Microfluidic chip-based protein capture from human whole blood using octadecyl (C18) silica beads for nucleic acid analysis from large volume samples.
We have previously described the development of a novel capillary-based photopolymerized monolith that offered unprecedented efficiency (approximately 85%) for DNA extraction from pre-purified human genomic DNA [J. Wen, C. Guillo, J.P. Ferrance, J.P. Lander, Anal. Chem. 78 (2006) 1673]. However, the major drawback associated with this phase was the limited binding capacity and low extraction efficiency (<40%) when purifying nucleic acids from a volume of whole blood greater than 0.1 microL. The limited DNA binding capacity, hypothesized to result from an overwhelming mass of protein overloading the monolith phase, severely limits the clinical utility, which will require a whole blood DNA capacity orders of magnitude larger. One proposed solution involved use of a protein capture bed to remove the majority of the protein present in blood before nucleic acid extraction was performed. To evaluate this, microchips with different channel configurations were designed and tested containing silica beads with various reversed phases, and their protein capture efficiency determined. Triton X-100 in the cell lysis buffer was found to be a critical component, greatly affecting the binding of proteins to the C18 reversed phase. An optimum Triton X-100 concentration of 0.1% was determined to enhance red and white blood cell lysis without adversely affecting protein binding to the C18 phase. A parallel 4-chamber design was found to be optimal, with 70% of the proteins (1020+/-45 microg) from a load solution containing 10 microL of whole blood captured on the C18 phase in a single microdevice. Electrophoretic analysis of the proteins in the flow-through of the C18 phase showed the absence of hemoglobin and larger proteins/peptides, indicating that they had been captured by the C18 phase, preventing these polymerase chain reaction inhibitory proteins from reaching and binding to the subsequent matrix which would be used for DNA capture.
Wen
Jian
J
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22904, USA.
Guillo
Christelle
C
Ferrance
Jerome P
JP
Landers
James P
JP
D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000097:blood*; D004867:Equipment Design; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D008832:Microchemistry / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques; D008863:Microspheres; D017830:Octoxynol / Q000737:chemistry; D017646:Organosilicon Compounds / Q000737:chemistry; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*
D001798:Blood Proteins; D008689:Methacrylates; D017646:Organosilicon Compounds; C026679:octadecylsilica; D012822:Silicon Dioxide; D017830:Octoxynol; D004247:DNA; C505504:3-(trimethoxysilyl)propyl methacrylate