Microarray detection method for pathogen genes by on-chip signal amplification using terminal deoxynucleotidyl transferase

Abstract

A microarray detection method based on on-chip signal amplification using terminal deoxynucleotidyl transferase (TdT) was developed to visualize pathogenic genes. Cyclic olefin copolymer (COC) substrate for microarrays was treated with oxygen plasma to induce hydrophilic surface properties. The capture probe DNA was immobilized on the COC surface by UV irradiation. The 3ʹ end of the capture probe DNA immobilized on the COC surface was modified with a phosphate group to provide resistance against the TdT reaction. Therefore, the TdT reaction was triggered only when the capture probe DNA acquired the target gene, and biotin-11-deoxyuridine triphosphate (b-dUTP) was continuously added to the 3ʹ end of the target gene. Thereafter, streptavidin-conjugated gold nanoparticles (s-AuNPs) tagged the poly uridine tails by the biotin–streptavidin interaction. The visual signal was amplified by silver enhancement in the presence of the s-AuNPs. The usefulness of this detection method was confirmed by analyzing four pathogens and allowing their visual identification.