on-chip aptamer-based sandwich assay for thrombin...
TRANSCRIPT
On-Chip Aptamer-Based Sandwich Assay For Thrombin Detection Employing Magnetic Beads And Quantum Dots
Dr. Hutanu Daniela
Diaspora în cercetarea ştiinţifică şi învăţământul superior din România 2010
(E-mail: [email protected])
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Presentation Overview
• About Life Technologies, and Oregon State University collaborator
• Introduction to the collaborative project
• Properties of reagents and devices
• Assay workflow and results
• Conclusions
• Acknowledgements
3
About Life Technologies
Life Technologies is a global
biotechnology tools company dedicated
to improving the human condition.
Life Technologies customers do their
work across the biological spectrum,
working to advance personalized
medicine, regenerative science,
molecular diagnostics, agricultural and
environmental research, and 21st century
forensics.
Each year, the company sponsors 20–25
collaboration projects that allow Life
Technologies researchers to connect with
external thought leaders and forge
relationships that last long after the six-
month compact ends.
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About Academic Collaborator from Oregon State University (OSU)
Microfluidic Nanofilitration
DeviceMacroscale Fixture Functionalized Magnetic
Nanoparticles
Microfluidics for In-situ
Water Quality Monitoring 3-D Image of a Microfluidic Channel
Lab-on-a-chip technology - fabrication and implementation of separations systems in
microchip format
Molecular recognition technologies - high selectivity sorbents for separations
Proteomics - separation and measurement of proteins from complex mixtures
Biothreat analysis
Environmental monitoring
Vincent T. RemchoEmail: [email protected] State University, Corvallis, OR
Professor of Chemistry and of Materials Science
Adjunct Professor of Biochemistry & Biophysics
Founding member of the Oregon Nanoscience and Microtechnologies Institute (ONAMI)
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Introduction to the Collaborative Research Compacts (CRC) Project with OSU• We report the development of an on-chip aptamer-based fluorescence
bio-sensor assay for protein detection and quantification based on
sandwich ELISA principles.
• Aptamer-functionalized magnetic beads were utilized to capture the
target analyte (alpha-thrombin), while a second aptamer,
functionalized with quantum dots, was employed for detection by
fluorescence microscopy in microchip format.
Magnetic field
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Why Quantum Dots?
655 605 585 565 525 nm
25
nm
Size of the nanocrystal determines the color.
Size is tunable from ~2-15 nm (±3%).
Size distribution determines the spectral width.
Highly fluorescent, nanometer-sized, single crystals of semiconductor materials.
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Qdot® Nanocrystal Structure and Properties
Core nanocrystal (CdSe)
Inorganic shell (ZnS)
Organic coating
Biomolecule15 - 18 nm
• Covalently attached to polymer
shell− Immunoglobulins
− Streptavidin, Protein A
− Receptor ligands
− Oligonucleotides
• Provides water solubility and
functional groups for conjugation
• Improves brightness and stability
• Determines color
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
4,000,000
4,500,000
5,000,000
400 450 500 550 600 650 700 750 800 850 900
Wavelength (nm)
Exti
ncti
on
Co
eff
icie
nt
(M-1
cm
-1)
Qdot 525 Conjugate Absorbance Qdot 525 Conjugate Emission
Qdot 565 Conjugate Absorbance Qdot 565 Conjugate Emission
Qdot 585 Conjugate Absorbance Qdot 585 Conjugate Emission
Qdot 605 Conjugate Absorbance Qdot 605 Conjugate Emission
Qdot 655 Conjugate Absorbance Qdot 655 Conjugate Emission
Qdot 705 Conjugate Absorbance Qdot 705 Conjugate Emission
Qdot 800 Conjugate Absorbance Qdot 800 Conjugate Emission
Advantages over fluorescent dyes
● Single source excitation
● Narrow emission (multiplexing)
● Excellent photostability
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TEM Images of Qdot® Nanocrystals
Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA
Qdot® 525
Qdot® 605
Qdot® 585Qdot® 565
Qdot® 655
5 nm + 20 nm Gold
~4 nm ~5 nm~5 nm
~5 x 12 nm ~8 x 15 nm
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Some Applications of Qdot® Nanocrystals
Live cell tracking
Imaging
In vivo imaging
Flow cytometry
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Example of Multiplexed Experiments with Qdot® Nanocrystals Golgi Fibronectin
Nuclei
Plasma
Membrane
Combo
Images provided by Jason Kilgore, Life Technologies.
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Why Dynabeads® Magnetic Particles
Magnetic parameters
Size: 1 m, 2.7 m, 4.5 m
Binding Capacity
Hydrophilicity / Hydrophobicity
Charge: -ve, neutral, +ve
Polarity: -, neutral, +
Attachment chemistry
Pre-coupled
e.g; Streptavidin, Antibody, Protein A/G
Signal/Noise
Readout Compatibility
Dynabeads® magnetic particles are superparamagnetic particles; they exhibit
magnetic properties when placed in a magnetic field, with no residual magnetism
once removed from the magnetic field.
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Dynabeads® Magnetic Particles Employed for Isolation
X
Specific nucleic
acids
Organelles
Cells
Surface activated
Primary Antibody
(Ab)
Secondary Ab
Protein A/G
Talon (His-tag)
Streptavidin
Oligo dT (deoxy-
thymine nucleotides)
Small molecules
Total nucleic acids
Peptides/proteins
Immunoassay
TTTTTAAAAA
TTTTTAAAAA
TTTTT
AAAAATTTTT
AAAAA
Oligo(dT)
Protein A/G
FlowComp™
(StA)
Surface
Activated
Silane
Talon
His-tag
Sheep-
anti-
rabbit
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Aptamers
• Single stranded DNA, or RNA molecules
• High specificity, comparable to antibodies
• Relative ease of synthesis & chemical modification
• Tailored binding affinity
• Resistance against denaturation
Microfluidics• Small sample and reagent volume (µL)
• Efficient washing in automated continuous flow
• Large surface area-to-volume ratio
• Decreased total analysis time (minutes)
• Inexpensive fabrication of disposable microchipsHutanu, D., and Remcho, V.T., Advances in Chromatography, 2007, 45, pp 173-196.
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Developed Assay Workflow
Washing
Ex Emmax
Washing
= Streptavidin-coated quantum dots
= Streptavidin-coated magnetic beads
= Biotin-Aptamer A
= Thrombin
= Biotin-Aptamer B= Magnet
Injection of
Unpurified Sample
Incubation
Automated Washes
Detection
and Readout
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Fluorescence Detection of Thrombin with Developed Assay
Side view
magnet
outlet
inlet
Detection
The magnetic beads were trapped by magnets
underneath the channel. Thrombin detection
was performed on a fluorescence microscope to
capture fluorescence images and intensity
measurements.
Dark field Fluorescence
Magnets
Immobilized beads
Qdot® 525 immobilized on 1 µm Dynabeads® MyOne™ Streptavidin C1 via
aptamer-based sandwich assay
Zeiss
Axioimager m1M
fluorescence
microscope
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Microchip Design for High-Throughput Thrombin Detection and Quantification
a
b
c
Reaction chamber
Magnet
To pump
A fully assembled device with a NanoPort for connection to a syringe pump.
A disposable plastic microchip, containing eight reaction chambers and a vacuum manifold.
A fixture to hold multiple NdFeB permanent magnets.
Tennico, Y., Hutanu, D., Koesdjojo, M., Bartel, C., and Remcho, V.T., Analytical Chemistry, 2010, 82 (13), pp 5591–5597.
Microchip components:
Top layer, made of
polycarbonate (PC) or
polymethylmethacrylate
(PMMA).
Middle layer, double-sided
adhesives, for channel
fabrication.
Bottom layer, made of PC or
PMMA, as the enclosure.
Channels were cut with laser
on 3M Optically Clear
Adhesive 8272 double-sided
adhesives, and then sealed
with plastic polymers on both
sides.
Patterning was done on a 5
watt 355nm ESI UV laser tool
designed for micro-
machining.
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On-Chip Thrombin Detection Results with Developed Assay
Brightfield Fluorescence
Fluorescence images of aptamer-coated beads
incubated with increasing concentrations of thrombin
(from left to right).
1.0 10.0 100.0 1000.0 10000.0
ng/mL [Thrombin]
RF
U
0.0 500.0 1000.0 1500.0 2000.0 2500.0 3000.0 3500.0 4000.0
ng/mL [Thrombin]
RF
U
On-chip dose-response curve for thrombin.
On-chip assay 96 well plate assay
minutes hours
µL mL
low medium
Linear range 100 – 1000 ng/mL 100 – 950 ng/mL
10 ng/mL 18 ng/mL
8% 14%
On-Chip 96 Well-Plate
Total assay time
Sample volume
Reagent consumption
Linear range
Limit of detection
Mean standard deviation
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Conclusions
• Successful application of on-chip aptamer-based sandwich assays,
with Qdot® nanocrystals and Dynabeads®, for detection of target
proteins of biomedical importance.
• Experimental conditions, such as reagent consumption and incubation
time, were optimized in the microchip platform for the lowest limit of
detection, highest specificity and shortest assay time.
• The microfluidic chip proved to be a rapid and efficient system for
aptamer-based thrombin assays, requiring only minimal (microliter)
reagent use.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic
use.
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Acknowledgements
• Funding: Life Technologies, 2009 Collaborative Research Compacts.
• CRC Collaborators: Yolanda Tennico and Cheryl Moody Bartel (Life
Technologies, Eugene, OR, USA); Myra Koesdjojo and Vincent
Remcho (Oregon State University, Corvallis, OR, USA).
• Reagents, instrumentation and assay development: Schuyler Corry,
Jason Dallwig, Jim Hirsch, David Wright, Kari Haley, Joe Bartel, Birte
Aggeler, Shawn Starkenburg, Dean Tsou, Vanessa Adams, Matt
Beaudet, Shula Jaron, Laurel Stone, Ameet Juriani (Life Technologies,
USA).
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© 2010 Life Technologies Corporation. All rights reserved.
The trademarks mentioned herein are the property of Life Technologies
Corporation or their respective owners
NOTICE TO PURCHASER: Limited Use Label License
The products shown in this presentation may be covered by one or more Limited
Use Label License(s). Please refer to the respective product documentation or
the Applied Biosystems website under www.appliedbiosystems.com for the
comprehensive license information. By use of these products, the purchaser
accepts the terms and conditions of all applicable Limited Use Label Licenses.
These products are sold for research use only, and are not intended for human or
animal diagnostic or therapeutic uses unless otherwise specifically indicated in
the applicable product documentation or the respective Limited Use Label
License(s).
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Extra Slides
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Dynabeads® Magnetic Particles Surfaces
Sheep -rabbit
Oligo dT / dX(Specific Capture)
SILANE
(Total Capture)
Nucleic acids
Tosyl
COOH
Epoxy
Amine
Surface Activated
Protein A Protein G
Coated beads
Sheep mouse
Streptavidin
(neutral)
Streptavidin
(neg. charge)
FlowComp Flexi
(”detachable biotin-StA”)
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Invitrogen™ Magnets
DynaMag™-2 DynaMag™-Spin
DynaMag™-50DynaMag™-15
(12302D)(12301D)
(123-21D) (123-20D)
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Relative Size of Qdot® Nanocrystals
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© 2010 Life Technologies Corporation. All rights reserved.
The trademarks mentioned herein are the property of Life Technologies
Corporation or their respective owners
NOTICE TO PURCHASER: Limited Use Label License
The products shown in this presentation may be covered by one or more Limited
Use Label License(s). Please refer to the respective product documentation or
the Applied Biosystems website under www.appliedbiosystems.com for the
comprehensive license information. By use of these products, the purchaser
accepts the terms and conditions of all applicable Limited Use Label Licenses.
These products are sold for research use only, and are not intended for human or
animal diagnostic or therapeutic uses unless otherwise specifically indicated in
the applicable product documentation or the respective Limited Use Label
License(s).
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Dynabeads (MyOne)®
Why Dynabeads® Magnetic Particles?Dynabeads® magnetic particles are superparamagnetic particles; they
exhibit magnetic properties when placed in a magnetic field, with no
residual magnetism once removed from the magnetic field.