Application of Capillary

Electrophoresis(CE) in
Cellular Analysis
Ong Siew Khim
(A0079832U)
P Sanjay Kumar
(A0087213J)
Pang Pee

Presenting..
O Introduction and why CE in cellular

analysis
Siew Khim
O Alternative methods Sanjay
O An automated application Pee
Wee
O One Application to achieve high
throughput
Xi Xian

Why the need to do cell analysis?

Cells are the fundamental unit of life, and studies

on cell contribute to reveal the mystery of life.

Since variability exists between individual cells
even in the same kind of cells, increased
emphasis has been placed on the analysis of
individual cells for getting better understanding on
the organism functions.

What is Capillary Electrophoresis?

Capillary Electrophoresis (CE)

is a technique designed to
separate ionic species based
on their size to charge ratio
in the interior of a small
capillary filled with electrolytes.

Why CE in Cellular Analysis?

Only a few conventional systems around that enable direct

intrinsic studies of single cells

Capillary electrophoresis is an excellent technique for
identifying and quantifying the contents of single cells.
A powerful separation technique with high resolution and
reproducibility
Has the capability to detect with high sensitivity even at low
sample concentrations.
The quantitativeness and accuracy, the exquisite sensitivity
and reduced background noise, has made the many methods
using CE highly versatile.

Several ways to sample cells.

The contents of individual cells can be sampled for CE

in several ways depending on the type of cell studied

and problems to be solved.
- Relatively large cells can be homogenized in a microvial
and the homogenate can then be prepurified and assayed.
For large cells, subcellular sampling is also possible.
- With small cells, a whole cell can be injected into the
capillary. In whole cell mode, the analysis consists of 4
major steps: (i) cell injection (ii) cell lysis, (iii) separation of
cellular contents and (iv) reconditioning of the capillary.
The quality of analysis requires optimization of all four
steps.

Not so good!
Although traditional CE and chip-

based CE (CE) are powerful

techniques for single-cell
analysis, a major impediment to
wider implementation of singlecell CE has been low throughput
for biologically relevant analytes.

Enhancements
Methods

Advantages

Automated technology
based on nanovolume
size-based separation

Higher sensitivity, a greater linear dynamic range

of different molecular weight proteins, high
reproducibility, the capacity for the higher
throughput screening of samples using small
sample input volumes

Microfluidic-based
electrophoresis

Higher throughputs;
Manipulation of a single cell and chemical reagent
handling could be easily realized;
Allows the integration of various tasks such as
reagent delivery, cell culture, sorting,
manipulation, lysis and separation, which enable
very rapid, highly efficient single-cell analysis to
be performed;
Many different detection schemes could be
integrated and multiple information from a single
cell could be obtained simultaneously;
Microfluidic devices can mimic the natural
physiological environment cells.

ALTERNATIVE METHODS

GC/MS for cancer cell analysis

VOCs (Volatile organic compounds) emitted by

the A549( human lung adenocarcinoma epithelial

cell line) cancerous cells and non-cancerous
HFB cells were studied

Cells were grown in a pollutant free air to reduce

background contamination.

Working principles of GC/MS

Traps are used to collect the
sample and pre-concentrate
for GC analysis.
Samples are mixed with dry
air to reduce moisture and
hence reduce interference
with analyte analysis.
Samples were de-sorped
from sorbents by heating at
3000C and injected into GC

Working principles of GC/MS

Cryfocusing , which is the condensation of analytes on a
cool surface, was performed.
Separates the VOCs by their affinity for stationary phase,
greater affinity of VOCs , longer the retention time.
Mass spectrometer detects the VOCs based on their
mass to charge ratio and fragmentation pattern.

Limitations of GC/MS
Only volatile components can be analyzed.
Upper temperature limit is set at 350-380oC and
hence analytes with high boiling points cannot be
analyzed.
Thermally labile analytes cannot be analyzed.
Quick changes in temperature might affect the
signal produced.
Environmental contamination present.

Analysis of macro-protein complexes

found in cells using 2D nano-HPLC
Ribosomes from cells were digested by Trypsin and the
macro-proteins were released.
72nL of sample was automatically injected.
Detection of analytes performed by ion trap mass
spectrometer.

Working principles of 2D nano

HPLC
Ion exchange chromatography in the first dimension and
reversed phase column in the second dimension
Ion exchange chromatography comprised of both cation
and anion exchange
For reversed phase chromatography, stationary phase is
non-polar whereby non-polar analytes get retained longer.

Working principles of 2D nano

HPLC
Linear gradient elution
Offline connection between both dimensions
Fraction connector connects both dimensions

Limitations of 2D nano-HPLC
Relatively difficult to operate
Time consuming process in order to obtain high resolutions
Lack of automation during procedure
Low salt buffer concentrations must be used to prevent salt

adducts from causing a decrease in signal intensity.

However, lower salt buffer concentration would result in
broader peaks and longer retention time.
Particle size of stationary phase can greatly affect the
resolution larger particle size poorer resolution obtained.

HIGH-THROUGHPUT CAPILLARYELECTROPHORESIS ANALYSIS OF THE

CONTENTS OF A SINGLE MITOCHONDRIA
Peter B. Allen, Byron R. Doepker, and Daniel T. Chiu

Principle of Method
Analysis of dye tagged contents of single mitochondria

using Capillary Electrophoresis (CE) and laser induced

fluorescence (LIF) detection:
Contents of Mitochondria labelled with membrane
permeable dye
Acidic intramitochondrial pH raised with
benzylethanolamine (BEA)
Photolyze individual mitochondria
Millisecond CE separation following single-pulse
photolysis to minimise diffusive dilution

Principle of Method
Sensitive Detection system required Native

Fluorescence
However, many chemical species within mitochondria
have no native fluorescence
label amine contents of mitochondria with fluorescent
dye, Oregon Green Diacetate Succinimidyl Ester (OGDASE)

Principle of Method
Diacetate group makes dye membrane-permeant
Dye cleaved by intracellular esterases and becomes

fluorescent
Reaction of free amines with Succinimidyl Ester (SE)
facilitated with basic pH BEA used to raise acidic
intramitochondrial pH
Single-pulse laser photolysis used to lyse cells
Ultrafast (few milliseconds) CE separation carried out

Key Experimental Details

Chip Design:
Short and shallow

separation channels
between 2 deep and
wide access channels
Electrical contacts at

opposite ends of chip

Key Experimental Details

Preparation and Labeling

of the Contents of Acidic

Vesicles

Key Experimental Details

Preparation of Mitochondria
Isolated mitochondria from B cells
Labeled the mitochondrial contents with OGDA-SE
Loaded mitochondria using pressure into outlet channels
Reverse voltage applied to introduce them into the

separation channels
When a surface density of ~5-10 mitochondria per
viewable frame was obtained, forward voltage applied to
pull clear buffer into the separation channel

Key Experimental Details

Photolysis of target

(mitochondria or
vesicles) followed
by bulk CE analysis

Results and Discussion

Verification of

Succinimidyl Ester
Labeling Strategy for
Acidic Organelles:

Results and Discussion

CE Analysis of
Single Mitochondria
Use of BEA resulted in
the presence of more
detected peaks
because of more
efficient labeling

Conclusion
Analysis of attoliter-volume samples using synthetic
vesicles
Able to separate and detect the contents of individual
mitochondria within seconds
Current duty cycle of about 1 min
With automation, the current duty cycle can be improved
to a few seconds.
Presence of variability in the contents of mitochondria

Future Work
Strategy can be used for other acidic organelles (eg

lysosomes)
More biologically meaningful studies with detailed
assignments of detected peaks

CONCLUSION
CE IS SUITABLE FOR
CELLULAR ANALYSIS