Naked eye detection of an amplified gene using metal particle-based DNA transport within functionalized porous...

The current study focuses on developing a system for visually detecting an amplified bacterial (Escherichia coli O157:H7) gene using a heavy metal particle (MP) and functionalized porous sepharose gel. To functionalize DNA-specificity to the MP, an avidin-modified MP was employed in combination with a biotin-conjugated primer. The porous sepharose matrix was functionalized with an amine-reactive group, such as N-hydroxysuccinimide (NHS), to achieve separation upon binding of the amplified gene. The pristine avidin-MPs strongly react with NHS-sepharose via imide bonds owing to the exposure of the amine group on the avidin-MP surface. Conversely, together with the amplified gene, the avidin-MPs are relatively less interactive toward the sepharose gel by steric hindrance of the amplified gene toward the imide bond between NHS and the amine groups. Owing to the higher molecular mass of the MP, those metal particles complexed with the amplified gene pass through the sepharose matrix when centrifugal force is applied. The MPs that are thus separated can be easily visualized by the naked eye owing to their inherent reddish-brown color. A polymerase chain reaction (PCR) product of E. coli O157:H7, present in concentrations ranging from 1.0 × 10¹ to 1.0 × 10⁶ colony forming units (CFU), in actual food sample was evaluated with high sensitivity and reproducibility. We expect that the MP-based sensing system, which allows for visual detection of PCR-amplified genes, can be clinically used as a point-of-care testing device.