06 Atomic Spectroscopy Module :17 SensingApplications of Quenching Principal Investigator: Dr.NutanKaushik, Senior Fellow The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Dr

12  Download (0)

Full text


Paper No. : 06 Atomic Spectroscopy

Module :17 SensingApplications of Quenching

Principal Investigator: Dr.NutanKaushik, Senior Fellow

The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry,

JamiaHamdard University, New Delhi

Paper Coordinator: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm.

Chemistry, JamiaHamdard, New Delhi.

Content Writer: Dr. MymoonaAkhtar, Associate professor, Dept. of Pharm.

Chemistry, JamiaHamdard, New Delhi.

Content Reviwer: Dr. A A Siddiqui, Professor of Pharm. Chemistry, JamiaHamdard University, New Delhi


Description of Module

Subject Name Analytical Chemistry / Instrumentation

Paper Name Atomic Spectroscopy


Module Id 17


Objectives In this module you will learn about the Sensing Applications of Quenching like Chloride-Sensitive Fluorophores, Intracellular Chloride Imaging, Chloride- Sensitive GFP.

Knowledge of the quenching mechanism that is valuable for rational design of fluorophores with the desired sensitivity.

In this module you will also learn about the Sensing application of quenching to Molecular Biology.

Keywords Fluorophores, Sensing Applications of Quenching, Chloride-Sensitive Fluorophores



Fluorescence is a kind of luminescence where the emission of photons is brought about by absorption of light and emission stops as the light is cut. Luminescence can be most conveniently defined as the radiation emitted by a molecule, or an atom, after it had absorbed energy to go to an exited state. Fluorescence and phosphorescence is alike in that excitation is brought about by absorption of photons. The lifetime of fluorescence is shorter than that of phosphorescence. Fluorescence occurs in simple as well as in complex gaseous, liquid and solid chemical system, or in biological systems; Simplest example is vaporized sodium atom 3s excited to 3p by absorption of λ5896 Å and 5890 Å and Jelly Fish. Fluorescence and phosphorescence is the ratio of the number of molecules that luminese to the total number of excited molecule. For highly fluorescent molecule such as fluorescein the ratio approaches unity. Fluorescence has been widely used for study of chemical and biological reactions, to understand the dynamics of the biological systems.

Similarly Quenching of fluorescence which means inhibiting or reducing the phenomena of Fluorescence has been used to study chemical and biological phenomena’s in research. Any process that can lead to a reduction in fluorescence intensity is referred Fluorescence quenching. The fluorescence quenching may be due to number of reasons including excited state reaction of the molecule, Mmolecular rearrangement, Ccollisional quenching i.e. loss of fluorescence by colliding with other ions or atoms. Energy transfer, charge transfer reactions or photochemistry may be also regarded as responsible for quenching of Fluorescence.

Fluorescence quenching has been widely studied both as a fundamental phenomena, and as a source of information about biochemical systems. These biochemical applications of quenching are due to the molecular interactions that result in quenching. A wide variety of substances have been developed as fluorophores and as quenchers of fluorescence. Best known collisional quenchers are molecular oxygen which quenches almost all known fluorophores. Mainly two types of quenching processes usually encountered Collisional (dynamic) quenching and Static (complex formation) quenching.Application of quenching of fluorescence is in study of different biological systems and their function. For example to study the diffusion coefficient of oxygen in membranes or to study the position of quenchers in the membrane.

Applications of quenching of fluorescence

The sensing application of Fluorescence quenching has been used for a wide variety of analytes including, NO, oxygen and heavy metals. The use of quenching for sensing is exemplified by chloride-sensitive fluorophores. Other applications include

1. Oxygen diffusion in membranes.

2. Localization of membrane-bound tryptophan residue by quenching.

3. Quenching of membrane probes using localized quenchers.

4. Parallax and depth – dependent quenching in membranes.

5. Boundary lipid quenching.

6. Effect of lipid-water partitioning on quenching.


7. Quenching in micelles.

8. Sensing Applications

Chloride-Sensitive Fluorophores

Chloride is an important biological anion that plays a role in neuronal processes, nerve conduction, fluid adsorption, and cellular pH. Chloride-sensitive fluorophores have been extensive developed for their analytical and biochemical applications. These fluorophores are based on a acridinium or quinolinium structure, with additional groups to modify their properties like solubility or sensitivity of the fluorophore to quenching. Usually chloride quenching is collisional type of quenching, as can be seen in Figure 1.

Figure 1: Chloride and iodide quenching of N-methylquinolinium iodide. The amount of iodide from the fluorophore is not significant. (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

This figure shows Stem-Volmer plots for quenching of N-methylquino linium iodide (MAl) by chloride or iodide. The equivalent decrease in intensity and lifetime shows that the quenching is collisional and complex formation does not occur. Eelectron transfer from the anion to the fluorophore seems to be the most probable mechanism of quenching, suggests that the quenching efficiency should depend on the oxidation potential of quencher. According to this iodide ion has the highest quenching efficiency as it gets easily oxidized. Whereas because of more stability of fluoride ion due to its electronegativity it is more difficult to get oxidized hence is much less efficient in quenching. Also even though the diffusion coefficients of chloride and iodide are similar, it is experimentally seen that iodide is a more efficient quencher than chloride and fluoride. Iodide is believed to quench strongly than chloride. Figure 2 shows bimolecular quenching constants for N-


methylquinolinium iodide and several quenchers. The quenching efficiency is highest for iodide, which is easily oxidized.

Figure 2: Bimolecular quenching constants for N-methylquinolinium iodide. (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

The quenching efficiencies for a charge-transfer mechanism is expected to depend on the free energy charge for the reaction. For a charge-transfer process the free energy change is given by

where Eox is the oxidation potential of the electron donor (the quencher) Ered is the reduction potential of the lectron acceptor (the fluorophore), E00 is the singlet energy of the fluorophore, C is a constant that accounts for the energy release due to the charge separation interaction of the charges with the solvent.

With increase in the oxidation potential, the value of ∆G also increase i.e. it becomes less negative and as a result less efficient quenching. The above equation does not correlate with the quenching efficiencies absolutely because other effects like electron exchange and heavy atom effects also have their effect on quenching. Although for this class of probes charge- transfer is the dominant mechanism for quenching by halides. Knowledge of the quenching mechanism is valuable because rational design of fluorophores with the desired sensitivity to halides depend on it. For example substitutions on the quinolinium ring that decrease the affinity for electrons are likely to decrease the quenching efficiency.

Intracellular Chloride Imaging

Chloride-sensitive fluorophores have been widely used for imaging the local chloride concentrations and measurements of intracellular chloride concentrations. Due to photobleaching and the unknown concentrations of probe at each position in the cells, the use of fluorescence microscopy for imaging is difficult. In contrast, calcium, and other cations collisionally quenched probes do not show spectral shifts. Earlier direct wavelength-ratiometric probes for chloride were not available. But the knowledge of the mechanism of chloride quenching has helped in the design of these probes. This consisted of linking two fluorophores, one which is quenched by chloride and one which is not sensitive to chloride


Figure 3 :direct wavelength-ratiometric probes for chloride

This probe contains two quinoline rings. The methoxy-substituted ring on the left readily accepts electrons and is quenched by chloride. Whereas amino group containing ring on right does not accept electrons from chloride and is not quenched.

Figure 4: a) Emission spectra of MQxyDMAQ b) Effect of increasing concentration of chloride on spectra of MQxyDMAQ (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

The graph figure 4 shows emission spectra of MQxyDMAQ in the presence of increasing chloride concentrations. The shorter-wavelength emission is quenched, but the longer wavelength emission is not sensitive to chloride. Examination of the spectra in Figure 4 suggests that there will be RET from the MQ to the DMAQ ring. In this probe some energy transfer occurs, which reduces the lifetime of the MQ ring and makes it less sensitive to quenching by chloride. However, the RET efficiency is modest, so that the MQ ring remains sensitive to chloride The sensitivity of a probe differs in a pure solution than its sensitivity when used in an intracellular environment. Therefore the chloride-sensitive probes were calibrated in cells by changing the intracellular chloride concentration. This is done by exposing the membrane permeability of the 3T3 fibroblast cells using valinomycin and other similar compounds, followed by exposing the cells to different chloride concentrations (Figure 5). This is reflected by decrease in intensity of the short-wavelength MQ emission at 450 nm with the increase in chloride concentration and the insensitivity of the intensity of the long-wavelength emission at 565 nm.


Figure 5. Structure and intracellular calibration of the chloride probe bis-DMXPQ (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

The wavelength-ratiometric chloride probes have been used to measure local chloride concentrations in CHO cells (Figure 6) by collecting the the intensity images at 450 and 565 nm. The intensity at 450 nm is sensitive to the local chloride concentration where as the image at 450 nm is not sensitive to chloride concentrations. Therefore the ratio of the emission intensities of the two can be used to determine the concentration of chloride in the cell. These images revealed uniform concentration of chloride in these cells, with a somewhat lower chloride concentration in the nuclei.

Figure 6. Fluorescence microscopy images of CHO cells labeled with the chloride probes.

The intensities of 450 and 565 nm are color coded for clarity. The images on the right are the chloride concentrations calculated from the intensity. (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

Chloride-Sensitive GFP

Green fluorescent protein (GFP) and its mutants are widely used to study gene expression and as sensors. A yellow variant of GFP (YFP) is known to be sensitive to chloride. The intensity of YFP-H148Q decreases progressively in response to added chloride in a manner and concentration range that suggests collisional quenching (Figure 7) The extent of quenching depends on pH, which is not expected for collisional quenching.


Figure 7. Fluorescence intensity of YFP-H148Q in the presence of NaCI. (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

The chromophore in GFP is buried in ß–barrel structure of the protein (Figure 8) and therefore is not accessible to freely diffusing chloride. These observations suggest that the chloride sensitivity of this YFP is not due to collisional quenching.

Figure 8: 3D structure of yellow fluorescent protein YFP-H48Q. (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

The solid line is the peptide backbone, and the green structure is the native chromophore and the red and blue surfaces show the location of bound iodide ions


Figure 9: Absorption spectra of YFP-Hl48Q in the presence of NaCl, pH 6.4. Binding of cbloride is thought to stabilize the right side structure in the upper panel (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

This figure shows the absorption spectra of YFP Hl48Q in the presence of chloride. With increase in [CI-] concentration a decrease in the absorption at the excitation wavelength 514 nm, and increases in the short-wavelength absorption is seen. There appears to be an isoelectric point near 430 nm, which indicates the presence of two species. It appears that chloride changes the structures or conformation of the protein or chromophore, decreasing the absorbance and giving the appearance of quenching.

The x-ray structure has been solved with bound iodide ions (Figure 8), one of which is next to the chromophore. An iodide or chloride next to the chromophore might result in static quenching. However, static quenching by chloride is not the reason for the chloride sensitivity of YFP. It appears that the quenching of fluorescence of YFP is due to a induction of change in the structure of the chromophore by chloride. The chloride is believed to binds near the chromophore in contact with the imidizolinone oxygen atom which force the shifting of the equilibrium to the left side (Figure 9). The larger amount of chloride quenching at lower pH (Figure 7) is consistent with protonation of the phenolic oxygen upon binding of chloride. Hence the apparent quenching of YFP by chloride in Figure 7 is not due to collisional or static quenching, but instead due to a specific binding interaction which changes the structure of the chromophore. A wide variety of fluorophores are available that can be used for intracellular sensing. It is difficult to use these probes in an intracellular environment because they are likely to bind to macromolecules that can shift the analyte calibration curves. GFP and its variants are promising intracellular sensors because the chromophore is buried in the J3-barrel and unable to interact directly with biomolecules. Due to this reason YFP-Hl48Q has been used to measure the intracellular pH of Swiss 3T3 fibroblasts. An advantage of GFP is that the probe is synthesized by the cell itself, and not added to the cells. The fibroblast are transfected with the gene for YFP-Hl48Q, which has a bright green fluorescence. The response of the protein to pH and chloride has been tested using ionophores to modify the internal ion concentrations. When excited at 480 nm the


emission intensity decreased with increasing pH and decreased with increasing chloride concentrations. These remarkable results show that it is now possible to insert genes that cause the cells to synthesize their own fluorophores, and the genetically engineered protein- fluorophore can be designed to have sensitivity to a desired analyte.

Application to Molecular Biology

Many advances in the development and use of sensing for DNA analysis have been during the past decade. A number of techniques make use of quenching. A typical situation is the need to detect a target oligomer of known sequence in a sample containing many sequences.

Detection of the target oligomer is usually accomplished by synthesis of a probe oligomer with the complementary sequence. The probe oligomer also contains bound fluorophores that display spectral changes upon hybridization with the target oligomers. The spectral changes can be due to RET, intercalation or quenching

Release of Quenching upon Hybridization

Figure 10:

One approach to target detection is to change the extent of quenching upon hybridization (Figure 10). In this case the fluorescein label is quenched by a nearby pyrene residue. In the absence of target DNA the fluorescence is quenched by the pyrene. Upon binding to the target sequence the pyrene intercalates into the double helical DNA, resulting in a several- fold increase in the fluorescein emission. This approach is general and could be used with a variety of fluorophore-quencher pairs.

Molecular Beacons in Quenching by Guanine

Many fluorophores are efficiently quenched by the nucleic acid bases Guanine probably due to photoinduced electron transfer. The other bases are much less efficient as quenchers.

Figure 11 shows Stern-Volmer plots for dGMP as the quencher with four fluorophores.


Figure 11: Structure of four fluorophores and quenching of these fluorophores by Guanine monophosphate (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

Figure 12: Quenching of fluorophores MR 121 by all the nucleotide monophosphate (Adapted from Principles Of Fluorescence Spectroscopy by J R Lakowicz)

From these result it can be seen that quenching by guanine monophosphate is not limited to a single fluorophore but multiple fluorophores can be quenched efficiently with it. Among the four probes studied MR121 has been reported to be most sensitive to quenching by dGMP. In the efficiency testing of all the nucleotide manophosphates, dGMP against MR 121 has been found to be the most efficient quencher. This Quenching property of guanine was used to design a molecular beacon (Figure 13). The fluorophore JA242 was positioned at one end of the beacon , and the other end was terminated with several guanine residues. The residues quench the fluorophore when in the folded state. The emission intensity increases about fivefold when the beacon is unfolded and hybridized with the target sequence.

Figure 13: Absorption and emission spectra of a molecular beacon based on guanine quenching of JA243

Binding of Substrates to Ribozymes

Quenching by guanine has also been applied to structural studies of RNA. Compared to DNA, RNA is flexible and frequently forms base-paired regions within a single strand of RNA. (Figure 14). Single strands of RNA like peptide chains fold can adopt well defined three-dimensional structures in solution by folding. Ribozymes is one type of structural RNAs, that display catalytic enzyme-like behavior. The catalytic properties of these highly structured RNAs was first reported in 1982, showing that certain RNAs displayed autocatalytic activity. Numerous examples of studies related to ribozymes and their catalytic behavior are reported in literature. One example is the hairpin ribozyme that cleaves single-stranded RNA (Figure 8.61).


Figure 14: Structure of hairpin ribozyme (HpRz) and fluorescent labeled substrate. The substrate binding region is the region adjacent to the substrate

The observations of quenching by Guanosine nucleotides suggested the use of guanine quenching to study substrate binding to ribozymes. The substrate contained a fluorescein residue covalently linked 3’ end. The hairpin ribozyme contains a guanosine residue at the 5N end. The fluorescein emission is quenched when the substrate nucleotide binds to the ribozyme, (Figure 15). Quenching also occurs when the fluorescein-labeled substrate binds to the substrate-binding strand (SBS) that contains a 5N-guanosine residue (G-SBS).

The guanosine residue is needed for quenching, and the emission of fluorescein is unchanged in the presence of the substrate-binding strand without a 5' terminal guanosine residue (not shown). The example shows how fundamental studies of nucleotide quenching have found useful applications in modern biochemistry

Figure 15: Emission spectra of the fluorescein labeled substrate analogue (dA-1) in solution and when bound to hairpin ribozome (HpRz) or the guanine containing substrate binding strand


In this module you learned about the Sensing Applications of Quenching like Chloride- sensitive fluorophores. Mechanism for rational design of fluorophores with the desired sensitivity was cover along with how the position of chromophore affects the mechanism and sensing ability of the ion quencher. In this module the students also learnt about how hybridization leads to release of quenching, what are molecular beacons, their making and working, and the use of ribozomal RNA in sensing application.




Related subjects :