title: Microchip-based macroporous silica sol-gel monolith for efficient isolation of DNA from clinical samples.
abstract: Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.
authors.affiliation: Department of Chemistry and Department of Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: Qirong
authors.initials: Q
authors.lastname: Wu
authors.forename: Joan M
authors.initials: JM
authors.lastname: Bienvenue
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hassan
authors.forename: Yien C
authors.initials: YC
authors.lastname: Kwok
authors.forename: Braden C
authors.initials: BC
authors.lastname: Giordano
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Norris
authors.forename: James P
authors.initials: JP
authors.lastname: Landers
authors.forename: Jerome P
authors.initials: JP
authors.lastname: Ferrance
mesh_terms: D004247:DNA / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification; D004279:DNA, Viral / Q000134:cerebrospinal fluid / Q000302:isolation & purification; D006801:Humans; D046208:Microchip Analytical Procedures / Q000379:methods*; D017646:Organosilicon Compounds; D044367:Phase Transition; D016133:Polymerase Chain Reaction; D016062:Porosity
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D017646:Organosilicon Compounds; C500739:tetramethyl orthosilicate; D004247:DNA