Post on 09-Feb-2022
Integrated Digital Microfluidic
Biochips
R.B. Fair
Department of Electrical and Computer Engineering
Duke University
Durham, N.C.
Department of Electrical and
Computer Engineering 2
• Background and motivation
– Integrated disposable microfluidics
– Integrated microfluidic systems: past and present
• Microfluidic integration issues
– Architectural choices
– Integrated detectors
• The digital microfluidic options and examples
– Implications of droplet architecture
– Examples of integration
• Analog/Digital Hybrid Microfluidic Chip For DNA & RNA Analysis
• Cytotoxicity Screening
• Protein Crystallization
• Summary and conclusions
Outline of Presentation
Department of Electrical and
Computer Engineering 3
Background & Motivation
Test tubes
Robotics
Microfluidics Automation
Integration ?
Miniaturization
Automation
Integration
Miniaturization
Automation
Integration
Miniaturization
Department of Electrical and
Computer Engineering 4
Start of the Art Commercial Disposable
Microfluidics
Department of Electrical and
Computer Engineering 5
BioSite Biochip
Department of Electrical and
Computer Engineering 6
Disposable Chip Paradigm
Cell Filter Reaction ChamberMetered plasma
volume
Blood
Fluorescent Antibodies
Analyte capture
Wash
Fluorescent
detection
Department of Electrical and
Computer Engineering 7
Fluidigm 8.96 Screening Chip
Department of Electrical and
Computer Engineering 8
Concept of Disposable Integration
Application Devices
MICROFLUIDICPROCESSING/ANALYSIS
Department of Electrical and
Computer Engineering 9
Promise of Biochips
How important is a fully integrated chip?
Department of Electrical and
Computer Engineering 10
Historical Electronic/Fluidic Integration
• Trend has been to integrate the fluidics on the
electronics
Man, 1997, UM
Department of Electrical and
Computer Engineering 11
Current Integrated Microfluidic Devices
Department of Electrical and
Computer Engineering 12
Hybrid Integration
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Computer Engineering 13
Integration Issues • Can only integrated simple fluidic functions on an
IC!
• Option: integrate the electronics on the fluidic
platform (Motorola, 2004)
Department of Electrical and
Computer Engineering 14
Integration Compatibility Issues
Fluidic Platform IC Platform
Department of Electrical and
Computer Engineering 15
Microfluidic Functions
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Computer Engineering 16
Architectural Choices
• Fixed data path (application specific)
• Reconfigurable (multiple applications)
– Shared elemental operations
– Microfluidic instruction set
– Programmable
– Reusable
Basic Component
Integration
Basic Component
Integration
Reconfigurable
Microliquid
Handling
Architecture
Reconfigurable
Microliquid
Handling
Architecture
Capillary
Electrophoresis
Capillary
Electrophoresis
Microelectrofluidic
System (MEFS)
Processor
Microelectrofluidic
System (MEFS)
Processor
Channels and Flow Sensors
Reaction
Reservoirs
Dispensing
Reservoirs
Agent
Detection
Composition
Measurement Catalysts
Acquisitio
nReservoirs
Process Control
Pumps
Channels and Flow Sensors
Reaction
Reservoirs
Reaction
Reservoirs
Dispensing
Reservoirs
Dispensing
Reservoirs
Agent
Detection
Agent
Detection
Composition
Measurement
Composition
Measurement CatalystsCatalysts
Acquisitio
nReservoirs
Acquisitio
nReservoirs
Process ControlProcess Control
PumpsPumps
Instruction
Decode
Rea
ctio
n U
nit
Sto
rag
e
Unit
Sto
rag
e
Unit
Department of Electrical and
Computer Engineering 17
Where Are We?
Basic Component
Integration
Basic Component
Integration
Reconfigurable
Microliquid
Handling
Architecture
Reconfigurable
Microliquid
Handling
Architecture
Capillary
Electrophoresis
Capillary
Electrophoresis
Microelectrofluidic
System (MEFS)
Processor
Microelectrofluidic
System (MEFS)
Processor
Channels and Flow Sensors
Reaction
Reservoirs
Dispensing
Reservoirs
Agent
Detection
Composition
Measurement Catalysts
Acquisitio
nReservoirs
Process Control
Pumps
Channels and Flow Sensors
Reaction
Reservoirs
Reaction
Reservoirs
Dispensing
Reservoirs
Dispensing
Reservoirs
Agent
Detection
Agent
Detection
Composition
Measurement
Composition
Measurement CatalystsCatalysts
Acquisitio
nReservoirs
Acquisitio
nReservoirs
Process ControlProcess Control
PumpsPumps
Instruction
Decode
Rea
ctio
n U
nit
Sto
rag
e
Unit
Sto
rag
e
Unit
Commercial Research
Department of Electrical and
Computer Engineering 18
Present Status Summary • 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 integrated microfluidics? – 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
– Integrated fluidic I/O
– Integrated low voltage CMOS control incompatible with current fluidic operating voltages and footprints
– Detector integration a priority
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Computer Engineering 19
PCR Integrated System
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Detection Methodology
Opaque
solid
DROPLET
TRANSPORT
SAMPLE
LOADING
DROPLET
DISPENSING
MIXING &
REACTORS DETECTION
Chemoluminescence underneath the
TeflonAF coated photodetector
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Computer Engineering 21
Detector Integration
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Computer Engineering 22
Integrated Microdisk Sensor
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Complexity of Diverse Applications
Reduced to a Manageable Set of
Fluidic Operations
Biomedical FluidicFunctions: Func.1, Func.2,...…,Func.n
Elemental Set of Operations: Op.1, Op.2,.........…,Op.i
•Agent Detection
• Precision Dispensing
• Enzyme Analysis
• Electrochromatography
• Capillary Electrophoresis
• Molecular/Protein Analysis
• Isotachophoretic Separation
•Transport
• Mixing
• Flushing
• Filtering
• Analysis
• Detection
• Monitoring
• Buffers
•Channels
•Valves
•Mixers
Elemental Set of
Components Comp. 1, Comp. 2,…,Comp. n
Department of Electrical and
Computer Engineering 24
Microfluidic Architecture
• Extensive biomedical analysis technology base needs to
be leveraged by expanding integration of microfluidic
operations into a complete system
– Key is integration of sample preparation processes on chip.
Hybrid integration option possible.
– Alternative: interfacing to off-chip systems
Reagent Mixing
Chemical Separation
Filtration
Heating
Detection
Electrophoresis
Immunoassay
DNA Assay
Mass Spec
Department of Electrical and
Computer Engineering 25
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 26
Integrated Operation - Serial
• Serial protocol
• One glucose
assay at a time
• Much simpler
• Does not
require
detector
multiplexing
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Computer Engineering 27
Implications of Droplet Architecture
• Droplets allow microfluidic functions to be reduced to a set of basic operations
• Numerous elemental fluidic operations can be accomplished with a common set of elemental components
• Array can be partitioned into “cells” that perform fluidic functions
• Functional cells dynamically reconfigured at least once per clock cycle
Department of Electrical and
Computer Engineering 28
Integrated Lab-on-a-Chip
Systems
• Digital microfluidic toolkit demonstrated
• Can digital microfluidics deliver a true integrated
lab-on-a-chip technology that is adaptable to
numerous applications?
• Examples from ECE299 (Duke Univ. Fall
2006/2007)
– Analog/Digital Hybrid Microfluidic Chip For DNA & RNA
Analysis
– Cytotoxicity Screening
– Protein Crystallization
Department of Electrical and
Computer Engineering 29
Analog/digital hybrid biochip (A. Garcia, G. Pan, J. Zhang)
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Computer Engineering 30
Fluidic Platform
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Computer Engineering 31
Floor Plan of the DMW
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Computer Engineering 32
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 33
Previous Work
Anal. Chem. 2005, 77, 667-672
Toxicology in Vitro 2007, 21, 535–544 Lab on a Chip 2007, 7, 740-745
Department of Electrical and
Computer Engineering 34
Architecture
Media Compound to
Test Cells + Media 1. Dispense buffer and compound
droplets, mix.
Assay
Reagent
To
Waste
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Computer Engineering 35
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 36
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 37
Inputs, Outputs, and On-Chip
Function Inputs
(1) Cell suspension, (2) Cell media for dilutions, (3) Solution of compound to be tested for cytotoxicity, (4) Reagents for the cytotoxicity assay
If portable: include Lithium ion battery
On-chip functions
Create droplets of input liquids, split and mix droplets, incubate droplets for programmed length of time, detect intensity of droplet color or presence of stained cells.
Outputs
Color intensity of droplets or presence of stained cells.
Department of Electrical and
Computer Engineering 38
Cell concentration after dispensing
Biotechnology and bioengineering, Vol 38, Iss. 9, 1007-1011.
Cytotoxicity assay result
Cells + Media
DetectDetect If not in range,
send back.
Color intensity detector
Most cells alive Most cells dead
OR
Dead cellAlive cell
Image
acquisition
and
processing
Output:
# cells alive in
droplet
# cells dead
in droplet
Department of Electrical and
Computer Engineering 39
Protein Crystallization on an Array (H. Fang, M. Shafir, T. Xu)
Glucose isomerase crystals on chip – 20× Proteinase K crystals on chip – 40×Glucose isomerase crystals on chip – 20× Proteinase K crystals on chip – 40×
Department of Electrical and
Computer Engineering 40
Protein Crystallization
• Major applications of proteins crystallization – Structural biology and drug design
– Bioseparations
– Controlled drug delivery
• Requires large number of experiments to get the correct parameters for the crystallization of proteins
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Phase Diagram
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Integrated Array Chip Layout
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Implementation
Multi-well-plate
Transportation pathways
Protein/
reagents
Well electrode
Segregation Wall
Transportation pathways
Protein/
reagents
Well electrode
Segregation Wall
Department of Electrical and
Computer Engineering 44
Sample Droplet Splitting and Dilution
Scheme
Protein Stock
Solution Crystallization
Reagents
Incubate in well / Dilution can be
performed by routing further water
droplets Route one droplet to an
adjacent well / We can split
the droplets to a uniform
volume
Well Electrode
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Computer Engineering 45
Architectural Block Diagram
45
Sample Injection
Sample Mixing
Sample Incubation
Detection
Waste Handling
Sample Dilution
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Pin-constrained Design
• 1284 pins→133 pins
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Computer Engineering 47
Efficient loading of condition
solutions • Shuttle-passenger-like well-loading
5 1 2 3 4 5 1 2 3 4 5
2 4 2 4 2
4
3 4
3 4
1 2 Well
1 2 Well
1
3 1
3
3
5 1 2 3 4 5 1 2 3 4 5
2 4 2 4 2
4
3 4
3 4
1 2 Well
1 2 Well
1
3 1
3 1
3
5 1 2 3 4 5 1 2 3 4 5
Reservoir 2 2 2
2 2 2
1
2
3
4
5
Loading step I
Department of Electrical and
Computer Engineering 48
Remarks on Applications • Extensive biomedical application base can
leverage microfluidic operations in an electrowetting system.
• Based on: – Shared elemental fluidic operations
– Reconfigurability
– No cross-contamination
– Multitasking by components
– Few 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 49
Summary and Conclusions • Integration of lab-on-chip microfluidics on IC’s may happen at the
femtoliter scale (1µm)
– Requires sample in/result out integration
– High sensitivity detector
• Electrowetting-based digital microfluidics is good candidate for multifunctional microfluidics
– Programmability
– Reconfigurability
– Multifunctional
• 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
Department of Electrical and
Computer Engineering 50
Acknowledgements
• NSF
• NIH
• DUHS
• ECE299 students