physics in molecular biology-2010

7/28/2019 Physics in Molecular Biology-2010

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PHYSICS IN MOLECULAR BIOLOGY

1. SINGLE MOLECULES TRANSPORT

2. ELECTROPORATION

3. CHROMATOGRAPHY4. FLOW CYTOMETRY


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OBJECTIVES

OBJECTIVES

THIS STUDIES WILL DEVELOP THE STUDENTS ABILITY

AND PROPERLY APPLIED PHYSICS IN MOLECULAR

BIOLOGY.

THIS STUDIES WILL DEVELOP THE STUDENTS ABILITY

AND PROPERLY INSTRUMENT FOR MEASUREMENTMOLECULAR BIOLOGY.


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OUTLINE

OUTLINE 1. WHY SINGLE MOLECULE TRANSPORT

2. SIMPLE THEORY

3. HOW TO MEASURE COMPLEMENTARY MEHODS

4. CAN WE USE A MOLECULAR FUNCTIONALITY


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MECHANISMS OF INTERACTION

MECHANISMS OF TWO INTERACTIONS OF THE MOLECULE 1. WEAK INTERACTION IS CALLED van der WAALS

OR RESIDUAL , FOR EXAMPLES IONIC, COVALENT ORMETALLIC BONDING

2. STRONG INTERACTION OF MOLECULES OCCURSWHEN AN ORGANIC MATERIAL IS ENCOURAGES TO

POLYMERIZE.MECHANISMS OF INTERACTION CAUSE BONDING BETWEEN

MOLECULES DERIVE FROM ELECTRICAL ATTRACTION ANDREPULSION. THE DIFFERING STRENGTHS AND DIFFERING TYPES

OF BOND ARE DETERMINED BY THE PARTICULARELECTRONIC STRUCTURE OF THE MOLECULESINVOLVED.


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WEAK AND STRONG INTERACTION

THE IMPORTANT OF van der WALLS BONDING IT IS A WEAKATTRACTIVE BETWEEN MOLECULES

FOR EXAMPLE : A GAS WHICH REPRESENT THE PROPERTIES OF

REAL GASES RATHER BETTER THAN THE IDEAL GAS LAW. AN EXPLANATION OF THE ATTRACTIVE FORCE, SUCH ATOMS AND

MOLECULES ARE ATTRACTED TO OTHERS BY ELECTRICAL FORCES.

FAR MORE TYPICALLY , van der WALLS FORCES BIND SATURATEDMOLECULES TOGETHER,

AND FOR MOLECULES WITHIN MUCH STRONGER MECHANISMSARE AT WORK.


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SINGLE MOLECULE TRANSPORT

A V

MOLECULES


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MEASURES

WHAT IS THE I(V) CURVE FOR A MOLECULE DEVICE

A

V

I(V) IS NON LINEAR

GOLDGOLD

MOLECULES


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MEASURES

METHOD USED TO MEASURE MOLECULES IN MEDICINE. THEABILITY TO MEASURE MOLECULES THE BIOLOGICAL ANDCHEMICAL PROCESSES. WE CAN MEASURE DIFFERENT MOLECULESIN USED DIFFERENT DEVICE.


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ELECTROPORATION

ELECTROPORATION

Cells are exposed to high intensity electric field

Specific regions of the cell are destabilized

Resulting structural rearrangement forms temporal pores in the membrane

Poration increases the permeability of the cell membrane: they increase thediffusive, electrophoretic, and pressure driven flux of water solublemolecules and ions.

Poration leads to ion leakage, the escape of metabolites, and increaseduptake of drugs, molecular probes, and DNA by the cell.

Electroporation results in a physical reduction of the biological systembarrier.


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ELECTROPORATION

Electroporation occurs when the transmembrane potential inducedby

the electric field is greater than a threshold voltage. Phase one: The formation of pores occurs at the cell membrane

facing the positive electrode. That is because the negative interior of the

cell is where the capacitance of the membrane first exceeds when an external electric field is applied. Phase two: The formation of pores at cell membrane facing the

negative electrode. Pore formation happens within microseconds, membrane resealing happens over a range of minutes, allowing the transfer of

materials into and out of the cells.


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PROCESS ELECTROPORATION

Fig.1. Illustration of the process of electroporation.

(a) Before the electric field is applied, (b) Application of the electricfield and the formation of membrane pores and (c) When theelectric field is removed and membrane reseals


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DIELECTROPHORESIS

External electric fields can induce formation of pores in membranes, move cells by

dielectrophoresis, and fuse membranes

The interaction of the external electric fields with the polarized material results in forces

which can then induce motions inside particles

The motions inside the material can result in structural rearrangements or even mechanical

fracture, which can subsequently lead to membrane electroporation and electrofusion


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PRINCIPLES OF OPERATION

Electroporation is the process in which a cell is subjected to an electrical current

pulse. This pulse creates temporary openings in the cell membrane and allows

molecules or particles to enter the cell

The electropores are located primarily on the surfaces of cells that are closest to the

electrodes

If the electric field pulse has the proper parameters, the pored cells can recover and

continue to grow

PRINCIPLES OF OPERATION


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ADVANCES OF ELECTROPORATION

ADVANTAGES OF ELECTROPORATION

Electroporation has a number of advantages over the conventional methodsof cell permeabilization.

A noninvasive, non-chemical method

Does not change the biological structure or function of the target cell.

Nontoxic as compared with the other chemical or biological methods.

Greater efficiency: electroporation is generally better than most alternative methods

Can be applied to a much wider selection of cell types


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ELECTROPORATOR

This electroporator consists of a computer, pulse generator, voltage amplifier, current

amplifier and low voltage pulse generator. The user can select the pulse parameter throughthe computer. All pulse parameters, except pulse amplitude, are then transferred to the pulse

generator. This subunit generates digital signal that is then amplified in the voltage amplifier

to the value that is set by external potentiometer. The amplified signal is then pass through

the current amplifier to create enough power demanded by the load at the output The

sample is placed between the electrodes for electroporation

block diagram of an

electroporator

obtained from Puc M., Flisar

K., Reberek S. and Miklav.i.

D. Electroporator for in

vitro cell permeabilization.,

Radiol Oncol2001; 35(3):

203-7.


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ELECTROFUSION

A frequent application of electroporation is electrofusion

Cell fusion may occur during the electroporation process if the cells are brought

together prior to the delivery of the pulsed electric field

The AC current causes a dielectrophoresis and bring target cells into contact

After delivery of the direct current pulse, pores that have been formed in close

alignment may reseal upon one another

ELECTROFUSION


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APPLICATIONS

APPLICATIONS

1. Introduction of foreign DNA or RNA into living cells for gene transfections Gene therapy

2. Fusion of cells Embryo Manipulation Hybridoma Formation Plant Protoplast Fusion

3. Insertion of proteins into cell membranes

4. Improving drug delivery Chemotherapy of cancerous cells

5. Transdermal delivery of drugs


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CHROMATOGRAPHY: SEPARATIONMECHANISM

CHROMATOGRAPHY IS USED TO SEPARATE COMPONENTSDISSOLVED IN SOLUTION BY CONDUCTING THE SEPATIONPROCESS AT A HIGH VELOCITY WITH A PRESSURE DROP.

Adsorption

Partition

Ion - Exchange & Ion - Interaction

Size Exclusion

Affinity (antibody-antigen interactions; chemical interaction;attraction)

Complexation - Chelation

Ion exclusion (Separation of weak acids)


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CHROMATORAPHY : SEPARATIONMECHANISMADSORPTION PARTITION ION EXCHANGE


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CHROMATOGRAPHY: SEPARTIONMECHANISM

AFFINITY SIZE EXCLUSION


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EQUILLIBRIUM

Chromatography - Equilibrium

A MOBILE A STATIONARY


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MOLECULAR DIFFUSION

Molecular diffusion (B) in mobile phase

proportional to time analyte spends in

a column affected by diffusion coefficient of

analyte in mobile phase

affected by temperature and pressure

not important in LC low diffusioncoefficient

inversely affected by mobile phase

velocity


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RESISTANCE

Resistance to mass transfer (C):

Mass transfer in mobile and stationary

phase Lack of equilibrium moving phase

Affected by thickness of liquid phase

Affected inversely by the diameter ofparticles or inner diameter of capillarycolumn

Lower at higher temperatures (viscosity)


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CONCLUSIONS

Conclusions:

Minimum value for H is achieved when:

stationery phase thickness is minimal

column packed with the smallest particles

capillary columns have the smallest

internal diameter mobile and stationary phases have low

viscosity and high diffusion coefficient


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FLOWCYTOMETRY AND CELL SORTING

Definition: A technique of rapidly measuring physical and chemical characteristics ofcells as they flow in single file through a sensing region


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INTRODUCTION

Three Stages:

Fluidics Control: Positioning of cellsample stream by hydrodynamic orelectrokinetic focusing

Optical Detection: Analysis of scatteringeffects and fluorescence emitted afterillumination by light beam

Cell Sorting: Aerosol droplet sortingusing electrokinetics

Figure adapted

from [1]

Figure adapted from [2]


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FLUIDICS CONTROL : HYDRODYNAMICS

Theory: Two sheath fluid lines are

used to focus the samplestream.

Circuit model where flow =current and pressure =voltage

Design: Adjust the injection rates of

the sheath and samplereservoirs to change widthof sample core


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MICROFABRICATED: FLOWCYTOMETRY


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FLUIDICS CONTROL :ELECTROKINETICS

Theory

A particle in field Eexperiences an electrostatic force, qEwhen acharge qis placed on it

Balanced by a friction force controlled by fluid flow.

Change potential difference across electrodes to change the flowof sample streams.

Design

Pt or Al electrodes used to apply external electrical field.


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CELL SORTING :DROPLET

Theory Fluid stream is vibrated to

form drops that are uniformlyseparated.

Depending on itscharacteristics, each drop ischarged by a strong electricalpulse.

External electrical field

deflects desired cells intocollecting reservoir.

Design Piezoelectric transducer used

to generate periodicvibrations.


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CONCLUSIONS

CONCLUSIONS

FLOW CYTOMETRY:

Simple idea but complicated instrumentation:Fluidics Control

Optical Detection

Cell Sorting

physics in molecular biology-2010

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