1
Miniaturization of
Bioanalytical Instrumentation
DNA analysis – genomics
Protein analysis – proteomics
Metabolite analysis – metabolomics/metabonomics
Glycomics, …
New technologies
Microfluidics – Mass Spectrometry
Frantisek Foret, Institute of Analytical Chemistry
Academy of Sciences of the Czech Republic, Brno
Capillary gel electrophoresis
Separation of nerve cell proteins
H. Hydén et al. Anal.Biochem, 17, 1-15, 1966.
1973
Instrumentation Miniaturization
Technology
Producst
Consequences
Microelectronics Microfluidics
?
Microfluidics?
Speed of
analysis
Space saving
Cost cutting
Mass production
Control of electric current control of fluid flows
Stephen Quake, Dept. Bioengineering, Stanford University, http://thebigone.stanford.edu/index.html
MICROFABRICATED DEVICES
* Sensors - accelerometers, glucose monitors, ...
* Genomics - first commercial applications
* Proteomics - sample processing
separation
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http://www.ncbi.nlm.nih.gov/PubMed
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
(as of April 19, 2010)
Incidence of the word “MICROFLUIDIC” in PubMed
1. Expose 2. develop and etch
mask
photoresist
protecting layer
(Cr, Au)
Substrate (glass, silicon, ...)
3. Remove the protection layer 4. Close the structure
(thermal bonding)
Photolithography
• Substrates up to 100 x 100 mm2
• Structures down to 1 μm
• Address grid down to 40 nm
• 3D exposure mode
• Standard or UV laser source
mPG 101 Tabletop Laser Pattern Generator
SILICON - ANISOTROPIC ETCHING
* Anisotropic etching – direction dependent etch rate
* Etch rate slower perpendicularly to the crystalline planes
with the highest density
* Typical etches: KOH, Tetramethyl Ammonium Hydroxide (TmAH)
Ethylene Diamine Pyrocatechol (EDP)
Alternative technologies
Hot embossing
Injection molding - production scale
Casting - polymeric resins, PDMS
Plasma etching
Laser machining
System Integration
1cm
sample in
waste inject
+ HV wash
liquid junction
(spray liquid)
ESI capillary in
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Diffusion limited mixing Spatial flow focusing
Capillary force filling
Exact volume metering on the nl level
sample
Defined volume (nl)
Hydrophobic patch
Seth Fraden et al., J. AM. CHEM. SOC. 2007, 129, 8825-8835.
Droplet deneration in nl-pl volumes
Microfluidics
Smaller size – faster analysis
Microchannel junctions without dead volume
Parallel systems for high throughput
Disposable parts
Phenomena unimportant on a macro scale become dominant
Space saving
Fluid (liquid) phase handling
4
Small volume problem
2.15 mm -> 10 ml ~ 10-15 M
1 mm -> 1 ml ~ 10-14 M
0.1 mm -> 1 nl ~ 10-11 M
0.001 mm -> 1 fl ~ 10-5 M
Example: LOD = 100 molecules
www.gyros.com
Point-of-care analysis
i-Stat -> Heska -> www.abbottpointofcare.com
Examples
New approaches for DNA analysis based on:
massively parallel PCR and pyrosequencing
(www.454.com)
or
microfluidics
and
high sensitivity (single molecule) detection
Human genome for $ 1000?
J. Craig Venter
At present ~1000 x more
Microfluidics necessary
First system – 454.com
(www.454.com, Roche)
454 Massively Scaleable Sequencing
in Picoliter Volumes
1) Random DNA fragments
2) Emulsion amplification
on 28 µm beads
4) Sequencing using microbeads with
immobilized enzyme and
chemiluminiscence detection
3) Loading of the beads into
100 000 microchannel glass plate
1
44 μm
Each Well contains a
single DNA Bead &
hundreds of enzyme
beads
Three current plate sizes:
300K Wells (25x75 mm
2
)
860K Wells (40x75 mm
2
)
1.6M Wells (70x75 mm
2
)
Bead Loading - 454 PicoTiterTM Plate
5
1. add one of the four dNTPs and DNA polymerase pyrophosphate
(PPi) released stoichiometrically
2. ATP sulfurylase converts PPi to ATP in the presence of
adenosine 5´ phosphosulfate; ATP+luciferase ->luciferin to
oxyluciferin. Light emission proportional to the amount of
ATP.
3. Unincorporated nucleotides degraded by apyrase
Sequencing-By-Synthesis -
Pyrosequencing
Sepharose
DNA Capture Bead
Containing Millions of
Copies of a Single
Clonal Fragment
A A T C G G C A T G C T A A A
A G T C A
Anneal Primer
T
PP i
ATP
Light + oxy luciferin
Sulfurylase
Luciferase
APS
luciferin
191bp Perfect Read on 454 System
T
A
C
G
Cont.
2
tSMS -true Single
Molecule Sequencing
www.helicosbio.com
HeliScope™
Break
the DNA
ligate
fluorescent
universal
primer at
the 3’ end
attach on
the polyT
modified
surface
record
positions
remove
fluorescent tag
sequencing
reaction
using
fluorescent
dNTP’s
record
positions
remove
fluorescent
label and
repeat for
other dNTP’s
1
2
3
4
5
6
7
3
www.pacificbiosciences.com
SMRT™ single molecule, real-time sequencing
Direct electrical detection of DNA synthesis
Nader Pourmand, Miloslav Karhanek, Henrik H. J. Persson, Chris D. Webb, Thomas H. Lee,
Alexandra Zahradnı´kova´ , and Ronald W. Davis, PNAS 2006 vol. 103 6466–6470
Ion Torrent
3
6
DNA
nanopore
Nanopore DNA sequencing
http://www.nanoporetech.com
Protein Analysis
Much more complicated than DNA
Posttranslational modifications
1013 concentration range
No PCR
Separations + ESI/MALDI Mass Spectrometry
Ionization in mass spectrometry
ESI - concentration sensitive
(10 nL/min or 10 mL/min – similar sensitivity)
Charge competition
Different proton affinity
in the gas phase
Signal suppression
SEPARATION
MS IONIZATION - SIGNAL
SUPRESSION
(ESI and MALDI)
+
+ + +
+ +
+
condensed phase phase transfer- ionization gas phase
MIXTURE
SEPARATED
COMPOUNDS
+ + +
+ +
+
+
++
+
+
Separation and Signal Suppression
1 min infusion CE-ESI/MS
279.2 459.4 639.6 819.8
Mass (m/z)
530.62
591.61
810.17
674.16
449.77
99.0 279.2 459.4 639.6 819.8
Mass (m/z)
449.82
810.24
674.23
591.61
530.62
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Electrospray ionization
o
sample
~ 3000 V
Microfluidic channel
CHIP ESI/MS COUPLING
* flat surface electrospray
* microfabricated tips
* external (inserted) tips
* external interface with a
transfer capillary
* integrated pneumatic nebulizer
* integrated liquid junction
ESI for Microfluidics
100 µm
50-60 µm
Separation
channel
N2N2
to MS
1 mm
auxiliary
channel
separation
channel
Microfabricated nebulizer
300 µm
Channel exit ESI port
25× 75 µm
Nebulizer
channels
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nitrogen chanels
separation chanels
to MS
reservoirs
electrodes
Pneumatic nebulizer
50 mm
Pneumatic nebulizer
10-6M bradykinin, neurotensin and angiotensin
100 mm
Sputtering of gold tips
1 mm
Si wafer
SU-8
Le Gac S., Arscott S., Rolando C. ELECTROPHORESIS 2003 , 24, 3640-3647.
Micro-nib electrospray source
(SU-8 on a silicon wafer)
Sjödahl, J., Melin, J., Griss, P., Emmer, A., Stemme, G., Roeraade, J. Rapid Commun. Mass Spectrom. 2003, 17, 337—341.
ESI tips produced by DRIE in silicon ESI tip array
www.advion.com
DRIE in silicone Plasma etched
in polyimide
www.diagnoswiss.com
www.phoenix-st.com
Molded plastics
www.agilent.com
9
www.agilent.com
HPLC on a chip
Polyimide HPLC-chip, integrating an enrichment column, frits, a
laser ablated ESI tip and trapazoidal separation column
www.agilent.com
http://www.youtube.com/watch?v=oBXK29YsplU
Segmented column HPLC/chip
www.agilent.com
www.agilent.com
Three LC columns – length 130 mm
Each segment individually packed.
BSA digest separated with a 30min gradient on a 2 column segmented
chip, packed with 3.5μm particles
Segmented column HPLC/chip
www.agilent.com
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Trizaic System
nanoTile Assembly
Cut Ceramic Material Fired Wafer
Ceramic Microfluidic Fabrication
Final Diced Part
Green tape
Al2O3–MgO–SiO2 glass particles mixed with organic
binders and solvents to form glass ceramic
Product Description
951 Green Tape is a low-temperature cofired ceramic tape.
The 951 system comprises a complete cofireable family of Au and Ag
metallizations, buried passives, and encapsulants.
951 is available in multiple thicknesses for use as an insulating layer in:
•Multichip modules
•Single chip packages
•Ceramic printed wiring boards
•RF modules
http://www.dupont.com/mcm
Channel Geometries and Packing
1.7mm particles
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Channel shape effect
connections
Packed glass LC chip
Eksigent, part of AB SCIEX, www.eksigent.com
Miniaturized (microfabricated) mass spectrometers?
Miniaturized (microfabricated) mass spectrometers
Miniature Mass Spectrometers
Ouyang Z., Cooks, G.R., Annu.Rev.Anal.Chem. 2009, 2, 187-214.
Ouyang Z., Cooks, G.R., Annual Rev.Anal.Chem. 2009, 2, 187-214.
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Miniature Mass Spectrometer Systems
based on a Microengineered Quadrupole
Filter
A. Malcolm, S.Wright, R. R. A. Syms,
N. Dash, M.-A. Schwab, A. Finlay
Anal. Chem. 2010, 82, 1751–1758
A miniature mass spectrometer for liquid
chromatography applications
Rapid Communications in Mass Spectrometry
Volume 25, Issue 21, pages 3281-3288, 3 OCT 2011 DOI: 10.1002/rcm.5230
http://onlinelibrary.wiley.com/doi/10.1002/rcm.5230/full#rcm5230-fig-0002
Mass Analyzer ionchip® quadrupole mass spectrometer
Mass Range m/z 50-800 with ionchip®150
Mass Accuracy ± m/z 0.3 in full scan
Mass Resolution m/z 0.7±0.1 FWHM
Sensitivity 10pg of reserpine in SIM mode S/N ratio of 10:1 (R
www.microsaic.com
Advion expression Compact Mass
Spectrometer
www.advion.com
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NanotechnologyMicrofluidics
“… the potential of nanotechnology is so vast
that it has been easy for researchers to get
lost in the wilderness of possibilities.”
Stephen Empedocles co-founder of Nanosys Inc., Palo Alto, CA
“… (the researchers) say the quantum dots
can do amazing things. How do I make money
from them?”
Edward K. Moran, Nanotech Industry Practice, Deloitte Services LP, NYC
What is a patent
Invention disclosure
Does it make sense to patent?
Patent search
Resources
Patent? Patent!
What Is a Patent?
A patent for an invention is the grant of a property right to the inventor,
issued by the United States Patent and Trademark Office. Generally, the term of a new
patent is 20 years from the date on which the application for the patent was filed in
the United States or, in special cases, from the date an earlier related application was filed,
subject to the payment of maintenance fees. U.S. patent grants are effective only
within the United States, U.S. territories, and U.S. possessions. Under certain
circumstances, patent term extensions or adjustments may be available.
What is granted is not the right to make, use, offer for sale, sell or import, but the right to
exclude others from making, using, offering for sale, selling or importing the invention. Once
a patent is issued, the patentee must enforce the patent without aid of the USPTO.
There are three types of patents:
1) Utility patents may be granted to anyone who invents or discovers any new and
useful process, machine, article of manufacture, or composition of
matter, or any new and useful improvement thereof;
2) Design patents may be granted to anyone who invents a new, original, and ornamental
design for an article of manufacture; and
3) Plant patents may be granted to anyone who invents or discovers and
asexually reproduces any distinct and new variety of plant.
Patentable subject
1. Does not fall under the laws of nature, natural phenomena or abstract ideas
2. Utility requirement - invention must be useful in association with machines,
human-made products, compositions of matter or processing methods
3. Novelty the idea must not be presented to the public before the filing
4. Nonobviousness – it must be unrecognizable to a skilled person
in the field of invention
5. Clarity of the description included in the application
Patent je zákonná ochrana vynálezů zaručující vlastníkovi
patentu výhradní právo k průmyslovému využití vynálezu.
V České republice udělování patentů upravuje zákon 527/1990. Podle něj se patenty udělují na
vynálezy, které jsou nové, jsou výsledkem vynálezecké činnosti a jsou průmyslově
využitelné.
Vynález se považuje za nový, jestliže není součástí stavu techniky.
Stavem techniky je všechno, co bylo zveřejněno přede dnem přihlášení patentu, ať již v České
republice nebo v zahraničí.
Za vynálezy se naopak nepovažují zejména :
objevy, vědecké teorie a matematické metody,
pouhé vnější úpravy výrobků,
plány, pravidla a způsoby vykonávání duševní činnosti,
programy počítačů,
pouhé uvedení informace
Majitel patentu má výlučné právo vynález využívat (tj. výrobek vyrábět, uvádět do oběhu nebo
upotřebit postup), dále poskytnout souhlas k využívání vynálezu jiným osobám (např. licenční
smlouvou) a má právo převést patent na jinou osobu.
Proto, aby patent zůstal v platnosti, je nutno platit tzv. udržovací poplatky, a to v každém
státu zvlášť. Maximální možná délka patentové ochrany je 20 roků.
http://cs.wikipedia.org/
14
www.uspto.gov
http://www.epoline.org/
http://isdvapl.upv.cz http://cz.espacenet.com/
15
Companies offering microfluidics solutions
Abbott Laboratories http://www.abbott.com
Advanced Liquid Logic http://www.liquid-logic.com/
Agilent Technologies http://www.agilent.com
Applied Biosystems http://www.appliedbiosystems.com/
Aviva Biosciences http://www.avivabio.com/
Biacore http://www.biacore.com
Bioident http://www.bioident.com/
Bioprocessors http://www.bioprocessors.com/
Bio-Rad http://www.bio-rad.com
Biotrove http://www.biotrove.com
Caliper Life Sciences http://www.caliperlifesciences.com/
Cellix http://www.cellixltd.com
Cepheid http://www.cepheid.com
Ciphergen http://www.ciphergen.com
Cole-Parmer http://www.coleparmer.com
Dionex http://www.dionex.com
Eksigent Technologies http://www.eksigent.com
Erie Scientific Company http://www.eriemicrofluidics.com/
Evotec Technologies http://www.evotec-technologies.com/
Fluidigm http://www.fluidigm.com
Gyros http://www.gyros.com
Handy Lab Inc. http://www.handylab.com
Helicos Biosciences Corporation http://www.helicosbio.com
Hewlett-Packard http://www.hp.com
Ibidi Integrated BioDiagnostics http://www.ibidi.de/
Invitrogen http://www.invitrogen.com
Iq Micro Inc. http://www.iq-micro.com/
Liquidia Technologies http://www.liquidia.com/
Micralyne Inc. http://www.micralyne.com/
Microchip Biotechnologies Inc http://www.microchipbiotech.com
Microfluidics http://www.microfluidicscorp.com/
Micronics http://www.micronics.net
Micronit Microfluidics BV http://www.micronit.com/
Monogram Biosciences http://www.monogrambio.com/
Nanogen http://www.nanogen.com
Nanostream http://www.nanostream.com
Nanoterra http://www.nanoterra.com/
Network Biosystems http://www.networkbiosystems.com
Orchid Biosciences http://www.orchid.com
Pyrosequencing AB http://www.pyrosequencing.com
Roche 454 http://www.roche.com
Spin X Technologies http://www.spinx-technologies.com/
Surface Logix http://www.surfacelogix.com
Tecan http://www.tecan.com
Tronics Microsystems http://www.tronics-mst.com/