Digital Microfluidic Chips for
Chemical and Biological Applications
R.B. Fair
Department of Electrical and Computer
Engineering
Duke University
Durham, N.C., USA
Department of Electrical and
Computer Engineering 2
• Background and motivation
– Digital microfluidics
• Chip architecture issues
• Toolkit
• Applications of digital microfluidics
– Sample preparation
– DNA sequencing
– Enzymatic assays
– Cytotoxicity screening
• Summary and conclusions
Outline of Presentation
Department of Electrical and
Computer Engineering 3
Microfluidic Functions
Department of Electrical and
Computer Engineering 4
Background and Motivation
• The reality of current lab-on-a-chip technologies... – Highly application specific
– Commercial trend: simple, disposable devices that interface with expensive control boxes
– Disposable devices may perform limited set of steps
• What is required for a diagnostic lab-on-a-chip? – Leverage devices into multiple applications
– Complexity of diverse applications reduced to a manageable set of fluidic operations
– Modular architecture gives flexibility of choosing fundamental operations
– Top-down design
Department of Electrical and
Computer Engineering 5
Digital Microfluidics
• Droplet-based microfluidic devices
– Droplets are moved in “virtual channels”
defined by electrodes
– Programmable electrodes in an array directly
control discrete droplet operations – dispense,
transport, mix, split, incubate – to perform any
liquid-based test
Features
• Electrowetting
– Modulation of solid-liquid interfacial
tension by the application of an
electric field
• Works with or without a top plate
– Newly developed coplanar
electrowetting method
How It Works
Voltage Off:
Voltage On:
Department of Electrical and
Computer Engineering 6
Digital Microfluidic Toolkit
Reservoirs droplets
Dispensers electrode sets
Pumps electrode sets
Valves electrode sets
Reaction vessels droplets
Mixers electrode sets
Collection scanning droplet
Implementing numerous applications on a
elemental set of components:
Department of Electrical and
Computer Engineering 7
Diagnostic Lab-on-a-Chip
Digital microfluidic
lab-on-a-chip
DROPLET
TRANSPORT
SAMPLE
LOADING
DROPLET
DISPENSING
MIXING &
REACTORS DETECTION
Analog
input
Analog-to-
Digital
Digital Digital-to-
Analog
Analog
INTEGRATE
Department of Electrical and
Computer Engineering 8
World to Chip Interface
• well plate interface
• 384 well spacing
• reservoir inputs from microliters to milliliters
Department of Electrical and
Computer Engineering 9
High Speed Continuous Droplet
Dispensing
Advanced Liquid Logic, Inc.Advanced Liquid Logic, Inc.
Department of Electrical and
Computer Engineering 10
35 Picoliter Droplet Dispensing
Department of Electrical and
Computer Engineering 11
On-Chip Processing
• Demonstrated transport of whole blood, plasma, serum, urine, saliva,
and sweat.
• Colorimetric glucose assay on Plasma, Serum, Saliva and Urine
Clinical Diagnostics
82.56
107.77
26.67 25.31
80.30
99.63
24.25
8.79
0
20
40
60
80
100
120
PLASMA SERUM SALIVA URINE
Glu
cose C
oncentr
ation in m
g/d
l Reference method
Electrowetting
Whole blood transport @ 20 Hz
Department of Electrical and
Computer Engineering 12
Droplet Mixing
• Mixing in ~5 seconds by shuttling on linear array
for 1µL (1.5mm scale) droplets
• Shuttling reverses flow causing un-mixing
– unidirectional motion is preferred
• Scaling down volume decreases mixing time
– mixing of two 25nL droplets was complete in 0.8
seconds at 10Hz switching @ 50V
DROPLET
TRANSPORT
SAMPLE
LOADING
DROPLET
DISPENSING
MIXING &
REACTORS DETECTION
Department of Electrical and
Computer Engineering 13
Detection Methodology
Opaque
solid
DROPLET
TRANSPORT
SAMPLE
LOADING
DROPLET
DISPENSING
MIXING &
REACTORS DETECTION
Chemoluminescence underneath the
TeflonAF coated photodetector
Department of Electrical and
Computer Engineering 14
Reconfigurable Lab-on-a-Chip Status
• Digital microfluidic toolkit demonstrated
– All fluidic functions demonstrated
– Lacking molecular separation method
• Commercial prototypes soon available (ALL)
• Examples from current research
– DNA extraction from blood
– DNA sequencing by synthesis
– Enzyme assays
– Cytotoxicity screening
Department of Electrical and
Computer Engineering 15
Microfluidic Operations • How are key operations performed on chip?
– Sample preparation (fluidic function) • Cell separation
• Cell lysis
• DNA/RNA extraction
• Purification
– Mixing and transport
– Sample/reagent storage
– PCR (fluidic function) • Mixing
• Temperature cycling
• Transport
• Detection
Sample loading
Transport
Mixing
Dilution/washing
Filtering
Separation
Cell Separation
Cell Lysis
DNA or RNA
Extraction
Purification
Blood Sample
Department of Electrical and
Computer Engineering 16
Cell Lysing Area
From Blood Input
In the Cell Lysis Area, Blood will be
inputted in 25 nL sized droplets from
the blood storage reservoir. This will
be mixed with a 5 nL drop of Rose
buffer solution which lyses the cells.
The end solution will be mixed with a
5 nL drop of magnetic beads. Then a
5 nL drop of solution will be pulled
from the lysis area.
[click for example]
Red – blood
Blue – Rose buffer
Yellow – mag. beads
Department of Electrical and
Computer Engineering 17
DNA Extraction
Permanent
Magnet
Magnet
Wash droplet
Heater – 27C to 96C
Wash Droplets
Magnetic
Separation
Washing
Solvent
Input: Droplet containing dna
strands, cell fragments,
magnetic particles attached
to A’B’ dna
Drop with magnetic beads, cell
fragments, dna, etc… enters
Magnetic
Beads
held by
permanent
magnet
Final droplet enters, pauses, temperature increases,
releasing A’B’ DNA, and droplet splits and moves on…
Output: Droplet containing
A’B’ dna
Department of Electrical and
Computer Engineering 18
Magnetic Bead Washing
One complete wash cycle
1X speed video of first wash cycle
800 wash cycles (condensed to 26 s)
Still images taken after completion of
each wash cycle are played at 30 fps
Department of Electrical and
Computer Engineering 19
Steps involved
- Dispense beads (sample)
- Wash beads
- Add dNTP mix to beads
- Add enzyme mix to beads
- Mix & Detect
On-Chip Pyrosequencing Protocol
Department of Electrical and
Computer Engineering 20
Results • 17-bp sequencing of 211-base long C.albicans DNA
template with 20-base primer attached
0
200
400
600
800
1000
1200
1400
0 200 400 600 800 1000 1200
Experiment time, s
Lu
min
es
ce
nce
, c
ou
nts
pe
r 0
.1s
a c g T A c G T a C g T AA CC g
Correct sequence: TAGGTCTAACCAAAAACATT
t AAAAA C g t A T
Department of Electrical and
Computer Engineering 21
Digital Multiplexed Assay Chip
Waste Sample
Glucos
e
Calibrant
s
Controls Lactate
Urea Buffer
G
G
L U
s
M
M M
s
L U B
C
S
C 4 phase bus
2 phase bus
mixing
detectio
n
G
s
L
U
B
G
L
U
M
C
Sample
Glucose
Lactate
Ure
a
Mixed product
Buffer
Control/
Calibrant
Duke University
Department of Electrical and
Computer Engineering 22
Multiple Glucose Assays
• 9 consecutive
assays
• 3 glucose
concentrations
• 60 seconds
absorbance
measurement at
same spot
Data more
noisy for 40
and 80mg/dL
Kinetic Data - Glucose Assays - 40, 80, 120 mg/dL
y = 1.47E-03x
R2 = 0.98
y = 1.10E-03x
R2 = 0.964
y = 6.01E-04x
R2 = 0.918
y = 1.49E-03x
R2 = 0.981
y = 1.06E-03x
R2 = 0.961
y = 6.21E-04x
R2 = 0.892
y = 1.49E-03x
R2 = 0.979
y = 1.09E-03x
R2 = 0.954
y = 6.04E-04x
R2 = 0.883
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0 60 120 180 240 300 360 420 480 540
Time in seconds
Ab
so
rba
nce
in
AU
RUN 1 RUN 2 RUN 3
120mg/dL 80mg/dL 40mg/dL120mg/dl 80mg/d
L
40mg/dL 40mg/dL80mg/dL120mg/dl
Department of Electrical and
Computer Engineering 23
Cytotoxicity Screening
• Large market: Drug discovery, product testing, experimental research, and more.
• Researchers and companies must determine how their drug or other compound effects cells before animal and human trials can begin.
• Limited supply (1-10 mg) of compound to test, many compounds (up to 1 million) need tested Miniaturization and automation provided by biochips is attractive.
Department of Electrical and
Computer Engineering 24
On-chip Dilution Tree for Cytotoxicity
Screening (Y. Zhao, A. Wang, Y. Yamanaka)
Grow cells in 96 well plateAdd various concentrations of
compound to be tested to cells
Wait specified
length of time
Add Cytotoxicity Assay reagent
1, incubate, add reagent 2Use plate reader to measure color
intensity (proportional to survival)
Department of Electrical and
Computer Engineering 25
Architecture
Media Compound to
Test Cells + Media 1. Dispense buffer and compound
droplets, mix.
Assay
Reagent
To
Waste
Department of Electrical and
Computer Engineering 26
Architecture
Media Compound to
Test Cells + Media 1. Dispense buffer and compound
droplets, mix.
2. Split. One droplet stays for
further dilution, one droplet gets
mixed with cells.
3. Dispense cell solution. Optical
absorbance check of
concentration (optional). Mix with
diluted compound droplet.
Assay
Reagent
To
Waste
Department of Electrical and
Computer Engineering 27
Architecture
Media Compound to
Test Cells + Media 1. Dispense buffer and compound
droplets, mix.
2. Split. One droplet stays for
further dilution, one droplet gets
mixed with cells.
3. Dispense cell solution. Optical
absorbance check of
concentration (optional). Mix with
diluted compound droplet.
4. Split. Both droplets go to
holding.
Assay
Reagent
To
Waste
with previous dilution drop.
REPEAT to test
multiple dilutions
Electrode
number will
determine
throughput.
5. Incubate desired length of time.
6. Transport droplets to integrated
on-chip functions (lysis, PCR, etc)
Paths to other functional units of integrated biochip.
Department of Electrical and
Computer Engineering 28
Remarks on Applications • Extensive biomedical application base can
leverage microfluidic operations in an electrowetting system.
• Based on: – Shared elemental fluidic operations
– Reconfigurable functional units and programmable control
– No cross-contamination/wash droplets
– Multi-tasking
– Recongfigures around bottlenecks
• Wide diversity of applications can be parsed into manageable components and assembled into a programmable, reconfigurable and reusable architecture.
Department of Electrical and
Computer Engineering 29
Summary and Conclusions
• Electrowetting-based digital microfluidics is good candidate
for multifunctional microfluidics
– Programmability
– Reconfigurability
– Multifunctional reliable EWD actuator operation if V≤ Vsat
• Open issues:
– On-chip sample preparation
– Lack of a molecular separation method
• Capillary electrophoresis
– Accurate on-chip dilution an open issue
– Scalable, compatible detector technology needed