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Aswathy Bose et al / International Journal of Advances in Pharmaceutical Analysis 2018; 08(01): 01-08. 1

International Journal of Advances in Pharmaceutical Analysis

ISSN: 2277-9353 (Online)
Journal DOI: https://doi.org/10.7439/ijapa Review Article

Fluorescence spectroscopy and its applications: A Review

Aswathy Bose*, Irene Thomas, Kavitha G and Elessy Abraham

Nazareth College of Pharmacy, Othera p.o, Thiruvalla, India

QR Code *Correspondence Info:

Aswathy Bose,
Nazareth College of Pharmacy,
Othera p.o, Thiruvalla, India

*Article History:
Received: 12/01/2018
Revised: 11/02/2018
Accepted: 14/02/2018
DOI: https://doi.org/10.7439/ijapa.v8i1.4578
Abstract
Fluorescence spectroscopy is a rapid, sensitive method for characterizing molecular environments and events
samples. Fluorimetry is chosen for its extraordinary sensitivity, high specificity, simplicity and low cost as compared to
other analytical techniques. It is widely accepted and powerful technique that is used for a variety of environmental,
industrial, medical diagnostics, DNA sequencing, forensics, genetic analysis and biotechnology applications. It is a
valuable analytical tool for both quantitative and qualitative analysis. This article presents a brief overview of the theory of
fluorescence spectroscopy, together with examples of applications of this technique in organic and inorganic chemistry,
medical diagnosis, medical science etc.
Keywords: Fluorimetry, DNA sequencing.
1. Introduction 2. Types of luminescence
Alzheimer's Fluorescence and phosphorescence a) By Mechanism:
are photon emission processes that occur during molecular i) Fluorescence
ii) Phosphorescence
relaxation from electronic excited states. These photonic
b) By Excitation Source:
processes involve transitions between electronic and i) Chemiluminescence
vibrational states of polyatomic fluorescent molecules ii) Cathodoluminescence
(fluorophores). iii) Electroluminescence
Fluorophores play the central role in fluorescence iv) Photoluminescence
spectroscopy. Fluorophores are the components in Fluorescence spectroscopy is a sensitive optical
molecules that cause them to fluorescence. Majorly emission technique in which sample molecules are excited
fluorophores are the molecule which contains aromatic with a photon source. Those molecules that relax by radiant
rings such as Tyrosine, Tryptophan, and Fluorescein etc. emission can be subsequently detected by measuring the
Luminescence is emission of light by a substance intensity of that emission. [1]
not resulting from heat is thus a form of cold body
radiation. It can be caused by chemical reactions, electrical 3. Principle of fluorescence spectroscopy [1,2]
energy, subatomic motions, or stress on a crystal. There are Absorption of UV or visible radiation causes
two pre-requisites for luminescence: transition of electrons from singlet ground state to the
 The luminescent material must have a semiconductor singlet excited state. As this state is not stable, it emits
structure with a nonzero band gap. [Metals do not provide energy in the form of UV or visible radiation and returns to
luminescence if they have no band gap]. singlet ground state. Fluorescence emission occurs as the
 The energy must be imparted to this material before fluorophore decay from the singlet electronic excited states
luminescence can take place. to an allowable vibrational level in the electronic ground
state.

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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 2
The fluorescence excitation and emission spectra 5.3 Nature of substituent groups
reflect the vibrational level structures in the ground and the Electron donating groups like amino, hydroxyl
excited electronic states respectively. groups enhance fluorescence activity. Electron
withdrawing groups like Nitro, carboxylic group reduce
fluorescence. Groups like SO3H or on NH4+ have no effect
on fluorescence intensity.
5.4 Effect of temperature
Increase in temperature leads to increase in
collisions of molecules and decrease in fluorescence
intensity while decrease in temperature leads to decrease in
collisions of molecules and increased fluorescence
intensity.
5.6 Viscosity
Increase in viscosity leads to decreased
collisions of molecules which will enhance fluorescence
Figure 1: Jablonski diagram intensity while decrease in viscosity causes increased
collisions of molecules which cause decreased fluorescence
4. Instrumentation of spectrofluorometer
intensity.
5.7 Oxygen
Oxygen decreases the Fluorescence intensity in
two ways: Oxidises fluorescence substances to non
fluorescence substances. It quenches fluorescence because
of paramagnetic properties.
5.8 Effect of pH
a. Aniline: Neutral or alkaline medium shows visible
fluorescence while acidic conditions give fluorescence in
UV region only.
b. Phenols : Acidic conditions do not give fluorescence
while alkaline conditions gives good fluorescence.[2,4]

Figure 2: Schematic diagram of a spectrofluorometer 6. Effect of concentration on fluorescence intensity

Spectroflourometer mainly consists of Fluorescence intensity = Q x Ia
A. Source of light Where,
 Mercury vapour lamp Q = Fluorescence efficiency
 Xenon arc lamp Ia = Intensity of absorbed light
 Tungsten film Since emission is proportional to absorption, Ia has
B. Filters and monochromators to be knownWhere,
 Primary filters and secondary filters Io = Intensity of incident light and It = Intensity of
 Excitation monochromators and Emission incident light
monochromators It is known that,
C. Sample cells, Detectors[2-4] It = I0e-act [from beer lamberts law]
Ia = I0 [1-e-act]
5. Factors affecting fluorescence Ia = I0 x act
5.1 Conjugation Fluorescence intensity = Q x Ia
Molecule must have unsaturation i.e. it must have F = QI0 act
π electrons so that UV/vis radiation can be absorbed. If In this equation,
there is no absorption of radiation, there will not be Q = Constant for a particular substance
fluorescence. Ia = Constant for an instrument
a = Molecular extinction coefficient
5.2 Rigidity of structures
t = path length
Rigid structures will produce more fluorescence, c = concentration of substances
while flexible structure will produce less fluorescence. Fluorescence intensity is directly proportional to
the substances. [2,4]

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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 3
7. Advantages 10. Fluorescent Compounds
 It’s one of the newer methods and its potentialities are Table 1: Fluorescent compounds [4]
still largely unexplored. Wavelength[nm] Minimum
Compound pH
Fluorescence concentration
 It also affects precision. Up to 1% can be achieved easily
Adrenaline 1 335 0.1
in Flourimetric. Allyl morphine 1 355 0.1
 The method is very sensitive and also possesses Amylobarbitone 14 410 0.1
specificity because there is a choice of wavelength not Chloroquine 11 400 0.05
only for the radiation emitted, but also for the light which Chlor promazine 11 480 0.1
Cinchonidin 1 445 0.01
excites it. Cinchonine 1 420 0.01
Cyanocobalamine 7 305 0.003
8. Limitations Ergometrine 1 465 0.01
 Careful buffering is necessary as fluorescence intensity Folic acid 7 450 0.01
Noradrenaline 1 320 0.006
may be strongly dependent
Oxytetracycline 11 520 0.05
 Ultraviolet light used for excitation may cause Pamaquine 11 530 0.06
photochemical changes or destruction of the fluorescent Procaine 11 345 0.01
molecule . Procainamide 11 385 0.01
Proflavine 1 510 0.01
 The presence of dissolved oxygen may cause increased
Physostigmine 1 360 0.04
photochemical destruction. Quinine 1 450 0.002
 Traces of iodide and nitrogen oxides are efficient Reserpine 1 375 0.008
quenchers and therefore interfere. Riboflavine 6 520 0.01
Salicylic acid 11 435 0.01
 The method is not suited for determination of major
Thiopentone 13 530 0.1
constituents of a sample, because the accuracy is very Thymol 7 300 0.1
less for large amounts. Vitamin A 470 0.01
 The extent of applicability of this technique is limited,
because of the fact that all elements and compounds are 11. Compounds readily converted to fluorescent
unable to exhibit fluorescence.[13,15] derivatives
Table 2: Compounds readily converted to
9. Precautions fluorescent derivatives. [4]
Compound Reagent Excitation Emission
 Fluorescence analysis is especially applicable to trace Wavelength Wavelength
substances, care must be taken to eliminate [nm] [nm]
Adrenaline I2or NaOH 420 530
contaminations of sample. Chlordiazepoxide Photo oxidation 380 480
 Rubber and cork stoppers contain fluorescent materials Hydrocortisone 70% H2SO4 470 520
and these are extracted if the solvent touches them. 5Hydroxytryptamine Phthalaldehyde 365 495
Zinc Rhodamine B 366 580
 Filter paper also contains fluorescent material which is Primaryamines Flourescamine 390 485
extracted by solvents. and
secondary amines
 Grease from stop cocks and other sources is a fluorescent
contaminant.
 All glasses contain Al, Ca and SiO2 which may be 12. Fluorescent indicators
extracted. The intensity and colour of many fluorescent
 The most important consideration is the concentration of substances depends on pH. Some substances are so
the reagent. Concentration must be expressed in micro sensitive to pH that they can be used as pH indicators.
molecules so that the ratio of the reagent to metal may be These are termed as fluorescent or luminescent indicators.
estimated easily. Those substances which fluorescence in ultra violet light
and change in colour or have their fluorescence quenched
 Large temperature change between unknown and
with change in pH can be used as fluorescent indicators in
standard should be avoided.
acid base indicators .The merit of such indicators is that
 It is also not desirable to expose the solution to ultra
they can be employed in the titration of coloured solution in
violet radiation for longerperiods.[4]
which the colour change of usual indicators would be
masked.

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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 4
Table 3: Some important fluorescent indicators [2,7] a complex that quenches the fluorescence. Any other
Indicators pH Colour change element of the platinum group can be present to the extent
Eosin 3.0 -4.0 Colourless to green of atleast 30mµ.mL-1, without interfering the determination
Fluorescence 4.0 -6.0 Colourless to green of ruthenium in the range of 0.3 -2.0mµ.mL-1.
Quinine sulphate 3.0 -5.0 Blue to violet 14.1.2. Determination of boron in steel
Acridine 5.2 -6.6 Green to violet blue It is determined by means of complex formed with
2 naptha quinine 4.4 -8.3 Blue to colourless
benzoin. The boron present in the acid solution of the
sample is first converted in to boric acid, which is then
13. Chemistry of bioluminescence separated from the other constituents by co-distillation with
The luciferin—luciferase reaction is the emission methyl alcohol. The resulting distillate containing boric
of light as a result of the enzyme (a luciferase) -catalysed acid is neutralised by NaOH and methyl alcohol is
oxidation (generally with oxygen) of a substrate (a evaporated off.
luciferin). 14.1.3. Determination of aluminium in alloys
At present there are only three luciferins whose The reagent used is dye pontachrome blue black F,
structures have been elucidated; namely. which is used at p H of 4.8 in buffered solution. It is
Firefly luciferin, suitable for the range of 0.01 – 1.00 % of acid soluble
aluminium in steel. The principle is the formation of
complex of aluminium with azo dye 2,2-dihydroxy – 1,1
Cypridina luciferin azo naphthalene – 4 sulphonic acid, sodium salt. After
removal of aluminium and other interferences by mercury
cathode electrolysis, the fluorescence of the complex is
measured at 4.9 p H.
14.1.4. Determination of chromium and manganese in
steel
Steel is dissolved in acid and the solution is
Latia luciferin oxidised with persulphate.CrO72- and MnO4-, ions, which
absorb in the violet and yellow green respectively. There is
slight overlapping of absorption and, but if a portion is
13.1 Synthesis of luciferin treated with NaNO2 which reduces MnO4- but not, Cr2O72-,
Condensation of 3-indolyiglyoxal with the a- a difference measurement can be used to estimate Mn. A
amino-amidine gives the 2-aminopyrazine derivative. measurement at λ = 4100 AO on the reduced portion will
give chromium.
14.1.5. Determination of uranium salts
The sample is first boiled with nitric acid and then
fused with sodium fluoride and uranium fluoride .Upon
cooling, the melt solidifies in to glass which can be
examined directly in a specially designed fluorometer.
Palladium forms a precipitate with the reagent which can be
removed by centrifuging .Iron should be absent because it
forms a complex that quenches the fluorescence.
14.1.6. Estimation of rare earth terbium
Fluorescent complex formation with EDTA and
sulpho salicylic acid.The excitation spectrum corresponds
to the absorption spectrum of sulpho salicylic acid.The
fluorescent spectrum shows peaks at 4850, 5450, 6300A0
which corresponds to the ion Tb.
14.1.7. Estimation of bismuth
14. Applications
The solutions are evaporated in an argon hydrogen
14.1 Applications in inorganic chemistry
14.1.1. Determination of ruthenium flame and then irradiated with iodine emission line at λ =
It is determined in the presence of platinum metal. 2061.63 A0 which is very close to the bismuth line at λ =
Palladium forms a precipitate with the reagent which can be 2061.70 A0 to absorb the radiation.
removed by centrifuging. Iron should be absent as it forms
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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 5
14.1.8. Determination of beryllium in silicates to the first, while equal amount of NaOH and isobutyl
The formation of fluorescent complex of beryllium alcohol are added to the both the aliquots. After shaking
with morin. The interferences such as iron and rare earths aqueous layer is removed and alcoholic layer is examined
are removed by mercury cathode electrolysis; the in a flurimeter.[4]
fluorescence of the complex is complexing with Estimation of quinine sulphate by fluorimetry
triethanolamine and diethylene triamine penta acetate.  For the construction of calibration curve: In to a series of
14.1.9. Estimation of 3,4 benzpyrene 10 ml volumetric flasks transfer 1ml, 2ml, 3ml, 4ml and
This carcinogenic material is extracted by solution 5ml of standard solution of quinine sulphate [1µg/ml] and
from tobacco or from the tobacco smoke deposits and dilute to mark with 0.1N sulphuric acid. Select the proper
separated out by the chromatography [Al2O3] and excitation and emission filters [365nm and 459 nm].
subsequent elution. The resulting fluorescent solution is Measure the fluorescence with a fluorometer.
then placed in a glass cell and irradiated with the mercury  For the analysis of tablets: weigh 20 tablets and reduce to
lamp and glass filter.[4] a fine powder. Weigh accurately a quantity of the tablet
14.1.10. Determination of zinc powder equivalent to 100mg of quinine sulphate and mix
The zinc complex of oxime fluoresces in ultra with 75ml of 0.1 N sulphuric acid in a 100 ml volumetric
violet light and this forms the basis of the following flask. Mix the contents thoroughly to dissolve the drug
method. By means of calibrated burette, 5.0, 10.0 ,15.0 and adjust the volume to 100 ml with 0.1N sulphuric acid
,20.0 ,25.0 ml of standard zinc solution in to separate 100ml and filter the contents.[6]
volumetric flask .To each flask add 10 ml of ammonium 14.3 Special fluorimetric applications
acetate solution ,4ml of the gum Arabic solution, dilute to 14.3.1 Investigation of chemical structures and
45 ml with distilled water and mix by swirling. Now add processes
exactly 0.40 ml of oxime solution and dilute to the mark Fluoremetric methods have successfully been
with distilled water. Shake gently and transfer immediately applied in the investigation of hydrogen bonding, cis trans
to the cell of fluorimeter for measurement. Employ dichloro isomerism polymerisation, tautomerism and rates of
fluorescein solution as the standard. Commence reactions etc.
measurements with most concentrated zinc solution. Plot  The free radicles can best be detected with a spectrograph
instrument readings against zinc contents [mg/ml] so that the whole spectrum of a short lived component
14.1.11. Determination of cadmium may be photographed at the same time.
Cadmium may be precipitated quantitatively in  Steric hindrance in diphenyl can be studied by
alkaline solution in the presence of tartarate by 2-[0- constraining the two phenyl group to move out of a single
hydroxy phenyl]-benzoxazole. The complex dissolves plane ,by substituting CH3 groups adjacent to the centre
readily in glacial acetic acid, giving a solution with an bond [ortho substitution].The molecule behaves like a
orange tint and a bright blue fluorescence in UV light. The two benzene-insulated chromophores.
acetic acid solution is used as a basis for fluorimetric 14.3.2 Chemical analysis
determination of cadmium This type analysis may be quantitative as well
Use an aqueous solution of the sample [25-50ml] as qualitative
containing from 0.1-2.0mg of cadmium and about 0.1 g of  Detection of impurity – solvents used for
ammonium tartarate. Add an equal volume of 95% ethanol, spectrofluorometry must be free of absorbing impurities.
warm to 600C, treat with excess of reagent solution. Adjust Thus , cyclohexane must be purified until it shows no
the pH to 9-11digest at 600C for 15 minutes, filter, wash trace of benzene bands at 2600 A0.
with 20-25 ml of 95% ethanol containing a trace of  If absorbing impurities are present in the vitamin, they
ammonia and dry the precipitate at 130C for 30-35 minutes. can be removed either by chemical method or utilizing
Dissolve the precipitate in 50.0ml of glacial acetic acid, and the change of shape of absorbing band in the presence of
measure the fluorescence of the solution. Evaluate the an impurity.
cadmium content. [7]  Estimation of fluorescent intensity – intensity of pure
14.2 Application in organic chemistry fluorescing component in sufficiently low concentration
Assay of thiamine: Assayed by blue fluorescence of its is proportional to concentration, but this condition is not
oxidation product, thiochrome. The sample such as meat, always easy to get. It is therefore desirable to measure the
cereals etc is first treated with acid and extract is treated intensity from a specimen.
with enzyme phosphatase. The latter causes hydrolysis of This method however, suffers some difficulties. They are;
phosphate esters of thiamine which are present in the food  If some of the exciting radation is absorbed by the
materials. An oxidising agent such as K4Fe[CN]6 is added impurities, the amount of radation left to actually
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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 6
irradiate the specimen is reduced by unknown amount. The phenomenon is most significant in the 270–400 nm
This is called quenching. range.
 Impurities present may also deactivate the excited 14.3.5 Accurate determination of glucose
molecules by collision before they have time to radiate Glucose is considered as a major component of
the fluorescence. This is called quenching. animal and plant carbohydrates in biological systems.
 If a major part of the exciting radiation is absorbed by a Furthermore, blood glucose levels are also an indicator of
specimen, further increase in concentration will cause human health conditions. The abnormal amount of glucose
very little increase in fluorescent intensity.[ 4] provides significant information of many diseases such as
14.3.3 Laser induced fluorescence spectroscopy of diabetes or hypoglycemia. Fluorophotometry was used
human tissues for cancer diagnosis widely owing to its operational simplicity and high
Cancer is one of the most dreaded disease. Early sensitivity.
tumours often arise from tissue which have a rapid Recently bio-molecule-stabilized Au nanoclusters
turnover of cells and are active in repair like transformed were demonstrated as a novel fluorescence probe for
mucosa on the surface of hollow organs (oral cavity, sensitive and selective detection of glucose.
gastrointestinal tract, female reproductive organs etc.). Fluorescence spectroscopy is a rapid, sensitive
Laser spectroscopic techniques have the potential for in- method for characterizing molecular environments and
situ, near real time diagnosis and the use of non-ionizing events. Fluorescence output is linear to sample
radiation ensures that the diagnosis can be made repeatedly concentration over a very broad range.[1]
without any adverse side effects. Laser Induced 14.3.6 A highly sensitive fluorescent immunoassay based
Fluorescence (LIF) has been used for diagnosing cancer in on avidin labeled nanocrystals
two ways. Nanocrystals of the fluorogenic precursor
One approach involves Systemic administration of fluorescein diacetate (FDA) were applied as labels to
a drug like hematoporphyrin derivative which is selectively enhance assay sensitivity. Each FDA nanocrystal can be
retained by the tumour. When photo excited with light of converted into ~2.6 x 106 fluorescein molecules, which is
appropriate wavelength the drug localized in the tumour useful for improving sensitivity and limits of detection of
fluoresces. This fluorescence is used for detection and immunoassays. There are four binding sites in each avidin
imaging of the tumour. molecule that can bind non-co-operatively up to four
Photo excitation also leads to populating the triplet molecules of biotin with a very high affinity .The four
state via intersystem crossing. The molecule in excited binding sites together with the high affinity of the avidin-
triplet state can directly react with bio-molecules or lead to biotin interaction serve as an aid in amplifying the
generation of singlet oxygen which is toxic to the host sensitivity of immunoassays .
tissue. The resulting destruction of the host tissue is The avidin-biotin complex is almost inseparable
exploited for photodynamic therapy of tumour. and even stable under strong chemically denaturant
14.3.4 Study of marine petroleum pollutants conditions over a wide pH range. The avidin-biotin
Fluorescence spectroscopy is one of the good technique is widely used to localize antigens in cells and
techniques to detection of oil slicks on the water surface, tissues and to detect biomolecules in immunoassays and
determination of petroleum contaminants in seawater and DNA hybridization techniques.
determination of particular petroleum derivative  In the immunoassay using the labeled avidin-biotin
compounds as well as identification of pollution sources. technique, biotinylated antibody and Neutr Avidin-
Main components of any oil are hydrocarbons. The other labeled FDA were used.
components are primarily derivatives of hydrocarbons  The biotinylated antibody was first incubated with the
containing single atoms of sulfur, oxygen or nitrogen. Only antigen.
few of hydrocarbons fluoresce, while the major of them  After washing, NeutrAvidin-labeled FDA was added.
show no ability to luminescence. The content of compounds  After further incubation and washing steps, the FDA
able to fluorescence1 rarely exceeds 10% of the oil mass. associated with the antigen was hydrolyzed and
At the same time the petroleum strongly absorbs radiation, dissolved. The fluorescence intensity was finally
especially the ultraviolet and blue light. measured.
In spite of this petroleum is a luminescent medium
and fluorescence is a phenomenon which allows testing
oils. Fluorescence of oils has wavelength over then 260 nm
and covers a spectral area of ultraviolet and visible light.

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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 7
conducted in solution phase. Unlike heterogeneous
immunoassays, homogeneous assays do not require the
separation of the free and bound tracer.
When a fluorophore in solution is exposed to
plane-polarized light at its excitation wavelength the
resulting emission is depolarized. The depolarization results
from the motion of the fluorophore during the processes of
excitation and emission. Because of this, the more rapid the
motion of the fluorophore the more the emission is
depolarized. The fluorescence emission can be segregated,
using polarizers, into horizontal and vertical components.

Figure no 4: Principle of a labelled avidin – biotin

fluorescent immunoassay
The analyte was first incubated with the capture
antibody preadsorbed on the microtiter plate and then
exposed to the biotinylated-antibody followed by FDA-
avidin conjugates.
High signal amplification was achieved after
solubilization, release, and conversion of precursor FDA in
to fluorescein molecules by the addition of DMSO and
NaOH. Figure 5: Measurement of fluorescent polarisation
14.4 Flourescence polarisation immunoassay of In order to make the assay specific for a toxin, the
mycotoxins toxin can be covalently linked to the fluorophore to make a
Immunoassays are routinely used in the screening fluorescent tracer .In this case the tracer competes with
of commodities and foods for fungal toxins (mycotoxins) toxin (from the sample) for a limited amount of toxin-
constructed in competitive heterogeneous formats. specific antibody. In the absence of toxin the antibody binds
Immunoassays have been realized in the development of the tracer, restricting its motion and causing a high
major groups of mycotoxins, including aflatoxins, group B polarization. In the presence of toxin less of the tracer is
trichothecenes (primarily deoxynivalenol), ochratoxin A, bound to the antibody and a greater fraction exists unbound
and zearalenone. In typical competitive enzyme-linked in solution, where it has a lower polarization. The
immunosorbent assay (ELISA) formats the signal polarization is inversely related to the toxin concentration.
developed depends upon the presence of an enzymatic The advantage of this format, relative to a competitive
tracer. ELISA format, is the lack of a need to separate the free
Generally the tracer is either the toxin that has from the bound tracer, potentially improving assay speed.
been labeled with an enzyme (often used in cases where
antibody is immobilized) or antibody labeled with an
enzyme (in cases where a toxin-protein conjugate is
immobilized). The same two configurations have been used
in many immunoassays and biosensors. Non-enzymatic
labels such as fluorescence, radioisotopes, colloidal gold,
etc. have also been used to facilitate detection of the
competitive event. Assays of this nature, which require
separation of the ‘free’ and ‘bound’ tracer are termed Figure 6: Fluorescent polarisation immunoassay
heterogeneous and encompass the vast majority of A cuvette is filled with dilute antibody solution, a
mycotoxin immunoassays. portion of sample extract is added and the fluorescence
The separation can be achieved in various ways, intensities of the blank are obtained. The tracer is then
from chromatographically (as in lateral flow test strips), added, mixed, and held for a period before re-introducing
washing (as in ELISAs), or reagent flow over a surface (as the cuvette into the instrument to obtain the fluorescence
in certain biosensors). polarization measurement. The holding period, generally
Fluorescence polarization immunoassay (FPIA) ranging from several seconds to several minutes can be an
differs from ELISA in that it is a homogeneous assay important factor in the assay.
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Aswathy Bose et al / Fluorescence spectroscopy and its applications: A Review 8
Example Reference
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likewise need to detect lower level.[8] [3]. Douglas A Skoog, Donald M west, F James holler,
Stanley R crouch, Molecular fluorescence spectroscopy
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bioluminescence are reviewed. 122.
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in this study. At first glance it seems easy to perform ,page no;855
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results. One needs to be constantly aware of the possibility Toxins, [2009]. Page no: 196-207.
of sample contamination, or contamination of the signal
from scattered or stray light. Collection of emission spectra,
and examination of blank samples, is essential for all
experiments.

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