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Paper No. : 06 Atomic Spectroscopy

Module :13 Fluorophores -Special and specific Fluorophores

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: Prof. Anees Ahmed Siddiqui, Professor of Pharm.

Chemistry, JamiaHamdard University, New Delhi


Description of Module

Subject Name Analytical Chemistry / Instrumentation Paper Name Atomic Spectroscopy

Module Name/Title Fluorophores -Special and specific Fluorophores

Module Id 13


Objectives From this module we will learn about fluorescent probes. How probes are used for study of specific functions like membrane potential DNA replication. Will also learn about other types of probes used and their application

• Chemical Sensing Probe

• Fluorogenic Probes

• Fluorogenic reagents for amines

• P-Galactosidase activity in cells

• Structural Analogues of Biomolecules

• Viscosity Probes and GFP

Keywords Fluorescent probes, Membrane Probes, Covalent labeling of membranes, DNA Probes, Chemical Sensing Probe



Fluorescent probes are molecules that absorb light of a specific wavelength and emit light of a different, typically longer, wavelength (a process known as fluorescence), and are used to study biological samples. The molecules, also known as fluorophores, can be attached to a target molecule and act as a marker for analysis with fluorescence microscopy. (Nature) Large number of Fluorescent probes have been developed for use in biological sciences to understand the functioning of biomolecules. General probes which are used non specifically, have been discussed in previous module on Fluorophores. In this section we shall be discussing about specific probes like membrane probes, DNA probes, long lifetimes probes and probes for metallic ions.

Membrane Probes

Membranes are made of lipid bilayer, proteins and some sugars. Lipids are long chain of fatty acids with one polar head. The lipid bilayer in a cell membrane are arranged in such a way that hydrophilic part is on the outer and inside of the cell whereas hydrophobic part faced to each other. Lipids are amphiphilic in nature and consist of a hydrophobic chain and a hydrophilic head group region. That means in principle there are two regions within the molecule to which fluorescent dyes can be covalently coupled. The majority of dyes those emitting in the visible light region used are hydrophilic.

Figure 1: Structure of Phospholipid molecule constituting the biological membrane


When they are attached in the chain region, the dye will change the hydrophilic/hydrophobic balance of the lipid molecule. Head group labeled lipids can also be prepared when properties of the chain regions are important, e.g., for phase determination in biological membranes.

Figure 2: Fluorescence approaches commonly used in biological membranestudies (Adapted fromA Kyrychenko, Methods Appl. Fluoresc. 3 (2015) 04, 2003)

Membrane Probes

Membrane Probes are used to study the lateral diffusion, fluidity, positioning of lipids and proteins in the lipid bilayer, and surface potential of membranes. It can be done by partitioning of water insoluble probes into nonpolar part of the membrane. Lateral diffusion is studied by excimer-forming fluorophores (dye– dye interaction) such as pyrene.Fluorescein is one of the fluorophore, used for studying lateral diffusion.

DPH (1,6-diphenyl-1,3,5-hexatriene) one of the most commonly used fluorophore serves as a sensor of the disordering of fatty acyl chains. The mobility of DPH within the lipid bilayer restricts the detection of its exact location and as a result limits the interpretation of data.

Addition of DPH to a membrane suspension results in complete binding, with no significant emission from DPH in the aqueous phase. All the emission from DPH is then due to DPH in


the membrane environment.DPH has often used as partitioning probe as it can be localized near membrane –water interface by attachment of the trimethylammonium group to one of the phenyl ring.

Covalent labeling of membranes Covalent attachment to lipids

• Useful with water soluble probes like Fluorescein and Rhodamine

• Localized by attachment to long acyl chain of phospholipid

• Depending on chemical structure of probe, fluorescent group can be positioned either on the fatty acid side chain (Fluorenyl – PC) or

• Membrane water interface (Texas Red – PE)

• Texas Red – PE used for its long wavelength and photostability Texas red Phosphatidylethanolamine

Figure 3: Fluorophores for covalent labeling of biomolecules


Figure 4: Structure of some other probes

• Pyrene-labeled-lipid probes

– Determine coefficient of the lateral diffusion within fluid lipid membranes, – utilizing their ability to form excited dimers or excimers .

• The excimer formation rate is a very sensitive measure of changes in membrane fluidity which is an important regulator of membrane functional proteins.

• Emission spectra id highly dependent on temperature

Membrane Potential Probes

Membrane Potential Probes are probes which are sensitive to difference in electrical potential across the membrane. They are useful for the tracking and imaging of membrane potential across excitable cells. Membrane potential is a result of differences in K+, Na+, and Cl- concentrations inside and outside the cells and is controlled by active transport processes.


These probes can be used to study important cell signaling processes, neurobiology, and muscle contraction processes. Microelectrodes can be applied, but their spatial resolution is limited and sampling requires a certain time. But use fluorescent probes has helped to overcomes these limitations.Potential sensitive probes differ in their response time and amplitude of signal. Other probes like fast-responding probes e.g. styrylpyridinium (signal amplitude is weak); Slow responding probes (signal amplitude is strong) have been developed according to the need

• Figure 5: Structure of some Examples of Probes sensitive to electric potential of membrane

Mechanism: A number of mechanisms are thought to be possible including

• Partitioning of dye from water to membrane or

• Reorientation of dye in the membrane or

• Aggregation of dye in the membrane or

• Inherit sensitivity of dye to electric field

• Carbocyanine dye respond to – Potential by partitioning or – Aggregation in the membrane

• Stryryl dye respond to

– Electric potential directly

• Merocyanine dye responds to membrane potential by both mechanisms.


DNA Probes

DNA is weak or nonfluorescent for practical purposes however number of probes are available to study the functioning and structure of DNA. DNA probes attach spontaneously and enhance emission and one of the most widely used DNA probe is Ethidium bromide (EB). Ethidium bromide is weak fluorescent in water with lifetime of 1.7ns but when bound to double helical DNA 30 times increase in fluorescence with lifetime of 20ns could be observed. The mode of binding of Ethidium bromide is intercalation of the planner aromatic ring between the base pairs of double helical DNA.

Figure 6: Structure of Ethidium bromide


Figure 7: Representation of Attaching Florescent Probe to DNA(Adapted from www.motifolio.com)

The DNA of interest extracted and digest with a restriction enzyme such as EcoRI or Hind III which cuts DNA at specific sites or positions. Separation of DNA fragments of different sizes is achieved by running on an agarose or polyacrylmide gel electrophoresis. Then isolate DNA of specific fragment from a particular band identified through southern blots by hybridization with specific labeled mRNA or cDNA molecules. This is followed by cloning of DNA in a vector and then allow chimeric vector to infect bacteria for multiplication where it can make billions of copies.

Other probes used DNA probes Acridine orange

Acridine orange is useful for determination of cell cycle. This probe interacts with DNA by intercalation. The excitation maximum of Acridine orange is at a wavelength of 502 nmand emission maximum is at 525 nm

Figure 8: Structure of Acridine orange and DAPI

DAPI (4',6-diamidino-2-phenylindole) is another probe used for DNA studies. It binds strongly into the minor groove of DNAand can pass through an intact cell membrane. It can be used for study of live as well as fixed cell systems. The absorption maximum of DAPI is at a wavelength of 358 nm and emission maximum is at 461 nm. The fluorescence of DAPI is highest when adjacent to A-T rich regions of DNA.

Other probes used include Hoechst 33342, Hoechst 33358, Ethidiumhomodimer 528/617, TOTO-1 etc.

Hoechst 33358 binds to specific base-pairs sequences in DNA. Ethidiumhomodimer is a high affinity dye of known DNA probes, TOTO-1 is an elongated positively charged dye and such dyes remain bound to DNA during electrophoresis and thereby increase the sensitivity of the DNA detection in the experiment.


Hoechst 33342

Ethidium homodimer 528/617

Figure 9: Structue of Hoechst 33342, Ethidiumhomodimer 528/617, and TOTO DNA Base Analogues

As we know the native bases of DNA are non fluorescent therefore are not useful in assays making use of external probe necessary. This problem can be overcome by use of DNA base analogues which make DNA fluorescent . DNA base analogues like 2-Amino purine (2-AP) an analogue of adenine, isoxanthopterine (IXP) an analogue of guanine. 2-Amino purine has a high quantam yield in solution and has single exponential decay time of 10ns. The 2-amino purine (2-AP) is sensitive to environment and is therefore suitable for studies of DNA conformation and dynamics. Fluorescence of 2-amino purine is partially quenched whenattached to double stranded DNA but this does not affects its potential for use as a DNA probe.

Another DNA base analogues is isoxanthopterine (IXP) which is also partially quenched when attached to double stranded DNA like 2-amino purine. But isoxanthopterine (IXP)


shows more fluorescence when in dinucleotide. Because of this property it is used in assay for integration of HIV DNA into host cell genome.

Figure 10: DNA Purine bases and their fluorescent analogues

Figure 11: Emission spectra of IXP nucleotide in a dinucleotide and oligonucleotide (Adapted from Principles of fluorescence Spectroscopy by J R Lakowicz)

Chemical Sensing Probe

Detect spectroscopically silent substances such as Cl-, Na+., or Ca2+ is very often desirable in various biological conditions. This is possible using sensing probes, some of which are shown in below figure.

• The probe MQAE is collisionally quenched by chloride according to the Stem- Volmer equation allowing the chloride concentration to be estimated from the extent of quenching

• Other probes allow measurement of free Ca2+, Probes such as Fura-2 display Ca2+- dependent spectral shifts.


• Such probes are called wavelength-ratiometric probes because the analyte (Ca2+) concentration can be determined from a ratio of intensities at different excitation or emission wavelengths.

Chemical sensing probes and their spectra


Figure 12: Structure of chemical sensing probes on left and their spectra on right (Adapted from Principles of fluorescence Spectroscopy by J R Lakowicz)

Calcium Green display a Ca2+- dependent increase in intensity but no spectral shift.

Wavelength-ratiometric and non-ratiometric probes are known for many species including H+, Na+, K+, and Mg2+, amines, and phosphate.These dyes typically consist of a fluorophore and a region for analyte recognition, such as on azacrown ether for Na+ or K+, or a BAPTA group for Ca2+. Such dyes are most often used in fluorescence microscopy and cellular imaging, and are trapped in cells either by hydrolysis of cell-permeable esters or by microinjection.

Fluorogenic Probes

Fluorogenic Probes are dyes that are non- or weakly fluorescent until some event occurs,such as enzymatic cleavage. 7-Umbelliferyl phosphate (7-UmP) is nonfluorescent as the phosphate ester, but becomes highly fluorescent upon hydrolysis. 7- UmPis used to measure the activity of alkaline phosphatase. This enzyme is often used as the basis of enzyme linked immunoadsorbent (ELISA) assays, and is also used in enzyme amplified DNA assays.

Figure 13: Structure of some Fluorogenic Probes(Adapted from Principles of fluorescence Spectroscopy by J R Lakowicz)

Measuring P-Galactosidase activity in cells

• It is often important to measure P-galactosidase activity in cells.


• This enzyme is often used as a gene marker in cells.

• Its activity can be detected by a galactoside of umbelliferone or 7-hydroxy-4- methylcoumarin

• An improved probe is shown in the lower panel.

• This fluorescein derivative contains a fatty acid chain that serves to retain the probe at the site of hydrolysis.

• This allows the cells with P-galactosidase activity to be identified under a microscope.

• Another class of fluorogenic reagents are those that are

• Initially nonfluorescent, and become fluorescent upon reacting with amines .

• While they have been used for labeling proteins, they are more commonly used in protein sequencing, determination of protein concentration, or for detection of low molecular weight amines in chromatography.

Fluorogenic reagents for amines


Figure 14: Structure of some Fluorogenic reagents for amines Structural Analogues of Biomolecules

Another approach used to design fluorophores involves making of shapr similar compounds to the parent biomolecule.Two examples such examples include Cholesterol and estradiol . Cholesterol an essential part of the cell membrane is as such are nonfluorescent. But their synthesized structural analogues display useful fluorescence. Dehydroergosterol the structural analogues of cholesterol displays absorption and emission maxima near 325 and 390 nm, respectively. Dehydroergosterol has been used as a probe for the interactions of steroids with membranes.

Fluorescamine MDPF




Figure 15 Nonfluorescent steroid cholesterol and estradiol, and fluorescent analogues dehydroergosterol and I ,3-diaza-9-hydroxy-5,6,11,12-tetrahydrochrysene

Viscosity Probes

Viscosityprobes are as a subclass of the TICT (Twisted Intramolecular Charge Transfer) probesthat distort in the excited state to form twisted intramolecular charge transfer states. In a highly viscous environment the molecule cannot distort as needed for charge transfer, and the decay is radiative and Vice versa. Ina less viscous environment the molecule displays internal rotation and charge transfer, which results in radiationless decay. Therefore the quantum yield depends on solvent viscosity. These probes have been used to study the viscosities of membranes, and the rigidity of binding sites on proteins. However there are probes whose Q Y depends only weakly on the viscosity of the medium e.g.

Diphenylhexatriene (DPH)For DPH the viscosity is determined from the anisotropy.

Figure 16: Fluorescence emission spectra and relative quantum yields of CCVJ (9-(2- carboxy-cyanovinyl)julolidine) in ethylene glycol/glycol mixtures of varying viscosity (Adapted from: Principles of fluorescence Spectroscopy by J R Lakowicz)


Green Fluorescent Proteins

An important and peculiar addition to the library of probes has been the Greenfluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria.The bioluminescence of the primary photoprotein aqueorin is blue. The bioluminescence from the jellyfish is green due to a closely associated green fluorescent protein. GFP contains a highly fluorescent group within a highly constrained and protected region of the protein. The chromophore is contained within a barrel of P-sheet protein. The remarkable feature of the GFP is that the chromophore forms spontaneously upon folding of the polypeptide chain without the need for enzymatic synthesis. As a result, it is possible to express the gene for GFP into cells, and to obtain proteins which are synthesized with attached GFP& it is even possible to express GFP in entire organisms.

Fig 17: Barrel structure of GFP. Side and top view.The chromophore is linked covalently to the protein. (Adapted from: Principles of fluorescence Spectroscopy by J R Lakowicz)


Figure 18: Spontaneous formation of the Fluorophore in GFP by the serine-tyrosine- glycine residues (Adapted from: Principles of fluorescence Spectroscopy by J R Lakowicz) GFPs with different spectral properties have been created by introducing mutations into the amino-acid sequence. Mutants are known that display longer absorption and emission wavelengths and have higher photostability.GFPs have good photostability and display high QY which is probably because the P-barrel structure shields the chromophore from the local environment. Earlier it was thought that GFP was only present in Aequorea victoria. It is known that similar naturally fluorescent proteins are present in a number of Anthrozoa species, in corals. A large number of fluorescent proteins are now available with emission maxima ranging from 448 to 600 nm because of the variety of fluorescent proteins the terminology has become confusing. E.g. The fluorescent proteins from coral are often referred to as yellow (YFPs) or red (RFPs) fluorescent proteins. However, this is a misnomer because green fluorescent proteins can also come from coral. It has been suggested that proteins derived from Aequores victoria be called AFPs to indicate their origin with this jellyfish. For simplicity all these proteins are called as GFPs


In this module we learned about Fluorescent probes….. Introduction. How probes are used for study of specific functions like membrane potential DNA replication. Will also learn about other types of probes used and their application. How different substance can be made useful in analysis by fluorescence. How change in Viscosity can be used for study of biological systems.Also learned about natural Fluorescent proteins like GFP.



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