Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

The overall goal of this procedure is to fabricate highly magnetically responsive lanthanide ion chelating polymolecular assemblies, and to easily monitor the magnetic response with birefringence measurements. Birefringence measurements can help answer key questions in the design of magnetically responsive polymolecular assemblies, such as bicelles, by providing an alternative and comparatively easy means of monitoring alignment. The main advantage of this polymolecular assemblies is the ability to deliver an unrefined and tunable magnetic response at field strength as low as one or two tesla by associating with numbers per magnetic lanthanide ions.

Although monitoring of the birefringence seeker provides important information for the bicelles design, it can also actually apply to the study of other magnetically responsive systems, such as neuro crystalline sedolos or amyloid fibers doped with iron oxide nanoparticles. To begin the procedure, use a 2.5 milliliter glass syringe and three milliliter snap cap vials to measure the needed quantities of DMPC, DMPE-DTPA, Cholesterol, and Thulium(III)chloride stock solutions. Combine the stock solutions in a 25 milliliter round bottom flask.

Rinse each vial into the flask with 2.5 milliliter of the solvent used in the corresponding stock solution. Clamp the flask on a rotary evaporator. Remove solvent from the mixture at 40 degrees Celsius under 30, 000 Pascals, until only a viscous transparent film remains.

Reduce the pressure to 100 Pascals and continue drawing the sample for a minimum of two hours to obtain a dry lipid film. Then, flush the flask with argon for one minute. Seal the flask with paraffin film and store the sample at 18 degrees Celsius.

When ready to rehydrate the lipid, add three milliliter of 15 millimolar pH 7.4 phosphate buffer to the flask. Rotate the flask in a liquid nitrogen bath until the sample is thoroughly frozen, as indicated by the liquid nitrogen no longer boiling. Then, rotate the flask for 5 minutes in a water bath set to 60 degrees Celsius.

Once the sample is completely liquid, vortex the sample for 30 seconds. After the last freezing step, stabilize the sample with two heating-cooling cycles. First, obtain track-etched polycarbonate membrane filters with the desired pore size for the extrusion.

Begin assembling a 10 milliliter lipid extruder with a jacketed sample vessel, aided by silicone-tipped tweezers. Use a few drops of phosphate buffer to wet the support mesh and the polyester drain disk for optimal placement of the membrane filter. Carefully lay the O-ring on the membrane filter.

Ensure that there are no folds or creases on the membrane. Finish assembling the lipid extruder and connect the jacketed sample vessel to a temperature controlled water circulator. Heat the circulator water bath to 60 degrees Celsius and start circulation.

Connect the extruder to a pressurized nitrogen gas bottle using high-pressure PVC tubing and quick-connect fittings. Set the extrusion pressure to the appropriate value for the membrane pore size. Use a two milliliter glass pipette to add the sample to the jacketed vessel.

Close the vessel and allow the sample to equilibrate for 30 to 60 seconds. Place the end of the sample outlet tube in the sample flask. Then, open the flow of nitrogen to the extruder while holding the sample outlet tube steady.

Once sample extrusion has finished, vent the extruder and use the same two milliliter glass pipette to transfer the extruded sample to the jacketed vessel. Perform 10 extrusion cycles in this way. Replacing the membrane filter whenever it begins to clog.

Repeat the extrusion through smaller pore sizes as desired. Turn on and set up the birefringence measurement apparatus. Then, place the sample in a temperature-controlled quartz cuvette with a 10 millimeter path length.

Connect the cuvette to an external water bath set to five degrees Celsius. Insert a 0.5 millimeter thick k-type thermocouple into the sample through an aperture in the cap. Adjust the water bath temperature based on the thermocouple reading until the sample reaches five degrees Celsius.

Place the cuvette in the bore of the magnet. Verify that the thermocouple is not in the laser path by holding a sheet of paper in the laser path beyond the cuvette and looking for shadows cast by the thermocouple. It is important to monitor the sample's temperature directly but the probe must not be in the way of the laser light.

This is readily checked with the white paper placed in the laser path after the cuvette and looking for shadows. Apply a steady flow of room-temperature dry compressed air at 10, 000 Pascals to the cuvette, to avoid the formation of condensation. Select the amplifier sensitivity multipliers, ensuring that the amplifier is not overloaded, and no more than four red bars appear on the display.

Next, autofaze both lock-in amplifiers. The sample's turbidity may change dramatically during a temperature cycle due to polymolecular rearrangements. The highest turbidity usually occurs at low temperatures and to avoid a signal overload upon heating, the sensitivity must not be set too high at five degrees Celsius.

Fill in the sample name and the lock-in amplifier sensitivities. Enter a file name for the measurement and start logging the data. Then, ramp the magnetic field to 5.5 Teslas using the measurement program.

Acquire birefringence signal data at various sample temperatures and magnetic field strengths as desired. The birefringence signal of cholesterol doped bicelles extruded through membranes with 800 nanometer pores increased with increasing magnetic field strength, with peak birefringence achieved at 5.5 Teslas. Successive extrusions at 60 degrees Celsius through membranes with decreasing pore sizes resulted in decreasing birefringence values at five degrees Celsius.

A reduction in absolute alignment factor was also observed. These results indicated that magnetic alignment could be tuned by adjusting the bicelle sizes by extrusion of their vesicle precursors. Once mastered, this technique can be done on a daily basis to effectively screen many samples and identify the most promising candidates, delivering an optimal magnetic rease bonds of the required temperature range.

Following the successful identification of the most promising sample with this procedure, are the methods like small angle neutron scattering or cryo transmission electron microscopy can be performed to answer additional questions about the structure of the physical chemical properties of the assemblies. When working with lipid assemblies, such as the bicelles presented herein, it is important to work with clean glassware in a calm environment. We recommend dedicating glassware for this work and washing with chloroform.