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