An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis

Abstract

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase

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Portable nucleic acid thermocyclers

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“…83 In comparison to an optical detection module which has similar detection capability, this is a large footprint. 80 Another issue to note is that capillary electrophoresis does not provide a detection method on its own. 31 This is a large footprint for a single well system.…”

“…While Fang X. et al implemented the contactless detection as part of a microfluidic chip, 37 moving the detection to the instrument would provide the advantage of a reduction in cost and complexity of the consumable. 80 Moving the detection electrodes outside the reaction chamber is an advantage because it removes the risk of inhibiting the amplification reaction. 37 In fact, Jha et al applied a DC voltage in order to generate hydroxide ions with the purpose of hydrolyzing the cell membrane.…”

Multi-Dimensional Nanostructures for Microfluidic Screening of Biomarkers: From Molecular Separation to Cancer Cell Detection

“…In comparing microfluidic immunoassays to microtiter plate immunoassays and protein microarrays, for the same level of performance, microfluidic immunoassays outperforms the other two, requiring less sample volume and shorter time-to-result 49 . Much research has been done to take conventional analytical methods and scaling them down to micro- and nanoscale regimes to improve performance, from centrifugal microfluidics 2,6,27,71 to electrophoretic microfluidics 18,36,66 to mass spectrometric microfluidics 37,53,65 . As a result of such advantages, microfluidics has been a tool of interest in a variety of biological applications, particularly in molecular and cellular separations and detection.…”