Microchip-based macroporous silica sol-gel monolith for efficient isolation of DNA from clinical samples.
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.
Wu
Qirong
Q
Department of Chemistry and Department of Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, Virginia 22904, USA.
Bienvenue
Joan M
JM
Hassan
Benjamin J
BJ
Kwok
Yien C
YC
Giordano
Braden C
BC
Norris
Pamela M
PM
Landers
James P
JP
Ferrance
Jerome P
JP
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
D004269:DNA, Bacterial; D004279:DNA, Viral; D017646:Organosilicon Compounds; C500739:tetramethyl orthosilicate; D004247:DNA