2013-04-08
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New Developments in Capillary
Electrophoresis with focus on
Bioanalysis
Lecture 6
Christian Nilsson
Lecture today
• Two-dimensional separation
• CE-MS
• Micro total analysis systems
• Biomarker analysis by CE
• Molecular Imprinted Polymers
Diagonal CE
• Two-dimensional CE
• Identical separation modes
• Enzyme-based microreactor
• Analytes that are unaffected by the
microreactor will be detected at the diagonal
in the 2D electropherogram
Diagonal CE
• Alkaline phosphatase
– Remove phosphate groups
– Most effective at high pH
– Present in the periplasmic space in bacterias
– Function?
• Generate phosphate group for uptake and use?
• Regulate uptake into cell? Phosphate groups usually
prevents organic molecules to pass through the
membrane
Diagonal CE
• Use of alkaline phosphatase to monitor
phophorylation of a mixture of peptides
• Could be used also for other post translational
modification
Diagonal CE
• Previous use:
– Paper chromatography to characterize disulfid
bonds
– Diagonal LC to characterize phosphorylation,
glycosylation and acetylation
• Use of diagonal CE:
– Easier to automate
– Faster
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Diagonal CE
• One fraction is separated in the second
dimension while the following fraction is in
the microreactor
Diagonal CE
• Microreactor
– Alkaline phosphatase immobilized on magnetic
nanoparticles
– Microreactor kept at right position by magnets
– 1.0 or 2.8 μm nanoparticles
– Replaced between each analysis
– Microreactor: 1-3 mm plug
Diagonal CE
• A fraction present for 45 s in the microreactor
• Several fractions present in the second
capillary simultaneously
– Possible as long as the fastest moving component
do not overtake the slowest moving component of
previous fraction
• Laser induced fluorescence for detection
– Peptides fluorescent labeled
Diagonal CE
• Separation of tryptic digest of casein
Proteomics
• Liquid chromatography routinely used for
bottom up proteomics to detect peptides
from tryptic digests
• Very long gradient elution separations often
produce extraordinary peak capacity and
resolution
Proteomics
• Desireable to have a complementary
technique to reversed phase chromatography
• CE have so far mostly been used for analysis of
standard peptides or tryptic digest of a few
standard proteins
• However, analysis of complex proteomic
samples are now developed
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CZE-MS/MS for proteomics
• Advantages
– Separation mechanism
– Faster
– Higher efficiency
• Disadvantages
– Speed and efficiency must be handled by the mass
spectrometer. The acquisition rate can be limiting
– Loading capacity is lower
Proteomics
• CZE-MS/MS as an alternative to UPLC-MS/MS
for proteomics studies
• 11 fractions were first separated by reversedphase
LC
• Each fraction was then separated by CZE-ESI-
MS/MS
• 250 ng of sample and 165 min of MS time was
used
Proteomics
• CZE similar to UPLC and the techniques were
complementary
• CZE favor basic, hydrophilic, small peptides
CE-MS for Proteomics
• Analysis of whole proteome
– Time
– Amount of sample
– Labor
• LC-MS usually employed
• Detection of 10 000 proteins?
• Large dynamic range of proteome
– 7 order of magnitude
• CE-MS?
CZE-ESI-MS/MS for Proteomics
• 50 min separation
• Identification of 1250 peptides in E Coli
• 1-100 ng protein digest
• Electrokinetically pumped nanospray interface
• Coated capillary
• Stacking conditions for injection
CZE-ESI-MS/MS for Proteomics
• Electrophoretically pumped sheath flow
• Very low flow rate
• Less dilution of sample
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CZE-ESI-MS/MS for Proteomics
• Comparsion to UPLC-ESI-MS/MS
– With 100 ng digest UPLC was able to identify more
pepides
– With 1 ng digest CZE was able to identify many
more peptides
• The methods were complementary
CZE-ESI-MS/MS for Proteomics
CZE-ESI-MS/MS for Proteomics
• Coated capillary (Linear polyacrylamide)
– Prevent capillary wall adsorption
– Reduce EOF / Increase separation time
• Sample buffer modified
– Facilitate sample stacking
– Injection of a larger amount of sample
CZE-ESI-MS/MS for Proteomics
CZE-ESI-MS/MS for Proteomics
• Sample stacking
– Varying amount of formic acid in the sample
buffer
• Either 0.05% or 0.1% formic acid
– Better sample stacking with 0.05%
– Reduce the effect of a larger sample plug
– Improve sample capacity
– More peptides identified
• 1132 instead of 815 peptides
CZE-ESI-MS/MS for Proteomics
• Different capillary length
– 40 or 60 cm
• Longer capillary and gave identification of
more peptides
– 1520 vs 1184
– Longer separation time
– More mass spectra generated
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CZE-ESI-MS/MS for Proteomics
• Complementary techniques
• Can improve coverage of proteins
CZE-ESI-MS/MS for proteomics
• CZE as a competitor to UPLC for proteomics
• Improvements by further optimizing
– Sample preparation
– Separation conditions
– Electrospray conditions
– MS parameters
CZE-ESI-MS/MS
• Analysis tryptic digests from cellular
homogenate
• 700 pg (volume of ~10 eukaryote cells) was
analyzed
• 10 proteins could be detected
Micro Total Analysis Systems for Cell
Biology and Biochemistry
• Rapidly maturing
• Still improvements of fabrication
• More focus on biological applications
Micro Total Analysis Systems for Cell
Biology and Biochemistry
• Fabrication
– Cost
– Robustness
– Surface Chemistry
– Optical properties
– Biocompatibility
– Ease of fabrication and integration
– Suitability for large scale production
Micro Total Analysis Systems for Cell
Biology and Biochemistry
• Polymers for production of devices
• Polymethylsiloxane (PDMS) extensively used
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PDMS
• Easy to fabricate
• Low cost
• Established procedures
• Absorption of hydrophobic molecules
• Swelling in organic solvent
Alternatives to PDMS
• Poly(methyl methacrylate)
• Polystyrene
• Polycarbonate
• Cyclic olefin copolymer
• Relatively complex fabrication
– Less suitable for prototyping at academic labs
• More suitable for industrial mass production
Glass and Silicon
• More complicated fabrication
• Glass
– Chemical inert, optical transparent, thermal
stability
• Silicon
– Derived from eletronics industry
Sample preparation
• A challenging step for biological samples
– Often complex mixtures of compounds
• Extensive off chip sample preparation reduce
the utility of a micro total analysis system
– Especially important for samples of low amount
and volume
Biology and μTAS
• Single cell analysis
– Large heterogenity within a population of a
certain cell type
– Understand cell variation
Analysis of intact cells
• Imaging cytometry
– Labeling specific cell components
– Microfluidics for high throughput sample
preparation
• Number of certain protein or mRNA differ
from cell to cell within the same population
• However, hard to detect low abundant
compounds in single cells
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Analysis of intact cells
• Yellow fluorescent protein (YFP) fusion library
• Particular genes tagged with YFP
• YFP can be detected with single molecule
sinsitivity in living cells.
• High throughput analysis of the different
stains on microchip
Analysis of intact cells
Single enzyme detection
• Fluorescence-based
• A microchip with an array of wells
• Dilute the sample so there will be a maximum
of 1 enzyme molecule per well.
• Reporter system
– Fluorescent reactant
– Fluorescent product
CE for biomarker analysis
• Proteins and peptides as biomarkers
• From biological fluids
• Handling of real samples
• NIH: Biomarker = a characteristic that is objectively measured
and evaluated as an indicatorof normal biological processes,
pathogenic processes or pharmacologic responses to a
therapeutic invention
CE for biomarker analysis
• Small sample volume
• High efficiency and resolution
• High sensitivity with LIF or MS detection
CE for biomarker analysis
• Difficulties
– Complex sample matrix
– Low concentration
• Low abundant substances may interact with other
molecules in the sample
– Often a sample preparation step necessary
• Remove non-interesting compounds
• Concentrate biomarker
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Biomarkers in tears without sample
pretreatment
• Advantage: Relatively pure
• Used for detecting lysozyme and lactoferrin by
CE-UV
• Sjögrens syndrome, an automimmune disease
• Acidic buffer and cationic coating
Basis of pretreatment methods
• Based on affinity for solid phase
• Based on immunoaffinity supports with
antibodies
• Membrane-based techniques: dialysis or
filtration
• Liquid-Liquid-based techniques: centrifugation
or precipitation
• Advantage if the technique can be
implemented on column
Detection of neuropeptides in urine
• Detection of angiotensin II and neurotensin
• Fab fragments from polyclonal antibodies
immobilized on glass beads at an microreactor
Capillary coating
• Avoid adsorption and tune EOF
• Preparation of capillary coating is often time
consuming and the stability is limited
• Often physically adsorbed neutral och
possitively charged polymers are used
– Possible to regernerate
Capillary coating
• Successive multiple ionic polymer layer (SMIL)
• Layers of polyelectrolyte are attached by
succesive rinsing steps with cationic and
anionic polymers
Detection sensitivity
• High sensitivity necessary for early diagnostics
• Use of flurescence (labeling often necessary)
or MS
• Preconcentration
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Example – CE-LIF of amyloid peptides
• Alzheimer disease
• Fluorescent labeled peptides
• From cerebrospinal fluid
• Offline immuno-capture were used prior to
the CE-LIF analysis
Example – CE-LIF of amyloid peptides
• Magnetic nanoparticles coated with
monoclonal antibodies was used
pretreatment
Molecular Imprinting
Making a lock to a molecular key...
Synthetically prepared
biomolecule mimics
For separation, catalysis, assays, sensors
Molecular
imprinting
Immobilisation
(Entrapment)
Enzymes, cells,
ligands
Proteins (Enzymes),
ligands
Covalent
immobilisation
HISTORY
Improvement of MIPs
By combining MIPs with a support material, namely silica,
I) target molecules were immobilized to solid silica supports
better binding sites
II) porous silica beads were filled with imprinted polymer
better particle shapes
Filling
FUNCTIONAL MONOMERS
Methacrylic acid Itaconic acid 4-Vinylbenzoic acid 2-Acrylamido-2-methyl-
1-propanesulfonic acid
N,N,N-trimethylaminoethyl
methacrylate
Trifluoromethylacrylic
acid
Hydroxyethyl-
methacrylate
1-Vinylimidazole 4-VinylimidazoleVinylpyridine
Acrylamide
NN
N NH
N
NH2
O
OH
O
OH
O
CF3 OH
O
O OH
H
N
O
SO3H
O
O
N+
O
O
OH
Allylamine
NH2
Methylmethacrylate
O
O
OH
O
Styrene
N
COOH
COOH
Vinylbenzyliminodiacetic
acid
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O
O
O
O
N
H
N
H
O O
Ethyleneglycol dimethacrylate
N,N'-Methylene diacrylamide
Divinylbenzene
3,5-Bis(acryloylpiperazine) N,N'-1,4-Phenylene
diacrylamide
CROSS-LINKERS
Trimethylolpropane trimethacrylate
H
N
O
N
H
O
O O
O
O O
O
N N
O
O
POROGENIC SOLVENTS
• Solubilise all components of the imprinting mixture
• Create porous structure
• Inert
• Provide an favourable environment for
non-covalent interactions
Chloroform r = 5
Dichloromethane r = 9
Toluene r = 2
Tetrahydrofurane r = 8
Acetonitrile r = 36
Water ? r = 80
RADICAL INITIATORS
2,2’-Azobis-isobutyronitrile (AIBN) UV 350 nm, or 65 °C
2,2’-Azobis-(2,4-dimethyl valeronitrile) (ABDV) 45 °C
2,2-Dimethoxy-2-phenyl acetophenone (DMPAP) UV 350 nm
CH3
CH3C
C
N N C
CH3
C
CH3
N N
CH3
CH3C
C N
.2
+ N2
UV 350 nm
AIBN
Polymerisation
Extraction
Pre-polymerisation
complex
Binding site
Polymerisation
Cross-linked
polymer
NON-COVALENT IMPRINTING
Flexible
polymer
chains
O
OH
OHO
OHO
N
H
NH3
O
O
O
O-
OHO
O OH
OHO
O
N
H
NH3
O
O
O
O-
OHO
O OH
OHO
OH
OH
O
+ +
N
H
NH3
O
O
O
O-
OHO
O OH
OHO
+
IMPRINTING EFFECT
0
25
50
75
100
Bound 3H-theophylline, %
0.01 0.1 1 10
Polymer, mg/ml
no T
1:5000
1:500
1:100
1:12
1:4
P50
T:M
OH
O
CF3Functional
monomer
(M)
Template
(T)
N
H
NN
N
O
O
Theophylline
Trifluoromethyl
acrylic acid
Cross-linker
Divinylbenzene
N2
CH3CN
Methacrylic acid
Vinylpyridine
EDMA
ABDV
Template
45°C
TYPICAL PROCEDURE
Monomers, porogen and template
are mixed, initiator is added
The mixture is purged
with nitrogen
Polymerisation is
initiated
The polymer is removed
from the tube...
...and mechanically ground The polymer particles are sieved... ...and sedimented The template is extracted
Methanol/
acetic acid
CONFIGURATIONS
• Fragmented polymer monoliths
• Polymer beads prepared by suspension, emulsion or precipitation polymerisation
• Composite polymer beads
• In situ-prepared polymer rods
• Polymer particles bound in thin layers
• Thin polymer membranes or films
• Surface-imprinted polymer substrates
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CHARACTERISTICS
Feature Characteristics
Physical Stability Resistant against mechanical stress, high
pressures and elevated temperatures
Chemical Stability Resistant against acids, bases, various
organic solvents and metal ions
Storage Endurance > 1 year without loss of performance
Imprint Memory Repeated use >100 times without reduction
Recovery of imprint molecule > 95%
Capacity ≈ 10 mg imprint molecule/g polymer for
baseline separation of racemic mixtures
Substance class Examples
Amino acids Free and derivatised amino acids
Peptides Enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH)
Steroids Cholesterol, cortisol, testosterone
Carbohydrates Derivatised sugars, glycosides
Nucleotides NAD
+
Nucleotide bases Adenine
Dyes Safranine O, rhodanile blue
Pesticides Atrazine, 2,4-D
Metal ions Ca
2+
, Cu
2+
, Eu3+
Drugs Propranolol, theophylline, morphine, nicotine, penicillin
Proteins Transferrin, RNase A, urease, HGH, myoglobin, IgG
Bacteria cells Staphylococcus aureus, Listeria monocytogenes
Crystals Calcite
TEMPLATE MOLECULES
APPLICATIONS
• Antibody / receptor binding site mimics
Immunoassays
Drug development / screening
• Biomimetic sensors
• Tailor-made separation materials
• Facilitated synthesis / Catalysis (enzyme mimics)
• Biomedical
Slow release matrices
In-situ or extracorporeal removal of unwanted molecules
SEPARATION
Chromatography
Solid-phase extraction
Capillary electrophoresis
Thin layer chromatography
Membrane-based separation
• Chiral separation
• Separation of closely related compounds
Tailor-made affinity separation materials
CHIRAL CHROMATOGRAPHY
Pre-determined elution order
L
D
L-imprinted polymer
D+L
Non-imprinted polymer
L
D
D-imprinted polymer
SOLID-PHASE EXTRACTION
Sample clean-up
Analyte preconcentration
Possible alternative for
• Solvent extraction
• Extraction with non-specific matrices
• Immunoextraction
Imprinted
polymer
Sample
with
analyte
Extraction of
the sample
Elution of the
analyte from
the polymer
Analysis of the eluate
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SCREENING OF COMBINATORIAL LIBRARIES
Imprinting of a polymer with a target compound
Screening of a combinatorial library for binding to the
imprinted artificial receptor
• Ligands
• Potential drugs
• Enzyme inhibitors
Selection of from
• Chemical combinatorial libraries
• Biological combinatorial libraries
Imprinted polymers could be useful
• Because of their high selectivity
• Because of their robustness
• If natural receptor does not exist or is poorly characterised
• If natural receptor is difficult to purify
1 11--Hydroxyprogesterone
2 11--Hydroxyprogesterone
3 17--Hydroxyprogesterone
4 Progesterone
5 4-Androsten-3,17-dione
6 1,4-Androstadiene-3,17-dione
7 Corticosterone
8 Cortexone
9 11-Deoxycortisol
10 Cortisone
11 Cortisone-21-acetate
12 Cortisol-21-acetate
COMBINATORIAL STEROID LIBRARY
O
18
O
HO
1
2
3 4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
21
20
Template:
Ramström, Ye, Krook, Mosbach (1998) Anal. Comm. 35, 9-11.
Time, min
Screening on imprinted polymer
0 30 60
3,4,5,
6,8
2,11
7,9,12
10
1
1 2 3 4 5 6 7 8 9 10 11 12
ANTIBODY MIMICS
• If biological antibodies are difficult to obtain:
Small and non-immunogenic molecules (no conjugation required!)
• Use in difficult environments (organic solvents, high temperatures…)
Rigid
Insoluble, large structures
Flexible
Soluble, small in size
Imprinted polymers Biological antibodies
Specific binding of a target molecule
Possible alternative or complement
Dissolution
of silica
IMMOBILISED TEMPLATES
N
H
N N
N
O
O
O
OH
CF3
HO CF3
O
OHO
F3C
Immobilised template
Functional monomers
Crosslinker
Silica
Polymerisation
Polymer with binding site
Advantages
• Reduced tumbling rate of template
• Homogeneous orientation of binding sites
• Better accessibility of binding sites
• Tagging of analyte possible
N
H
N N
N
O
O
O
OH
HO
O
OHO
O
OH
HO
O
OHO
Yilmaz, Haupt, Mosbach (2000) Angew. Chem. Int. Ed. 39,
2115-2118.
Model analyte: 2,4-dichlorophenoxyacetic acid
N
Functional monomer
Methanol/water 4:1
Porogen
Template :
2,4-D
O
O
O
O
Cross-linker
Ethyleneglycol dimethacrylate 4-Vinylpyridine
Polymer :
O COOH
Cl
Cl
Cl
O COOH
Cl
2,4-D
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Radioliganddisplacement assay
Radiolabelled probe: 14C-2,4-D
Polymer titration Standard curve for 2,4-D
0
50
100
Bound 14C-2,4-D, %
0 500 1000 1500 2000 2500
Polymer concentration, µg/ml
Imprinted
Controls
0
20
40
60
0.001 0.01 0.1 1 10 100
2,4-D, µg/ml
Bound 14C-2,4-D, %
Imprinted
Cross-reactivities of structurally related compounds (%)
15 2
10 1
2 14
<0.1 <0.1
MIP MAb
Buffer CH3CN Buffer
100 100 100
24 50 0.5-3
MIP MAb
Buffer CH3CN Buffer
95 2 1.5-20
7 1 30-160
?
? ?
?
Cl
O COOH
Cl
2,4-D
CH2OH
Cl
O COOH
O COOH
O
Cl
Cl
COOH
Cl
COOCH3
Cl
O
Cl
O
Cl
COOH
Cl
COOH
OR
O
RN3
N
N N
R
O
O R
N
N N
R
O
O
R
+
+
anti-isomer
syn-isomer
OHO
OH
O
OH
O
OHO
N
N N
O
O C2H5
N
H H OH
O
OHO
N
N N
O
O C2H5
N
H H
EDMA
remove
template
A Molecular Imprinted Polymer as the Nanoreactor for
Regioselective 1,3 – Dipolar Cycloaddition of Azides and
Alkynes
Chlorendic anhydride
O
Cl
Cl
Cl
Cl
S
O
O
O
Cl
Cl
OO
Cl
Cl
S
O
O
O
Cl
Cl
Cl
Cl
O
O
+
SO2 O
O
Catalysis: Diels-Alder reaction
Template:
Transition state analogue
Tetrachlorothiophene
dioxide
Maleic
anhydride
Imprinted polymer:
Methacrylic acid
Ethyleneglycoldimethacrylate
Chloroform
O
O
O
Cl
Cl
Cl
Cl
Cl Cl
WHY USE IMPRINTED POLYMERS?
• Tailor-made receptor
• Fast preparation
• Chemical and physical stability
• Possible use in organic solvents
• Storage and reuse possible over a long period
• Easy integration in industrial fabrication process
Problems to be addressed
• Heterogeneity of binding sites
• Imprinting of polymers in aqueous solvents
• Imprinting of larger templates (proteins, cells...)
Separation
• Chiral separation
• Closely related
compounds
• Sample preparation
Application areas of Molecularly Imprinted Polymers
Organic synthesis
and catalysis
• Protecting groups
• Directed synthesis
• Equilibrium shifting
• Artificial enzymes
Artificial receptors
(drug development)
• Library screening
• Directed synthesis
• Dynamic combinatorial
chemistry
Biomedical
• Slow drug release
• Extracorporeal devices
(toxin removal)
• Polymeric drugs
Antibody mimics
• Biosensors
• Immunoassays
?
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Antiideotypic Imprinting of a
Kallikrein inhibitor
Direct Molding Imprinting
Biological receptor Assembly and chemical linkage of
(eg enzyme) building blocks in receptor cavity
Imprint building blocks
New inhibitor
Dissociation of new ligand
from enzyme
Biological receptor Assembly and chemical linkage of
(eg enzyme) building blocks in receptor cavity
Imprint building blocks
New inhibitor
Dissociation of new ligand
from enzyme
In this approach novel ligands to a
receptor or enzyme can be formed
directly by assembly of reactive
building blocks within the binding cavity
of the biological receptor, thus using
the latter as a molecular scale reaction
(or nano) vessel.