The overall goal of the following experiment is to monitor ligand receptor interactions with fluorescence resonance energy transfer. The method presented here utilizes an interesting property of PDA BLUESHIFT in the UV vis electronic absorption spectrum of PDA. After an analyte interacts with receptors attached to PDA first polymer D acetylene or PDA is synthesized, and PDA liposomes are prepared.
Next R domine tagged BSA is bound to the PDA liposome surface. When fluorescence resonance energy transfer is then measured. The emission spectrum of liposome bound BSA Rod Domine overlaps with the absorption spectrum of PDA.
This satisfies the resonance requirement for the fret mechanism. Thus, data can be obtained that indicate interactions between ligands and receptors. The main advantage of this technique over the existing methods like Eliza is this method is very simple and inexpensive.
Since fluoresce is intrinsically more sensitive than calorimetry, we can have the detection limit at very low sub nano molar or lower concentrations. The implication of this technique extends toward bacterial and viral sensing because it can selectively sense those microbes at very, very low concentrations. We first had the idea of this method when we found the effectiveness of threat visit PAL line sensors in 2010.
Let's say we have two molecules, donor and acceptor, and they both molecules are coupled to each other. Then when we give some amount of energy to the donor, donor gets excited from ground state to the excited state, and then this energy gets transferred to the acceptor. So the acceptor in the ground state accepts this energy and gets excited in the fluorescence resistance energy transfer.
The same thing happens, but in terms of fluorescence, where the donor is excited, but the energy gets transferred to the acceptor and acceptor is the one that gives fluorescence. It's like you are having two bulbs together where you give the electricity to one of the bulb, but the other bulb blows without any electricity. Here we want to show the distance dependence of fret.
Fret only works when donor and acceptor are very close to each other, which is about 10 nanometers. Let's excite the donor molecules here, but as you can see, the acceptor molecule is far away. So even if we give some amount of energy to the donor, it does not get transferred to the acceptor molecule.
So F Fred doesn't work. So F Fred is distance dependent. An hydroxy CIN aide, D acetylene, or N-H-S-P-C-D-A is an essential component in the preparation of liposomes.
Throughout this procedure, the PDA solution should be protected from light. Using aluminum foil wrapping on every container, begin the synthesis in a chemical fume hood. In a round bottom flask combine 10 12 Pentecostal diic acid, end hydroxy CIN amide, and one three dimethyl amino propyl.
Three ethyl carbimide hydrochloride in a final volume of 20 milliliters of dry chloro methane in a round bottom flask. Stir the solution at room temperature for two hours. Make sure that the flask is tightly closed.
Then transfer the flask to a rotary evaporator to remove the solvent. Once the solvent has been removed, a dry thin film will remain under a fume hood. Add a solution consisting of 25 milliliters of dathyl ether and 25 milliliters of water to the round bottom flask and gently swirl for about a minute.
To reconstitute the residue, transfer the solution to a separating funnel and invert several times to mix. Once the ethyl ether and water layers separate, open the spigot to remove the aqueous layer to a beaker. Next, add 25 milliliters of water to the separating funnel and mix.
Repeat the extraction process as before for a total of three extractions. Combining the organic layers from each extraction following the last extraction, dry the organic layer with one gram of magnesium sulfate for half an hour. Using watman filter, grade filter paper, filter the solution.
Then remove the ethyl ether by rotary evaporation to obtain a white solid powder of N-H-S-P-C-D-A. Next, transfer two to three milligrams of powder to deuterated chloroform in an NMR tube. Analyze the N-H-S-P-C-D-A using nuclear magnetic resonance as shown here.
New NMR peaks are seen at 2.842. To prepare in a round bottom flask dissolve PCDA to P-C-D-A-N-H-S to one two di aristo SN glycerol three phosphocholine eight to one to one in 20 milliliters of di chloro methane while stirring. Next, place a watman filter paper inside a funnel and pour the solution through to remove aggregates.
Collecting it in a round bottom flask. Transfer the flask to a rotary evaporator, then evaporate the solvent completely to yield a thin film of monomers. Using a vacuum dry a thin film overnight.
The next day, add 50 milliliters of deionized water to make a liposome solution at 0.65 to one millimolar. Then transfer the solution to a new 50 milliliter beaker and sonicate the resultant suspension with a probe ator at 76 degrees Celsius for about 18 minutes. Carefully pass the solution through a watman filter paper in a funnel to remove the lipid aggregates and collect it in a flask.
Place the solution at four degrees Celsius overnight to promote self-assembly of the monomers. The next day solutions should be optically clear polymerize the self-assembled dia acetylene monomer liposomes by irradiation with 254 nanometers of UV radiation for about two minutes. Using a pen ray UV source in air, the transparent solution will turn blue after exposure to UV light.
The liposome solution will be stable at room temperature for at least two weeks and will be more stable if refrigerated. To bind BSA rod domine to the liposome surface dissolve BSA rod domine in PBS to a final concentration of 1.2 micromolar of BSA Rod Domine solution, add two milliliters of BSA rod domine to 10 milliliters of liposome solution prepared in the previous section at room temperature. A classical reaction for the binding of amine groups from the lysine residue of proteins to carboxylic acid activated by NHS group has been followed.
N-H-S-P-C-D-A was designed for covalently binding protein molecules with liposomes using NHS Amin reactions. NHS is an excellent leaving agent that drives the Amin carboxylic acid reaction in the forward direction. The yield of this reaction under appropriate conditions should be quantitative.
To remove free BSA Rod Domine soak a spectra pore, biotech cellulose ester membrane with a molecular weight cutoff of 100, 000 in deionized water for 15 minutes in a one liter beaker to perform dialysis to eliminate unreactive BSA Rumine, which has a molecular weight of about 66, 000. Daltons carefully transfer the solution to the dialysis membrane and submerge it in 800 milliliters. Deionized water in the dark, change the water at 2 8 14 24 and 36 hours after 48 hours, transfer the final solution to a vial covered in aluminum foil.
Store the vial at four degrees Celsius to monitor protein binding using fret BSA. Rod Domine tagged liposomes before and after polymerization were analyzed with UV V and fluorescent spectroscopy as can be seen here for the isolated donor and accepter. The fret efficiency is highly dependent on donor acceptor distance and J.The quenching in the emission is observed because of fret between r domine and PDA due to the appearance of electronic absorption.
Spectrum of blue PDA after photo polymerization. Here, fret efficiency is zero for un polymerized liposomes and rod domine because there is zero spectral overlap for un polymerized liposomes in the visible region. To monitor fret between rod Domine and PDA liposomes prepared as described in this video, UN polymerized and polymerized biotin tagged liposomes were analyzed using UV vis and fluorescence spectroscopy.
As can be seen here, the emission of rumine SR 1 0 1 is decreased by about 45%after polymerization suggesting emission quenching due to fret with the addition of streptavidin to the solution. The spectral overlap indicated by the J value decreases as seen here. The fret efficiency decreased with the increase in streptavidin concentration.
The R domine emission was increased after the addition of streptavidin due to the decrease in the J value for the sofo R domine emission spectrum and PDA absorbent spectrum following biotin streptavidin interactions. This suggests that at the molecular level, the biotin streptavidin interactions lead to subtle changes in the effective conjugation length of the PDA, which results in a decrease in the blue PDA form to a more thermodynamically stable red PDA form. After watching this video, you should have a good understanding of how to selectively monitor ligand preceptor interaction using threat Following this procedure.
Other methods like Eliza can be performed to answer additional questions like the selective binding of proteins.
