Week Seven Progress

This week, the group's main focus was to create samples of hydrogel for testing. As we set out to test absorbance values, we developed a procedure that would allow us to create several samples of gel over a long period of time.

Table 1, below, was used to organize and label the samples during their preparation.

Table 1: Labeling Method Used for Preparing Samples

Table 2: Volumes Required for Mixture in Step 1

Procedure 1:

Mixing the Alginate Solution

1. In a 150-mL beaker, mix the volumes of water and sodium alginate required for the solution you are making, found in Table 2. (This step must be done for each of the 3 sodium alginate solutions. Then, the volumes for the 7% and the 2% solutions must be halved and mixed separately, only to be combined into a test tube at the end of this procedure).
2. Add .01 mL of FITC-BSA to the sodium alginate solution created in step 1.

Mixing the Calcium Chloride

3. Mix 25 mL of water and 5 mL of 30% calcium chloride into a beaker or large test tube. This can be used until it runs out, and if more is needed one can repeat this step.

Creating the Gel

4. Pour a thin layer of the 5% calcium chloride solution created in step 3 into a soap mold. Pour just enough so that the bottom of the soap mold is completely covered. 

5. Pour the sodium alginate solution prepared in step 2 into the soap mold containing the calcium chloride. It is best to do this using a thin hydrophobic surface that matches the width of the soap mold (for example a playing card). Start at one end of the soap mold and pour the solution over the playing card, trying to spread it evenly across the width. Then, move the playing card back slowly along the length of the soap mold as you pour in the solution. This will allow the gel to form in a thin sheet, rather than in clumps of spheres. 

Preparing the Sample

6. Remove the hydrogel created in step 5 from the soap mold, and place it into a test tube. Ensure that there is no extra liquid in the test tube. Cap it, and label it as shown in the Table 1. 

After following this procedure through the night (as it required lengthy intervals that would not allow us to do it during the day when we had classes), the group was excited to deliver the resulting samples to the lab and measure their absorbances. However, we were disappointed when we reached the lab and found out that the samples would not work. We had made 2 errors in Procedure 1 that would render our samples useless. These errors were:

1. We needed to use pure water as opposed to the tap water we had been using. The gel in our samples was not uniform because of impurities in the tap water, and this could cause our results to be obscured. 

2. Instead of delivering gel samples to the lab, we really needed to deliver the supernatant of 1-mL samples of gel that had been soaking in 10-mL of water since the time they were created. 

With this new information, the group set off to create the samples again, this time using the volumes in Table 3 and the steps in Procedure 2.

Table 3: Revised Volumes Required for Samples. Note that "Water" is pure water for this procedure.

Procedure 2:

Mixing the Alginate Solution

1. In a 150-mL beaker, mix the volumes of water and sodium alginate required for the solution you are making, found in Table 3. (This step must be done for each of the 3 sodium alginate solutions. Then, the volumes for the 7% and the 2% solutions must be halved and mixed separately, only to be combined into a test tube at the end of this procedure).
2. Add a tiny speck of FITC-BSA to the sodium alginate solution created in step 1. This speck should be about the size of the tip of a mechanical pencil.

Mixing the Calcium Chloride

3. Mix 25 mL of pure water and 5 mL of 30% calcium chloride into a beaker or large test tube. This can be used until it runs out, and if more is needed one can repeat this step.

Creating the Gel

4. Pour a thin layer of the 5% calcium chloride solution created in step 3 into a soap mold. Pour just enough so that there is a small circle, about 3 cm in diameter, of solution in the mold.

5. Pout the sodium alginate solution prepared in step 2 into the soap mold containing the calcium chloride. Because the volume of sodium alginate solution is so small, materials such as a spoon or stirring rod may need to be used to ensure that all of the solution has been transferred to the soap mold.

Preparing the Sample

6. Remove the hydrogel created in step 5 from the soap mold, and place it into a test tube.

7. Add 10 mL of pure water to the test tube, and label it according to Table 1. 

Collecting the Supernatant

8. Around 3 hours after the last batch (Round C) of samples is created, pipette about 0.5 mL of the liquid in the test tube, and transfer it to another test tube, labelled the same way. 

After following this procedure through a second night, the group was finally able to turn in some samples. We are currently awaiting the results. We predict that the results will show higher absorbance values for corresponding solutions in the A round than the C round, because the gel from A round has had a longer time to sit, and the protein has had a longer time to seep through the pores of the gel and diffuse into the supernatant. We also predict that of solutions in the same round, the solutions with the higher concentrations of sodium alginate will have lower absorbance values than those with lower concentrations, because the higher concentration gels have smaller pores and it was therefore harder, and took longer, for the protein to seep through.