Fluorescence spectroscopy is an analytical technique used to examine the characteristics and interactions of molecules. It is founded on the idea that when a molecule absorbs a photon of light and becomes excited, it emits light at a longer wavelength, a phenomenon known as fluorescence. This emitted light can be collected and studied in order to determine the composition, structure, and dynamics of the molecule.

The study of biological systems is one of fluorescence spectroscopy’s most important applications. It is typically employed to investigate the structure and function of proteins, nucleic acids, and other biomolecules. It can also be used to investigate the interactions between biomolecules and their surroundings, such as the binding of medicines to receptors and the activity of enzymes.

The history of fluorescence spectroscopy dates back to the nineteenth century. Sir George Stokes made the first observations on fluorescence in 1852, when he observed that certain minerals emitted light when activated by ultraviolet light. In 1921, Francis Perrin produced the first fluorescence spectrophotometer. Since then, the technology has been continuously expanded and enhanced by the introduction of additional fluorescent dyes, lasers, and sensing techniques.

In recent years, the field of fluorescence spectroscopy has continued to expand and develop, with the introduction of new techniques like fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM), and fluorescence-based imaging techniques like confocal and two-photon microscopy. Researchers are now able to examine complicated systems with high spatial and temporal resolution, yielding fresh insights into how cells and organisms function.

Also Read: Fluorescence Spectrophotometry – Definition, Principle, Parts, Advantages, Uses

Applications of Fluorescence Spectroscopy in inorganic chemistry

1. Determination of ruthenium

• It is determined with platinum metal present. With the reagent, palladium forms a precipitate that can be separated by centrifugation.
• Because iron produces a compound that quenches fluorescence, it should be missing. At least 30mµ.mL-1 of any other platinum group element can be present without interfering with the detection of ruthenium in the range of 0.3-2.0mµ.mL-1.

2. Determination of boron in steel

• It is identified through the formation of a compound with benzoin. Boron in the sample’s acid solution is first transformed to boric acid, which is then co-distilled with methyl alcohol to separate it from the other components.
• The resultant distillate, which contains boric acid, is neutralised with NaOH, and methyl alcohol is evaporated.

3. Determination of aluminium in alloys

• The utilised reagent is the dye pontachrome blue black F, which is used in a buffered solution with a p H of 4.8. It is suitable for acid-soluble aluminium concentrations ranging from 0.01% to 1.00% in steel.
• The principle is the creation of an aluminium complex with sodium salt of 2,2-dihydroxy-1,1-azo-naphthalene-4-sulphonic acid. Fluorescence of the complex is measured at 4.9 p H, following elimination of aluminium and other interferences by mercury cathode electrolysis.

4. Determination of chromium and manganese in steel

• Steel is dissolved in acid, which is then oxidised using persulphate. CrO7 2- and MnO4 – are ions that absorb violet and yellow-green light, respectively.
• There is a minor overlap between absorption and, but a difference measurement can be used to estimate Mn if a sample is treated with NaNO2, which reduces MnO4 – but not Cr2O7 2-. A measurement of the decreased part at λ = 4100 AO will yield chromium.

5. Determination of uranium salts

• The sample is initially heated with nitric acid, followed by fusion with sodium fluoride and uranium fluoride. Upon cooling, the molten substance hardens into glass, which may be directly viewed with a fluorometer.
• With the reagent, palladium forms a precipitate that can be separated by centrifugation. Because it produces a compound that quenches fluorescence, iron should be absent.

6. Estimation of rare earth terbium

• Formation of a fluorescent compound with EDTA and sulpho salicylic acid. The spectrum of excitation correlates to the spectrum of absorption of sulpho salicylic acid. The fluorescence spectrum has ion Tb peaks at 4850, 5450, and 6300A0.

7. Estimation of bismuth

• In order to absorb the radiation, the solutions are evaporated in an argon-hydrogen flame and then irradiated with the iodine emission line at λ = 2061.63 A0, which is quite close to the bismuth line at λ = 2061.70 A0.

8. Determination of beryllium in silicates

• The development of a luminous beryllium-morin complex. Mercury cathode electrolysis removes interferences such as iron and rare earths; the fluorescence of the complex is complexing with triethanolamine and diethylene triamine pentaacetate.

9. Estimation of 3,4 benzpyrene

• This carcinogenic substance is recovered from tobacco or tobacco smoke deposits by solution and then separated by chromatography [Al2O3] and elution.
• The fluorescent solution is then placed in a glass cell and exposed to the mercury lamp and glass filter.

10. Determination of zinc

• The fact that the zinc complex of oxime fluoresces under ultra violet light is the basis for the following procedure. Using a calibrated burette, transfer 5, 10, 15, 20, and 25 millilitres of standard zinc solution to separate 100 millilitre volumetric flasks. In each flask, combine 10 ml of the ammonium acetate solution, 4 ml of the gum Arabic solution, 45 ml of distilled water, and a vigorous swirling motion.
• Now add precisely 0.40 ml of oxime solution and dilute with purified water to the desired concentration.
• Shake gently and transfer directly to the measuring cell of the fluorimeter. Standardize with dichlorofluorescein solution. Start measurements with the most concentrated zinc solution possible. Graph instrument results versus zinc concentration [mg/ml].

11. Determination of cadmium

• 2-[0-hydroxy phenyl]-benzoxazole can precipitate cadmium quantitatively in alkaline solution in the presence of tartarate. The complex quickly dissolves in glacial acetic acid, resulting in a solution with an orange hue and a brilliant blue fluorescence under ultraviolet light.
• The acetic acid solution serves as the basis for fluorimetric cadmium determination. Use a sample aqueous solution [25-50ml] containing 0.1-2.0 mg of cadmium and approximately 0.1 g of ammonium tartarate. Add an equal volume of 95% ethanol, heat to 600°C, then treat with reagent solution in excess.
• Adjust the pH to 9-11, digest at 600 degrees Celsius for 15 minutes, filter, wash with 20-25 ml of ethanol containing a trace of ammonia, and dry the precipitate at 130 degrees Celsius for 30-35 minutes. Fluorescence is determined by dissolving the precipitate in 50 cc of glacial acetic acid and measuring the resulting solution. Assess the cadmium concentration.

Application in organic chemistry

Assay of thiamine

• Assayed by the blue fluorescence of the thiochrome oxidation product. The sample, such as meat or grain, is first treated with acid, and then the extract is treated with phosphatase enzyme.
• The latter results in the hydrolysis of phosphate esters of thiamine found in dietary items. To the first aliquot, an oxidising agent such as K4Fe[CN]6 is added, while equal amounts of NaOH and isobutyl alcohol are added to both aliquots.
• After shaking, the aqueous layer is discarded and the alcoholic layer is analysed using a fluorometer.

Estimation of quinine sulphate by fluorimetry

• Transfer 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml of standard solution of quinine sulphate [1 g/ml] to a series of 10 ml volumetric flasks and dilute to mark with 0.1N sulphuric acid for the building of a calibration curve. Select the appropriate excitation and emission filters [365nm and 459 nm, respectively]. The fluorescence is measured with a fluorometer.
• For the analysis of tablets, 20 tablets should be weighed and ground into a fine powder. Combine 100 mg of tablet powder corresponding to quinine sulphate with 75 mL of 0.1 N sulphuric acid in a 100-mL volumetric flask. Adjust the volume to 100 ml with 0.1N sulphuric acid and filter the contents after adjusting the volume to dissolve the medication.

Special fluorimetric applications

1. Investigation of chemical structures and processes

The examination of hydrogen bonds, cis-trans isomerism, polymerisation, tautomerisation, and reaction rates, among other phenomena, has been conducted with success using fluorometric techniques.

• Free radicals are best identified using a spectrograph so that the entire spectrum of a short-lived component can be captured simultaneously.
• It is possible to study steric hindrance in diphenyl by preventing the two phenyl groups from moving out of a single plane by substituting CH3 groups next to the central bond [ortho substitution]. The molecule behaves like two chromophores with benzene insulation.

2. Chemical analysis

This type analysis may be quantitative as well as qualitative

• Solvents used for spectrofluorometry must be devoid of contaminants that absorb light. Therefore, cyclohexane must be refined until no benzene bands are seen at 2600 A0.
• If absorbing contaminants are present in the vitamin, they can be eliminated either by chemical approach or employing the change of form of absorption band in the presence of an impurity.
• Estimation of fluorescence intensity – the intensity of a pure fluorescing component at a sufficiently low concentration is proportional to concentration, but it is not always possible to obtain this condition. Therefore, it is desirable to assess the intensity of a sample. This approach, however, has significant limitations. If some of the exciting radiation is absorbed by contaminants, the amount of radiation left to actually irradiate the specimen is diminished by an undetermined amount. This is known as slakement.
• There may also be impurities present that deactivate the excited molecules by collision before they can emit fluorescence. This is known as slakement.
• If a substantial portion of the exciting radiation is absorbed by a specimen, increasing the concentration will have a negligible effect on the fluorescence intensity.

3. Laser induced fluorescence spectroscopy of human tissues for cancer diagnosis

• Cancer is among the most feared illnesses. Early tumours frequently emerge from rapidly dividing and actively repairing tissue, such as altered mucosa on the surface of hollow organs (oral cavity, gastrointestinal tract, female reproductive organs etc.).
• Laser spectroscopic techniques have the potential for in-situ, near-real-time diagnosis, and the use of non-ionizing radiation guarantees that repeated diagnoses can be accomplished without unwanted side effects. Laser Induced Fluorescence (LIF) has been used in two methods to diagnose cancer.
• One method involves the systemic delivery of a medication, such as a hematoporphyrin derivative, that is retained exclusively by the tumour. The medication in the tumour fluoresces when stimulated with light of the relevant wavelength.
• This fluorescence is employed for tumour identification and imaging. Photoexcitation also results in triplet state population via intersystem crossing.
• The excited triplet state of a molecule can directly react with biomolecules or generate singlet oxygen, which is harmful to the host tissue. Utilizing the ensuing degradation of host tissue for tumour photodynamic treatment.

4. Study of marine petroleum pollutants

• Fluorescence spectroscopy is one of the best methods for detecting oil slicks on the water’s surface, determining petroleum pollutants in saltwater, identifying specific petroleum derivative compounds, and identifying pollution sources. Hydrocarbons are the main constituents of all petroleum.
• The remaining components are predominantly hydrocarbon derivatives having a single atom of sulphur, oxygen, or nitrogen. Only a small number of hydrocarbons glow, while the vast majority lack this property.
• Rarely reaches 10% of the oil’s bulk in terms of fluorescence-producing substances. In addition, petroleum absorbs radiation strongly, particularly ultraviolet and blue light.
• Despite this, petroleum is a luminous material, and fluorescence is a phenomena that permits oil testing. The wavelength of oil fluorescence exceeds 260 nm and encompasses both ultraviolet and visible light. The phenomena is particularly prominent between 270 and 400 nanometers.

5. Accurate determination of glucose

• In biological systems, glucose is a key component of animal and plant carbohydrates. In addition, blood glucose levels are an indicator of health issues in humans.
• The abnormal glucose level is indicative of numerous disorders, including diabetes and hypoglycemia. The widespread usage of fluorophotometry was due to its operating simplicity and great sensitivity.
• Recent research has established biomolecule-stabilized Au nanoclusters as a new fluorescence probe for the sensitive and selective detection of glucose. Fluorescence spectroscopy is a quick and sensitive technique for identifying chemical environments and events.
• The relationship between fluorescence output and sample concentration is linear throughout a vast concentration range.

6. A highly sensitive fluorescent immunoassay based on avidin labeled nanocrystals

As labels, nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were utilised to increase the sensitivity of the experiment. Each FDA nanocrystal may be transformed into approximately ~2.6 x 106 fluorescein molecules, which is beneficial for enhancing the sensitivity and detection limits of immunoassays.

Each avidin molecule contains four binding sites that can bind non-cooperatively with very high affinity to four biotin molecules. The four binding sites along with the high affinity of the avidinbiotin interaction serve as an assistance in enhancing the sensitivity of immunoassays .

The avidin-biotin complex is nearly inseparable and even stable in harsh chemically destabilising circumstances and throughout a broad pH range. The avidin-biotin approach is frequently used to detect biomolecules in immunoassays and DNA hybridization techniques and to locate antigens in cells and tissues.

• Biotinylated antibody and Neutr Avidin-labeled FDA were utilised in the immunoassay employing the labelled avidin-biotin method.
• The antigen was first treated with the biotinylated antibody. Following washing, NeutrAvidin-labeled FDA was applied.
• Following several steps of incubation and washing, the FDA associated with the antigen was hydrolyzed and dissolved. Finally, the fluorescence intensity was measured.

The analyte was initially treated with the capture antibody that was preadsorbed on the microtiter plate, followed by exposure to the biotinylated-antibody and FDAavidin conjugates. With the addition of DMSO and NaOH, precursor FDA was solubilized, released, and converted into fluorescein molecules, resulting in signal amplification.

7. Flourescence polarisation immunoassay of mycotoxins

Immunoassays developed in competitive heterogeneous formats are regularly used to screen commodities and foods for fungal toxins (mycotoxins). Immunoassays have been developed for important mycotoxin groups, including aflatoxins, group B trichothecenes (mainly deoxynivalenol), ochratoxin A, and zearalone. In conventional competitive enzyme-linked immunosorbent assay (ELISA) formats, the signal generated is contingent on the presence of an enzymatic tracer.

Typically, the tracer is either a toxin that has been labelled with an enzyme (typically utilised in situations where the antibody is immobilised) or an antibody that has been labelled with an enzyme (in cases where a toxin-protein conjugate is immobilized). Numerous immunoassays and biosensors make use of the same two designs. Additionally, non-enzymatic markers such as fluorescence, radioisotopes, colloidal gold, etc. have been utilised to aid in the detection of the competitive event.

The vast majority of mycotoxin immunoassays consist of heterogeneous assays, which require the separation of the “free” and “bound” tracer. chromatographically (as in lateral flow test strips), by washing (as in ELISAs), or by reagent flow over a surface (as in certain biosensors). Fluorescence polarisation immunoassay (FPIA) is distinct from ELISA in that it is a solution-based homogenous assay. Homogeneous tests, unlike heterogeneous immunoassays, do not require the separation of the free and bound tracer.

When a solution of a fluorophore is subjected to plane-polarized light at the wavelength of its excitation, the resultant emission is depolarized. The depolarization is caused by the mobility of the fluorophore during the excitation and emission processes. Because of this, the emission is increasingly depolarized the faster the fluorophore is moving. Using polarizers, the emission of fluorescence can be separated into horizontal and vertical components.

To make an assay specific for a poison, the toxin can be covalently attached to a fluorophore to create a fluorescent tracer. In this instance, the tracer and toxin (from the sample) compete for a limited amount of toxin-specific antibody. Antibody binds the tracer in the absence of toxin, inhibiting its mobility and creating a high degree of polarisation. In the presence of toxin, a smaller proportion of the tracer is bound to the antibody, and a larger proportion of the tracer exists unbound in solution, where it has a lower polarity. Polarization is inversely proportional to the concentration of the poison.

This format’s advantage over competitive ELISA formats is the elimination of the requirement to separate the free tracer from the bound tracer, which may increase test speed. A cuvette is filled with a diluted antibody solution, a sample extract is added, and the fluorescence intensities of the blank are measured.

Then, the tracer is added, mixed, and stored for a time before the cuvette is reintroduced into the device to acquire the fluorescence polarisation measurement. The holding time, which typically ranges from a few seconds to a few minutes, might be a crucial aspect of the assay.

Example

Aflatoxins are relevant to human and animal health at lower quantities than many other mycotoxins due to their potency as carcinogens, and the regulation standards for these toxins in foods and feeds are likewise lower (ppb level). Consequently, tests for these poisons must detect lower concentrations.