A functionally integrated thermoplastic microdevice for one-step solid-phase-based nucleic acid purification and isothermal amplification for facile detection of foodborne pathogen.
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.
Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si 461-701, Gyeonggi-do, Republic of Korea.
Minh Luan
ML
Ha
Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si 461-701, Gyeonggi-do, Republic of Korea.
Yu
Y
Zhang
Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si 461-701, Gyeonggi-do, Republic of Korea. nylee@gachon.ac.kr.|Gachon Medical Research Institute, Gil Medical Center, Inchon 405-760, Republic of Korea. nylee@gachon.ac.kr.
Nae Yoon
NY
Lee
D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004867:Equipment Design; D019544:Equipment Failure Analysis; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005504:Food Analysis / Q000295:instrumentation*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology / Q000295:instrumentation; D006361:Heating / Q000295:instrumentation; D008904:Miniaturization; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D011076:Polycarboxylate Cement / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018511:Systems Integration
D004269:DNA, Bacterial; D011076:Polycarboxylate Cement; C005506:polycarbonate
epoxy-functionalized poly(dimethylsiloxane) (PDMS); helicase dependent isothermal amplification; polycarbonate microdevice; polyethylenimine (PEI); solid-phase-based nucleic acid purification; surface hydrophilic/hydrophobic coating