Download Lecture 20 Lab on a Chip

Lab-on-a-Chip
EE C245
Dr. Thara Srinivasan
Lecture 20
Picture credit: Anderson et al.
Lecture Outline
• Reading from reader
• Auroux, P.-A., Manz, A. et al. , “Micro Total Analysis
Systems,” (2002) pp. 2637-52.
• Krishnan, M., et al., “Microfabricated Reaction and
Separation Systems,” (2001) pp. 92-98.
• Quake, S., R, and A. Scherer, “From Micro- to
Nanofabrication Using Soft Materials,” (2001) pp. 1552-69.
• Today’s Lecture
EE C245
• Lab-on-a-Chip Concept and Examples
• Application to Proteomics
• Lab-on-a-Chip Subunits
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U. Srinivasan ©
Sample handling
Reactors
Separation Methods
Detection
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Lab-on-a-Chip
• Micro total analysis system (µ-TAS)
• Vision proposed by Manz, Widmer
and Harrison in early ’90’s
• Perform sample addition,
pretreatment and transport, chemical
reactions, separation, and detection
on a microscope slide or credit card
size chip
• Annual conference, MicroTAS, had
700 attendees in ‘02
• Saves reagents and
labor
• Increases testing
throughput
• Creates portable
systems
EE C245
• Applications
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Genomics and proteomics
Environmental assays
Medical diagnostics
Drug discovery
Chemical production
Cellular analysis
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Affymetrix
Lab-on-a-Chip
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• Multiple operations
performed
• Cell lysis
• Sample
concentration
• Enzymatic
reactions such as
reverse
transcription, PCR,
DNAse digestion
and terminal
transferase
labeling
• Dilution,
hybridization, and
washing
• Dye staining
U. Srinivasan ©
Anderson et al.
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U of M Lab-on-a-Chip
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• Mastrangelo and Burns
groups’ integrated device
• Nanoliter liquid
injector
• Sample mixing
and positioning
system
• Temperaturecontrolled PCR
reaction
chamber
• Electrophoretic
separation
• Fluorescent
photodetector
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EE C245
Microscope-on-a-Chip
U. Srinivasan ©
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Proteomics
• A “proteome” is the set of proteins encoded by a gene
• Proteomics
• Identifying all the proteins made by a given cell, tissue or organism
• Determining how the proteins network among themselves
• Finding out precise 3D structures of the proteins
• Proteins more complex than genes
EE C245
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DNA: 4 bases, proteins: 20 amino acids
Even with a protein’s sequence, its function and networks still unknown
3D shape of folded protein difficult to predict
All human cells have same genome, but differ in which genes are
active and which proteins are made
• ~40,000 human genes, each gene can encode several proteins (typical
cell makes 100,000’s proteins)
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Scientific American April 2002
EE C245
U. Srinivasan ©
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Necessary Subunits for µ-TAS
• Sample handling
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Extraction
Mixers
Valves
Pumps
EE C245
• Reactors
• Separation
• Detection
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Sample Extraction
• Means for extracting samples from dilute
solutions required
EE C245
• At macroscale, centrifugal force is used
• For microfluidics, sample extraction is interface to
macroscale
• Most of the power consumption is spent at this
step
• Methods include
• Filtration
• Chromatography
U. Srinivasan ©
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Extraction Using Filters
• Microfabricated filters
• Mechanically robust to withstand high pressure drops
for filtering µm-sized particles
• Very uniform pore sizes determined by
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• Photolithography
• Sacrificial layer thickness
C.-M. Ho group, UCLA
Keller et al., UCB
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Solid-Phase Extraction
• As in chromatography,
• Desired components
bind reversibly to a
coated porous solid
and are later flushed
out by a change in
solvent
• Hydrophobic coatings
bind nonpolar
compounds in aqueous
flow
EE C245
• Bead chambers
• Hydrophobic beads
trapped in a flow
chamber
U. Srinivasan ©
Harrison group,
Univ. of Alberta
Stemme group,
Sweden
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Extraction Using Porous Polymers
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Porous polymers increase available surface
area for binding interactions
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Fill channels with polymerization mixture ~
monomers, initiator, and porogenic solvent
Irradiate chip with UV light through
photomask
Surface chemistry may be varied widely
Fréchet group, UCB
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Extraction by Diffusion
• Mixing in low Re flows is nearly reversible
• Two flows that have been stirred together may be
“unstirred”—except for any mixing by diffusion—by
reversing the driving force
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• Can we use irreversibility of diffusive mixing in
reversibly stirred flows to separate chemical
species based on size?
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Extraction by Diffusion
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• As two parallel
laminar flows
contact, diffusion
extracts certain
components
• Components with
higher diffusivity
extracted
• Micronics H-filter
pull elements out
of sample into
diluent
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Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
U. Srinivasan ©
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Mixing
• Mixing of particles, cells and molecules often
determines the system efficiency
• PCR, DNA hybridization, cell lyses…
• Diffusion, the mechanism of mixing at the microscale, still
requires relatively long times for thorough mixing.
EE C245
• How to assist mixing?
• Repeated lamination of
flows increases contact
area and decreases
diffusion length
U. Srinivasan ©
C.-M. Ho Group, UCLA
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• Chaotic flows
can be very
efficient mixers
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• Changing
surface
topography of
microchannel
floor induces
chaotic flows
U. Srinivasan ©
Stroock et al.,
Whitesides Group, Harvard
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Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
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Pumping Mechanisms
• Pressure gradients
• Electrokinetic forces
• Surface tension forces
• Electrowetting
• Thermocapillary
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• Surface acoustic waves
• Magnetohydrodynamic
• Dielectrophoresis
U. Srinivasan ©
C. M.Ho
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Centrifugal Forces
EE C245
• Gyros, Sweden
• When CD spins,
centrifugal force causes
liquids on their surface to
move outwards.
• The force can drive
liquids through
microchannels…
• …even breaking through
hydrophobic barriers in
the channels, releasing
different chemicals
selectively
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Electrowetting
• Electrical potential can control surface tension on a
dielectric solid surface
EE C245
• Asymmetric contact angles generate internal pressure imbalance,
leading to movement
• Fluidic operations can be done on discrete droplets
• Low voltages: 25 V DC for v = 30 mm/s; 100V AC for v = 200 mm/s
CJ Kim group, UCLA
U. Srinivasan ©
cosθ (V ) = cosθ 0 +
εε 0V 2
2γ LV t
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Thermocapillary
Pumping
• Thermocapillary effect
• Local heating reduces surface tension, pulling liquid
towards cooler surface
• Surface temperature manipulated by embedded heaters
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• Results
• v = 600 µm/s for liquid PDMS
+ Low operating voltage (2-3 V)
+ Works with polar and non-polar
liquids
• Thermocapillary mixer ~1000×
faster than diffusion
Troian group,
Princeton U. 23
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Thermocapillary
Mixer
EE C245
• ~1000× faster than diffusion
U. Srinivasan ©
Troian group,
Princeton U. 24
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Surface Acoustic Waves
EE C245
• More on ultrasonic fluidic devices
at http://wwwbsac.eecs.berkeley.edu/fluidics/
White group, BSAC
U. Srinivasan ©
Sandia Labs
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Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
U. Srinivasan ©
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Elastomer Valves
• A good valve needs flexibility and a valve seat that closes
completely
• Microfabricated poly-Si valves: microactuator forces limited, so stiffness limits
minimum size
• For elastomers, Young’s Modulus can be tuned over 2 orders of magnitude…
• PDMS valves and pumps
made by replica molding
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Crossed channel layout;
channels 100 µm wide, 10 µm
high
When P is applied to upper
channel, membrane deflects,
closing lower channel
Response time 1 ms, applied P
= 100kPa
Dead volume is zero for on-off
valve
Unger et al.,
Quake group
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Valves and Pumping
• Peristaltic pumping with
elastomer valves
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3 valves on a single channel (closing
pattern: 101, 100, 110, 010, 011, 001)
2.35 nL/s at 75 Hz, 1 mN force
Avoids drawbacks of EO pumping
• Dependence on medium
• Electrolytic bubble formation
• Difficulty setting voltages when many
junctions present
EE C245
• Flow stops and gas vents
• Hydrophobic patches
• Hydrophobic membrane vents
• Thermally-generated bubbles
U. Srinivasan ©
Unger et al., Quake group,
Caltech
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Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
Jensen group, MIT
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U. Srinivasan ©
Immunoassay Reactor
• Immunoassays
• Important analytical method for
clinical diagnostics,
environmental analyses, and
biochemical studies.
• Antigens and antibodies are
fixed onto a solid support
• ELISA = Enzyme-Linked
ImmunoSorbent Assay
EE C245
• Point of care testing using
microfluidics
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Enhanced reaction efficiency
Simplified procedures
Reduced assay time
Lower sample & energy
consumption
U. Srinivasan ©
Sato et al., University of Tokyo
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Clinical Diagnosis On-Chip
EE C245
• Diagnosis of colon cancer by
detection of human carcinoembryonic antigen (CEA) in serum
on-chip
• Polystyrene beads coated with
antibody in microchannel, antigenantibody complex detected optically
• Liquid handling significantly
simplified
• Assay time reduced to ~1% (45 h to
35 min)
• Compared to conventional ELISA,
detection limit dozens of times lower
• High throughput analysis using
branching channels for simultaneous
analysis
Sato et al., University of Tokyo
U. Srinivasan ©
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Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
U. Srinivasan ©
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Separation by Electrophoresis
• Current standard method for protein sizing
• Sodium Dodecyl Sulfate-PolyAcrylamide Gel
Electrophoresis (SDS-PAGE)
• SDS denatures proteins and gives them charge;
PAGE separates by size
• Protein electrophoresis on chip
• Steps: sample loading (protein + SDS), dye labeling
(staining), separation, SDS dilution and destaining,
and detection
EE C245
• Staining and SDS dilution steps occur in 100’s ms,
104 × faster than macroscale
• Sequential analysis of 11 samples, sizing accuracy
>5%, sensitivity 30 nM
Video clip at
http://www.chem.agilent.com/scripts/generic.asp?lPage=1566&indcol=N&prodcol=Y
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Separation by Isoelectric Focusing
• Isoelectric focusing (IEF) is electrophoresis in a pH
gradient (cathode at higher pH)
EE C245
+ IEF downscales well since
resolution is independent of
channel length, in contrast to CE
• EP focusing effect counteracted
by diffusion, yielding Gaussian
band distribution
U. Srinivasan ©
∆pI min = 3 D
Dilute acid
lower pH, (+)
• Issues
Dilute base
Higher pH, (-)
• A protein’s isoelectric point (pI) is the pH at which it has neutral
charge
• Charged species stop moving when EP pushes them to their pI
• Linear pH gradient built up using ampholytes
• IEF concentrates and separates
dpH
dµ
L V
dx
dpH
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IEF On-Chip
• Advantages
• Sample mobilization unnecessary
• No injection plug so separation does not depend on initial
sample shape
• Short channel length gives rapid analysis and…
• Full field detection by imaging with inexpensive CCD
• Challenge
EE C245
• High field with shorter separation length leads to increased
Joule heating
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Separation by
Entropic Traps
• Channels with nanoscale constrictions
• Require long DNA to repeatedly change
conformation, costing entropic free
energy
• Longer DNA has higher mobility
• Separation
EE C245
• No sieving medium needed
• 5-kbp sample at 80 V/cm in 30 min
• Longer channels for better separations;
resolution not as good as CE
• Sample concentration
• At low E, DNA is trapped into band
U. Srinivasan ©
Craighead group, Cornell
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Separation by Diffusion
• Using 2-D “obstacle course” and electric
field in –y direction
• Asymmetric obstacles rectify Brownian
motion (diffusion) of molecules
• Faster-diffusing species move more in
+x direction
EE C245
• Results
• Obstacles: 1.5×6 µm² at 45° angle
• No sieving medium; low E (1.4 V/cm);
may be applied to DNA, proteins, cells,
etc.
• v = 1-15 µm/s, for a 10 cm sieve
• Bandwidth = 200 µm for 15 kbp DNA
(RG = 0.31 µm)
U. Srinivasan ©
Chou, Austin groups, Princeton,
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Craighead group, Cornell
Necessary Subunits for µ-TAS
• Sample handling
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Preparation
Mixers
Pumps
Valves
EE C245
• Reactors
• Separation
• Detection
U. Srinivasan ©
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Detection: Chemiluminescence
• Chemiluminescence (CL) or electrochemiluminescence
(ECL)
• Ru(bpy)3+2 oxidized chemically or electrochemically to Ru(bpy)3+3
which…
• Reacts with amines, amino acids, glucose, PCR products, etc…
• …and emits light at 620 nm
• Advantages
• Laser not required
• Instruments much simpler than for LIF
• Low to zero background signal; sensitivity high
• Scaling benefits ~ microphotodetector for on-chip detection
EE C245
• Challenges
• Need for robust and/or universal probes
• Isolation of ECL electrodes from CE high voltage
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Electrochemical Detection
• Electrochemical detection (EC)
• Control potential of working electrode and monitor current as samples
pass by
• Applied potential is driving force for electrochemical reactions of
sample analytes, current reflects concentration of compounds
• Benefits and challenges
EE C245
• On-chip detection; truly portable
• Chemistries need to be developed
• Rossier et al. integrated screen-printed carbon ink electrodes into
plastic microchannels and demonstrated detection limit of ~1 fmol
for ferrocenecarboxylic acid (2001). (EPL, Lausanne)
U. Srinivasan ©
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Mass Spectrometry
• Mass spectrometry (MS) measures
mass-to-charge ratios (m/z) of species
fragments
EE C245
• Dilute solution of analyte (10-4-10-5 M) is
sprayed from capillary tip at high potential
(3-4 kV)
• Liquid forms Taylor cone, fine jet of tiny
charged droplets which blow apart due to
charge repulsion
• “Nanospray” uses smaller glass
capillaries for lower flows (20-50 nL/min)
2-30 µm
U. Srinivasan ©
New
Objective
• Electrospray ionization spectrometry
(ESI) is recent, powerful technique
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Proteomics-on-a-Chip
• Integrated chromatography + CE + ESI
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Photolithography and wet-etching of Corning 0211 glass
Nanospray emitter placed into a flat-bottomed hole drilled into the exit of
separation channel
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Bead channel for sample
concentration
800 µm wide, 150 µm deep, 22
mm long, etched into the cover
plate (2.4 µL volume)
Filled with bead suspension
slurry
Low flow resistance of bead
channel allows sample loading
without perturbing CE channel.
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Results
• Flowrate ~ 2 µL/min
• Throughput ~ 5 min/sample
• Sensitivity ~ 25 fmol (5 nM)
U. Srinivasan ©
Harrison Group, U. of Alberta
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ESI On-Chip – 2
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Fabrication
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Results
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EE C245
Polymer chip embossed from silicon
master
Electrospray tip is flat parylene C
triangle (5 µm thick) sandwiched
between channel chip and sealing
cover
Tip is wet by analyte, helping to
form and fix position of Taylor cone
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Low dead volume connection
Stable ion current 30-40 nA
measured using 2-2.8 kV potentials
Analyte liquid is completely confined
on triangular tip
Cone volume estimated as 0.06 nL
Craighead group,
Cornell U
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More Topics
• Cell culturing
• Cell handling
EE C245
• Dielectrophoresis
• Optical tweezers
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U. Srinivasan ©
Protein crystallization
Interfacing between micro-macroworlds
Materials and surfaces
Microfluidic/nanofluidic components, modeling
Applications
Many more…
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