Objectives
- Perform pressure tests on prepared hydrogels with Rhodamine 6G added.
- Prepare microspheres using a new preparation method.
Microsphere preparation
- After viewing the microspheres prepared on 2013/02/15, it appears as if all prepared microspheres formed a layer at the bottom of the beaker, creating a textured film as opposed to microspheres. Complete separation of the aqueous and organic layer occurred. The emulsion and suspension of the microspheres did not occur, perhaps due to the high freezing point of mineral oil. As such, a new procedure was used for the preparation of microspheres. This procedure was adapted from [1].
General Protocol:
- Measure out the desired mass of PVA and clay. The total mass should not exceed 1g.
- Place the PVA and clay in a clean, 50mL beaker with a magnetic stir bar.
- Add ~25mL distilled H2O to the beaker. While stirring, heat the contents of the beaker to at least 100°C and allow the PVA/clay to completely dissolve. (Allow the contents to heat for at least 1 hour. If necessary, more distilled H2O may be added. The exact amount of distilled H2O should be recorded for future Rhodamine 6G addition).
- After complete dissolution of PVA/clay, remove the magnetic stir bar and pour solution into a blender.
- Add at least 35mL safflower oil to the blender. If more than 25mL distilled H2O was added to the PVA and clay, then the same volume increase should used in the addition of safflower oil.
- Blend the safflower oil and PVA/clay solution on high for 5 minutes.
- Quickly pour the contents of the blender into 20mL glass vials.
- Quickly freeze the PVA/clay and safflower oil emulsion in liquid nitrogen. The vial should be held in the liquid nitrogen until the sample is completely frozen throughout. If the sample has been sitting for a short period of time and it appears as if the microsphere particles are separating out of the organic layer, use a sonicator prior to freezing the sample to ensure that the microsphere particles are frozen in the organic layer.
- Place the frozen samples in a freezer at -20°C for 24 hours. After 24 hours, remove the samples from the freezer and allow the samples to thaw for 24 hours.
- Repeat step 9 three times.
Note:
Extreme Caution: Liquid nitrogen is extremely cold. Extra care should be used when handling liquid nitrogen.
Prepared Microspheres:
- 90:10 PVA MW 146,000-186,000:Lamponite microspheres were prepared. The sample was dissolved in 35mL distilled H2O and 45mL safflower oil was added.
- Due to the dyes affinity for the organic layer, the Rhodamine 6G will be added at a later date.
Hydrogel Pressure testing
- After performing diffusion tests on all prepared hydrogels on 2013/02/15, it was determined that the hydrogels prepared with DMSO and dye added prior to the freeze thaw method did not leak dye at a fast rate. As such, pressure tests will be performed using these hydrogels because all collected dye will be caused solely by sheer pressure.
- First, the most effective pipette for pressure testing was determined.
General Protocol:
- A 9 in. Corning disposable, non-sterile Pasteur pipette was heated using a Bunsen burner and was bent in various ways(multiple bends at the stem of the pipette and the top, bends only at the top, etc.). Note: Pictures of bent pipettes will be uploaded for reference.
- Using a razor blade, the hydrogel for testing was cut into small cubes. The mass of each tested sample should be approximately 0.1g. If the hydrogel was too firm for safe cutting, the hydrogel was placed in 3mL distilled H2O for approximately 20 minutes. The time in distilled H2O can not exceed 2 hours(definitive amount of time that was determined where minimal leakage of dye occurred).
- 3mL of distilled H2O were added to the pipette and using a ribber bulb squeezed out of the pipette. The 3mL of H2O were collected in a beaker. Note: When adding the distilled H2O, the tip of the pipette should be completely covered with a finger or a hand to prevent any H2O from going straight through the pipette.
- Fluorescence was then run on the collected sample.
- The same hydrogel sample will be used to test the various Pasteur pipettes. As such, the sample with the largest amount of fluorescence will indicate the most effective manner in which to bend the Pasteur pipette to create the most sheer pressure.
Pressure samples:
| 90:10 PVA MW 146,000-186,000:110% Lamponite hydrogel pressure tests
|
'
|
|
|
| Shape of pasteur pipette |
Amount of hydrogel used(g)
|
| Plain pasteur pipette. No modifications |
0.1575
|
| Stem of pipette bent at angle slightly less than 90 degrees |
0.1134
|
| Stem of pipette bent at angle slightly less than 90 degrees right at the start of the stem of pipette |
0.1397
|
| Pipette with three pockets at the top of the pipette |
0.1039
|
| Pipette bent twice in the stem |
0.1158
|
| Pipette bent at the top of the pipette at close to 90 degree angle. Sides of pipette almost touching |
0.1108
|
| Pipette with a twist in the middle of the pipette |
0.0993
|
| Pipette bent twice in the middle of the pipette |
0.1416
|
| Pipette with two pockets in the top portion of the pipette |
0.1041
|
| Top bent so sides almost touching at 90 degree angle |
0.1258
|
| Plain pasteur pipette. No modifications. Run #2 |
0.121
|
Figure 1. Spectra for the pressure testing of 90:10 PVA MW 146,000-186,000:110% Lamponite
- It appears as if using an unmodified pipette or a pipette with a bend at the top of the pipette so the sides are almost touching produced the maximum amount of sheer pressure. Further testing will be done for verification.
- In comparison to the 0.25μM Rhodamine 6G control, this method of sheer pressure does not perform a significant amount of sheer pressure or does not elicit the diffusion of a significant amount of dye. Another method to test the sheer pressure will be considered.
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Project name
