A PCR reactor with an integrated alumina membrane for nucleic acid isolation

Abstract

Recently, there has been a growing interest in point-of-care devices capable of detecting nucleic acids (NA) in clinical and environmental samples. Nucleic acid detection requires, however, various sample preparation steps that complicate device operation. An attractive remedy is to integrate many, if not all, sample preparation operations and nucleic acid amplification into a single reaction chamber. A microfluidic chip that integrates, in a single chamber, polymerase chain reaction (PCR) amplification with solid-phase extraction of nucleic acids using a nanoporous, aluminium oxide membrane (AOM) is described. Samples suspected of containing target bacteria and/or viruses are mixed with lysis agents and a chaotropic salt and loaded into a plastic chip housing a nanoporous, aluminium oxide membrane. The nucleic acids in the lysate bind to the membrane. The membrane is then washed, the chamber is filled with the PCR reaction reagents, and the chamber's temperature is cycled to amplify the captured nucleic acids and produce detectable products. Both DNA and RNA (with reverse-transcription) isolation and amplification are demonstrated. Due to the dry membrane's high resistance to liquid flow, a specialized flow control system was devised to facilitate sample introduction and membrane washing.

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

“…The development of a fully functional integrated LOC microfluidic system that can perform cell lysis, nucleic acid extraction amplification, and detection with on-chip reagents is still a great challenge [2]; however, discrete examples of subfunctions of a complete system have been demonstrated in chip format over the last number of years and tested predominantly with cell line material, bacterial cultures, whole blood, or saliva specimens [27–30]. …”

“…The requirement to dilute the nucleic acid was completely dependent on the quality of the individual sample extract, with one sample amplifying equally well at the 1 : 5 as at the 1 : 10 dilution (Sample 508: HPV33) and another sample giving consistently bad results at either dilution. Many studies including those describing LOC devices have highlighted the amplification problems associated with contaminating ethanol and salts [27, 28]. …”

Miniaturized devices for point of care molecular detection of HIV

“…Most of commercially available portable molecular diagnostic systems, such as Cepheid GeneXpert, IQuum LIAT analyzer, utilize real-time RT-PCR assay to quantify HIV virus due to the high sensitivity, specificity and reproducibility. Early microfluidic-based molecular diagnostic devices used PCR technology to detect HIV [56,61]. However, PCR technology requires precise temperature control and rapid thermal cycling between 55 and 95°C, which is cumbersome to implement in resource-limited settings.…”

“…These have used multiple nucleic acid binding matricies, such as porous isolation membranes [50,51], silica-coated microchannels [52], monolithic sol-gels [53], and chitosan-coated silica beads [54]. Liu et al [55] reported a multifunctional amplification reactor chip (Fig.…”

Microfluidic “Pouch” Chips for Immunoassays and Nucleic Acid Amplification Tests

“…Pouches P 2 and P 3 are depressed in succession to wash the membrane-bound nucleic acid. For more detailed description regarding the structures and operations related to NA isolation, PCR, and detection, see Wang et al [19], Chen et al [5], and related work of Kim et al [22,23], Liu et al [24,25], and Mauk et al [26].…”