Miniaturized polymerase chain reaction device for rapid identification of genetically modified organisms

Abstract

In this study, a polycarbonate (PC) microdevice functioning as a reactor for the polymerase chain reaction (PCR) was fabricated for rapid identification of genetically modified organisms (GMOs). The PC microdevice was fabricated by first modifying its surface with an amine-functionalized alkoxysilane, namely bis[3-(trimethoxysilyl)propyl]amine (bis-TPA), to obtain a hydrophilic surface. Coating of bis-TPA on PC was realized by forming a urethane linkage between the amine terminals of the bis-TPA with the carbonate backbone of PC by aminolysis. This surface enabled the thermal bonding of two PC substrates at a relatively low temperature and atmospheric pressure, thereby maintaining the structure of the microchannel in high resolution. Next, the surface of the microchannel was further treated with a fluorosilane, namely tridecafluoro-(1,1,2,2-tetrahydrooctyl)-triethoxysilane (FTES), to obtain a hydrophobic surface inside the microchannel. This modification was realized by the hydrolysis and subsequent condensation of the alkoxy terminals of both bis-TPA and FTES to form a robust siloxane (Si–O–Si) bond. The hydrophobic microchannel improved the PCR performance by stabilizing the fluid flow, particularly under heated conditions, when the flow-through PCR was conducted on a microdevice. Using the microdevice, the 35S promoter sequences and bar gene, which are commonly used for the identification of GMOs, were successfully amplified, resulting in the detection of 234 and 504 bp gene fragments for the 35S promoter sequences and 261 bp gene fragment for the bar gene from the genomic DNA extracted from the leaves of GM soybean. This process took approximately 30–35 min, which was approximately 3-fold faster than when using a conventional thermal cycler.