Company LOGO A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process Pichit Sirikriangkrai ( 李俊榮 ), ME November 5 th, 2012

Topics 1.Introduction 2.Working principle and design 3.Fabrication process 4.Experimental result 5.Conclusions

1. Introduction 1.1) What is benefit of DEP 1.DEP devices can be fabricated easily 2.Eliminate clogging problem that previously occur in filtration method 3.Wide range for using in area of  food pathogen screening  environmental monitoring  biomedical research for drug discovery

1. Introduction 1.2) DEP Process DEP device can be used to study the particle biological cells such as proteins or DNA that applying knowledge of electrical, kinetic, and knowledge of biology at the cellular level integration. The device is a small laboratory at the cellular level that can monitor and track changes in the cell. Or the type of cell division immediately.

1. Introduction 1.2) DEP Process Conceptual view of hydrodynamic dielectrophoresis (DEP) process.

2. Working principle and design 2.1) Working principle The essence of the phenomenon of electrophoresis applications is to control the motion of spherical particles under an electric field by changing the concentration of the electric field. Direction of cell movement depending on DEP response: (a) positive DEP cells and (b) negative DEP cells.

2. Working principle and design 2.2) Dielectrophoresis force F DEP = 2π r 3 ε m {Re[K(ω)]} · ∇ |E rms | 2 F DEP = (volume)·(dielectric permittivity of medium)·(Clausius- Mossoti function)·(field gradient) Clausius-Mossoti function => Re[K(ω)] The Clausius–Mossotti factor can be expressed in terms of complex permittivities where ε* is the complex permittivity (ε* = ε – jσ/ω) σ is the conductivity ω is the electric field frequency. and subscripts p and m mean cells and the medium, respectively. Re[K(ω)] > 0 means that cells show pDEP response while Re[K(ω)] < 0 means nDEP response.

2. Working principle and design 2.2) Dielectrophoresis force Numerical simulation of the electric field distribution over the electrodes at the cross-section, A–A

3. Fabrication process 3.1) Fabrication process Fabrication process of present devices illustrating the cross-section, A–A’

3. Fabrication process 3.1) Fabrication process Picture (a) is overall view compared with a penny Picture (b) is enlarged view of microchannel.

4. Experimental results 4.1) Sample preparation Yeast cells are grown at 30 ◦C for 24 h in culture medium. viability of yeast cells is color by a methylene blue stain. Nonviable yeast cells are prepared by heat treatment (90 ◦C for 20 min) Conductivity of the medium is adjusted by adding a small amount of NaCl and conductivity is measured by HI8733 (HANNA instruments).

4. Experimental results 4.2) Experimental set-up Using syringe pump for cell mixture and buffer flow-rate control. Observe the cell separation by CCD camera from microscope. In order to prevent cell trapping at the electrode edges by the pDEP, we use pulsed DEP force that there is no DEP force during the half of a period.

4. Experimental results 4.3) DEP response test In order to obtain the optimal separation condition, we adjust the medium conductivity and the electric fields frequency The brighter stripes are electrodes and the darker are glass substrate (a) viable yeast cells at 10 kHz (b) viable yeast cells at 5 MHz (c) nonviable yeast cells at 10 kHz (d) nonviable yeast cells at 5 MHz

4. Experimental results 4.3) DEP response test DEP response at the electric field frequency of 5 MHz, the medium conductivity of 5 S/cm, At this conditions Viable yeast show as pDEP response Nonviable yeast show as nDEP response

4. Experimental results 4.4) Continuous separation At the mixture flow rates of 0.1–1 l/min. We have purity % of viable cell 64.5–74.3% of nonviable cell

4. Experimental results 4.4) Continuous separation Microscopic images of yeast cells: (a) yeast cells mixture before separation (b) separated viable yeast cells at outlet 1 (c) separated nonviable yeast cells at outlet 2.

5. Conclusions 5.1) Conclusions The purity of the separated viable and nonviable yeast cells has been measured in the range of 95.9–97.3% and 64.5–74.3%, respectively, at the mixture flow-rates of 0.1–1 l/min. The present chip is promising for applications to high-throughput integrated biological analysis systems

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