an investigation into the “world-to-chip” interface...drop ejection into a pipette water...
TRANSCRIPT
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An Investigation into the “World-to-chip” InterfaceWorld to chip Interface
Sammy Datwani, Ph.DJune 17, 2009
TFUG Meeting, San Jose, CA
Move Liquids With Sound
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HPLC on-a-chip
Overview • Challenges of small scale chromatography• Advantages of microfluidics • Component development for LC on-chipp p p• Separations
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Shrinking an HPLC
Goal: Miniaturize HPLC and maintain performance minimize system dispersion
σ2 = σcol2 + σinj
2 + σtube2 + σfittings
2 + σdet2
σext2
Loss of separation efficiency from components
• Large injection volumesd = 1 mm σext2 = 45,300 nL2
• Connection tubing• Fittings and connectors• Detector cell
extd = 0.3 mm σext
2 = 370 nL2
d = 0.1 mm σext2 = 14 nL2
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Why Microfabricate?
Benefits of Microfabricated LC• Reduce number of system components• Reduce human intervention • Improve system-to-system reproducibility• Increase performance• Green operation• Scalability of costScalability of cost• Expand capabilities of HPLC
– Sample handling and transferPortability– Portability
• On-line reaction monitoring• Field-portable HPLC systems
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Required Technology
• MicrofabricationMicrofabrication
• Precision Fluid Delivery
• Detection
• Integration
• Chip-to-World Interface
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Microfabrication
• High pressure compatible structures (>5,000 psi)
• Chemical compatibility
• High temperature operation• High temperature operation
• On-chip functionality:–Valves (pL volumes)–Injectors (precise 5 nL injections)–Columns (variety of phases)–Detectors (UV, Echem, MS, …)–Connectors (high pressure, user-friendly)–Sample Preparation (filters, traps, …)
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Chip to World Interface - Connectors
• Double-side etching in silicaC li d i l h l
Injector
– Cylindrical channels
– Separation columns
– Edge connectors
– Injectors
– Detection sensitivity
– Overall system performance
• Better pressure performance – 6000 psi routine Column Connector port
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Connectors – Alignment
GasketFixture
Lithography-controlled alignment of multiple simultaneous connections
ChipCapillaryJig
Chip
Jig
Chemically-Etched Shim Stock
A ld k S S i C h “ i fl idi C i ” S A li i # S200 0 6321A1 CD. W. Arnold, K. R. Hencken, P. P. Leung, S. S. Datwani, D. R. Cyr , J. E. Rehm.“Microfluidic Connections” US Application: # US2005056321A1; PCT
Application: # WO2005096751A3; EP Application: # EP1740984A3; CA Application: # 2561508A1.
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Connectors – Micro injection molded gasketmolded gasket
Material: Polypropylene for mechanical and chemical properties
Critical Dimensions: 150 um through vias on 1 mm pitchthrough vias on 1 mm pitch
Two per 6-port connection
Brass shim stock for retainer
Excellent performanceFluidic channel
capillaryJig chip
Working fluidic chip
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Connectors – Assembly performance
Robust connection for parallel channels to outside world
Excellent performanceExcellent performance• > 7200 psi demonstrated• 50+ make/breaks for 6 connections to
4500 psiMi il i f dd d li• Microcantilever springs for added alignment
precision - excellent• Chip caddy• Micro-injection molded gaskets and j g
lithographically defined retainers
Minor issues with chipping of parts being dd daddressed.
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Precision Fluid Delivery
Microfluidic Flow Controller (MFC)
• Active Flow Control - feedback control on
Microfluidic Flow Controller (MFC)
rapid, electronically adjusted variable pressure source • Flow meter measures the flow rate (nL/min to 10s of µLs/min)• Flow rate is maintained regardless of system back pressure or viscosity
Hi h f li bl i l fl• High performance, reliable microscale flow source• Fast, reproducible gradients
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Microfluidic Flow Controller
402
n) 1 nL/min
398
400
Rat
e (n
L/m
i 1 nL/minstep
2 nL/minstep
0 100 200 300
396
Flow
9000 Qa(nL/min) Qa_profile(nl/min) Qb(nL/min) Qb_profile(nl/min)
/min
)
0 100 200 300
Time (sec)
3000
6000
w R
ate
(nL/
0 10 20 30 400
3000Fl
ow
0 10 20 30 40
Time (min)
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Microfabricated Sample Injector
Concept Example - Check Valve High Pressure injector –Two check valves and a pipettorp p
Ch k l W t
Open
Check valve
Actuation
Waste
bypass
Closed
Open
Sample Inlet1mm
Mobile Phasepiston
bypassOpen
40 nL pipettor To column1mmClosed
J. E. Rehm, T. J. Shepodd and E. F. Hasselbrink, MicroTAS 2001 Proceedings, pp. 227-229
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Injector performance
High-pressure operation (6000 psi)E ll h i l ibiliExcellent chemical compatibilityExcellent reproducibility (manufacture and operation)
0.8 % RSD peak height1 3 % RSD peak area1.3 % RSD peak area
40 nL injections; 10 mM Thiourea (in MeOH)200,000 injections; on shelf for 6 months; 200,000 injections200,000 injections; on shelf for 6 months; 200,000 injectionsDetection: 239 nm
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Detection – UV Absorbance
Microfabricated UV Flow Cell (silica)• Fiber optic illumination/collectionFiber optic illumination/collection• 45 nL cell volume• 4 mm path length• Standard cell for ExpressLC® systems• Standard cell for ExpressLC systems• Improved versions under development
• Higher sensitivity• Lower volume 14000 Butyrophenone
8000
10000
12000
k A
rea
0
2000
4000
6000P
eak
0 50 100 150 200 250 3000
Concentration (μg/mL)
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Columns
• Columns microfabricated with 125, 150, 200 and 300 µm diameters• Particles retained by microfabricated and/or synthesized frits• Conventional separation particles
• Various particle manufacturers, phases, sizes p p• Method flexibility• Facilitate method transfer to microfabricated system
• Easy cHiPLC™-to-conventional LC comparison y p• Temperature control facilitated by low thermal mass and
device geometry• Isocratic and gradient methods
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Column-to-column performancece 3 columns
Abs
orba
nc 3 columns 4-5 replicate runs each25 nL injections (cHiPLC™ injector)
-2 0 2 4 6 8 10 12 14
Time (min)140000
Phenone mix
Separation conditions:200 µm x 6 2 cm column
80000
100000
120000
es/m
200 µm x 6.2 cm column 3 µm Luna C18 1 µL/min Isocratic 40 % H2O : 60 % ACN
0
20000
40000
60000
Plat
e ACNDetection: 240 nm
0 1 2 3 40
k'
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Integrated Inj-Col-DetColumn Diameter
Flow Rate (μL/min)
Injection Volume
(nL)
Detection Volume
(nL)•150 mm – Detection volume too small•300 mm – Serpentined columns have150 μm 1.0+ 10 10
200 μm 1.8+ 20 20
300 μm 3.5+ 40 40
•300 mm Serpentined columns have too much dispersion
•200 mm – Preferred balance of integrated systemintegrated system characteristics
Pipettor
Actuation
Sample Waste
Actuation
Sample• No connections in critical analysis region• Low volume / small dimensions around turn• Gentle expansion into larger diameter region of
Check valves
Mobile Phase
ColumnColumn Fill
column• Balance dispersion and pressure drop
Critical integration region
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Integrated Microfabricated System
160
g y
100
120
140 Compound Theoretical Plates/m
Symmetry
Factor
Uracil 75030 0.84
40
60
80
mA
U
Acetophenone 99660 0.94
Propiophenone 103580 0.94
-20
0
20 Butyrophenone 101050 0.97
Valerophenone 98260 0.99
0 2 4 6 8
Time (min)
200 µm x 10 cm column 3 µm Luna C18 1 2 µL/min
Hexanophenone 97030 1.00
Heptanophenone 96420 1.00
1.2 µL/min Isocratic - 30 % H2O : 70 % ACN20 nL injection Detection: 245 nm
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Reproducibility of Gradient Separations
6000 Peak 30.7 % RSD peak height
4000
0.8 % RSD peak area< 0.1 % RSD retention time
Peak 7
Abso
rban
ce 0.7 % RSD peak height0.9 % RSD peak area< 0.1 % RSD retention time
2000
A A: H2O ; B: ACN%B – 90%-100%; 6 sec 200 µm x 10 cm column 3 µm Luna C18
0 2 4 6 8
0
Time (min)
1.2 µL/min 20 nL injection Detection: 245 nm
Time (min)
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Test Sample Mix
cHiPLC™ A: 0.1%TFA/H2OB: 0.085% TFA/ACN
ce
Gradient: 0-30 min %B – 10%-90%
200 µm x 6.2 cm cHiPLC™ column3 μm Luna C1825 nL injection
Abs
orba
nc
0 2 4 6 8 10
ExpressLC®
25 nL injection2 μL/minDetection: 214 nm
A: 0.1%TFA/H2OB: 0.08% TFA/ACN
Gradient: 0-30 min %B – 10%-90%
300 µm x 15 cm Eksigent® column3 μm Luna C18
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
μ40 nL injection4 μL/minDetection: 210 nm
( )Theophylline; Sulfamerazine; Amphetamine; Hydrocortisone; Diazepam; Amitriptyline; Bumetanide; Thioridazine; Fenoprofen
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Acoustic Droplet Ejection
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History of Acoustic Dispensing
• 1979: Recognition Equipment Inc.D t i ti li ti
• 1920’s: No focus at surface– Document printing applications
• 1985: Xerox Corp.– Document printing applications
– Uncontrolled drop size and trajectory
• 1973: Focus at surface – IBMC t ll d d i d
• 1993: NASA– Part manufacturing
• 2000: Picoliter Inc
– Controlled drop size and trajectory
• 2000: Picoliter Inc.– Life science applications
• Acoustic dispensing
• 2005: Labcyte LLC/Picoliter Inc. merger to form Labcyte Inc.– Instruments offered as well as
acoustically qualified consumablesIBM TECHNICAL DISCLOSURE BULLETIN, Sept. 1973, p. 1168
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Non-invasive Acoustic EjectionEjection
Lens
Quartz
TransducerRF ->
AcousticEpoxy
Piezo
Coupling fluid
MHz RF Power
fluid
System
Immersion Non-invasive
(via “coupling Immersion (via coupling fluid”)
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Drops
• Drop volume is tunable
– inverse of RF frequency
1 MHz 200 nL
100 MHz 10 pL
• Multiple drops for larger volumes
• Fluid properties have secondary influence oversecondary influence over drop volume
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Single Ejection Event, Multi-Strobe CaptureStrobe Capture
• Water Ejection– 5 MHz
Low HighPower
gPower
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Changing Rep. Rate
750 Hz 1180 Hz750 Hz 1180 Hz
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Well Plate to Well Plate Transfer
Receiving well plate or substrateReceiving well plate or substrate
Fluid transferred
Source well plate
Piezoelectric transducer for the production of focused acoustic waves
Acoustic coupling fluid
•Well plates used: Greiner, Nunc, Whatman, and Corning Costar
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Sample Transfer Results
• DMSO 100%, 95%, 90%, 85%, 80%, 75% and 70%• Various fill heights• 5 nL and 50 nL transferred with fluorescein5 nL and 50 nL transferred with fluorescein• PE EnVision fluoresence reader (Excellent Linearity)
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Drop Ejection from a 1536 Well PlatePlate
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Microfluidic Device Fluid TransferTransfer
Unloading processed materials:Loading diversity:
Well plate to microfluidic device
Microfluidic device to well plate
Microfluidic device to analytical instrument
Microfluidicdevice
Microfluidicdevice
• What do microfluidic device “inlets” and “outlets” look like?
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Drop Ejection into a Pipette
Water ejection into 1550 μm hole of Pasteur pipetteWater ejection into 1550 μm hole of Pasteur pipette- Outer diameter 1840 μm
Images captured with multiple strobe flashes of a single droplet event
9.2 nL7 MHz
19.2 nL5 MHz
Images captured with multiple strobe flashes of a single droplet event.
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Drop Ejection into a Caliper
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Drop Ejection into a Capillary
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Materials we can transfer
• What can we transfer?– DMSO (100% - 0%)– Aqueous solutions
• Water• Water• Salt solutions, buffers• Surfactant solutions (Triton X-100, SDS)• DNA Primers ProbesDNA, Primers, Probes• Proteins (BSA, Lysozyme), Fetal Calf Serum• Glycerol• Alcoholco o
– Polymeric solutions• Acrylamide• OthersOthers
– Ionic Liquids
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Low Surface Tension Fluids
Ethanol 0.005% Triton X-100 Water
γ = 25 mN/m γ = 45 mN/m γ = 73 mN/m
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Acrylamide/ Mixed Fluid Systems
Less Dense More Dense
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Interesting Phenomena
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Fluid Detection
• Acoustics enable “echo• Acoustics enable echo detection” of fluid
• Drop formation• Drop formation– Delivery of energy to the focal
spotspot
• Echo detectionE fl t d b k t th– Energy reflected back to the transducer
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Acoustic Path: Attenuation
Aco stic energ is lost as the• Acoustic energy is lost as the waves travel from the source to the focusto the focus
a
Af Af Attenuation through fluid
A = Af + Ap + Ac
af
aw
apAp
A af
ApAc
Attenuation through plate Attenuation through coupler
Fluid attenuation (dB/mm for frequency F)Ac af
ac
ap
Fluid attenuation (dB/mm for frequency F)Plate attenuation (dB/mm for frequency F)Coupler attenuation (dB/mm for frequency F)
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Acoustic Path: Reflections
• Acoustic energy is reflected gyfrom interfaces where acoustic impedance of materials do not matchmaterials do not match
Z
Ra/f R1/2 =( Z1 - Z2 )2
(Z1 + Z2 )2Zf
Zc
Zp
Rf/p
R /
Ra/fRf/pR
Reflection from air/fluid interfaceReflection from fluid/plate interface
(Z1 Z2 )
Rp/c
ZfZp
Rp/c Reflection from plate/coupler interface
Fluid acoustic impedancePlate acoustic impedance
Zc
pCoupler acoustic impedance
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Acoustic Path: Transmission
• Acoustic energy is gytransmitted through interfaces based on degree of acoustic impedance match
ZT4 Z1 Z2
2T1/2 =Zf
Zc
Zp
Tp/c
Tf/p
Tf/pTp/c
Reflection from fluid/plate interfaceReflection from plate/coupler interface
(Z1 + Z2 )21/2
Zf
Zc
Zp
p pFluid acoustic impedancePlate acoustic impedanceCoupler acoustic impedancec Coupler acoustic impedance
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Acoustic Path
• Drop formationp
• Detection
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Fluid Depth Detection
• Acoustic Echo Signal Singlepoint
dit– 384 well in polypropylene plate
– 10 μL DMSO fill
audit
0.1
0.15
e [V
] Well Bottom DMSO
0 05
0
0.05
mpl
itude
-0.1
-0.05
25.00 25.50 26.00 26.50 27.00 27.50
Am
0.751 us = 0.595 mm
Time [us]
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Meniscus Detection
• Acoustic Echo Signal Multiplepoint
dit– 384 well in polypropylene plate
– 10 μL DMSO fill
audit
Acoustic Echo Image of Meniscus Surface [Vertical Axis = Echo Delay time]
1 2 3 4 5 6 7
S1 S
4 S726.3
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Well Plate Sonogramg
1536 Well Plate
h
o t
ime
E
ch
M43 M44 M45 M46 M47 M48M42
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Acknowledgements
Rick Stearns, Ph.DRich Ellson, CTO