Studies On Preparation Of ELISA Chips Using Silicon Microwell Array

Microarray chip technology can significantly reduce the amount of immunoassay samples and reagents,and enable high parallelization and multiplexing,a hot spot in immunoassay research.Micromospheres are common biomolecule carriers,whose surface can be chemically modified by a variety of reactive groups to immobilize biomacromolecules,such as nucleic acids and proteins.Therefore,they are widely used in nucleit acids hybridization and sequencing,and protein-protein interaction,etc.The objective of this article is to prepare ELISA chips in silicon microwell arrays based on microspheres.First,sequencec of hairpin DNA are designed.Then,cross-linking agent is selected for immobilization of DNA and protein molecules onto the surface of microspheres,and followed by co-immobilization of the DNA and the proteins onto the microspheres.Finally,ELISA chips in microwell arrays are fabricated.Accordingly,the following researches have been performed.Sequences of amino-labeled single-stranded DNA are designed so that hairpin structure of DNA molecules can conformed through intrastrand base-pairing during renaturation,which is verified by non-denaturing polyacrylamide gel electrophoresis.Two conventional cross-linking agents,glutaraldehyde and EDC/sulfo-NHS,are compared for their immobilization efficiency of DNA and protein onto microspheres.Equal molar ratio of hairpin DNA and human TNF-? capture antibody are immobilized onto amino-coated and carboxyl-coated microbeads with a diameter of 4 ?m,using glutaraldehyde and EDC/sulfo-NHS,respectively.The results show that cross-linking efficiency of glutaraldehyde is better than that of EDC/sulfo-NHS,particularly for protein immobilization.Therefore,glutaraldehyde is chosen to cross-link Cy3-labeled hairpin DNA and R-PE with the microspheres,and the resultant conjugates are investgated under the fluorescence microscope.Then,adjusted molar ratio of FAM-labeled hairpin DNA to R-PE are co-immobilized on the surface of microbeads,and the conjugates investigated under the fluorescence microscope.This would lay a foundation for high-throughput immunoassay by encoding various biomarkers with DNA sequences.Based on previous work on microwell and microbeads arrays of our laboratory,microsphere-based DNA and protein microarrays have been fabricated.Molecules of human CD40 capture antibody are immobilized onto the microspheres.Then,the immunocomplexes are formed on the surface of these microspheres after sequential capturing of the human CD40 antigen,the biotin-labeled human CD40 detection antibody and either streptavidin-labeled R-PE or streptavidin-labeled ?-galactosidase.The immunocomplexes are loaded into the microwell arrays to obtain the fluorescent immuno-chips or ELISA chips.The ELISA reaction in the closed micro-wells are performed.The detection potential of the ELISA chip has been investigated in order to lay a foundation for high multiplexing,high parallelizing,and low limit of detection ELISA assays.Microfluidics cells for such microarrays are also designed and fabricated,providing possibilities for automation of ELISA detection in the future.