title: Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device.
abstract: A silica monolith used to support both electro-osmotic pumping (EOP) and the extraction/elution of DNA coupled with gel-supported reagents is described. The benefits of the combined EOP extraction/elution system were illustrated by combining DNA extraction and gene amplification using the polymerase chain reaction (PCR) process. All the reagents necessary for both processes were supported within pre-loaded gels that allow the reagents to be stored at 4 degrees C for up to four weeks in the microfluidic device. When carrying out an analysis the crude sample only needed to be hydrodynamically introduced into the device which was connected to an external computer controlled power supply via platinum wire electrodes. DNA was extracted with 65% efficiency after loading lysed cells onto a silica monolith. Ethanol contained within an agarose gel matrix was then used to wash unwanted debris away from the sample by EOP (100 V cm(-1) for 5 min). The retained DNA was subsequently eluted from the monolith by water contained in a second agarose gel, again by EOP using an electric field of 100 V cm(-1) for 5 min, and transferred into the PCR reagent containing gel. The eluted DNA in solution was successfully amplified by PCR, confirming that the concept of a complete self-contained microfluidic device could be realised for DNA sample clean up and amplification, using a simple pumping and on-chip reagent storage methodology.
authors.affiliation: Department of Chemistry, The University of Hull, Cottingham Road, Kingston upon Hull, HU6 7RX, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Oakley
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: Shaw
authors.forename: Peter T
authors.initials: PT
authors.lastname: Docker
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Dyer
authors.forename: John
authors.initials: J
authors.lastname: Greenman
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Greenway
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Haswell
mesh_terms: D053523:Amelogenin / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D053841:Electroosmosis / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D000431:Ethanol / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D053523:Amelogenin; D005782:Gels; D000431:Ethanol; D012822:Silicon Dioxide; D004247:DNA