A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids.

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.

[1]  A simple dual pressure-ejection system and calibration method for brief local applications of drugs and modified salines , 1995, Journal of Neuroscience Methods.

[2]  Ofer Feinerman,et al.  A picoliter ‘fountain-pen’ using co-axial dual pipettes , 2003, Journal of Neuroscience Methods.

[3]  E. Delamarche,et al.  Patterned delivery of immunoglobulins to surfaces using microfluidic networks. , 1997, Science.

[4]  Kit T. Rodolfa,et al.  Two-component graded deposition of biomolecules with a double-barreled nanopipette. , 2005, Angewandte Chemie.

[5]  Kit T. Rodolfa,et al.  Nanoscale pipetting for controlled chemistry in small arrayed water droplets using a double-barrel pipet. , 2006, Nano letters.

[6]  T. Matsue,et al.  Electrochemical response at microarray electrodes in flowing streams and determination of catecholamines , 1990 .

[7]  C. Cotman,et al.  A microfluidic culture platform for CNS axonal injury, regeneration and transport , 2005, Nature Methods.

[8]  G. Whitesides.  The origins and the future of microfluidics , 2006, Nature.

[9]  A. Manz,et al.  Micro total analysis systems. Latest advancements and trends. , 2006, Analytical chemistry.

[10]  P. Kissinger,et al.  Difference mode detection with thin-layer dual-electrode liquid chromatography/electrochemistry , 1985 .

[11]  T. Matsue,et al.  Dual Imaging of Topography and Photosynthetic Activity of a Single Protoplast by Scanning Electrochemical Microscopy , 1999 .

[12]  Satoshi Takahashi,et al.  Multiple-sheathflow capillary array DNA analyser , 1993, Nature.

[13]  Y. Hirano,et al.  Topographic, electrochemical, and optical images captured using standing approach mode scanning electrochemical/optical microscopy. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[14]  Horiuchi,et al.  Subnanoliter volume wall-jet cells combined with interdigitated microarray electrode and enzyme modified planar microelectrode , 2000, Analytical chemistry.

[15]  Shuichi Takayama,et al.  Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics. , 2007, Lab on a chip.

[16]  H. Shiku,et al.  Regulation and characterization of the polarity of cells embedded in a reconstructed basement matrix using a three‐dimensional micro‐culture system , 2007, Biotechnology and bioengineering.

[17]  C. Larabell,et al.  Reversion of the Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies , 1997, The Journal of cell biology.

[18]  David Juncker,et al.  Multipurpose microfluidic probe , 2005, Nature materials.

[19]  Takashi Anazawa,et al.  Multiple Sheath-Flow Gel Capillary-Array Electrophoresis for Multicolor Fluorescent DNA Detection , 1994 .

[20]  P. Unwin.  THE MARLOW MEDAL LECTURE Dynamic electrochemistry as a quantitative probe of interfacial physicochemical processes , 1998 .

[21]  O. Lev,et al.  Scanning capillary microscopy/mass spectrometry for mapping spatial electrochemical activity of electrodes. , 2001, Analytical chemistry.

[22]  G M Whitesides,et al.  Patterning cells and their environments using multiple laminar fluid flows in capillary networks. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Tomoyuki Yasukawa,et al.  Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis. , 2007, Analytical chemistry.

[24]  E. Delamarche,et al.  Microfluidics for Processing Surfaces and Miniaturizing Biological Assays , 2005 .

[25]  Yoshiyuki Miwa,et al.  Automatic positioning of a microinjector in mouse ES cells and rice protoplasts. , 2006, Bioelectrochemistry.

[26]  Owe Orwar,et al.  Scanning electroporation of selected areas of adherent cell cultures. , 2007, Analytical chemistry.

[27]  Jayanta Debnath,et al.  Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. , 2003, Methods.

[28]  Nancy L Allbritton,et al.  CRITICAL REVIEW www.rsc.org/loc | Lab on a Chip Analysis of single mammalian cells on-chip , 2006 .

[29]  Joe W Gray,et al.  Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo. , 2006, Cancer research.

[30]  D J Harrison,et al.  mRNA isolation in a microfluidic device for eventual integration of cDNA library construction. , 2000, The Analyst.

[31]  T. Matsue,et al.  Multichannel electrochemical detection system for flow analysis , 1990 .

[32]  Matsuhiko Nishizawa,et al.  Localized chemical stimulation to micropatterned cells using multiple laminar fluid flows. , 2003, Lab on a chip.

[33]  J. Jorgenson,et al.  Microcolumn separations and the analysis of single cells. , 1989, Science.

[34]  Liming Ying,et al.  Multicomponent submicron features of biomolecules created by voltage controlled deposition from a nanopipet. , 2003, Journal of the American Chemical Society.