UV-Assisted Hyperbranched Poly(β-amino ester) Modification of a Silica Membrane for Two-Step Microfluidic DNA Extraction from Blood.

Abstract

Integrating nucleic acid extraction in amplification-based point-of-care diagnostics will be a significant feature for next-generation point-of-care virus detection devices. However, extracting DNA efficiently on a microfluidic chip poses many technological and commercialization challenges, including manual steps, multiple instruments, pretreatment processes, and the use of organic solvents (ethanol, IPA) that inhibit detection, which is not viable with routine testing such as viral load monitoring of transplant patients for post-operative care. This paper presents a microfluidic system capable of two-step DNA extraction from blood using a UV-assisted hyperbranched poly(β-amino ester) (HPAE)-modified silica membrane for cytomegalovirus (CMV) detection in a rapid and instrument-free manner without the presence of amplification inhibitors. HPAEs of varying branch ratios were synthesized, screened, and coated on a silica membrane and bonded between two layers of poly(methyl methacrylate) (PMMA) substrates. Our system could selectively extract DNA from blood with an efficiency of 94% and a lower limit viral load of 300 IU/mL in 20 min. The extracted DNA was used as the template for real-time loop-mediated isothermal amplification (LAMP)-based detection of CMV and was found to produce a fluorescent signal intensity that was comparable with commercially extracted templates. This system can be integrated easily with a nucleic acid amplification system and used for routine rapid testing of viral load in patient blood samples.