Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

We present the design of phosphorescent probes for oxygen based on platinum and palladium porphyrin DERs. The probes consist of metal porphyrin cores encapsulating dendron and peripheral hydrophilic polyethylene glycol layer construction of the probes and their calibrations will be illustrated in this paper. Hi, my name is Luis Sinks.

I work with Professor Sergei OV here in the Department of Biochemistry and biophysics at the University of Pennsylvania. I am Emmanuel Losis, also from the Vina. I'm fat also from the, And I'm Sergei V.Biological Oxygen measurements by phosphorous and quenching make use of exogenous phosphorous and probes, which are introduced directly into the medium of interest.

Blood or interstitial fluid probes are the only invasive component of the measurement scheme requiring special attention to their design. Today we'll show you a procedure for the synthesis and calibration of dendritic phosphorescent nano probes for oxygen measurement and biological systems. We use this procedure to synthesize and characterize probes For semi and two-fold oxygen measurements.

So let's get started. Before we begin constructing probes, let's first go over the basic theory behind probe phosphorescence. Phosphorescence originates from the long lived triplet state.

The probe molecule must be designed to give high quantum yield of the triplet state and to emit phosphorescence instead of fluorescence. Excitation of the probes occurs by the one photon or in the case of special two photon enhanced probes by the two photon mechanism. One photon excitation generally provides less spatial resolution, but requires simpler instrumentation and can be used in single point measurements with LED based fiber optic phos perimeters.

While in the triplet state, the probe can experience collisional encounters with molecules of oxygen, which can deactivate the triplet state a process called quenching. Therefore, in the presence of oxygen, the phosphorescence lifetime is shortened. As a result of the quenching, the dependence of the lifetime of the triplet state on the amount of oxygen in the environment is characterized by the stern volmer equation.

In in vivo experiments, the probe is delivered into the blood or interstitial fluid of an animal, and the surface of the tissue is illuminated with light of an appropriate wavelength to bring the probe into Its excited triplet state. In order to measure the phosphorescent lifetime, the emitted phosphorescent photons are bi in time. For example, after the excitation pulse, 3, 609 photons might be collected in the first five microseconds, 1, 421 photons collected in the next five microseconds and so on until no photons are collected.

The numbers in the bins plotted against the time give the phosphorescence decay, which is analyzed to yield the phosphorescence lifetime. In imaging, this procedure is applied to every pixel of the image resulting in phosphorescent lifetime maps. Measurements of lifetimes are insensitive to the heterogeneities of probe distribution throughout the object, which is common for biological samples.

Now let's see how to construct probes. Begin the synthesis procedure by adding an aromatic aldehyde to a 0.01 molar solution of tetra hydro iso indole. Stir the reaction mixture for 10 minutes in the dark at room temperature.

Then add boron, tri fluoride dathyl, ate, and keep stirring for an additional two hours. Next, add dichloride cyan, benzo quinone or DDQ, which results in the color. Change from pale red to deep green and leave the mixture overnight under continuous stirring the next day, wash and dry the solution, and then concentrate it in a vacuum.

Reg crystallization of the residue gives the target as a green powder. Yields are typically around 50%Next, treat the freebase porphyrin with palladium acetate. Monitor the conversion by UV vista spectroscopy.

The conversion is complete after the soap band of the Dion between 468 and 472 nanometers disappears. The porphyrin is isolated by column chromatography on silica gel. To prepare palladium tetra benzo porphyrin oxidize the palladium tetra cyclo porphyrin.

During reflexing, the color changes from dark red to deep green evaporate the solvent, dilute the residue with di chloro methane wash, dry and concentrate the organic phase in a vacuum. After chromatography on silica gel isolate palladium tetra benzo porphyrin as a bluegreen powder. Next hydrolyze the peripheral etro groups of the palladium trab benzo porphyrin.

First treat the tetra benzo porphyrin Esther with base in tetra hydro purin. Then continue the hydrolysis and aqueous base, precipitate the porphyrin by addition of hydrochloric acid and dry it in a vacuum. This completes the synthesis of the porphyrin.

Now let's see how to synthesize entrons. Before the probes are assembled, the entrons, which are the branches of the Denmark need to be pre synthesized. We use aero gly, same DERs, which can be conveniently prepared from inexpensive studying materials using chromatography free methods.

Therons with amino groups at their focal points are then attached to the caril groups on the phy, which we just showed you how to make. Then the ester groups on the periphery of the DME are hydrolyzed similarly to the carboxylic groups on the periphery of the porphyrin. At this point, starting from the Porphyrin DME poly carboxylic acid, either one or two photon probes can be synthesized to synthesize the two photon probes.

First divergently attach a few two photon antenna fragments to several carboxyl groups on the demer periphery. Now proceed with modification of the remaining carboxylic acid residues on the demer. Start by adding a 1.25 fold excess of HBTU to a highly concentrated solution of the porphyrin rimer and stir the reaction mixture at room temperature for 10 minutes.

Now, add di isopropyl ethylamine and methoxy polyethylene glyco lamine. Stir the reaction mixture for two days at room temperature and then add ethyl ether. Separate the form to precipitate by centrifugation and re precipitate it from tetrahedran a few times by adding dathyl ether.

Finally, purify the probe by size exclusion chromatography on polystyrene beads using tetrahedran as a solvent. Now let's look at probe characterization and calibration. The absorption and emission spectra of the probe are obtained using one micromolar probe solutions under ambient conditions with a standard spectrophotometer and steady state fluorimeter.

Next to obtain the stern Ulmer calibration plot, which allows us to relate the lifetime of the probe to the oxygen concentration, place a solution of the probe into a special cylindrical vete. The vete is positioned inside a temperature controlled chamber inside a light impermeable cage with ports for excitation and emission optical fibers. Close the vete with a stopper, which has an inserted highly sensitive Clark type oxygen electrode.

The stopper also has two needle ports for inlet and outlet of Argonne. Set the temperature to 36 to 37 degrees Celsius and leave the solution stirring until it reaches equilibrium. Connect the excitation fiber to the excitation pour of a digital phospho perimeter controlled by a pc.

The light source and the phos perimeter is a high power LED, whose output is controlled by a 333 kilohertz digital to analog board. The emission fiber is connected to another optical port of the phos perimeter, which is coupled to an infrared sensitive avalanche photo diode. The output of the diode is amplified and fed into the ad channel of the same control board, allowing synchronization between the excitation and emission channels.

The home written control software generates excitation pulses of any desired length, followed by the collection of phosphorescence decay. The output of the oxygen electrode is amplified and directed into another analog digital board on the same pc. This is a low frequency board, one kilohertz max, which is used to record the electrode current at selected time points, typically 10 times a second.

Once the solution temperature is equilibrated, both the phos perimeter and the electrode programs are initialized at the same time. To perform measurements every 10 seconds, their outputs are logged synchronously into two separate files. After that, Argonne is connected to the inlet port on the vet stopper.

As Argonne flows over the surface of the stirred solution, it gradually replaces oxygen. This results in a decrease of the electrode current and an increase in the phosphorus lifetime, which is measured by the phos perimeter. Usually oxygen is displaced from the solution entirely.

After about two hours after the titration run, the electrode data and the phosphorus lifetimes are imported into a standard analysis program, which creates a plot of the inverse phosphorus lifetime versus oxygen partial pressure. This plot is fitted with a straight line using the least squares method to give the oxygen quenching constant as its slope. Phosphorescent lifetime is obtained either from the same fit or directly from the measurement at zero oxygen.

The titration can be repeated using a solution of the probe in the presence of albumin, a protein present in the blood plasma in order to emulate the conditions met in the blood of an animal in vivo. The obtained stern Ulmer plots should be identical if the ER is protecting the probe. Well, and the peg groups isolate the probe from contact with albumin.

The suggestion here, selected steps in the synthesis of oxygen, then treated probs and their calibration. When doing the calibration. It's important to make sure that the conditions are as close to that of the biological system of interest.

It's important to make sure that the probe molecule is unaffected by biomolecules, such as albumin. So that's it. Thanks for watching and good luck with your experience.