Microfluidics and Nanofluidics

, Volume 5, Issue 6, pp 827–836 | Cite as

An optimal three-dimensional focusing technique for micro-flow cytometers

Research Paper


This study presents a novel three-dimensional (3-D) hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is initially compressed in the horizontal direction by two sheath flows such that it is constrained in the central region of the microchannel. The sample stream is then focused in the vertical direction by a second pair of sheath flows and subsequently passes over a micro-weir structure positioned directly beneath an optical detection system. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations to examine the effects on the sample stream distribution of the vertical and horizontal focusing ratios, the entrance angle of the second set of sheath flow channels, and the width and depth of the second set of sheath flow channels. The results indicate that the horizontal and vertical sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure results in the separation of the cells/particles in the vertical direction and ensures that they flow in a sequential fashion through the detection region of the microchannel and can therefore be reliably counted. It is shown that the 3-D focusing technique can achieve a focused sample stream width of between 6 and 15 μm given an appropriate value of the horizontal focusing ratio. Thus, the viability of the microflow cytometer for the counting and detection of individual biological cells is confirmed.


Micro-flow cytometer Three-dimensional hydrodynamic focusing Micro-weir structure Numerical simulations 



The current authors gratefully acknowledge the financial support provided to this study by the National Science Council of Taiwan under Grant NSC95-2314-B-020- 001-MY2.


  1. Bown MR, Meinhart CD (2006) AC electroosmotic flow in a DNA concentrator. Microfluid Nanofluid 2:513–523CrossRefGoogle Scholar
  2. Chang CC, Yang RJ (2007) Electrokinetic mixing in microfluidic systems. Microfluid Nanofluid 3:501–525CrossRefMathSciNetGoogle Scholar
  3. Chang YH, Lee GB, Huang FC, Chen YY, Lin JL (2006) Integrated polymerase chain reaction chips utilizing digital microfluidics. Biomed Microdevices 8:215–225CrossRefGoogle Scholar
  4. Chang CC, Huang ZX, Yang RJ (2007) Three-dimensional hydrodynamic focusing in two-layer polydimethylsiloxane (PDMS) microchannels. J Micromech Microeng 17:1479–1486CrossRefGoogle Scholar
  5. Chen D, Du H (2007) A dielectrophoretic barrier-based microsystem for separation of microparticles. Microfluid Nanofluid 3:603–610CrossRefGoogle Scholar
  6. Cheng IF, Chang HC, Hou D, Chang HC (2007) An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting. Biomicrofluidics 1:021503CrossRefGoogle Scholar
  7. Dittrich PS, Schwille P (2003) An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles. Anal Chem 75:5767–5774CrossRefGoogle Scholar
  8. Emmelkamp J, Wolbers F, Andersson H, DaCosta RS, Wilson BC, Vermes I, van den Berg A (2004) The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems. Electrophoresis 25:3740–3745CrossRefGoogle Scholar
  9. Erickson D (2005) Towards numerical prototyping of labs-on-chip: modeling for integrated microfluidic devices. Microfluid Nanofluid 1:301–318CrossRefGoogle Scholar
  10. Fu LM, Lin CH (2007) A rapid DNA digestion system. Biomed Microdevices 9:277–286CrossRefGoogle Scholar
  11. Fu LM, Yang RJ, Lee GB, Pan YJ (2003) Multiple injection techniques for microfluidic sample handling. Electrophoresis 24:3026–3032CrossRefGoogle Scholar
  12. Fu LM, Lee GB, Lin YH, Yang RJ (2004a) Manipulation of microparticles using new modes of travelling-wave-dielectrophoretic forces: numerical simulation and experiments. IEEE ASME Trans Mechatron 9:377–383CrossRefGoogle Scholar
  13. Fu LM, Yang RJ, Lin CH, Pan YJ, Lee GB (2004b) Electrokinetically driven micro flow cytometers with integrated fiber optics for on-line cell/particle detection. Anal Chim Acta 507:163–169CrossRefGoogle Scholar
  14. Fu LM, Tsai CH, Lin C H (2008) A high-discernment micro-flow cytometer with micro-weir structure. Electrophoresis (in press)Google Scholar
  15. Hardt S, Schonfeld F (2003) Laminar mixing in different interdigital micromixers: II. numerical simulations. AIChE J 49:578–584CrossRefGoogle Scholar
  16. Holmes D, Morgan H, Green NG (2006) High throughput particle analysis: combining dielectrophoretic particle focusing with confocal optical detection. Biosens Bioelectron 21:1621–1630CrossRefGoogle Scholar
  17. Huang MZ, Yang RJ, Tai CH, Tsai CH, Fu LM (2006) Application of electrokinetic instability flow for enhanced micromixing in cross-shaped microchannel. Biomed Microdevices 8:309–315CrossRefGoogle Scholar
  18. Hunt HC, Wilkinson JS (2008) Optofluidic integration for microanalys. Microfluid Nanofluid 4:53–59CrossRefGoogle Scholar
  19. Kohlheyer D, Unnikrishnan S, Besselink GAJ, Schlautmann S, Schasfoort RBM (2008) A microfluidic device for array patterning by perpendicular electrokinetic focusing. Microfluid Nanofluid (in press)Google Scholar
  20. Lacharme F, Gijs MAM (2006) Single potential electrophoresis microchip with reduced bias using pressure pulse injection. Electrophoresis 27:2924–2932CrossRefGoogle Scholar
  21. Lee GB, Hung CI, Ke BJ, Huang GR, Hwei BH, Lai HF (2001) Hydrodynamic focusing for a micromachined flow cytometer. Trans ASME 123:672–679Google Scholar
  22. Lee GB, Lin CH, Chang GL (2003) Micro flow cytometers with buried SU-8/SOG optical waveguides. Sensors Actuator A 103:165–170CrossRefGoogle Scholar
  23. Lee GB, Chang CC, Huang SB, Yang RJ (2006) The hydrodynamic focusing effect inside rectangular microchannels. J Micromech Microeng 16:1024–1032CrossRefGoogle Scholar
  24. Lee CY, Chen CM, Chang GL, Lin CH, Fu LM (2006) Fabrication and characterization of semicircular detection electrodes for contactless conductivity detector—capillary electrophoresis microchips. Electrophoresis 27:5043–5050CrossRefGoogle Scholar
  25. Lin CH, Lee GB, Fu LM, Chen SH (2004a) Integrated optical-fiber capillary electrophoresis microchips with novel spin-on-glass surface modification. Biosens Bioelectron 20:83–90CrossRefGoogle Scholar
  26. Lin CH, Lee GB, Fu LM, Hwey BH (2004b) Vertical focusing device utilizing dielectrophoretic force and its application on microflow cytometer. J Microelectromech Syst 13:923–932CrossRefGoogle Scholar
  27. Mao X, Waldeisena JR, Huang TJ (2007) “Microfluidic drifting”-implementing three-dimensional hydrodynamic focusing with a single-layer planar microfluidic device. Lab Chip 7:1260–1262CrossRefGoogle Scholar
  28. McClain MA, Culbertson CT, Jacobson SC, Ramsey JM (2001) Flow cytometry of Escherichia coli on microfluidic devices. Anal Chem 73:5334–5338CrossRefGoogle Scholar
  29. McClain MA, Culbertson CT, Jacobson SC, Allbritton NL, Sims CE, Ramsey JM (2003) Microfluidic devices for the high-throughput chemical analysis of cells. Anal Chem 75:5646–5655CrossRefGoogle Scholar
  30. Prakash R, Kaler KVIS (2007) An integrated genetic analysis microfluidic platform with valves and a PCR chip reusability method to avoid contamination. Microfluid Nanofluid 3:177–181CrossRefGoogle Scholar
  31. Rodriguez-Trujillo R, Mills CA, Samitier J, Gomila G (2007) Low cost micro-Coulter counter with hydrodynamic focusing. Microfluid Nanofluid 3:171–176CrossRefGoogle Scholar
  32. Stiles T, Fallon R, Vestad T, Oakey J, Marr DWM, Squier J, Jimenez R (2005) Hydrodynamic focusing for vacuum-pumped microfluidics. Microfluid Nanofluid 1:280–283CrossRefGoogle Scholar
  33. Sundararajan N, Pio MS, Lee LP, Berlin AA (2004) Three-dimensional hydrodynamic focusing in polydimethylsiloxane (PDMS) microchannels. J Microelectromech Syst 13:559–67CrossRefGoogle Scholar
  34. Tsai CH, Yang RJ, Tai CH, Fu LM (2005) Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips. Electrophoresis 26:674–686CrossRefGoogle Scholar
  35. Tsai CH, Chen HT, Wang YN, Lin CH, Fu LM (2007) Capabilities and limitations of 2-dimensinal and 3-dimensional numerical methods in modeling the fluid flow in sudden expansion microchannels. Microfluid Nanofluid 3:13–18CrossRefGoogle Scholar
  36. Tůma P, Samcová E, Opekar F, Jurka V, Štulík K (2007) Determination of 1-methylhistidine and 3-methylhistidine by capillary and chip electrophoresis with contactless conductivity detection. Electrophoresis 28:2174–2180CrossRefGoogle Scholar
  37. Van Doormal JP, Raithby GD (1984) Enhancements of the SIMPLE method for predicting incompressible fluid flows. Numer Heat Transfer 7:147–163CrossRefGoogle Scholar
  38. Wang Z, Hansen O, Petersen PK, Rogeberg A, Kutter JP, Bang DD, Wolff A (2006) Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency. Electrophoresis 27:5081–5092CrossRefGoogle Scholar
  39. Wolff A, Perch-Nielsen IR, Larsen UD, Friis P, Goranovic G, Poulsen CR, Kutter JP, Telleman P (2003) Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter. Lab Chip 3(1):22–27CrossRefGoogle Scholar
  40. Wu Z, Liu AO, Hjort K (2007) Microfluidic continuous particle/cell separation via electroosmotic-flow-tuned hydrodynamic spreading. J Micromech Microeng 17:1992–1999CrossRefGoogle Scholar
  41. Xia N, Hunt TP, Mayers BT, Alsberg E, Whitesides GM, Westervelt RM, Ingber DE (2006) Combined microfluidic-micromagnetic separation of living cells in continuous flow. Biomed Microdevices 8:299–308CrossRefGoogle Scholar
  42. Xuan X, Li D (2005) Focused electrophoretic motion and selected electrokinetic dispensing of particles and cells in cross-microchannels. Electrophoresis 26:3552–3560CrossRefGoogle Scholar
  43. Yang RJ, Fu LM (2001) Thermal and flow analysis of a heated electronic component. Int J Heat Mass Transfer 44:2261–2275MATHCrossRefGoogle Scholar
  44. Yang AS, Hsieh WH (2007) Hydrodynamic focusing investigation in a micro-flow cytometer. Biomed Microdevices 9:113–122CrossRefGoogle Scholar
  45. Yang R, Feedback DL, Wang W (2005) Microfabrication and test of a three- dimensional polymer hydro-focusing unit for flow cytometry applications. Sens Actuators A118:259–267Google Scholar
  46. Yu C, Vykoukal J, Vykoukal DM, Schwartz JA, Shi L, Gascoyne PRC (2005) A three-dimensional dielectrophoretic particle focusing channel for microcytometry application. J Microelectromech Syst 14:480–487CrossRefGoogle Scholar
  47. Zhuang GS, Li G, Jin QH, Zhao JL, Yang MS (2006) Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE. Electrophoresis 27:5009–5019CrossRefGoogle Scholar
  48. Zou H, Mellon S, Syms RRA, Tanner KE (2006) 2-dimensional MEMS dielectrophoresis device for osteoblast cell stimulation. Biomed Microdevices 8:353–359CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Chien-Hsiung Tsai
    • 1
  • Hui-Hsiung Hou
    • 2
  • Lung-Ming Fu
    • 2
  1. 1.Department of Vehicle EngineeringNational Pingtung University of Science and TechnologyPingtung Taiwan
  2. 2.Department of Materials EngineeringNational Pingtung University of Science and TechnologyPingtung Taiwan

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