Microarray preparation based on oxidation of agarose-gel and subsequent enzyme immunoassay

Abstract

Based on molecular assembly technique, a thin film with double functions was prepared by using agarose gel as matrix on surfaces of glass slides. This process involved drying, then oxidation with NaIO 4 and subsequent coupling with glutaraldehyde. The aldehyde groups, derived from the film of the gel–matrix, could covalently link with the amino-groups in the molecule of various antigens (e.g. recombinant peptide, phospholipid, DNA), horseradish peroxydase and antibodies through formation of Schiff’s bases. AFM characterization and assay results demonstrated that the gel assembly film was a surface of three-dimensional structure, and had a higher capacity for protein loading and linking than that of two-dimensional surfaces. In the light of the above principles, a biomolecular microarray was generated on this surface. By optimizing 100 mg/l was chosen as the concentration and 50 nl as the volume for spotting samples of diversified biomolecules. The enzyme–immunoassay system on the surface of gel–matrix by which the interactions of antigen–antibody and enzyme–substrate molecules were detected sensitively and specifically, was successfully developed by various experimental set-ups. The two main distinct advantages of the microchip are: (a) saving on volume of specimen and reagent, and (b) simultaneous analysis for multi-autoantibodies. Since the enzyme–substrate (HRP–DAB–H 2O 2) was used as the display system of signals from the microchip, the results were directly visible and more stable in comparison with fluorescent immunoassay.