User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/03/30

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Objective

  • Make new AuNP fibers using the higher ratio of gold:lysozyme
  • Make a standard curve for the UV-Vis absorbance of Rhodamine as a function of its concentration in an AuNP fiber supernatant (minimal incubation in the fibers)
  • Add Rhodamine to the lysozyme AuNP fibers synthesized yesterday
  • Measure the UV-Vis absorbance of the supernatants of the samples we made yesterday after they have incubated in Rhodamine for 2 hours

Protocol

Making AuNP Fibers

We made 10mL AuNP fiber samples in glass test tubes using the following reagents:

Screen Shot 2016-04-06 at 8.08.45 PM.png

We then added the following to each sample:

  • 977.33µL lysozyme
  • 741.31µL gold
  • 8281.36µL DI water

We put the samples in the oven and ran them on cycle P1.

Measuring the UV-Vis Absorption of AuNP Fiber Supernatants After 2 Hour Incubation in Rhodamine

We added the following amounts of Rhodamine to our lysozyme samples that we synthesized yesterday:

Screen Shot 2016-04-06 at 8.12.37 PM.png

We then covered our samples in Parafilm, inverted them gently to mix in the Rhodamine, and allowed them to incubate at room temperature in Rhodamine for 2 hours.

After 2 hours, we spun down the samples, pipetted 3.5mL of the supernatants into polystyrene cuvettes, and measured the UV-Vis absorption of each.

Measuring the UV-Vis Absorbance of Rhodamine as a Function of its Concentration in AuNP Fiber Supernatant

Since we only used 9 samples for our 2 hour incubation, we used 10 of the remaining lysozyme samples that we synthesized yesterday in order to make a standard curve for the UV-Vis absorption of Rhodamine as a function of its concentration in the supernatant of our fiber samples.

We pipetted the following volumes of Rhodamine from our 1390µM stock of Rhodamine into each of 10 samples in order to make standards:

Screen Shot 2016-04-06 at 8.18.07 PM.png

We then covered the samples in Parafilm, inverted them, and spun them down for 2 minutes. We pipetted 3.5mL of each supernatant into polystyrene cuvettes and measured the UV-Vis absorbance.


Analysis

All of the BSA fibers we tried to synthesize yesterday failed. However, the lysozyme worked. We will use this higher ratio of gold:lysozyme in our samples moving forward.

UV-Vis Absorption of AuNP Fiber Supernatants After 2 Hour Incubation in Rhodamine

Copied from Michael's notebook (as of April 6, 2016):

Rhodamine B 2 Hour Incubation Raw Data.png

Figure 2: The table above shows the raw and calculated data including the peak absorbance, the concentration that this absorbance corresponds to, the change in concentration after incubation, and the percent change in concentration.

Rhodamine B Percent Change Bar Graph.png Figure 3: The chart above displays the percent change in concentration of Rhodamine B in solution after incubation for 2 hours. Blue bars correspond to 10 μM initial Rhodamine concentrations, red bars correspond to 5 μM initial Rhodamine concentrations, and green bars correspond to 2 μM initial Rhodamine concentrations. As expected, the concentration of Rhodamine B in solution decreases indicating that Rhodamine B is likely being incorporated into the AuNP fibers.

Standard Curve for the UV-Vis Absorbance of Rhodamine as a Function of its Concentration in AuNP Fiber Supernatant

Also copied from Michael's notebook (as of April 6, 2016):

UV-Vis Rhodamine B Calibration Curve in Fiber Supernatant.png Figure 1: The graph above is a UV-Vis calibration curve relating absorbance of Rhodamine B at 556 nm when dissolved in AuNP fiber supernatant to the concentration of Rhodamine B. The equation of the line is included on the graph. The high R2 value indicates the strong fit of the trendline/equation to the data.



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