Fabrication of 3D continuous-flow reverse-transcription polymerase chain reaction microdevice integrated with on-chip fluorescence detection for semi-quantitative assessment of gene expression. Issue 23 (15th October 2018)

• Record Type: Journal Article
• Authors: Pham, Quang Nghia; Trinh, Kieu The Loan; Tran, Nguyen Khoi Song; Park, Tae-Sik; Lee, Nae Yoon
• Is Part Of: Analyst. Volume 143:Issue 23(2018)
• Page Start: 5692
• Page End: 5701
• Publication Date: 2018-10-15
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/c8an01739e
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : A 3D microdevice equipped with a portable pump and a single heater was fabricated integrating RNA amplification and detection functionalities. Abstract : We fabricate a three-dimensional (3D) microdevice operated with minimal peripheral accessories, including a portable pump for semi-automated sample delivery and a single heater for temperature control, for performing reverse transcription polymerase chain reaction (RT-PCR) integrated with a downstream fluorescence detection module for semi-quantitative assessment of gene expression. The microdevice was fabricated by wrapping a polytetrafluoroethylene (PTFE) tube around a pre-designed polycarbonate mold to create a seamless microchannel for both the reverse transcription (RT) of RNA and the amplification of complementary DNA. In addition, a silicone tube, which underwent a two-step surface modification mediated by polyethyleneimine and glutaraldehyde coating, was connected at the outlet to capture amplicons downstream of the PTFE tube for on-site fluorescence detection. This fabrication method enabled continuous-flow RT-PCR (CF RT-PCR) using the 3D CF RT-PCR microdevice as a reactor, a single heater for the temperature control of both RT and PCR processes, and a disposable plastic syringe for semi-automated sample delivery. The microdevice was successfully implemented for the identification of the β-actin gene, a constitutively expressed gene in all cells, and the sphingosine-1-phosphate lyase 1 gene, a potential pharmacological target gene in the diagnosis of cancer, diabetes, and atherosclerosis. This portable integrated microdevice offers a potential approach towards preliminary studies of gene expression and identification of RNA viruses.