Flurometry

Done by : Samyah Alanazi

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 different but specific wavelength.

Types of fluorophores
1- Organic Dye : Fluorescein and ist derivatives. 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 fluorescence when excited at a wavelength that is dependent 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 converts to fluorescent derivatives like: steroides,metals by 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 hits flurophore in its absorption spectrum, which lead to 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 power of -15. The electron are not stable in the vibrational level so, it will seek a semi stable level which is S1. This transition is called internal conversion (IC).It is a non-radiative process and occurs in less than 10-11 second .Now from S1 the molecule return to ground state by any of the following paths.

 Path I : The molecule may lose rest of the energy also in the form of heat so that the complete path is non-radiative. Path II: Molecule emit energy in the form of light or uv-radiation. This is called Fluorescence Path III : Some energy may be lost in Tranfer from S1 to T1 in the form of heat. It is called intersystem crossing (ISC). This path is nonradiative.

 Path IV : After ISC, the molecule may lose energy in the form of light in going from the excited triplet state to the ground state. This is called phosphorescence. Phosphores, solid materials that exhibit phosphorescence .It is used in TV tubes where beams of electron bombard them and excite them to emit light. Path V : the molecule go back and forth between ISC and lowest level of excited state and then lose energy in the form of light in going from the lower excited state to the ground state. This is called delayed fluorescence.

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 quenching compounds.

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 on daily 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 to 100% scale of transmission. Aabsorbance of 0.1 is only allowed for standards to form a curve .

Fluorescence attenuation assay : Absorption > 0.1. Dye added to test and control . Analyte causing reaction in which a light absorbing compound produced. Graeter amount of colored reaction product , the smaller amount of light absorbed by dye, therefore a decrease in light passing through solution , decrease in intensity and then decrease in conc.

Time- delayed fluorescence

- Done to improve sensitivity of the technique. - It will increase the specificity of analysis. - Specialized instruments used this technique to illuminate the sample for a time, stop illumination and measure the emitted fluroscence over a specified time from 400 microsec. To 800 microsec. After illumination.

Fluorescence polarization
When a fluorescent molecule is excited with plane polarized light, light is emitted in the same polarized plane, provided that the molecule remains stationary throughout the excited state (which has a duration of 4 nanoseconds for fluorescein). If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitation plane.

 P= Ivv Ihv /Ivv+Ihv. Ihv ... Intensity with polarizers parallel. Ivv ... Intensity with polarizers perpendicular.

 Factors affecting final polarization are: 1- viscosity. 2- Size of molecule.

Advantages and disadvantages

Advantages: Flourscence polarization measurments can be maid very accurately because they are less affected by variations in fluroscence measurments.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 electrons transitioning back to the ground state. E.g. the reaction of luminol with oxygen produce 3aminophthalate which possesses a fluorescence spectrum of the product of the chemical reaction. *In this reaction, the resulted emission in the range of 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 adding enhancer molecules (luciferin, 6hydroxybenzothiazole) to the reaction in the present of peroxidase . As a result, the reaction can be followed for many minutes (30 or more) with a several thousand- fold increase in 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 system work 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
Its 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 in which 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 is required as a source of energy for excitation, no light source and monochromator are required .Luminometry can be performed with a rather simple set-up, where a reaction is started in a cuvette or mixing chamber, and the resulting light is detected by a photometer. Photo-multiplier tube is needed to 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 with high efficiency.

Bioluminescence: (luciferase): 1- Determine concentration of ATP . 2- Determination of electron transfer cofactor.