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,
`*Department of 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 is 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 two carrier 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
`was also 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 in practical use.
`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 and finite element fluidic analysis to realize 3-D sheath flow are described in this paper.
`The first 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.1 (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
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`element fluidic 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
`sample inlet part. In this case, a C—shape microstructure is formed near the sample inlet.
`
`53 mple Flow
`Buffer Flow
`{Sheath Flow]
`
`
`
`amnp'l- i]: m
`Carrier Flow
`_——/
`
` Carrier Flor-\-
`
`(a) Ideal sheath flow (Cylindrical channel)
`
`(b) Proposed 3-D sheath flow
`
`Fig.1 Schematic of the particles and biomolecles handling micro flow system
`
`Glass or other materials
`
`Pyrex Class
`
` ‘— h‘~—/‘
`
`
`Carrier Inlet |
`Sample Ink-t
`(Tamer Inlet |
`Outlet
`
`
`
`Fig.2 Cross-sectional View of the proposed micro flow cell
`
`Sump!" |n|.."l
`
`(a) Model
`
`(b) Result (with C shaped wall)
`
`Fig.3 The result of finite element microfluidic analysis of
`the sample inlet part using a C—shaped wall
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`3. Measurement of the first prototype
`
`A first prototype of the micro flow cell (400 pm 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 ul/min to 20
`til/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
`
`(luM, Molecular Probe Co.: T-6027) was used as the carrier and 1 um 41 microspheres in red
`
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`211
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`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
`(CSUlO, Yokogawa Electric 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 mm apart from the both side of the channel wall. These results
`indicate that the lateral and vertical sheath flow was realized 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
`_
`18
`strongly depends on the
`second
`carrier flow rate.
`In order to achieve
`
`optimum design of the second prototype,
`comparison between the
`simulation
`results and the actual flow have been
`
`currently carried out.
`
`
`
`and
`
`#717
`
`0mm light
`i
`.
`
`3mm”
`me Laser .\
`
`ExcitationfiGan
`_ _
`- Obpecuve Lens
`
`game \
`Confocal Unit
`Carrier:
`Tmmethylmodarnine-S-maleimida
`{Molecular Probe 180”}
`Sample:
`1pm 9 miuoanpom in red fluorescent
`
`_
`
`_.
`_.
`Detemion:56?nrn
`1
`
`Fig.4 Setup of the flow observation system
`
`t. 21m he: —
`
`- sage Wall (3:) "
`'
`.
`‘ irricr Inch
`
`
`
`Fig.5 Schematic of the confocal scanner
`with microlens and pinhole arrays
`
`Fig.6 Video image of the lateral microshpere
`flow distribution in the micro channel
`(400 um in width and 100 mm in depth)
`
`151 Currier How ' Slut-"mm! Sample. Flow 3 [ZZqumin]
`.—
`
`mo
`
`
`030 [------ -.-
`------
`1'
`
`100 ml1
`
`|
`
`
`
`i
`
`J
`i
`l
`
`Relativeintensityofthefluorescence
`
`
`
`.
`
`..
`
`0.20
`
`- -
`
`000
`
`- +2ndi'arrichlow 5[u|.-'min]
`- - - -:
`+3nd{'nrri1:r Flow ][|J|"|TIII1]
`I
`: +2nd ('nrner Flow 3[u|-'nnn]
`I
`r
`.
`
`- -
`
`-
`
`(a) 10 pm deep
`
`(b) 50 pm deep
`
`(e) 90 mm deep
`
`25
`
`[“3”
`
`“
`
`Fig.7 Video images 0f the microsphere flOW at
`different depth (“the mICFO channel
`(400 um in width and 100 um in depth)
`
`Fig.8 Normalized vertical distribution of
`the microsphere flow under different 2nd
`carrier flow conditions
`
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`212
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`4. Micro flow switch
`
`One inlet 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 Ramstance
`
`Flow Resistance
`Decreased
`
`/l
`
`
`
`carrier
`
`
`
`Flowr Resistance
`Increased
`
`
`
`(all on
`
`{b} On
`
`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 sample distribution
`design optimization of the structure around the sample inlet and the second carrier inlet is
`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.
`
`a
`
`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
`
`1. P.Telleman, U.D.Larsen, J.Philip, G.Blankenstein, A.Wolff, “Cell sorting in microfluidic systems”,
`Micro Total Analysis Systems’98, Kluwer Academic Pub., (1998), 39-44
`sorting in a
`2. A.Wolff, U.D.Larsen, G.Blankenstein, J.Philip, P.Telleman, “Rare event cell
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`Kluwer Academic Pub., (1998), 77—80
`3. U.D.Larsen, G.Blankenstein, J.Branebjerg, “A novel design for chemical and biochemical liquid
`analysis system”, Proc. Micro Total Analysis Systems’96, (1996), 1 13-1 15
`4. G.Blankenstein, L.Scampavia, J.Braneberg, U.D.Larsen, J.Ruzica, “Flow switch for analyte
`injection and cell/particle sorting”, Proc. Micro Total Analysis Systems’96, (1996), 82-84
`5. R.Miyake,
`l.Yamazaki, H.0hki, T.Kaneko, “New sample transfer system for flow cytometer”,
`Proc. 4''1 Int. Symp. On Fluid Control, Fluid Measurement & Visualization, (1994), 870-874
`6. A.lchihara, T.Tanaami, K.lsozaki, Y.Sugiyama, Y.Kosugi, K.Mikuriya, M.Abe,
`I.Uemura,
`“High-speed confocal fluorescence microscopy using a nipkow scanner with microlenses for 3-D
`imaging of single fluorescent molecule in real time”, Bioimages 4(2), (1996), 57-62
`7. H.Sato, S.Shoji, E.Kim, K.Miura, “Partly disposable switching and injection microvalves for
`medical applications”, Proc. ASME Micro-Electro-Mechanical Systems, (1999), 241—245
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`ABS Global, Inc. and Genus plc - Ex. 1011, p. 212
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