Published on March 14, 2014
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 medium AGE (Agarose gel electrophoresis) PAGE (Poly-acrylamide gel electrophoresis) Paper strip electrophoresis (cellulose acetate paper/membrane) Based on mode of technique Slide/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 Speed 4x – 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
INSTRUMENTATION Separation 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 detection Amperometric 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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