Flu Rome Try

8/13/2019 Flu Rome Try

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Principle

• Fluorescence is an emission phenomenon

where an energy transition from a higher to a

lower state is accompanied by radiation. Only

molecules in their excited forms are able to

emit fluorescence ; thus, they have to be

brought into a state of higher energy prior to

the emission phenomenon.



Which molecules?

• A fluorophore, is a part of a molecule which

makes a molecule to be fluorescent. It is

similar to a chromophore, the element of a

molecule accountable for its color. Generally,

flurophore is a functional group in a molecule

which absorbs energy of a particular

wavelength and emits energy at a differentbut specific wavelength.



Types of fluorophores

• 1- Organic Dye : Fluorescein and istderivatives.

• 2- Biological fluorophores : as GFP (green

flourescent protein) was synthesized from the jelly fish Aequorea victoria.

• 3-Quantum dot: Quantum dots are 2-50nm

sized semiconductors which emit fluorescencewhen excited at a wavelength that isdependent on the size of the particle.



• 4- Aromatic and heterocyclic compounds.

• 5- Compounds with multiple conjugated groups.

• 6- compounds with electron donating groups as

OH, NH, OCH3.• 7- Polycyclic compounds like Vat K, Purines,

nucleosides, Vat A.

• 8- NADH fluorescence.

• 9- Non- fluorescence compounds when convertsto fluorescent derivatives like: steroides,metalsby chelating, absorbance



Principle continue

• Fluorescence activity can be schematically

illustrated with the classical Jablonski

diagram, first proposed by Professor

Alexander Jablonski in 1935 to describe

absorption and emission of light.



Jablonski diagram



• The first step is that a photon of light hitsflurophore in its absorption spectrum, which leadto electrons transition from lower ground state to(S0) to any vibrational level of excited singlet

state (S1) this will took a time of 10 to the powerof -15. The electron are not stable in thevibrational level so, it will seek a semi stable levelwhich is S1. This transition is called internal

conversion (IC).It is a non-radiative process andoccurs in less than 10-11 second .Now from S1 themolecule return to ground state by any of thefollowing paths.



• Path I : The molecule may lose rest of the energyalso in the form of heat so that the complete pathis non-radiative.

Path II: Molecule emit energy in the form of lightor uv-radiation. This is called Fluorescence

Path III : Some energy may be lost in Tranfer fromS1 to T1 in the form of heat. It is calledintersystem crossing (ISC). This path is non-radiative.



• Path IV : After ISC, the molecule may lose energy in theform of light in going from the excited triplet state tothe ground state. This is called phosphorescence.Phosphores, solid materials that exhibit

phosphorescence .It is used in TV tubes where beamsof electron bombard them and excite them to emitlight.

• Path V : the molecule go back and forth between ISCand lowest level of excited state and then lose energy

in the form of light in going from the lower excitedstate to the ground state. This is called delayedfluorescence.



Instrumentation

• 1- Light source : generally is mercury arc

lamp(gas discharge) or other suitable UV- visible

light. Xenon lamp for continues source of energy.

• 2- Monochromators: two gratings, two Prisms

,two filters for isolation of radiation. One for

tuning the WL. Of the excitation beam and the

second for the analysis of fluoresce emission.• 3- Two slits :After first monochromator and

before and after second monochromator.



• 4- Sample cuvette .

• 5- Detector: placed perpendicular to the

exciting pathway to reduce chance of

background incident light from reaching

detector which allow greater sensitivity.



Instrumentation



Applications

• 1- lightening.

• 2- Analytical chemistry.

•3- Fluorescence spectroscopy.

• 4- Biochemistry.

• 5- FRET.

• 6- DNA detection.• 7- DNA sequencing…… etc.



Limitations

• Fluorescence signal is affected by:

• 1- Solvent.

•2- pH.

• 3- Temp.

• 4- Absorbance of the solution.

• 5- presence of interfering or quenchingcompounds.



Standardization is not the absolute procedure in

absorption spectroscopy because fluorescence

varies depending on:

• 1- Intensity of incident light.

• 2- The amount of light intercepted by the

detector as controlled by the slits.

• 3- The bandwidth of the analyzed light.

• 4- The efficiency of the detector.



• The emission of light usually varies ondaily basis due to any change in the pH,temp. and solvent therefore the relative

fluorescence measurement will be taken.• For fluorometric assay zero only used for

setting reagent blank. No equivalent to100% scale of transmission.

• Aabsorbance of 0.1 is only allowed forstandards to form a curve .



• Fluorescence attenuation assay :

• Absorption > 0.1.

• Dye added to test and control .

• Analyte causing reaction in which a lightabsorbing compound produced.

• Graeter amount of colored reaction product ,the smaller amount of light absorbed by dye,

therefore a decrease in light passing throughsolution , decrease in intensity and thendecrease in conc.



Time- delayed fluorescence

- Done to improve sensitivity of thetechnique.

- It will increase the specificity of analysis.

- Specialized instruments used thistechnique to illuminate the sample for atime, stop illumination and measure the

emitted fluroscence over a specified timefrom 400 microsec. To 800 microsec. Afterillumination.



Fluorescence polarization

• When a fluorescent molecule is excited with plane polarizedlight, light is emitted in the same polarized plane, providedthat the molecule remains stationary throughout the excitedstate (which has a duration of 4 nanoseconds for fluorescein).

If the molecule rotates and tumbles out of this plane duringthe excited state, light is emitted in a different plane from theexcitation light. If vertically polarized light is exciting thefluorophore, the intensity of the emitted light can be monitoredin vertical and horizontal planes (degree of movement ofemission intensity from vertical to horizontal plane is related to

the mobility of the fluorescently labeled molecule). If amolecule is very large, little movement occurs duringexcitation and the emitted light remains highly polarized. If amolecule is small, rotation and tumbling is faster and theemitted light is depolarized relative to the excitation plane.



• P= Ivv –Ihv /Ivv+Ihv.

• Ihv ... Intensity with polarizers parallel.

•Ivv ... Intensity with polarizersperpendicular.



• Factors affecting final polarization are:

• 1- viscosity.

•2- Size of molecule.



Advantages and disadvantages

• Advantages:

• Flourscence polarization measurments can bemaid very accurately because they are lessaffected by variations in fluroscencemeasurments.Thus precision is readily achieved.

• Disadvantages:

• Is limited to assays that can use fluoroscence dye.

•Less flexible than absorption spectroscopy.

• Crucial to control viscosity and temp.



Luminescence

• Luminometry is the technique used to

measure luminescence .

• Lluminescence is the emission of

electromagnetic radiation in the energy

range of visible light as a result of a

reaction.



1- Chemiluminescence

*It arises from the relaxation of excited electronstransitioning back to the ground state.

• E.g. the reaction of luminol with oxygen produce 3-aminophthalate which possesses a fluorescencespectrum of the product of the chemical reaction.

*In this reaction, the resulted emission in the rangeof 400 to 450 nm.



The low photon yield of this reaction has:

1- limited its sensitivity

2- limit its application.

However this problem tackled by addingenhancer molecules (luciferin, 6-hydroxybenzothiazole) to the reaction in thepresent of peroxidase . As a result, thereaction can be followed for many minutes (30or more) with a several thousand- fold increasein photon output .



Advantages and disadvantages

• Advantage:

• very sensitive.

•Disadvantage:

• Reaction performed in a heterogeneous

system.



2- Bioluminescence

• It describes the same phenomenon , only the reaction leading to

fluorescent product is an enzymatic reaction. The most commonly

used enzyme is Luciferase.

• Bioluminescence is a highly sensitive method, due to the high

quantum yield of the underlying reaction . Some luciferase systemwork with almost 100% efficiency . For comparison, the

incandescent light bulb loses about 90% of the input energy to heat.

• Because Luminescence does not depend on any optical excitation,

problems with auto fluorescence in assays are eliminated.

• quantum phenomenon: the interaction of electro-magnatic

radiation with matter which depend on properties of radiation and

properties of the matter (sample structure).



3- Electrochemiluminescence

• It’s a process that based on the formation

of an excited-state chemical intermediate

that returns to the ground state by emitting

photon . This is different from those inwhich an excited state is achieved by

absorption of a photon. In this case the

excited state achived by chemical reaction.



• 1- Ru (complex) 2+ (electrode) = e- + Ru(complex)3+.

• 2- TPA (electrode) = e- + TPA *+ =TPA* +

H+.• 3- Ru (complex)3+ + TPA*+ e- = TPA

degradation products + excited Ru

(complex)2+.• 4- excited Ru (complex)2+ = Ru

(complex)2+ +hv (light at 620 nm).



Instrumentation

• Since no electromagnetic radiation isrequired as a source of energy for excitation,no light source and monochromator are

required .Luminometry can be performed witha rather simple set-up, where a reaction isstarted in a cuvette or mixing chamber, andthe resulting light is detected by a

photometer. Photo-multiplier tube is neededto amplify the output signal prior to recording.Temperature must be controlled why??



Applications

• Chemiluminescence: (luminol)• 1- Competitive binding assays.

• 2- phagocytosis.

•3- Detect molecules and compounds withhigh efficiency.

• Bioluminescence: (luciferase):

• 1- Determine concentration of ATP .• 2- Determination of electron transfer co-

factor.

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