One-step DNA purification and amplification on an integrated plastic microdevice for on-site identification of foodborne pathogens

Abstract

In this study, we demonstrate a simple and facile method to enable DNA purification and amplification in a continuous step inside a thermoplastic microdevice. The innate property of thermoplastics was adopted to simplify DNA purification because negatively charged DNA can electrostatically interact with the induced positively charged surfaces of thermoplastics as a sample flows inside a microchannel; thus, the DNA purification step can be eliminated. We report the use of natural plastics for the selective adsorption of DNA and conduct a subsequent flow-through polymerase chain reaction (PCR) using a thermoplastic microdevice. Four thermoplastics-poly(methyl methacrylate) (PMMA), polycarbonates (PC), polystyrene (PS), and polypropylene (PP)-were selected as analytical models, and DNA adsorption phenomena on their pristine surfaces were examined. DNA was successfully immobilized onto the pristine thermoplastic surfaces, and the results were confirmed by fluorescent measurement and contact angle measurement and by performing PCR using a thermocycler. The electrostatically attached DNA was subsequently released from the pristine thermoplastic surface using a PCR reagent, which contained a low ionic strength salt as a component that could be used for DNA elution. The eluted DNA was then seamlessly amplified in the subsequent microchannel designed to perform flow-through PCR. The PCR products were collected by a microchamber connected at the end of the microchannel for on-site fluorescence detection. The PMMA microdevice was used to successfully purify and amplify a 210 bp target amplicon from a foodborne pathogen, Escherichia coli O157:H7, within 35 min. This study is expected to pave the way for developing an integrated monolithic thermoplastic microdevice suitable for rapidly identifying foodborne pathogens with reduced operation, decreased manufacturing cost, and enhanced device disposability. (C) 2018 Elsevier B.V. All rights reserved.