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
2
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
3
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
7
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
8
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
10
(A) 30min total run time
(B) 70min total run time
Peak capacity with a two-segment chip
www.agilent.com
Waters microfluidic HPLC
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
Trizaic System Manufacturing process
The stack of green tape is laminated at
elevated temperature and pressure
11
nanoTile Assembly
High Voltage
Connection
Liquid Connection Ports
ESI Capillary NanotechnologyMicrofluidics
There's Plenty of Room at the Bottom
An Invitation to Enter a
New Field of Physics
by Richard P. Feynman
December 29th 1959 at the annual meeting of
the American Physical Society at the
California Institute of Technology (Caltech)
Fluorescent quantum dots
semiconductor inorganic crystals (1-10 nm)
core from elements of II. and VI. or III. and V. group
(ZnS; ZnSe; PbS; CdSe; CdTe)
Surface groups -COOH, -NH2
Preparation
4 NaBH4 + 2 Te + 7 H20 → 2 NaHTe + Na2B4O7 + 14
H2
2nd step: quantum dots formation
CdCl2 + NaHTe + MPA or MA + heating
MPA: 3-mercaptopropionic acid: HS-CH2-CH2-COOH
MA: 2-mercaptoethylamin : HS-CH2-CH2-NH2
3rd step: coating
CdS: CdTe + CdCl2 + Na2S + MPA or MA + heating
ZnS: CdTe/CdS + ZnCl2 + Na2S + MPA or MA + heating
1st step: preparation of hydogen telluride
Quantum dot CdTe nanocrystal
36 crystal elements, 650 atoms, 78 000 Da
3.5 nm
0.8 nm
MPA
fluorescence
emision
600 nm
12
High resolution transmission electron microscopy
CdTe QDs excitation and emission spectra
wide excitation spectra
narrow and symmetric
emission spectra
with maximum at 600 nm
bandwidth 58 nm
at half height
58 nm
relativefluorescenceEmission spectra
PVA coated capillary 20/30 cm, i.d. 75mm, separation buffer 3% LPA 10 MDa in 50 mM TRIS/TAPS
buffer, pH = 9, QD 2.8 and 3.7 nm (525 and 610 nm 1:1), injection time 10 s, separation voltage 3 kV
Electrophoresis in replaceable sieving media
CH3
HN N NH
+
CH3
O
O
O
CH3
O
OH
R
CH3 N
C
N N
+
Cl-
CH3
CH3
H
N
O
O
OH
SO3Na
N
O
O
O
SO3Na
CH3
O
NH
O
R
I
Carboxylic Group
EDC
Sulfo - NHS
Sulfo-NHS Peptide Bond Formation
Molecule Containing
Primary Amine Group
N
O
O
OH
SO3Na
CH3 NH
C NH N
+
Cl-
CH3
CH3
H
O
CdTe
CdTeCdTe
CdTe
CdTe
NH2
Sulfo-NHS Ester Intermediate
O - acylurea reactive intermediate
N - acylurea by-product
Conjugation of antibodies with quantum dots
- carboxylic group on
surface
- zero length linkers:
(carbodiimide) EDC
(succinimide) HNS
- amine group from
antibody
- formation of peptide
bond
7 nm
3.5 nm
6 nm
3.5 nmQD
15 nm
Ab Ag
Size comparison of antiovalbumin, ovalbumin and CdTe QD
13
Conjugation of CdTe to Ab PCNA protein
(Immunofluorescence microscopy of cultivated mouse embryo tissues)
Fluorescence microscope Leica
20 x objective
100 W Hg lamp
530nm 600 nm
Luca Andor EMCCD camera
Proliferating Cell
Nuclear Antigen
DNA repair and
replication
protein
CE immunoassay
LIF detection 488/610 nm
uncoated capillary length15/20 cm, i.d. 75 µm
buffer 100 mM TRIS/TAPS, pH 8.3
voltage 6 kV, eof mobility 66 .10-9 m2/(Vs)
EOF
crude conjugation
reaction mixture
Ag addition
QD addition
Ab - anti-
ovalbumin
QD - CdTe
(610nm)
Ag -
ovalbumin
Antibodies - Immunoassays
Spectroscopic methods
● UV-vis absorbance spectroscopy
● Laser Induced Fluorescence
Total Internal Reflection Fluorescence (TIRF)
Multiple Photons Absorption
● NMR best structure information – miniaturization limits
● IR spectroscopy
● Scanning Probe Microscopy (SPM), Near Field Microscopy
………
Raman Spectroscopy
Structure related spectra
Examination of minerals
Objects of art
Proteins, cells and organs …
Inherently insensitive
Surface Enhanced Raman Scattering (SERS)
Surface Enhanced Resonance Raman Scattering (SERRS)
Coherent Anti-Stokes Raman Scattering (CARS) …
14
Surface-enhanced Raman scattering
(SERS)
Martin Fleischmann
Van Duyne in 1970’s
Silver or gold surface – typically colloid
Surface plasmons of the metal excited by laser
Increase in the electric fields surrounding the metal
Raman intensities proportional to the electric field
Signal enhancement over 1011
Surface-enhanced Raman scattering
1.
Molecule in contact with
metal (colloid,
nanoparticle …)
2.
Irradiation
3.
Surface Plasmon
Excitation
4.
Raman scattering enhanced Raman signal
nanoparticle
(Au,Ag)
analyte molecule
irradiation
(laser)
SPR
Surface-Enhanced Raman Scattering (SERS)
 qualitative analysis – vibrational bands unique to
molecular structures
 quantitative analysis
 metal nanomaterial:
metal nanoparticles (NPs),
roughened surfaces,
metal tips,...
 enhancement factor 106 to 1012 => high sensitivity
Laboratory Detection System
16-bit deep cooled
back-illuminated
CCD camera
(iDus, Andor, UK)
spectrograph
(Shamrock SR-303i,
Andor, UK),
diffraction grating 600 l/mm,
blazed for 500nm
epifluorescence
microscope
microscope
objective
50x (0.95 NA)
He-Ne laser
(λ=632.8 nm, 15 mW)
Raman notch filter
(λ=632.8 nm)
dichromatic mirror
(λ=650 nm)
Dependence of SERS on NP’s concentration
Laser 632.8 nm, Ag particles 50 - 100 nm
LOD ~ 2·10-8 M
Rh. 6G
Detection window
Ag nanoparticles deposited on inner fused-silica surface
Laser beam 632.8 nm
(7 mW)
50 µm
15
CE with SERS detection
2D analysis of rhodamines 123 and B as model samples
CZE:
fused-silica capillary ID 50 μm, 15/25 cm
BGE: 40 mM CAPS with 20% methanol, pH 10
Voltage 6 kV
Sample: 5 x 10-5M Rhodamine 123
2.5 x 10-5M Rhodamine B
SERS:
exposure time 0.5 s
SERS Spectra of Peptides
2 x10-5 M KWK
5 x10-5 M HCKFWW
Phosphopeptide identification
~ 10-5 M
Structure information
Very small detection volume
Very good mass sensitivity
– fg – ag
(significant potential for improvement)
Concentrating techniques
– IEF, ITP
Conclusions
Miniaturization 2011
F. Foret & J.P. KutterCECE 2012 Brno
www.ce-ce.org
“… 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
16
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/
www.uspto.gov
http://www.epoline.org/
17
http://isdvapl.upv.cz http://cz.espacenet.com/
18
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/