Micropillar array chip for integrated white blood cell isolation and PCR.

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.

[1]  E. Beutler,et al.  Interference of heparin with the polymerase chain reaction. , 1990, BioTechniques.

[2]  S. Narayanan Overview of principles and current uses of DNA probes in clinical and laboratory medicine. , 1992, Annals of clinical and laboratory science.

[3]  W. Al-Soud,et al.  Purification and Characterization of PCR-Inhibitory Components in Blood Cells , 2001, Journal of Clinical Microbiology.

[4]  L J Kricka,et al.  Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis. , 1996, Journal of chromatography. A.

[5]  Mann A. Shoffner,et al.  Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chambers. , 1998, Analytical biochemistry.

[6]  S. Quake,et al.  An Integrated Microfabricated Cell Sorter , 2022 .

[7]  R. Bashir,et al.  Dielectrophoretic separation and manipulation of live and heat-treated cells of Listeria on microfabricated devices with interdigitated electrodes , 2002 .

[8]  H. Carlson,et al.  Sorting of White Blood Cells in a Lattice , 1997 .

[9]  Vincent Studer,et al.  A nanoliter-scale nucleic acid processor with parallel architecture , 2004, Nature Biotechnology.

[10]  Kiumi Akingbehin,et al.  Biochip technology , 1984, SIGB.

[11]  Larry J Kricka,et al.  Surface Effects on PCR Reactions in Multichip Microfluidic Platforms , 2004, Biomedical microdevices.

[12]  Jeffery H. Fenton,et al.  A miniature integrated device for automated multistep genetic assays. , 2000, Nucleic acids research.

[13]  Robin H. Liu,et al.  Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. , 2004, Analytical chemistry.

[14]  L J Kricka,et al.  Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips. , 1996, Nucleic acids research.

[15]  Increased amplification efficiency of microchip-based PCR by dynamic surface passivation. , 2004, BioTechniques.

[16]  L J Kricka,et al.  Evaluation of DNA fragment sizing and quantification by the agilent 2100 bioanalyzer. , 2000, Clinical chemistry.

[17]  S. Quake,et al.  A microfabricated fluorescence-activated cell sorter , 1999, Nature Biotechnology.

[18]  S. Narayanan Concepts, principles, and applications of selected molecular biology techniques in clinical biochemistry. , 1996, Advances in clinical chemistry.

[19]  P. Wilding,et al.  Microchip module for blood sample preparation and nucleic acid amplification reactions. , 2001, Genome research.

[20]  A. R. Kaiser,et al.  Microfabricated structures for integrated DNA analysis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.