ABS Global, Inc. and Genus plc - Ex. 1011, cover 1
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`ABS Global, Inc. and Genus plc - Ex. 1011, cover 2
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`ABS Global, Inc. and Genus plc - Ex. 1011, cover 3
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`ABS Global, Inc. and Genus plc - Ex. 1011, cover 4
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`DESIGN AND SIMULATION OF PARTICLES AND
`BIOMOLECULES HANDLING MICRO FLOW CELLS WITH
`THREE-DIMENSIONAL SHEATH FLOW
`
`Koichi Tashiro, Tetushi Sekiguchi, Shuichi Shoji, Takashi Funatsu™,
`Wataru Masumoto, Hironobu Sato
`
`Department of Electronics, Information and Communication Engineering,
`*Departmentof Physics, Waseda University, 3-4-1 Ohkubo, Shinjuku,
`169-8555 Tokyo Japan
`E-mail:shojis@mn.waseda.ac.jp
`
`Keywords: Micro Flow Cell, 3D-Sheath Flow, Biomolecules Handling, Fluidic CAD
`
`Abstract
`In order to realize high performance particles and biomolecules handling systems, micro
`flow cell realizing three-dimensional (3-D) sheath flow was designed and the first prototype
`was fabricated. A finite element fluidic analysis is applied to realize optimal design of the
`flow cell. Especially, the structure of the sample injection part was considered to realize 3-D
`sheath flow. The high resolution flow monitoring method was studied and was applied to
`evaluate the flow behavior in the first prototype. The results indicated that the two steps
`introduction of the carrier flow is quite effective to put the sample flow away from the
`channel wall. This is very useful to avoid adsorption of the particles and the biomolecules on
`the wall due to the small flow rate near the wall. A simple micro flow switch for biomolecular
`separation 1s also proposed.
`
`1. Introduction
`Particles and cell handling micro fluidic devices have been developed using laminar
`behavior in microfabricated flow channels. These devices have ‘two-dimensional’ structures
`which have twocarrier inlets and one sample inlet located in the middle. [1,2,3] The sample
`flow was controlled by the flow ratio of the right and left carriers. A multi outlet flow switch
`wasalso demonstrated with the similar principle. [4] In these micro flow systems, the samples
`flow along the top and bottom wall of the channel where the flow rate is small. Unexpected
`adsorption of the particles or the bio molecules on the wall can be a problem inpracticaluse.
`A miniaturized sample transfer system realizing perfect sheath flow was developed for flow
`cytometers.
`[5] It has optimized structure to reduce sample dispersion and to realize low
`pressure loss compared to the conventional systems. We propose particles and biomolecules
`handling micro flow cells with simpler structure fabricated by simple micromachining. The
`design andfinite elementfluidic analysis to realize 3-D sheath flow are describedin this paper.
`Thefirst prototype of the flow cell was fabricated and flow in the channel was investigated. A
`micro flow switch having a simple structure for biomolecular separation is also proposed.
`
`2. Design and simulation
`The coaxial sheath flow in the cylindrical channel (Fig.1 (a)) is used in the conventional
`flow cytometer. The schematic of the proposed micro flow cell to realize three-dimensional
`sheath flow is illustrated in Fig.l (b). To realize the vertical sheath flow with simple inlet
`structure of carrier and sample, two steps introduction of carrier flows was considered. The
`side view of the designed micro flow cell is shown in Fig.2. Since the flow distribution of the
`sample is critically changed with the pressure and the flow rate of the carrier, a 3-D finite
`209
`
`A. van den Berg et al. (eds.), Micro Total Analysis Systems 2000, 209-212.
`© 2000 Kluwer Academic Publishers.
`
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`210
`
`elementfluidic analysis is indispensable to design the micro flow cell. A FlumeCAD system
`(Microcosm Technologies,
`Inc.) was used for this purpose. One of the results of the
`microfluidic simulations is shown in Fig.3: (a) the model and (b) the simulation result of the
`sampleinlet part. In this case, a C-shape microstructure is formed near the sample inlet.
`
`Sample Flow
`
`Buffer Flow
`
`
`
`
`Sample Flow
`
`
`(a) Ideal sheath flow (Cylindrical channel)
`
`Carrier Flaw
`
`(b) Proposed 3-D sheath flow
`
`Fig.1 Schematic of the particles and biomolecles handling micro flow system
`
` ”\ (Connector \\comecog”
`
`
`
`
`Carrier Inlet ]
`Sample Inlet
`CarrierInlet |
`Outlet
`
`Fig.2 Cross-sectional view of the proposed micro flow cell
`
`(Sheath Flow) Carrier Flow
`
`RT ea
`
`(b) Result (with C shaped wall)
`(a) Model
`Fig.3 The result of finite element microfluidic analysis of
`the sample inlet part using a C-shaped wall
`
`3. Measurementofthefirst prototype
`A first prototype of the micro flow cell (400 um in width and 100 pm in depth) was
`fabricated by both side etching of a silicon wafer and bonding with a Pyrex glass. Small
`syringe micropumps under computer control (controllable flow range from 0.1 l/min to 20
`pl/min) were used to control the sample and carrier flow. The flow in the cell was observed
`using the optical
`flow detection system as
`shown in Fig.4. Diluted fluorescent
`(1uM, Molecular Probe Co.: T-6027) was used as the carrier and 1 um 6 microspheres in red
`
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`21)
`
`fluorescent was used as the sample. The fluorescence images of the optical sections of 2 um
`are obtained at video-rate by using a confocal scanner with microlens and pinhole arrays
`(CSU10, YokogawaElectric Corp.) whose system principle is illustrated in Fig.5. [6] Fig.6 is
`lateral flow distribution of the microsphere observed from the topside. Fig.7 is the tomograms
`of the microchannels at just the lower part the second carrier inlet. Very few microspheres
`were observed within 10 um apart from the both side of the channel wall. These results
`indicate that the lateral and vertical sheath flow wasrealized by the simple structure. Fig.8 is
`the normalized vertical flow distribution
`of the microsphere estimated from the
`intensity of
`the
`fluorescent
`under
`different carrier flow conditions. The
`vertical flow distribution of the sample
`is
`strongly depends on the
`second
`carrier flow rate.
`In order to achieve
`
`
`
`Q Infrared light
`|
`alsa
`YAG Laser 2,
`
`
`hs
`ff”
`Excitation:532nm
`
`Ee Objective Lens
`
`ae
`csuio
`Confocal Unit Carrier:
`(Molecular Probe T-6072)
`Sample:
`dpm @ microshpere in red fluorescent
`
`Detection:567nm
`
`Fig.4 Setup of the flow observation system
`
`simulation
`comparison between the
`results and the actual flow have been
`currently carried out.
`
`Yo
`
` |M
`
`optimum design of the second prototype, Tetramethylrhodamine-5-maleimide
` *
`s
`
`fis
`
`Relativeintensityofthefluorescence
`
`acy
`‘arrier Inlet
`
`Outlet >
`
`Fig.5 Schematic of the confocal scanner
`with microlens and pinhole arrays
`
`Fig.6 Video image ofthe lateral microshpere
`flow distribution in the micro channel
`(400 um in width and 100 um in depth)
`
`Ist Carrier Flow : S{ul/min] Sample Flow: [2ul/min]
`
`i}
`
`1
`'
`
`1
`
`100um
`
`
`
`—O— 2nd Carrier Flow: 5[ul/min]
`4 —— 2nd Carrier Flow|3[ul/min] }
`
`
`: te 2nd Carrier Flow:2[ul/min] |
`0,00
`*
`E
`+
`!
`DhacalaeNeuen
`-
`a
`Fig.8 Normalized vertical distribution of
`the microsphere flow under different 2nd
`carrier flow conditions
`
`(a) 10 umdeep (b) 50 umdeep (c) 90 yum deep
`Fig.7 Video images of the microsphere flow at
`different depth of the micro channel
`(400 um in width and 100 um in depth)
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`212
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`4. Micro flow switch
`Oneinlet and two outlets micro flow switch using 3-D sheath flow is proposed. The
`schematic of the outlets is shown in Fig.9. Pneumatic actuators are considered to control the
`variable flow resistances of the outlets. [7] The pneumatic actuator using 4 um thick CPTF
`polymer (Cytop: Asahi Glass Co.) membrane was fabricated and their basic characteristics
`were evaluated.
`
`Pneumatic Valuable
`Flow Resistance
`
`Flow Resistance
`Decreased
`
`
`
`i\
`
`
`
`3
`
`—s
`
`
`
`
`Flow Resistance
`Increased
`
`(b) On
`(a) Off
`Fig.9 Schematic of the micro flow switch for particles and biomolecles handling
`(with pneumatic variable flow resistances)
`
`5. Conclusions
`Micro flow cell realizing three-dimensional (3-D) sheath flow was designed and the first
`prototype was fabricated and evaluated. Three-dimensional sheath flow was realized in the
`fabricated micro flow cell. In order to reduce sample dispersion and finer sampledistribution,
`design optimization of the structure around the sample inlet and the second carrier inletis
`currently carried out using finite element flow analysis. Since higher resolution of the
`fluorescent image can be obtained with the same optical flow detection system, the principle
`of 3-D sheath flow will be applied to realize biomolecules observation and handling systems.
`
`Acknowledgement
`Authors thank to Mr. I.Kinoshita and Mr. S.Miyashita of Marubeni Solution Co. for their
`advices on Flume CAD. A part of this work was supported by the Grant-in-Aid for University
`and Society Collaboration No. 11794006, Micro Machine Center of Japan, Electromechanical
`Technology Advancing Foundation and Takahashi Foundation.
`
`References
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`Micro Total Analysis Systems’98, Kluwer Academic Pub., (1998), 39-44
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`3. U.D.Larsen, G.Blankenstein, J.Branebjerg, “A novel design for chemical and biochemical liquid
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`4. G.Blankenstein, L.Scampavia, J.Braneberg, U.D.Larsen, J.Ruzica, “Flow switch for analyte
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`5. R.Miyake, 1.Yamazaki, H.Ohki, T.Kaneko, “New sample transfer system for flow cytometer”,
`Proc. 4" Int. Symp. On Fluid Control, Fluid Measurement & Visualization, (1994), 870-874
`6. A.Ichihara, T.Tanaami, K.Isozaki, Y.Sugiyama, Y.Kosugi, K.Mikuriya, M.Abe,
`[.Uemura,
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`7. H.Sato, S.Shoji, E.Kim, K.Miura, “Partly disposable switching and injection microvalves for
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`ABS Global, Inc. and Genusple - Ex. 1011, p. 212
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