Solid-Phase PCR on Film Biochips with Brush Polymer Cells, “Lab-On-A-Chip”

Abstract

Objective: To develop a multifactorial, highly sensitive nucleic acid analysis method on biological microarrays within an environmentally sealed system. This approach eliminates the need for transferring biological material between vessels and avoids adding components during the assay, thereby reducing contamination risk and enabling process automation. Methods: Polyethylene terephthalate (PET) film substrates for biochip fabrication were treated with corona discharge. A thin layer of photoactive polyvinyl acetate was deposited on the surface by spin coating. A matrix of cells composed of brush polymers was obtained through photoinitiated radical polymerization of monomers “from the surface” using photolithographic patterning under UV irradiation through a photomask. Reactive carboxyl groups on polymer chains were activated, and primers with C6-amino modification at the 5′-end were immobilized within biochip cells. PCR with extension of immobilized primers using Cy5-fluorescently labeled nucleotides was performed within the biochip cells. Results and Discussion: A method for nucleic acid analysis by solid-phase PCR with immobilized primer extension in a closed film biochip during thermocycling was successfully developed. Detection was achieved through endpoint digital fluorescence microscopy monitoring Cy5-fluorescently labeled nucleotide incorporation. The developed “film biochip” consists of PET film with an internal chamber, brush polymer cells containing immobilized primers, and channels for solution supply and removal. The biochip features low heat capacity and high thermal conductivity of thin-film components, thermocycling capability, and result detection by digital fluorescence microscopy through the lid and washing solution layer without biochip disassembly in an environmentally isolated lab-on-a-chip system. The method′s performance and functional suitability were demonstrated by analyzing samples containing DNA from pathogenic bacteria Staphylococcus aureus and Legionella pneumophila. Conclusions: Film biochips made of commercially available PET film with brush polymer cells show promise for further development and application in solid-phase PCR for multiplex nucleic acid analysis, lab-on-a-chip microanalysis technologies, and clinical laboratory applications.