TB Conference Presentation , Virginia Beach

September 22-24, 2008

Novel Microfluidic Technologies for Portable Diagnostics Systems

Delivering TB Chips to the Field
Professor Abe Lee Biomolecular Microsystems & Nanotransducers (BioMINT) Lab Micro/nano Fluidics Fundamentals Focus (MF3) Center Director BME and MAE Departments UC Irvine

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Outline
Background and motivation of microfluidics Microfluidics for TB serodiagnostics Microfluidics platforms for cell and biomolecular assays DARPA-Industry Micro/Nano Fluidics Fundamentals Focus (MF3) Center Summary

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TB Conference, VA Beach, September 23, 2008

Field Deployment Point of Care

Engineering love to solve/Build Miniaturization microfabrication Automation integration of pumps and sensors Multiplexing and multifunctional Minimal volume of sample and reagents Speed - mixing Low power Data storage

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TB Conference, VA Beach, September 23, 2008

Lab-On-A-Chip: Size, Speed, Accuracy

Today In 3 years
Biochip today

In 6 years
BioMEMS, BioChip, mTAS

electronics

optics

Days to hours

1 hour

Minutes to seconds

Time from sample to CORRECT answer

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TB Conference, VA Beach, September 23, 2008

Chip-Scale Integration for harnessing the power of electrons, photons and biomolecules
electronics photonics MEMS Microfluidics/LOC

Bot

1 mm
Data Com

Microfluidics

CMOS

Gene chips Optical Bench

Silicon Carbon Nitride Si SOI SiGe GaAs InP GaN SiC

VCSEL

Micro Optical Bio-detection

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TB Conference, VA Beach, September 23, 2008

Lab on a Chip
Is it possible?

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TB Conference, VA Beach, September 23, 2008

Advantages of Microfluidics
Miniaturized channels and reservoirs High surface to volume ratio Low Reynolds number Increase speed of reaction Reduce cost of reagents Reduce power consumption Precise mixing/dosage and heating

Integration Reduce cost of manufacture Minimize dead space, void volume Minimize sample carryover Multiplex capability: increased number of parameters monitored per assay

20 m
Hong & Quake, Nat. Biotech 2003

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TB Conference, VA Beach, September 23, 2008

Lab on a Chip or Micro Total Analysis Systems

Motivation: The awesome scale of potential molecular testing
>6 billion people on our planet needing all forms of molecular diagnosis including cancer screening, risk profiling and detection Potential pathogens drawn from ~1.5M species of fungi ~800,000 species of bacteria All possibly with drug resistance genes Food, water, agricultural and environmental testing needs

Perhaps the need for >15 billion molecular tests per year
Courtesy: Peter Gascoyne - UT MDACC

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TB Conference, VA Beach, September 23, 2008

1900s-2000 Centralized laboratories to which samples are brought and processed by skilled operators
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TB Conference, VA Beach, September 23, 2008

1950s 1970s Centralized computers to which the data is brought and processed by skilled operators

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Courtesy:TB Conference, VA Beach, September 23, 2008 Peter Gascoyne - UT

Microprocessors

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TB Conference, VA Beach, September 23, 2008

Lab testing/Diagnostics

Third world country health

Point-of-care Patient monitoring

Surgery and treatment Biodetection, homeland security

Microfluidic Processors

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Drug Discovery,Targeted drug delivery, Personalized medicine

Real-time food, water, and environmental monitoring

TB Conference, VA Beach, September 23, 2008

Microarray-based Genetic Analysis

Crude Sample
Filtration Centrifugation Magnetizable beads Beakers Cell PreconCell Isolation centration Cell Cell Lysis Purification
Lab Cartridge

Centrifugation Thermocyclers

DNA Isolation Cell Lysis

Amplification

Hybridization

Separation Hybridization
Conventional DNA Microarray

Readers

Detection

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Answer

TB Conference, VA Beach, September 23, 2008

Projects
Acoustic agitator active mixing Electrical detection of protein binding Molecular spotting in microchannels Colorimetric imaging by scanner Other projects
Droplet DNA analysis Droplet lipoplex Cell sorting

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TB Conference, VA Beach, September 23, 2008

Proteome Microarrays in Felgners Lab

Serodominant Antigen Profiling
ELISA IgM IgG stage-> ---- - - - + + - - + + + + + + + + + + + - - + + + + + + + + + + + + + + - - - - - +++++ - + - - - ++++++++++++++ - - + - - - - - - - - - - - - - - - - - - - ++ - ++ - - - + - + - - - - +++++++++++++++
0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 1 1 1 1 2 1 1 1 1 2 1 1 2 2 1 1 2 2 1 1 1 1 1 1 1 1 1 3 3 2 3 3 3 3 2 2 2 3 3 3 Antigen ID

Disease Stage ID
0 Healthy Control 1 Primary Infection 2 Disseminated Disease 3 Chronic Disease

The Protein Microarray Laboratory (PML) at UC Irvine has developed a high-throughput genome cloning, expression and printing platform to generate protein microarrays on a genome-wide scale for vaccine and serodiagnostic antigen discovery. High density whole microorganism protein microarrays are printed on nitrocellulose coated glass slides and probed with human infected sera to identify serodominant antigens potentially useful for diagnostic tests, and vaccine discovery.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

1.0

4.0

8.0

Antigen Reactivity Scale

Lyme Disease
TB Conference, VA Beach, September 23, 2008

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Traditional Microarray
Protein Microarrays 100-200 micron antigen spots or biomarkers on a finger-nail sized nitrocellulose pad 250-4000 biomarkers per pad Approximately 24hrs for complete assay to be run Uses fluorescent scanner for antigen-antibody detection

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TB Conference, VA Beach, September 23, 2008

Protein Array Immunodiagnostic Chip

Protein array chip to specifically detect multiple diseases
Alkaline Phosphatase Conjugated Secondary Ab
Ft Bb Bp TB Pf Vv

Cy3 Conjugated Secondary Ab

Diagnostic array size compared to a dime

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TB Conference, VA Beach, September 23, 2008

Platform 1: Flow-induced Electrical Admittance Biosensor (FEAB) A microfluidic electrical sensing platform that is capable of detecting antigen/antibody binding in real time. The development of this platform will allow for faster detection of certain illnesses and diseases within a patients sample compared to standard techniques such as ELISA.

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TB Conference, VA Beach, September 23, 2008

Microchannel Sealed Microarrays

Protein microarrays inside sealed microchannels with electrical detection Integrate and automate the assay protocols with microfluidic control

Presented at uTAS 2007

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TB Conference, VA Beach, September 23, 2008

Microfluidic Colorimetric Platform using Acoustic Micromixing

The acoustic micromixer is based off of an acoustic mixer design for enhancement of DNA hybridization detection. (Liu et al)

Acoustic energy applied to system causing bubble to resonate

Acoustic Mixer Time Lapse

Time 0 sec

Time 1 sec

Time 3 sec

Time 6 sec

Thiolene device: 4x4 array of 400 micron cavities at 400 micron depth within an channel approximately 1000microns deep

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TB Conference, VA Beach, September 23, 2008

Colorimetric Enhancement

Colorimetric Results

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Lab in a Droplet!
Advantages of Droplets Rapid mixing Homogeneous reactions Precise control of volumes Precise control of concentrations Mimic cellular reactions

Analog

Digital microfluidics
TB Conference, VA Beach, September 23, 2008

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Nanomedicine Carriers Monodispersed droplets, particles, vesicle

vs

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Droplet Arrays for Combinatorial Assays

VS

Microarray

Droplet array

Droplet arrays provides a versatile screening platform for biochemistry and molecular biology.
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Serial Droplets Detect and Quantitative Fluorescence Intensity

Fluorescence Saturation Zone

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Rapid DNA Hybridization Inside Micro Droplets

To product reservoir

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Presented at microTAS 2005, Boston

TB Conference, VA Beach, September 23, 2008

Non-viral Gene Carrier Formulation in Microfludic System for Gene Transfection

Picoliter volume micro reactor and incubator (PMRI) system.

Preliminary Transfection Results

Comparison of Lipoplex sizes.

TB Conference, VA Beach, September 23, 2008

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DUAL-LAYER MICROBUBBLE LIPOSPHERES AS DRUG DELIVERY VEHICLES

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TB Conference, VA Beach, September 23, 2008

Neural Stem Cell Sorting based on DEP

Label-less assays Rapid flow through sorting Dielectric properties Cost effective cell sorting

Sorting based on signature peaks

Sort predifferentiated neural stem cells based on dielectric properties

Acquisition of impedance spectrum signatures

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TB Conference, VA Beach, September 23, 2008

Microfluidic DEP device

Flanagan et al., Stem Cells 2008 Abe Lee, Lisen Wang, Jente Lu UCIrvine
TB Conference, VA Beach, September 23, 2008

Distinct DEP profiles of stem cells, neurons, astrocytes

NSPCsneural stem/precursor cells Jente Lu UCIrvine

TB Conference, Flanagan et al., Stem Cells, in press VA Beach, September 23, 2008

Separation of 293 and N115 cells in vertical device

efieldy 1.00E+005 --

1:3 ratio electrodes

0.00E+005 --

-1.00E+005 --

1:1 ratio electrodes

Abe Lee, Lisen Wang UCIrvine

TB Conference, VA Beach, September 23, 2008

Integration of in vitro Diagnostics with in vivo Therapy Changing the current healthcare paradigm
Biomedicine Pull:
-Disease diagnostics -Disease treatment -Disease monitoring -Disease prevention Detection

Detection
Sensing Signaling

Diagnosis
Effector Action Data Analysis

Treatment

Technology Push:
- Lab on a chip - Drug discovery - Drug delivery - Implants - Surgical tools - Biosensors - Imaging Treatment

Judgement

Diagnosis

Source: National Cancer Institute

TB Conference, VA Beach, September 23, 2008

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Other Applications for Microfluidics in TB Diagnostics

Pediatric-specific devices Rapid analyses in the field for epidemiology studies Broad distribution of new assays to biological labs Study hybridization of molecules on microarrays Point-of-care genetic analysis combined with immunodiagnostics for third world countries

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TB Conference, VA Beach, September 23, 2008

Plenty of Room at the Bottom!

Need more engineers to harness your science and speed up delivery of solutions Holistic medicine is enabled by lab-on-achip technologies that integrate diagnostics and therapeutics functions Digital (droplet) microfluidics enables high throughput cellular and molecular assays Cell sorting enables more practical cellular analyses and therapies

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TB Conference, VA Beach, September 23, 2008

Collaborators
Dr. Phil Felgner, molecular biology/UCI Prof. Paul Dayton BME/U. of North Carolina Prof. Sadik Esener, EECS/Nanotumor/UCSD Prof. Hugh Fan ME/University of Florida Dr. Lisa Flanagan, pathology/UCI Prof. Jamie Jester, opthalmology/UCI Dr. Peter Lee, BME/Singapore DSO/NUS) Prof. Ed Monuki, pathology/UCI Prof. Ken Shea, chemistry/UCI Prof. Jeff Wang ME/Johns Hopkins Prof. Dan Nicolau ChemE/U of Liverpool

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TB Conference, VA Beach, September 23, 2008

Biomolecular Microsystems & Nanotransducers (BioMINT) Lab

Funding Sources: DARPA, NIH, Industry, UC Discovery

Graduate students: Jeff Fisher, Lisen Wang, Albert Hsieh, Kanaka Hettiarachchi, Jason Hung, Tim Tseng, Armando Tovar, Robert Lin, Shia-Yen Teh, Uland Liao, Javier Lopez, Rajtarun Mandago, Chia-Sheng Liao, Roger Shih Undergrads: Patrick Pan, Jeff Draper, Kinman Lin, Joseph Hazani

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TB Conference, VA Beach, September 23, 2008