Development Of A Microfluidic Protein Microarray Based High-throughput Detection System For Genetically Modified Organisms

Along with the fast development of genetically modified organisms (GMOs) techniques, the species of GMO crops and the planting area of GMOs are increasing every year. However, the safety concerns with GMOs have also been seriously aroused so that the strategy to monitor GMOs has become an important task during the development of this new technique. With the growth of GMO species, the pattern of traditional GMO monitoring, which are based on one or several targets in one test, is hampered by labor and time consumption, and fall short of the feasibility to screen all GMO components in an unknown sample. Under this circumstance, high-throughput detection platform is an important trend for new GMO monitoring techniques. Although a variety of gene-based high-throughput platforms have currently been developed, the development of high-throughput analytical platform for GM proteins is still limited. In this study, we constructed a microfluidic protein microarray based on an oligonucleotide chip combined with an immobilizing approach for antibody through complementary oligonucleotides hybridization. This platform is aimed to provide a fast and sensitive platform for high-throughput GM protein analysis. The main results are listed as following:1. Antibody-Oligonucleotides Conjugates and Characterization of the ConjugatesIn this study, we conjugated antibody and oligonucleotides by modifying both molecules and conjugating them by forming bis-aryl hydrazone linkage. The process of conjugation at each step of reaction, purification, blocking and so on was monitored by UV/vis and SDS-PAGE. The conjugation protocol was developed and the formed linkage was verified at the end. In an analysis of the formed conjugate, an average of3-4oligonucleotides was attached on each antibody. A comparison of binding efficiency of antibody between the stage of start, hydrazine-modified, and oligonucleotides-conjugated forms using ELISA method showed antibody-antigen binding capability was not significantly influenced by the substitution of the amino on antibodies, but was reduced after attachment of oligonucleotides. The reason of this phenomenon was probably because of the steric hindrance from oligonucleotides.2. The Immobilization of Antibody on μParafloTM Oligonucleotide ChipSix conjugates against different GM proteins were synthesized and bound onto the oligonucleotide chip through complementary oligonucleotides hybridization. The location of antibodies was tracked by Cy3-secondary antibody bound onto the immobilized antibodies. A protocol for hybridization between conjugates and the underlying oligonucleotides array achieving high specificity was obtained through optimization, which was hybridization using25%formamide contained buffer for2h at30℃, followed by0.2%SDS contained washing buffer to remove extra conjugates. Gradient concentrations of conjugates were tested to hybridize with the underlying oligonucleotide array; an optimized concentration of conjugates at50nM was adopted after a same concentrated GM protein solution were detected respectively on formed arrays and the signal intensities yielded thereby were compared.3. GM Protein Detection on Formed ArrayThe condition of antigen detection on formed protein arrays to reach highest intensity was optimized, and4℃for overnight was decided as the antigen incubation condition. Based on the developed hybridization method, gradient concentrations of six antigens were tested on the formed array to examine the sensitivity of the platform. The detection limit for the majority of antigens was between0.1-0.5ng/mL. By a comparison with ELISA detection, it was observed that the sensitivity on the formed protein array was comparable to ELISA methods, and even improved to a significant level for some analytes having poor performance with ELISA. This result evidenced the feasibility of the formed protein array being applied to transgenic protein detection.4. Reversibility and Repetitive Utilization of Protein ArraysThe protein array was reversed to oligonucleotide array and the feasibility of protein array regeneration was validated through detecting antigen solution on regenerated protein arrays. The coefficient of variation (CV) between three times of detection on regenerating protein arrays was10.52%. However, the sensitivity of regenerated protein array was not significantly influenced, verifying the feasibility of reversibility and multiple utilization of this platform.5. The Flexibility of Protein Array FormationA new protein array containing "GMO" characterized layout was formed on the same oligonucleotide chip, successful detection of multiple antigens was achieved on this array, verifying the feasibility of forming flexible protein arrays on this platform.