PHYSICS IN MOLECULAR BIOLOGY

1. SINGLE MOLECULES TRANSPORT 2. ELECTROPORATION 3. CHROMATOGRAPHY 4. FLOW CYTOMETRY

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 MEASUREMENT MOLECULAR BIOLOGY.

OUTLINE

OUTLINE

1. WHY SINGLE MOLECULE TRANSPORT 2. SIMPLE THEORY 3. HOW TO MEASURE COMPLEMENTARY MEHODS 4. CAN WE USE A MOLECULAR FUNCTIONALITY

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

OR RESIDUAL , FOR EXAMPLES IONIC, COVALENT OR METALLIC BONDING 2. STRONG INTERACTION OF MOLECULES OCCURS WHEN AN ORGANIC MATERIAL IS ENCOURAGES TO POLYMERIZE. MECHANISMS OF INTERACTION CAUSE BONDING BETWEEN MOLECULES DERIVE FROM ELECTRICAL ATTRACTION AND REPULSION. THE DIFFERING STRENGTHS AND DIFFERING TYPES OF BOND ARE DETERMINED BY THE PARTICULAR

ELECTRONIC STRUCTURE OF THE MOLECULES INVOLVED.

WEAK AND STRONG INTERACTION

THE IMPORTANT OF van der WALLS BONDING IT IS A WEAK

ATTRACTIVE 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 SATURATED MOLECULES TOGETHER, AND FOR MOLECULES WITHIN MUCH STRONGER MECHANISMS ARE AT WORK.

SINGLE MOLECULE TRANSPORT

A V

MOLECULES

MEASURES
WHAT IS THE I(V) CURVE FOR A MOLECULE DEVICE
I(V) IS NON LINEAR
GOLD

GOLD

MOLECULES A

MEASURES
METHOD USED TO MEASURE MOLECULES IN MEDICINE. THE
ABILITY TO MEASURE MOLECULES THE BIOLOGICAL AND CHEMICAL PROCESSES. WE CAN MEASURE DIFFERENT MOLECULES IN USED DIFFERENT DEVICE.

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 the
diffusive, electrophoretic, and pressure driven flux of water soluble molecules and ions. uptake of drugs, molecular probes, and DNA by the cell. barrier.

Poration leads to ion leakage, the escape of metabolites, and increased Electroporation results in a physical reduction of the biological system

ELECTROPORATION
Electroporation occurs when the transmembrane potential induced

by 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.

PROCESS ELECTROPORATION

Fig.1. Illustration of the process of electroporation. (a) Before the electric field is applied, (b) Application of the electric
field and the formation of membrane pores and (c) When the electric field is removed and membrane reseals

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

PRINCIPLES OF OPERATION
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

ADVANCES OF ELECTROPORATION
ADVANTAGES OF ELECTROPORATION
Electroporation has a number of advantages over the conventional methods
of 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

ELECTROPORATOR
block diagram of an electroporator obtained from Puc M., Flisar K., Reberek S. and Miklav.i. D. Electroporator for in vitro cell permeabilization., Radiol Oncol 2001; 35(3): 203-7. This electroporator consists of a computer, pulse generator, voltage amplifier, current amplifier and low voltage pulse generator. The user can select the pulse parameter through the 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

ELECTROFUSION
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

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

CHROMATOGRAPHY: SEPARATION MECHANISM

CHROMATOGRAPHY IS USED TO SEPARATE COMPONENTS DISSOLVED IN SOLUTION BY CONDUCTING THE SEPATION PROCESS AT A HIGH VELOCITY WITH A PRESSURE DROP. Adsorption Partition Ion - Exchange & Ion - Interaction Size Exclusion Affinity (antibody-antigen interactions; attraction) Complexation - Chelation Ion exclusion (Separation of weak acids)

chemical interaction;

CHROMATORAPHY : SEPARATION MECHANISM

ADSORPTION PARTITION ION EXCHANGE

CHROMATOGRAPHY: SEPARTION MECHANISM

AFFINITY SIZE EXCLUSION

EQUILLIBRIUM

Chromatography - Equilibrium
A
MOBILE

A STATIONARY

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 diffusion coefficient inversely affected by mobile phase velocity

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 of particles or inner diameter of capillary column Lower at higher temperatures (viscosity)

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

FLOWCYTOMETRY AND CELL SORTING

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

INTRODUCTION

Figure adapted from [1]

Three Stages:

Fluidics Control: Positioning of cell sample stream by hydrodynamic or electrokinetic focusing Optical Detection: Analysis of scattering effects and fluorescence emitted after illumination by light beam Cell Sorting: Aerosol droplet sorting using electrokinetics

Figure adapted from [2]

FLUIDICS CONTROL : HYDRODYNAMICS

Theory:
Two sheath fluid lines are used to focus the sample stream. Circuit model where flow = current and pressure = voltage Adjust the injection rates of the sheath and sample reservoirs to change width of sample core

Design:

MICROFABRICATED: FLOWCYTOMETRY

FLUIDICS CONTROL :ELECTROKINETICS

Theory
A particle in field E experiences an electrostatic force, qE when a charge q is placed on it Balanced by a friction force controlled by fluid flow. Change potential difference across electrodes to change the flow of sample streams. Design Pt or Al electrodes used to apply external electrical field.

CELL SORTING :DROPLET

Theory
Fluid stream is vibrated to form drops that are uniformly separated. Depending on its characteristics, each drop is charged by a strong electrical pulse. External electrical field deflects desired cells into collecting reservoir.

Design
Piezoelectric transducer used to generate periodic vibrations.

CONCLUSIONS

CONCLUSIONS
FLOW CYTOMETRY: Simple idea but complicated instrumentation: Fluidics Control Optical Detection Cell Sorting