Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

Abstract

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with ~270 μm, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 μL of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/μL (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of λ-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Disposable polyester–toner electrophoresis microchips for DNA analysis

“…While the inherent low EOF is clearly advantageous for separations like DNA, and circumvents the need for coating the channels, they stated that PeT microchips presented a major drawback over the other chip substrates -low separation efficiency/poor resolution and the length of injection plug. In the same work, Coltro and co-workers demonstrated that the contribution of the channel wall, s 2 wall , accounted for almost 90% of the total variance, where in the glass chip, this parameter was <30%. Since the geometries of all devices compared were essentially identical, the poor performance of the PeT chips was attributed to the roughness of the channel wall.…”

“…3 In addition to the effects of the wall, not surprisingly, the injection plug length also has a significant influence on the separation efficiency. The contributions to total variance, s 2 T , in microchip electrophoresis are:…”

“…1 Polyester-toner (PeT) electrophoresis devices have exhibited a great potential for bioanalytical analysis. 2 PeT chips can be fabricated in a matter of minutes using a direct-printing process, which makes possible the production of tens of devices on a single transparency sheet (letter/A4 size) with consumables that cost less than 1.0 USD. The microfluidic architecture is defined by the white regions of a drawing, which is interpreted by a laser printer as an instruction to avoid the deposition of toner particles.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…The rapidly growing field of soft-lithography driven microfluidic technology has resulted in the development a plethora of micro-devices for extraction [75] and amplification and analysis [31,[76][77][78][79][80][81] of nucleic acids. The main idea of microarray technology, namely spatial separation of microarray elements and their simultaneous subjection to one sample of interest can be easily realized using highly parallel architectures of micrometresized channels available/affordable due to microfluidics.…”

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

“…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. Despite some commercially available systems (e.g.…”