MICROCHIP ELECTROPHORESIS

- Dr.Arun Babu.N.B

II year MD (Biochemistry)

V.M.K.V.Medical College

INTRODUCTION

DEFINITION

Technique used to separate various proteins, nucleic

acids and other charged molecules under the influence of

an electric field.

PRINCIPLE

In an electric field, the charged molecules move towards

oppositely charged electrode at different rates based on

electrical charge & molecular size.

1st Electrophoresis method- to study proteins- was Free

solution/ moving boundary method- By TISELIUS

It was used to measure electrophoretic mobility and to study

protein-protein interaction.

It was able to resolve serum proteins into 4 component

mixtures, with α1 fraction incompletely separated from

albumin.

An ampholyte/ zwitterion becomes +vely charged in a

solution that is more acidic than its isoelectric point, and

migrates to cathode and vice versa.

Electrophoretic mobility is directly proportional to net

charge & inversely proportional to molecular size and

viscosity of the electrophoresis medium.

Mobility may be +ve or -ve, depending on whether the

protein migrates in the same or opposite direction as the

electrophoretic field, which is from anode to cathode.

Factors affecting electrophoresis are:

Electric Field (voltage, current, resistance)

Sample (charge, size, shape)

Buffer (composition, concentration, pH)

Supporting medium (electroendosmosis)

TYPES

Based on nature of supporting mediumAGE (Agarose gel electrophoresis)

PAGE (Poly-acrylamide gel electrophoresis)

Paper strip electrophoresis (cellulose acetate paper/membrane)

Based on mode of techniqueSlide/Slab gel electrophoresis

Disc electrophoresis

Isoelectric focusing electrophoresis

Capillary electrophoresis

Microchip electrophoresis …..

MICROCHIP ELECTROPHORESIS

Recently undergone substantial development like integrated microchip designs, advanced direction systems

DNA & proteins

ADVANTAGES

High Speed4x – 10x faster than conventional capillary electrophoresis

1 order of magnitude faster than slab gel electrophoresis

Simplicity

Potential for automation

DISADVANTAGES

Limited separation efficiency of zone electrophoretic

measurements

Imprecise injection

Low sensitivity of absorption detection(UV/Vis

absorption detection)

Early stage of commercialization

INSTRUMENTATIONSeparation channels

Sample injection channels

Reservoirs

Sample preparation reactors

Pre and post column reactors

Truly multi-functional, “integrated” analytical device embedded in a single monolithic substrate.

Fabricated onto the surface of the microchip, using photolithographic processes.

Usually cross T design [double T(larger injector region)]

Has a short (injection) channel & longer (separation)

channel

1 reservoir each at each end

2 for introduction of sample & background electrolyte (buffer

solution)

2 serving as waste reservoirs

Channel dimensions (depth=15-50μm, width=50-200μm and length of separation channel=1-10cm)

The volume of the separation channel is 1 order of

magnitude smaller than conventional capillary systems.

Sample volume injected varies from 100-500pL.

With the decrease in volume requirements, pressure

injection is more challenging. Hence sample is injected

electro kinetically, by applying an electric field across the

sample channel.

All reservoirs are connected to electrodes

An injection voltage of several 100V is applied across the

sample and sample waste reservoirs- to migrate the

sample to the injection cross

Separation voltage (1-4kV) is then applied to separation

channel, which induces the separation of analyte zones

before they reach detection window downstream.

Portion of the sample present in the intersection

represents the injection plug, which is subjected to

separation when electric field is applied across separation

channel

Detection on microchip is usually made at opposite end

of separation channel, most commonly by LIF (Laser

Induced Fluorescence) due to its sensitivity.

Typical microchip separation time- 50-200seconds.

DETECTION

LIF (Laser Induced Fluorescence) – Most commonly used method on chip due to its high sensitivity.

Most analytes are not fluorophores & have to be derivatized to be detected by LIF.

LIF is much larger than the microfabricated separation device, which makes it unfavorable for portable analytical device.

Electrochemical detectionAmperometric detection

Voltametric detection

Conductiometric detection

Potentiometric detection

APPLICATIONS

For simultaneous separation of catecholamines & their

cationic metabolites.

To enhance sensitivity of on-chip amperometric detection

Carbon nanotube modified amperometry

Microchip Affinity Capillary electrophoresis (MC-ACE)

For Enzyme assays

Microchip isoelectric Focusing (MC-IEF)

To compare practical applicability of pharmaceuticals

FABRICATION

Microchips are constructed from substrates such as:

Glass (Pyrex-like or soda lime)

Silicon (as per microelectronic chips)

Polymeric materials (plastics)

Silicon-like materials (polydimethylsiloxane)

A buffered solution of HCl is used to etch the desired

structures into a glass wafer, thereby producing a series

of U-shaped troughs that interconnect appropriately.

Channels are U-shaped because of downward & lateral

etching by the etch solution.

After etching, the etched wafer is bonded to a 2nd piece of

glass, into which reservoirs have been drilled, to enclose

the chambers and channels of the device.

REFERENCES

Tietz Textbook of Clinical Chemistry & Molecular Diagnostics; 5th Edition

Microchip Capillary Electrophoresis-Methods & protocols: Charles.S.Henry; 2006 edn

S. Gotz, U. Karst, Anal. Bioanal. Chem. 387 (2007) 183

W.R.Vandaveer, S.A.Pasas-Farmer, D.J.Fischer, C.N.Frankenfeld, S.M.Lunte, Electrophoresis 25 (2004) 3528

M.A. Schwarz, P.C. Hauser, Lab Chip 1 (2001) 1

E.S. Roddy, H.W. Xu, A.G. Ewing, Electrophoresis 25 (2004) 229

www.micruxfluidic.com/archivos/videos/micrux_mce.swf

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