with an automatic pump system. The antibody loading capacity, Ab-Ag interaction efficiency and signal-to-noise ratio are investigated with the developed 3-D microfluidic array device and conventional 2-D microarray device.5. Tumor markers AFP and CEA were used as model proteins to evaluate the performance of 3-D microfluidic device and potential application in clinical detectionTumor marker AFP and CEA were utilized as model proteins to evaluate performance of the developed 3-D array device. The antibody loading capacity, Ab-Ag interaction efficiency and signal-to-noise ratio in 3-D microfluidic device are better than traditional 2-D microarray device. From the typical sigmoidal dose-dependent curves, LODs are determined to be 10 pg mL-1 for AFP and 100 pg mL-1 for CEA. In a word, the 3-D microarray device not only significantly increases the density of the sensing spots, but also greatly enhances the mass transport for rapid immunoassay when using in a flow-through device by the caused turbulent flow.