The overall goal of the following experiment is to fabricate sodium nanosensors for in vitro and in vivo applications. This is achieved by preparing an opto mixture containing the sensor components for the nanosensors. As a second step surfactant is sonicated in aqueous solution to evenly distributed, which will allow nanosensor formation when the opto is added.
Next, the opto mixture is added to the aqueous surfactant mixture under sonication to form the nanosensors in solution. This video demonstrates that the sensor response is tuneable. The sensors can be micro injected into cardiac myocytes and that the sensors can also be injected subcutaneously into mice.
The main advantage of this technique over existing methods like fluorescent indicator dyes, is that we can tune the sensors to a dynamic range that matches physiological environment, measure a wider range of analytes and perform ratio metric measurements for quantitative analysis. Generally, individuals new to this method can struggle because once the sensors are made, it can seem daunting to introduce em into a biological environment. Stocks of dry sodium ion four x dry sodium TFPB and chromo four three need to be prepared in advance of making the opto according to the written protocol.
In a fume hood, combine all the materials into a 1.5 milliliter glass vial. With a Teflon coated screw cap, gently vortex the vial until all the PVC has dissolved. The solution should appear red.
Begin making the nanosensors by adding 100 microliters of 10 milligrams per milliliter. Peg lipid into a four gram glass vial. Next, add four milliliters of the desired aqueous solution, such as 10 millimolar hippies.
Sync a fornicating tip seven millimeters into the vial using a laboratory jack and apply a low sonication power for 30 seconds. In a 0.6 milliliter tube, combine 50 microliters of the opto material and 50 microliters of di chloro methane using the pipette to ensure even mixing. Next, begin a three minute sonication.
As the sonication begins, add 100 microliters of the opto mixture by immersing the pipette tip and dispensing the complete volume in one quick even injection. The solution will turn blue if the pH rises above seven, and if there is no sodium to remove the residual THF and di chloro methane from the solution. Move the sonic hitting tip to a depth of two millimeters.
As the remaining three minutes elapse, the solution will become opaque. Once Sonication is complete, pull up the Nanosensor solution into a five milliliter syringe. Attach a filter and dispense the nanosensor solution into a glass vial with a screw top cap.
Begin by making 10 solutions with progressively increasing sodium concentration against which the sensitivity of the nanosensor can be measured. Combine zero molar and one molar sodium stock solutions in 50 milliliter conical tubes. Now in an optical bottom 96, well plate add 100 microliters of each concentration of sodium to three wells in a column.
Then add 100 microliters of freshly prepared nanosensors to each well load the plate into a microplate fluorimeter and enter the excitation emission and cutoff wavelengths that are ideal for the chromophore being used. The choice of measured wavelengths depends on the application. These wavelengths provide for intracellular fast dynamics measurements or noise reduction.
To obtain alpha values, divide the ratio of intensity at 570 nanometers by the intensity at 670 nanometers or 680 nanometers. Plot the alpha values against the log of the sodium concentration. Setting the log of zero sodium concentration to minus one.
Now fit a sigmoidal curve to the response and from the curve, calculate the sodium concentration where alpha value is 0.5. This represents the sodium concentration at which the sensor responds optimally. Using this information, adjust the ratio of sodium iono four x sodium TFPB and chromo iono four three in the opto to create nanosensors with the optimal sodium concentration.
For example, hero response curves of five different nanosensor sets representing different opto formulations, varying only in the amount of sodium of four 10. For intracellular imaging, replace the cell media with a clear media like Tyro solution and transfer the cells to an imaging chamber. Now backfill a pulled glass pipette with nanosensor solution made in five milligrams per milliliter glucose in water.
Then mount the pipette to a micro manipulator and connect it to a pressure controlled injection system. Lower the pipette on top of the cell and into the cytoplasm. Sometimes it is necessary to tap the manipulator holder to pierce the membrane.
Inject a few hundred picoliters of nanosensor solution and quickly withdraw the pipette. Anesthetize a CD one nude mouse with isof fluorine using an anesthesia chamber. Once anesthetized, place mouse into disinfected imaging chamber.
Next, fill a sterile 31 gauge insulin syringe with 10 microliters of nanosensors with the appropriate KD in 10 millimolar.PBS. Make sure to remove all air bubbles using forceps. Grasp a small fold of skin along the back of the mouse at the desired injection site while grasping the skin.
Insert the syringe into the skin with the bevel facing up and the needle parallel to the skin. Now pull back on the syringe plunger to ensure the needle is not inserted into a blood vessel. To minimize the back pressure, gently move the needle right and left to create a pocket within the skin.
Then inject the sensors and gently remove the syringe. Gently apply pressure to the injection site and wipe away any solution that may have leaked out of the injection site. Typically, six evenly spaced injections are made along the back.
If performing on different mice, change the needle to minimize disease, transmission, and cross-contamination. After the injections, acquire brightfield and fluorescent images using filter sets that closely match the excitation emission spectrum of the nanosensors. This also minimizes the autofluorescence of the skin.
These neonatal cardiac myocytes were cultured on a cover glass and injected with sodium nanosensors. This fluorescent image is taken at a 639 nanometer excitation wavelength. This is a brightfield image of the same cells, and this is an overlay of the two.
There is some sensor clustering that occurs, but the cells have normal morphology. Sensors have diffused throughout the cytosol and there is no nuclear loading. This overlay of a bright field and fluorescent image shows nine different subcutaneous injections of sodium nanosensors into the subcutaneous space of a nude mouse.
Once Masten fabricating nanosensors can be done in minutes if performed properly and applied to any research to measure reversible real time analyte flu. After watching this video, you should have a good understanding of how to load nanosensors into the intracellular or subcutaneous space for quantitative measurements in a biological environment.
