Add Rhodamine B to the "After" samples synthesized on Wednesday. We are not using acetylsalicylic acid for the time being.
Create new AuNP fiber samples
Create AuNP samples for use in standard curves tomorrow
Protocol
We used the same chloroauric acid stock as last week. We made a new lysozyme stock solution that was 18.01µM.
Stock Solutions of Rhodamine B
We made three stock solutions of the following concentrations by diluting Rhodamine B in DI water in a volumetric flask:
139.03µM
13.903µM
1.3093µM
Adding Rhodamine B to the "After" Samples Synthesized on February 17, 2016
On February 17, we synthesized samples that were going to receive acetylsalicylic acid today after having been incubated in the oven. However, we decided to switch to Rhodamine instead of using acetylsalicylic acid (see Data, Observation, and Analysis).
The following table shows the amount of Rhodamine we added to each sample.
Table 1: Rhodamine B Added to "After" Samples Synthesized on 20160217
We then covered the samples in Parafilm and incubated them at room temperature overnight.
Creating New AuNP Fiber Samples
We made new AuNP fiber samples according to the following layout.
Table 2: Components of AuNP Fiber Samples Synthesized on February 23, 2016
We will add Rhodamine to the samples tomorrow (as indicated in the "Volume of Rhodamine to Add After Oven" column). The samples will be incubated in Rhodamine for 2 hours tomorrow before we measure the fluorescence of their supernatants.
Creating AuNP Samples
We made two identical samples of AuNP for use tomorrow in our calibration curve of the fluorescence of Rhodamine as a function of the concentration of AuNP that the Rhodamine is dissolved in. We made the AuNP samples as follows:
Table 3: Components of AuNP Samples
We incubated these samples in the oven at 80°C for four hours.
Data, Observations, and Analysis
Why We Changed Our Protocol to Include Rhodamine Instead of Acetylsalicylic Acid
Lysozyme fluoresces at about the same wavelength as acetylsalicylic acid but not at the same wavelength as Rhodamine B. In order to measure fluorescence of our samples, we decided to switch back to using Rhodamine B in place of acetylsalicylic acid for the time being. It will be important to note, however, that the gold in our nanoparticle fiber samples will absorb the same wavelength of light as the rhodamine, but it will not fluoresce. Thus, when measuring the fluorescence of Rhodamine in our samples, we will need to take into account that the fluorescence will seem to be less than would be expected for a given Rhodamine concentration. As a result, tomorrow we will measure the fluorescence of Rhodamine in solutions of AuNP of varying concentrations of AuNP. We will measure the fluorescence of Rhodamine as a function of the concentration of AuNP in solution. Then, when we measure the fluorescence of our samples, we can determine the actual concentration of Rhodamine B left in the supernatant after determining the concentration of AuNP.
Why We Changed Our Protocol To Incorporate Rhodamine After Instead of Before Incubation in the Oven
We consistently found that the addition of acetylsalicylic acid to our samples before placing the samples in the oven helped fiber formation. In this way, the acetylsalicylic acid was acting as a reducing agent and was being oxidized to a different substance. Most likely, anything with an aromatic ring would act in this way. This oxidation would explain why some of our Rhodamine-doped samples earlier in February had a peak shift when measuring fluorescence. As a result, we decided not to add any acetylsalicylic acid or rhodamine before incubating the samples. We would only add the substances after incubating the samples in order to prevent the substances from being oxidized.
In the future, we may try incubating our samples in Rhodamine for 2 hours. However, today we are incubating the samples in Rhodamine overnight.
Data, Observations, and Analysis of Samples Synthesized on February 17, 2016
Figure 1a: From Left to Right: Samples B_50uM_01; B_50uM_02; B_50uM_03 These three samples were synthesized on February 17, 2016. 50µM acetylsalicylic acid was added to each sample before incubation in the oven. This image was taken after the samples had incubated in the oven.Figure 1b: Sample B_50uM_04 This sample was synthesized on February 17, 2016. 50µM acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1c: From Left to Right: Samples B_100uM_01; B_100uM_02; B_100uM_03 These three samples were synthesized on February 17, 2016. 100µM acetylsalicylic acid was added to each sample before incubation in the oven. This image was taken after the samples had incubated in the oven.Figure 1d: Sample B_100uM_04 This sample was synthesized on February 17, 2016. 100µM acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1e: From Left to Right: Samples B_200uM_01; B_200uM_02; B_200uM_03 These three samples were synthesized on February 17, 2016. 200µM acetylsalicylic acid was added to each sample before incubation in the oven. This image was taken after the samples had incubated in the oven.Figure 1f: Sample B_200uM_04 This sample was synthesized on February 17, 2016. 200µM acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1g: From Left to Right: Samples A_50uM_01; A_50uM_02; A_50uM_03 These three samples were synthesized on February 17, 2016. When we were synthesizing the samples, we were planning to add 50µM acetylsalicylic acid to each sample; however, we decided today to add 0.01µM Rhodamine to the samples instead. This image was taken after the samples had incubated in the oven but before we added Rhodamine.Figure 1h: Sample A_50uM_04 This sample was synthesized on February 17, 2016. No acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1i: From Left to Right: Samples A_100uM_01; A_100uM_02; A_100uM_03 These three samples were synthesized on February 17, 2016. When we were synthesizing the samples, we were planning to add 100µM acetylsalicylic acid to each sample; however, we decided today to add 0.1µM Rhodamine to the samples instead. This image was taken after the samples had incubated in the oven but before we added Rhodamine.Figure 1j: Sample A_100uM_04 This sample was synthesized on February 17, 2016. No acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1k: From Left to Right: Samples A_200uM_01; A_200uM_02; A_200uM_03 These three samples were synthesized on February 17, 2016. When we were synthesizing the samples, we were planning to add 200µM acetylsalicylic acid to each sample; however, we decided today to add 1.0µM Rhodamine to the samples instead. This image was taken after the samples had incubated in the oven but before we added Rhodamine.Figure 1j: Sample A_200uM_04 This sample was synthesized on February 17, 2016. No acetylsalicylic acid was added to the sample. Instead of being placed in the oven, this sample was left on the bench top at room temperature. This image was taken after being left out at room temperature.Figure 1m: From Left to Right: Samples aspirin50uM_au; aspirin100uM_au; aspirin200uM_au; aspirin200uM_hcl; aspirin200uM_hcl_lys These blanks were synthesized on February 17, 2016. This image was taken after the samples had incubated in the oven.Figure 1n: From Left to Right: Samples au_lys_before; au_lys_after These blanks were synthesized on February 17, 2016. Both samples consisted of chloroauric acid, lysozyme, and DI water; neither sample received acetylsalicylic acid (or Rhodamine). This image was taken after the samples had incubated in the oven.
From observing the blanks, we could see that the acetylsalicylic acid was acting as a strong reducing agent toward the chloroauric acid, even in the absence of lysozyme. The blanks that included chloroauric acid and acetylsalicylic acid but not lysozyme (Figure 1m, samples aspirin50uM_au, aspirin100uM_au, and aspirin200uM_au) formed flecks of gold.
Just like the samples that were synthesized on February 16, 2016, the samples that were left on the bench at room temperature instead of being placed in the oven (Figures 1b, d, f, h, j, l) formed grey mesh-like substances.
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