A Simple, Valveless Microfluidic Sample Preparation Device for Extraction and Amplification of DNA from Nanoliter-Volume Samples

Abstract

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system

“…T here has thus been an effort to develop low-cost, automated diagnostic biosensors (e.g. nanotechnology-based sensors (Zhang et al 2005) or microfluidic systems, such as droplet -based , Chiou et al 2013, centrifugal (Cho et al 2007), paper (Martinez et al 2009, Fu et al 2010, Zuo et al 2013, capillary (T akayama et al 1999, Yager et al 2008, Gervais and Delamarche 2009, pneumatic (Legendre et al 2006, Mohan et al 2013 devices and lateral flow assays (Yager et al 2008, Chen et al 2010, You et al 2011). For the past decade, this has led to numerous publications about promising, early-stage technologies (Baeumner et al 2003, Leonard et al 2003, Kost 2006, Lazcka et al 2007, Lucchi et al 2010, Asiello and Baeumner 2011, Duarte et al 2011, Karlsson et al 2011, Schudel et al 2011, Matlock-Colangelo and Baeumner 2012, Lounsbury et al 2013, Mohan et al 2013.…”

Analysis of Electric Fields inside Microchannels and Single Cell Electrical Lysis with a Microfluidic Device

“…Such a development will be valuable for emergency responders, criminal investigators and forensic identification. In this work, electrical lysis of biological cells is investigated with a commonly used microchannel structure, where the electrode with excitation potential is positioned at one end of the microchannel, whereas the electrode at the other end of the microchannel is grounded [5][6][7][8][9][10]. The novelty of this work is the analysis of the estimation errors of electric fields of typical microfluidic devices used in such experiments and the models to reduce such estimation errors to achieve a high yield, which is critical for single-cell analysis.…”

“…This is the most commonly used expression to estimate the electric field strengths inside the microchannels of microfluidic devices [5][6][7][8][9], even though the assumptions involving dielectric constant, fringe field and parallel electrode surface are invalidated. Furthermore, solid-liquid interface develops an electrical double layer (EDL) with the characteristic thickness of the Debye length, which has significant influence on the behavior of ionic conductors.…”

“…The microchannel structure under consideration has a uniform cross-section throughout its length, and the electrodes are placed at both terminals of the microchannel. There are a number of reports that share this topology [5][6][7][8][9][10]15]. This work attempts to analyze the estimation error stemming from Expression (2) and proposes two analytical expressions to reduce the error.…”

Extraction of genomic DNA using a new amino silica monolithic column

“…Both high concentration salts and organic solvents are potent inhibitors to the downstream processes such as PCR. Legendre et al [18] reported that DNA in the initial fraction eluted from the silica beads was poorly amplified, and thus a longer elution time was required to remove the residual inhibitors. Witek et al [19] removed ethanol by drying the extraction device with nitrogen gas before the elution step.…”