August 28, 2009Soumya K Srivastava
| Soumya K Srivastava - A Three-Dimensional Dielectrophoretic Particle Focusing Channel for Microcytometry Applications
Choongho Yu, Jody Vykoukal, Daynene M. Vykoukal, Jon A. Schwartz, Li Shi, and
Peter R. C. Gascoyne
| | | | Review for Journal Club
August 28th 2009
Soumya K. Srivastava
Overview:
Here authors have fabricated a microdevice for focusing biological cells by dielectrophoresis for cytometry applications. Fabrication of an elliptic channel was by isotropic etching of glass wafers. Experiments were conducted using latex microbeads and human leukemia HL60 cells. Focusing of cells and latex beads occurred at an AC peak to peak voltage of 15 V and below 100 kHz frequency. Authors claim that this design eliminated bulky and complicated components compared with conventional cytometer which made it more portable. There have been several authors who have attempted to fabricate micro flow cytometers.
In this paper, the authors discuss about the concept of dielectrophoresis and how they use this technique to build a micro flow cytometer. They have replaced the hydrodynamic flow system with electrodes to generate DEP force. They simulated the system using Maxwell-2D software package from Ansoft Corp. From simulations, they found that the minima of the magnitude of gradient E2 was at the center of channel and all the particles were focused by negative DEP.
For the experiments, epifluorescent microscope with a CCD camera was used. To verify whether the particles were indeed focused or not, they changed the frequency and conductivity of the medium; thus observing that beads were not focused at this range. They also estimated temperature rise due to Joule heating and concluded that focusing of particles were not due to thermally induced flow. Mean flow velocity of the particles and cells were also calculated along with width of the particle streams. Authors also observed no cell damage at peak to peak voltage of 15 V and did not find any accumulation of peroxide due to the suspending medium.
Introduction:
1. Discussion of flow cytometry concepts was good.
a. Applications of flow cytometer
b. Focusing of particles
c. Conventional flow cytometer disadvantages- large, expensive, prone to misalignment, thermal expansion, vibration. Basically a complicate system- have to control flow rates and need of a reservoir
2. Possible solution of using MEMS to avoid drawbacks of conventional flow cytometer
a. Many researchers in the past have explored this option.
b. They were successful in reducing the size of the hydrodynamic focusing system, eliminating liquid reservoirs, eliminating mechanical pump and opting for electrokinetic flow.
c. Some authors tried to modify channels to focus the particles, but this often clogged their system due to small channel sizes.
3. Dielectrophoretic particle focusing system.
a. Definitions and concepts were well presented.
b. In the past, researchers have been successful in manipulating biological particles by DEP.
c. Some researchers modified the electrode geometry to achieve focusing of nano or micro particles.
d. Cheung et al. combined DEP with impedance for particle sizing.
4. In this study, 3-D electrode design in elliptic-like channel has been used to focus the particles via negative DEP.
a. Cells directed towards center of channel
b. Voltage applied between adjacent electrodes on the channel wall
c. 15 Vp-p AC voltage and frequency below 100 kHz was used for focusing of cells.
Theory:
1. Well explained equations.
2. Eq 5 and 6- is this the thin shell model which they talk about?
3. DEP affected by several factors
a. To obtain maximum focusing of particles, the operating regime should be below the cross over frequency
4. Homogenous particle model?
5. A very low frequency close to DC needs to be avoided to prevent hydrolysis of medium??? What is the value??
Design:
1. Electrode fabricated on the circumference of the elliptical-like channel
a. Minimum field gradient at the center of the channel
2. DC voltage of 7.5 V used for simulation?
Fabrication:
1. Soda lime glass wafers- washed with piranha- HMDS coating to promote adhesion of photoresist
2. Electrodes obtained using lift-off process in acetone?
a. Lift-off process is a method of creating structures of target material on the surface of substrate using a sacrificial material.
3. After wafer bonding, plastic tube inserted into ports and sealed with epoxy
Experiment:
1. Used DI water to dilute latex beads and then made up the conductivity to a desired value by PBS
a. Concentration of PBS used not mentioned, was it pure salts?
2. What is serum-free RPMI tissue culture medium?
3. Conductivity values different for latex beads and cells
4. Cells stored in sucrose and dextrose,
a. Reference would have been good as to how they estimated the concentration of sucrose and dextrose needed
b. Why both sucrose and dextrose mixed solution?
5. Wavelength (excitation and emission) information missing for blue light and particles
6. Frame speed of pictures taken?
7. Figure 9 should have been of better quality and more number of particles/ cells should be shown
8. Verification of focusing of particles/cells
a. by increasing conductivity of the medium and frequency was a good measure
b. Calculation of temperature rise in the system to prove that the focusing was not due to thermally induced flow.
9. Measuring width of focusing stream, how was this done?
10. Is peroxide accumulation found in PDMS devices?
Conclusion:
a. Nice concept of building a micro flow cytometer.
b. Very well written introduction and theory with well represented figures.
c. Method of calculating temperature rise is a good idea to see at what voltage Joule heating would occur in my system.
d. Dilution by DI water and then adjusting the conductivity could be used for latex beads but difficult for blood samples due to isotonicity and osmolarity constraints.
e. Accept with slight revisions.
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