7/24/2019 CM_4242_Group 10.pptx

1/37

Application of CapillaryElectrophoresis(CE) in

Cellular Analysis

Ong Siew Khim(A00798!")# San$ay Kumar

(A0087!%&)#an #ee

7/24/2019 CM_4242_Group 10.pptx

2/37

Presenting..O Introductionand why CE in cellular

analysis

Siew Khim

OAlternative methods

San$ayOAn automated application #ee

'ee

OOne Application to achieve highthroughput

i ian

7/24/2019 CM_4242_Group 10.pptx

3/37

Why the need to do cell analysis?

Cells are the fundamental unit of life, and studieson 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 cellsfor getting better understanding on

the organism functions.

7/24/2019 CM_4242_Group 10.pptx

4/37

What is Capillary lectrophoresis?

Capillary lectrophoresis !C"

is a techni#ue designed to

separate ionic species based

on their size to charge ratio

in the interior of a smallcapillary filled with electrolytes.

7/24/2019 CM_4242_Group 10.pptx

5/37

Why C in Cellular $nalysis?

%nly a few conventional systems around that enable directintrinsic studies of single cells

Capillary electrophoresis is an excellent techni#ue for

identifying and #uantifying the contents of single cells.

$ powerful separation techni#ue with high resolution andreproducibility

&as the capability to detect with high sensitivity even at low

sample concentrations.

'he #uantitativeness and accuracy, the ex#uisite sensitivityand reduced background noise, has made the many methods

using C highly versatile.

7/24/2019 CM_4242_Group 10.pptx

6/37

Several ways to sample cells(.

'he contents of individual cells can be sampled for C

in several ways depending on the type of cell studied

and problems to be solved.

) *elatively large cells can be homogeni+ed in a microvial

and the homogenate can then be prepurified and assayed.or large cells, subcellular sampling is also possible.

) With small cells, a whole cell can be in-ected into the

capillary. n whole cell mode, the analysis consists of /

ma-or steps0 !i" cell in-ection !ii" cell lysis, !iii" separation ofcellular contents and !iv" reconditioning of the capillary.

'he #uality of analysis re#uires optimi+ation of all four

steps.

7/24/2019 CM_4242_Group 10.pptx

7/37

1ot so good2

Although traditional CE and chip-based CE (CE) are powerful

techniques for single-cell

analysis, a maor impediment to

wider implementation of single-

cell CE has been low throughput

for biologically rele!ant analytes"

7/24/2019 CM_4242_Group 10.pptx

8/37

Enhancements#ethods Ad!antages

$utomated technology

based on nanovolumesi+e)based separation

3icrofluidic)based

electrophoresis

&igher sensitivity, a greater linear dynamic range

of different molecular weight proteins, highreproducibility, the capacity for the higher

throughput screening of samples using small

sample input volumes

&igher throughputs4

3anipulation of a single cell and chemical reagenthandling could be easily reali+ed4

$llows 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 tobe performed4

3any different detection schemes could be

integrated and multiple information from a single

cell could be obtained simultaneously4

3icrofluidic devices can mimic the naturalphysiological environment cells.

7/24/2019 CM_4242_Group 10.pptx

9/37

$5'*1$'6 3'&%7S

7/24/2019 CM_4242_Group 10.pptx

10/37

8C93S for cancer cell analysis

6%Cs !6olatile organic compounds" emitted by

the $:/;! human lung adenocarcinoma epithelial

cell line" cancerous cells and non)cancerous

&< cells were studied

Cells were grown in a pollutant free air to reducebackground contamination.

7/24/2019 CM_4242_Group 10.pptx

11/37

Working principles of 8C93S

'raps are used to collect the

sample and pre)concentrate

for 8C 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

=>>>C and in-ected into 8C

7/24/2019 CM_4242_Group 10.pptx

12/37

Working principles of 8C93S

Cryfocusing , which is the condensation of analytes on a

cool surface, was performed.

Separates the 6%Cs by their affinity for stationary phase,greater affinity of 6%Cs , longer the retention time.

3ass spectrometer detects the 6%Cs based on their

mass to charge ratio and fragmentation pattern.

7/24/2019 CM_4242_Group 10.pptx

13/37

5imitations of 8C93S

%nly volatile components can be analy+ed.

pper temperature limit is set at =:>)=@>oC and

hence analytes with high boiling points cannot be

analy+ed. 'hermally labile analytes cannot be analy+ed.

Auick changes in temperature might affect the

signal produced.

nvironmental contamination present.

7/24/2019 CM_4242_Group 10.pptx

14/37

$nalysis of macro)protein complexes

found in cells using B7 nano)&5C

*ibosomes from cells were digested by 'rypsin and themacro)proteins were released.

DBn5 of sample was automatically in-ected.

7etection of analytes performed by ion trap massspectrometer.

7/24/2019 CM_4242_Group 10.pptx

15/37

Working principles of B7 nano

&5C on exchange chromatography in the first dimension and

reversed phase column in the second dimension

on exchange chromatography comprised of both cationand anion exchange

or reversed phase chromatography, stationary phase is

non)polar whereby non)polar analytes get retained longer.

7/24/2019 CM_4242_Group 10.pptx

16/37

Working principles of B7 nano

&5C 5inear gradient elution

%ffline connection between both dimensions

raction connector connects both dimensions

7/24/2019 CM_4242_Group 10.pptx

17/37

5imitations of B7 nano)&5C

*elatively difficult to operate

'ime consuming process in order to obtain high resolutions

5ack of automation during procedure

5ow salt buffer concentrations must be used to prevent saltadducts from causing a decrease in signal intensity.

&owever, lower salt buffer concentration would result in

broader peaks and longer retention time.

article si+e of stationary phase can greatly affect the

resolution E larger particle si+e poorer resolution obtained.

7/24/2019 CM_4242_Group 10.pptx

18/37

7/24/2019 CM_4242_Group 10.pptx

19/37

7/24/2019 CM_4242_Group 10.pptx

20/37

7/24/2019 CM_4242_Group 10.pptx

21/37

7/24/2019 CM_4242_Group 10.pptx

22/37

7/24/2019 CM_4242_Group 10.pptx

23/37

7/24/2019 CM_4242_Group 10.pptx

24/37

&8&)'&*%8&' C$55$*F)

5C'*%&%*SS $1$5FSS % '&

C%1'1'S % $ S185 3'%C&%17*$Peter B. Allen, Byron R. Doepker, and Daniel T. Chiu

7/24/2019 CM_4242_Group 10.pptx

25/37

rinciple of 3ethod

$nalysis of dye tagged contents of single mitochondria

using Capillary lectrophoresis !C" and laser induced

fluorescence !5" detection0 Contents of 3itochondria labelled with membrane

permeable dye$cidic intramitochondrial p& raised with

ben+ylethanolamine !

7/24/2019 CM_4242_Group 10.pptx

26/37

rinciple of 3ethod

Sensitive 7etection system re#uired E 1ative

luorescence

&owever, many chemical species within mitochondria

have no native fluorescence

E label amine contents of mitochondria with fluorescent

dye, %regon 8reen 7iacetate Succinimidyl ster !%87$)

S"

7/24/2019 CM_4242_Group 10.pptx

27/37

rinciple of 3ethod

7iacetate group makes dye membrane)permeant

7ye cleaved by intracellular esterases and becomes

fluorescent

*eaction of free amines with Succinimidyl ster !S"

facilitated with basic p& E

7/24/2019 CM_4242_Group 10.pptx

28/37

Gey xperimental 7etails

Chip $esign%

Short and shallow

separation channels

between B deep and

wide access channels

lectrical contacts at

opposite ends of chip

7/24/2019 CM_4242_Group 10.pptx

29/37

Gey xperimental 7etails

&reparation and 'abeling

of the Contents of Acidic

esicles

7/24/2019 CM_4242_Group 10.pptx

30/37

&reparation of #itochondria solated mitochondria from < cells

5abeled the mitochondrial contents with %87$)S

5oaded mitochondria using pressure into outlet channels

*everse voltage applied to introduce them into the

separation channels

When a surface density of H:)I> mitochondria per

viewable frame was obtained, forward voltage applied to

pull clear buffer into the separation channel

Gey xperimental 7etails

7/24/2019 CM_4242_Group 10.pptx

31/37

&hotolysis of target

(mitochondria or!esicles) followed

by bul CE analysis

Gey xperimental 7etails

7/24/2019 CM_4242_Group 10.pptx

32/37

erification of

*uccinimidyl Ester

'abeling *trategy for

Acidic +rganelles%

*esults and 7iscussion

7/24/2019 CM_4242_Group 10.pptx

33/37

CE Analysis of

*ingle #itochondria

se of

7/24/2019 CM_4242_Group 10.pptx

34/37

Conclusion

$nalysis of attoliter)volume samples using syntheticvesicles

$ble to separate and detect the contents of individualmitochondria within seconds

Current duty cycle of about I min

With automation, the current duty cycle can be improvedto a few seconds.

resence of variability in the contents of mitochondria

7/24/2019 CM_4242_Group 10.pptx

35/37

uture Work

Strategy can be used for other acidic organelles !eg

lysosomes"

3ore biologically meaningful studies with detailed

assignments of detected peaks

7/24/2019 CM_4242_Group 10.pptx

36/37

C%1C5S%1

CE * *.A/'E 0+1

CE'''A1 A2A'3**

7/24/2019 CM_4242_Group 10.pptx

37/37