A functionally integrated thermoplastic microdevice for one‐step solid‐phase‐based nucleic acid purification and isothermal amplification for facile detection of foodborne pathogen

Abstract

In this study, we fabricate a functionally integrated monolithic thermoplastic microdevice for continuous operation of nucleic acid purification and amplification using polycarbonate (PC). A solid-phase-based purification and subsequent isothermal amplification, specifically, thermal helicase-dependent amplification (tHDA), was performed in a single operation in a valve-free manner. PC microdevice was assembled using modified thermal bonding process under relatively low temperature and pressure condition, realized by surface chemical modification of PC into hydrophilic property using amine-bearing polyethyleneimine (PEI). After the device sealing, only the microchannel parts were selectively modified to be hydrophobic, using epoxy-terminated poly(dimethylsiloxane) (PDMS) (epoxy-PDMS) on amine-coated surface for stepwise introduction of multiple reagents in a valve-free manner. Using the integrated PC microdevice, nucleic acids from genetically modified Escherichia coli (E. coli) O157:H7 were captured inside a chamber bearing amine functionality, by electrostatic interaction, and were subsequently amplified isothermally in the same chamber. Purified DNA captured inside the microchamber was detected directly inside the chamber by fluorescence measurement, and a 92-bp long EaeA gene, inserted into the E. coli O157:H7, was successfully amplified using the integrated PC microdevice in less than 90 min, paving the way for facile identification of foodborne pathogens with simple operation and reduced peripheral operations applicable for portable healthcare purposes. Biotechnol. Bioeng. 2016;113: 2614-2623. © 2016 Wiley Periodicals, Inc.

Irreversible bonding techniques for the fabrication of a leakage-free printed circuit board-based lab-on-chip in microfluidic platforms—a review

“…The formation of a siloxane structure on the mediator facilitated the molecular bonding in all of the cases. Several related studies also explored the application of amine-epoxy and thiol-epoxy bonds between two salinised substrates to seal various types of polymers [120][121][122][123][124][125]. The results from the studies are summarised in table 5.…”

“…Similar to those in previous works, all of the bonding experiments were performed at temperatures below the T g . In cases involving amine-epoxy and thiol-epoxy as the mediators, the experiments were performed at room temperature [120,121,125] and at the temperature range of 65 • C to 80 • C [122][123][124], respectively.…”

Microfluidic-Based Nucleic Acid Amplification Systems in Microbiology

“…Another on-chip DNA purification technique is solid-phase based DNA collection. Ha et al [221] employed an integrated thermoplastic microdevice for solid-phase based NA purification. The prepared chip consisted of three microchannels for washing solution, DNA purification, and amplification.…”

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection

“…A UV lamp for excitation and an i-Pad camera permitted obtaining limits of detection in the fM range comparable to the less cost-efficient epifluorescence microscope [52]. Based on the same electrostatic principle, DNA purification was achieved on polycarbonate modified with polyethyleneimine in a valve-free fashion in 30 min [61]. Device rotation sequentially propels the solutions from each of three reservoirs (for extraction, washing, and HDA reagents) to the chamber where DNA is electrostatically entrapped to perform the symmetric HDA amplification of E. coli DNA.…”