Disposable Microfluidic Devices for
Life-Science Applications
Luc Bousse
Director of Fluidics
QuantaLife, Inc.
Diversity in microfluidic devices
The term “Microfluidics” is used to describe devices with a broad variety of
properties. The most important variables are:
1. Channel dimensions: 100’s of um and microliter volumes vs. 10’s of um
and nanoliter volumes
2. Driving force: pressure, electrokinetic, centrifugal, integrated pumps
3. Device materials: silicon, glass, thermoplastic polymers, elastomeric
polymers (PDMS)
4. Channel fabrication method: etching, molding, embossing, casting, laser
ablation
5. Detection method: Fluorescence, optical absorption, electrochemical, mass
spec, or synthetic device only
These choices are connected, and there are many constraints - but there
are still many types of microfluidics devices. No broad standard has
emerged.
Choices in microfluidics with enclosed channels
Some typical choices:
1. Devices for synthesis, channels 100’s of
microns, molded polymer
2. Devices for CE separation, channels 10’s of
microns, molded polymer
3. Devices for CE separation, channels 10’s of
microns, glass or quartz
4. Devices cast from PDMS, channels 10’s of
microns, many research applications
What material to choose?
Quartz, Glass or Silicon:
• Excellent material properties
• Requires clean room, photolithography, etching, special equipment for
bonding, etc…
• Cost moderate to high in volume, depending on size and material
Thermoplastic Polymers:
• Good material properties
• Can be prototyped from an electroform, or injection molded
• Cost moderate for prototyping, to low for injection molding
Rubber-like Polymers (PDMS):
• Poor material properties
• Easy to prototype from various molds
• Difficult for volume manufacturing
• Wide range of mastering/prototyping methods
• Masters can be made complex, multi-level, etc..
without making the replication in polymer more difficult
• Low cost, high-volume manufacturing methods
–injection molding, hot embossing
• Disposable devices due to low cost
–reduced risk of cross contamination, no cleaning necessary
–Essential in many applications such as PCR, or clinical Diagnostics
• Material surface properties can be manipulated
Advantages of Polymeric Microfluidic Chips
Example 1: Disposable Glass Chips in the Agilent Bioanalyzer
Disposable Glass Chips: Protein Sizing
Example 2: Microfluidic integrated CE with ESI/MS detection
• Rapid electrophoretic separations (~12 minutes)
• Voltage driven injection and separations provide reproducibility and reliability
• Reproducible electrokinetic sample injection; low sample volume
• Recessed tip minimizes biohazard and fragility
• Single-use, plastic disposable – no carryover
• Separation channel coated to provide EOF and eliminate sample adsorption
• Integrated injection, separation, spray eliminate connections to give high reliability and reproducibility, efficient fabrication
• Multiple channels open onto the tip to provide electrical contact through solution rather than a tip electrode
Microfluidic integrated CE with ESI/MS detection
Two-Channel Electrospray Tip • Right channel
• Separation channel
• Positively coated, electro-osmotic flow, ~100 nL/min
• Left Channel
• Electrical contact channel
• Uncoated
• Spray voltage determined by voltage divider between right and left channels
Example 3: Wako biomarker detection with disposable polymeric chips
The Wako i30 is an FDA-approved microfluidic system for clinical diagnostics.
The first assays are for liver cancer biomarkers (AFP, AFP-L3%, and DCP). It
uses isotachophoresis for sample concentration, to achieve sub-picomolar
sensitivity.
QuantaLife Introduction ™
• System built on a decade’s worth of research and development
• A disposable microfluidic device is a key part of the system
• Beta program in progress; launch planned for 2011
Droplets serve as micro-reactors for making thousands of independent, digital measurements
One
measurement
Many thousands
of discrete measurements
Third Generation of PCR
First generation
Thermostable
Taq Polymerase
1987 1996 2010
Second generation
TaqMan Assays and
Real-time Detection
®
Third generation
Droplet Digital PCR ™
ddPCR Process ™
1. Make Droplets 2. Cycle Droplets 3. Read Droplets
Count positive droplets to estimate concentration of target
Sample 1 Sample 2
Sample 3
Sample 4
Low
concentration
High
concentration
NO
targets
Medium
concentration
Number of Positive Droplets is Directly Related to Concentration
Modeling as Poisson
copies per droplet = - ln (1 – p)
where p = fraction of positive droplets
* at 20,000 droplets per reaction
0. Prepare Sample and Reagent Mixture
Primers
& Probes
Nucleic
Acid Sample
Inexpensive
Consumable
QuantaLife
Master Mix
1. Make Droplets
Load consumable into the droplet generator
Generate eight sets of 20,000 droplets
Transfer emulsion
to plate
A.
B.
2. Cycle Droplets
Standard thermal cycler provides
scalability in standard 96-well format
and flexibility of cycling parameters
96-well plate Transfer
emulsion to plate
3. Read Droplets
Load the plate into the droplet reader A.
Stream droplets single-file past the detector B.
Your Results with Digital Resolution
S. aureus titration shows excellent reproducibility and linearity across multiple QuantaLife instruments
0.1
1
10
100
1000
10000
0.1 1 10 100 1000 10000
Measu
red
Co
ncen
trati
on
(co
pie
s/µ
L)
Theoretical Concentration (copies/µL)
Unit 03 Unit 05 Unit 06
™
900 copies/µL (18,000 copies / 20,000 droplets)
High Separation between Positives and Negatives
Detecting EGFR L858R Mutation in High Wildtype Background
• EGFR mutants are found in some non-small cell lung
carcinomas.
• Carriers of the mutation respond to the tyrosine kinase inhibitor
class of drugs.
• Detection of mutants in plasma is desirable, as many patients
have inoperable tumors.
• Sequencing experiments suggest significant heterogeneity
within solid tumors, but significance is unclear.
• Mutation testing can be used to monitor drug response.
Real-time PCR can only detect levels > 1%
Detecting EGFR L858R Mutation in High Wildtype Background
Mutant
Fraction L858R Wildtype
10% 24.5 24.8
1% 27.9 25.1
0.1% no call 24.9
0.05% no call 25.1
0.01% no call 25.0
0% no call 25.0
NTC Undetermined Undetermined
Cycle Threshold Values
10%
1%
0%-0.1% NTC
* Assay from Yung et al. 2009
Droplet Partitioning Effectively Dilutes the Background Relative to the Target
Starting sample
40,000 wildtype molecules
40 mutant molecules
20,000 droplets w/o mutant
40 droplets w/ mutant
Partitioned Sample
Target is at 0.1%
relative to wildtype
Target is at 33%
relative to wildtype
ddPCR System Readily Detects 1% EGFR L858R in Wildtype Background
1% EGFR L858R
™
Concentration
of mutant: 0.02
copies per
droplet
Concentration
of wildtype: 2
copies per
droplet
ddPCR System Readily Detects 0.1% EGFR L858R in Wildtype Background
0.1% EGFR L858R
Concentration
of mutant: 0.002
copies per
droplet
™
Concentration
of wildtype: 2
copies per
droplet
ddPCR System Readily Detects 0.05% EGFR L858R in Wildtype Background
0.05% EGFR L858R
Concentration
of mutant: 0.001
copies per
droplet
™
Concentration
of wildtype: 2
copies per
droplet
ddPCR System Readily Detects 0.01% EGFR L858R in Wildtype Background
0.01% EGFR L858R
Concentration
of mutant: 0.0003
copies per
droplet
™
Concentration
of wildtype: 2
copies per
droplet
ddPCR System Readily Detects 0.01% EGFR L858R in Wildtype Background
Mutant Fraction
in Sample
(as prepared)
Measured
mutant fraction
Measured mutant
concentration – 4 wells
(copies/µL)
Measured wild-type
concentration – 4 wells
(copies/µL)
NTC NA 0 0
0.00% 0.00% 0 1980
0.01% 0.01% 0.2 2037
0.05% 0.05% 1 2101
0.1% 0.10% 2.1 2078
1% 1.01% 22 2172
10% 9.78% 205 2097
™
© 2010 QuantaLife, Inc. All rights reserved. QuantaLife and ddPCR
are trademarks of QuantaLife, Inc. All other trademarks are the
property of their respective owners.
Thank you