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New Developments in Capillary
Electrophoresis with focus on
Bioanalysis
Lecture 3
Christian Nilsson
Lecture today - Content
• Example of protein adsorption.
• Example of lipid analysis.
• Introduction to microchip electrophoresis.
• Example of miniaturized analysis.
Protein CE
• Advantages compared to slab gel
– Automation
– Quantitation
– Fast
– High efficiency
Protein CE
• Disadvantages:
– Protein adsorption
• Can be prevented by coating of the capillary wall.
However, the reproducibility of the coating procedure
and the stability of coating have to be considered.
• Adsorption of proteins decrease reproducibility.
– Reproducibility?
Protein adsorption
• Adsorption on the negatively charged surface
of the capillary wall.
• Especially problematic for basic proteins.
• However, due to a non-uniform charge
distribution protein can adsorb even with a
negative net charge.
Protein adsorption
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Protein adsorption Capillary wall coating
• Review article
• Lucy et al., J. Chromatogr. A, 2008, 1184, 81-
105
Example of lipids analysis
• Non-aqueous CE (NACE).
• Separation of hydrophobic compounds
P
P -OH P -O-C2H5
Hydrolysis Transphosphatidylation
PethPA
Choline
PLD
EthanolWater
HV
Alcohol biomarker Peth
with NACE
Analysis of lipids
Analysis of alcohol biomarker Peth with NACE
- 0 ,0 0 5
0 ,0 0 0
0 ,0 0 5
0 ,0 1 0
0 ,0 1 5
0 ,0 2 0
0 ,0 2 5
0 1 2 3 4 5
T im e ( m in u t e s )
A200
P e t h
0 ,0 0 3 0
0 ,0 0 3 5
0 ,0 0 4 0
0 ,0 0 4 5
0 ,0 0 5 0
0 ,0 0 5 5
2 ,3 2 ,4 2 ,5 2 ,6 2 ,7
T im e (m in u te s )
A200
Blood lipids
Non Aqueous Capillary Electrophoresis
of the new Ethanol Consumption Biomarker Biphosphatidylethanol
Microchip electrophoresis
• First chip analysis published in 1979.
– Stanford
– Chip GC
• Next breakthrough in the beginning of the
90s.
– Liquid chromatography
– Microchip Electrophoresis
• Microfabrication techniques were developed
for electronics.
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Microchip electrophoresis
• Rapid development of the technique in 1990s.
• Now:
– Integration of different functions.
– Development of specific applications.
• Integration of functionalities on a single
device minimize transfer steps.
Microchip electrophoresis
• Development of micro total analysis system
(lab-on-a-chip).
– All necessary analytical functions on a single chip.
Diagnostics
Microchip electrophoresis
• Can pattern many different channels
– Possibility for high throughput parallell analysis.
• More flexibel than CE.
• Electrophoresis and electroosmotic flow a
good alternative for microchip analysis due to
the small dimensions.
Microchip electrochromatography
• A pressure-driven flow is easier to control.
• An electroosmotic flow is easy to apply and no
pump is necessary.
– No problems associated with increased
backpressure due to small channel dimensions.
Microchip construction
• First glass was used as substrate.
• The fabrication methods were already
developed.
• Glass has excellent optical properties
– Transparent at all useful wavelengths.
• Glass has similar surface chemistry as fused
silica.
– Methods for surface modification in CE can be
applied also here.
Glass Microchip
• The devices are fabricated one by one.
– High manufacturing cost per chip.
• Glass is expensive.
• Glass is fragile and easily broken.
• Research to develop polymeric microchips.
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Polymeric microchips
• Less expensive.
• Easier to fabricate.
– High throughput fabrication.
– Low cost material.
• Less known about the surface chemistry.
Polymer microchips
• However, detection can be problematic for
protein analysis.
Cyclic Olefin Copolymer (Topas®)
Microchip CE - Injection
• In CE, injection is performed by moving the
capillary to the sample solution, which is not
possible with a microchip method.
• New injection protocols.
Microchip CE – Integration of
functionalities
• Solid phase extraction.
• Two dimensional separation.
• Gradient systems.
Microchip CE - Injection
• Gated injection
• Pinched injection
Microchip CE – Gated injection
Changing the applied voltages to inject.
Injection dependent on the electrophoretic mobility of
the analytes.
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Microchip CE – Pinched injection
Injected sample volume is limited by the width of the channel.
Microchip CE - Detection
• Small detection volume.
• Few microchip CE approaches use UV
detection.
• Laser-induced fluorescence is often used due
to its high sensitivity.
• However, most analytes require modification
to allow fluorescence detection.
• Other detection techniques include
electrochemical detection and mass
spectrometry.
Microchip CE – Z-shaped detection cell Microchip CE – Electrospray MS
Microchip electrochromatography
• First conducted in 1994 in open-tubular
format.
– The surface of the microchip was modified by C18
material for reversed phase separation of neutral
analytes.
Microchip electrochromatography
• An advantage with the microchip format is the
possibility to produce tailor-made microchips.
– For example an array of pillars in the separation
channel.
• More homogenous separation column
• Smaller structures enable a larger surface area.
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Microchip electrochromatography Microchip CE – DNA separation
• Use of a polymeric gel.
• Laser induced fluorescence for detection.
• Shorter separation column compared to CE
give a shorter analysis time.
Microchip CE – DNA separation
Example of commercial instrument.
Microchip CE – Amplification of DNA
• Polymerase chain reaction (PCR) for
amplification of DNA.
• Miniaturization of PCR reduce the
amplification time.
Conventional PCR
The temperature of the sample need time to adjust.
This time is reduced by miniaturize the technique.
Microchip PCR
• The cycle time can be significantly reduced.
• As an example infrared heating was used
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Microchip CE – Protein Separation
• Fluorescent labeled proteins.
– Labeling might be inhomogeneous.
• Development systems for determining enzyme
kinetics and activities.
Single Cell Analysis
• For analysis of variability among cells.
Separation in polymer capillary
Without nanoparticles
With nanoparticles
Cyclic Olefin Copolymer (Topas®)
GFP variants In Topas® Capillary
With nanoparticles
Separation on polymer chip
Channel:
3.4 cm long
80*80 μm
Separation on polymer chip
0 20 40 60 80 100 120 140
0
5
10
15
20
25
30
35
40
45
50
time / s
Fluorescencesignal/arbitraryunits
GFP variants differing in one amino acid
GFP N212K GFP N212E
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Acoustic Levitation
wall-less test tube
1.2 cm
Levitated 500 nL drop
• Stable Sample Position
• No Special Sample Properties
• Easy access to sample
Acoustic Levitation
•The Surrounding Gaseous Medium
the Only Contacting Surface
• Miniaturisation Benefits
(volume range pL-mL)
Ink jet methods
Flow-through Drop-on-Demand Dispenser
• Fabricated in silicon
• 250-100 nL inlet to nozzle
• Piezoelectric multilayer element for actuation
• Nozzle dimensions 30-50 µm
• Typical droplet volume: 50-100 pL
• Dispense rate up to 9 kHz
Flow-through Drop-on-Demand Dispenser
Instrumental Set-up Adipocyte Reactions Single Cell
Type 2 Diabetes
-FFA H+FFA +
ISOPRENALINE
INSULIN
IRS-1
PI3K
reg
PI3K
cat
PY
PY
PY
-ARa
-ARb
Gs Gi
PS
PIP3
AC
cAMPATP AMP
PSPS
PS
PS PKB
kinase
PKB PKBP
P PDE3B PDE3B
PKA
HSL HSL
Fatty acids
+ glycerol
Triacylglycerol
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Adipocyte Lipolysis
6
6,5
7
7,5
8
0 50 100 150
pH
Time (s)
Two adipocytes
One adipocyte
Isoprenaline
Insulin
Two Adipocytes
One adipocyte
IncreasingLipolysis