User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/03/02

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Objective

Today's objectives were to:

  • Make a standard curve for the fluorescence of Rhodamine B as a function of concentration
  • Measure the fluorescence of all of the supernatants of the samples that had been synthesized on February 24, 2016 and then incubated in Rhodamine overnight
  • Incubate the samples that were synthesized on March 1, 2016 in Rhodamine for 2 hours and then measure the fluorescence of their supernatants

Protocol

Making a Standard Curve for the Fluorescence of Rhodamine as a Function of Concentration

First, we made 1mL samples of the following Rhodamine samples:

Table 1: Rhodamine Standard Solutions for Standard Curve

We then used a 200µL sample of each standard for fluorescence measurements. We cleaned the cuvette in between each run with DI water and methanol. We also measured a DI water blank.

For all the fluorescence measurements, we used the following parameters:

Start 540 nm End 700 nm Ex: 520 nm

Adding Rhodamine to the Samples Synthesized on March 1, 2016

We added Rhodamine to the samples that were synthesized on March 1, 2016 as follows:

Table 2: Rhodamine Added to Samples Synthesized on March 1, 2016 for 2 Hour Incubation

Note that a - indicates that we dod not use this sample because AuNP were formed instead of fibers. The samples that received 0µL of Rhodamine served as blanks.

Also note that we made the final concentrations of Rhodamine 1µM, 0.5µM, and 0.1µM instead of 1µM, 0.1µM, and 0.01µM like we had been doing in previous weeks. When we were taking measurements for our standard curve, we found that 0.01µM Rhodamine was too low of a concentration to get meaningful fluorescence measurements, so we increased the concentration to 0.5µM. In order to do this, we made a 69.52µM stock of Rhodamine by making a 1:1 dilution of some of the 139.03µM stock of Rhodamine in DI water.

Measuring Fluorescence of Supernatants

First, we pipetted 1mL of supernatant from each sample to be measured and put it in separate 1mL eppindorf tubes. This way, the samples that we measured last would not have incubated with fibers for longer than the samples that we measured first.

We then used 200µL of each sample for fluorescence measurements. We cleaned the cuvette in between each run with DI water and methanol. We also measured a DI water blank.

For all the fluorescence measurements, we used the following parameters:

Start 540 nm End 700 nm Ex: 520 nm

Data, Analysis, and Observations

Standard Curve for the Fluorescence of Rhodamine as a Function of Concentration

At first, our fluorescence measurements for the standard curve did not seem to make sense because the fluorescence of some of the lower concentration standards had stronger fluorescence than those of the higher concentrations. Additionally, when we took multiple measurements of some of the standards, the difference between measurements was up to 50%. We think this discrepancy could be caused by the formation of Rhodamine dimers. Going forward, we will use UV-Vis in order to make a standard curve for the absorbance of Rhodamine as a function of concentration. We'll then use UV-Vis to measure the supernatants of our samples.

Fluorescence of Supernatants

Because of time, we only measured the fluorescence of the supernatants of the samples that had been incubating in Rhodamine overnight. Since our standard curve turned out not to be useful, we can't really determine the concentration of the Rhodamine in the supernatants based on their fluorescence. We will be using UV-Vis absorbance spectroscopy in the future.

Images of Samples

Figure 1: Samples Synthesized on February 24, 2016 After 24 Hour Incubation in Rhodamine

The above image shows the samples that were synthesized on February 24, 2016 after the 24 hour incubation in Rhodamine. From left to right, the samples are:

  • A_1uM_01
  • A_1uM_02
  • A_1uM_03
  • A_1uM_04
  • A_0.1uM_01
  • A_0.1uM_02
  • A_0.1uM_03
  • A_0.1uM_04
  • A_0.01uM_01
  • A_0.01uM_02
  • A_0.01uM_03
  • A_0.01uM_04
Figure 2: Samples Synthesized on March 2, 2016 Before 2 Hour Incubation in Rhodamine

The above image shows the samples that were synthesized on March 2, 2016 before the 2 hour incubation in Rhodamine. From left to right, the samples are:

  • A_1uM_01
  • A_1uM_02
  • A_1uM_03
  • A_0.5uM_01
  • A_0.5uM_02
  • A_0.1uM_01
  • A_0.1uM_03
  • A_0.1uM_04
  • A_0.5uM_03
  • A_0.1uM_02
  • A_1uM_04
  • A_0.5uM_04

The four samples on the right all formed nanoparticles.



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