User:Sarah Burkhard/Notebook/471 Nano Notebook/2016/09/07

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Fluorescence

Objective

Draw conclusions on reaction speed of nanoparticle formation from emission spectra taken in small time intervals of 5 minutes over a period of 3 hours.

protein: BSA | pH: 4

Chemical background

BSA contains Tryptophan, which is fluorescent in the unfolded state. The excitation wavelength is 295 nm (more information read here http://www.physics.nus.edu.sg/~Biophysics/pc3267/Fluorescence%20Spectroscopy2007.pdf) With decreasing pH, the time it takes for BSA to unfold decreases as well. However, we still see an emission peak after the unfolding process: the newly formed gold nanoparticles are fluorescent. The time we see the emission peak around 435 nm.

Protocol

Prepare solutions. Gold, protein, acid , water .

  1. BSA Fluorescence
    1. 3 mL total
      1. 0.25 mM Au
      2. 3.125 μM BSA
      3. HCl or NaOH appropriate for your pH (pH = 4)
      4. water

M1V1 = M2V2 was used for calculations (Anneliese's spreadsheet)

A table of used concentration and volumes can be found here.

Protocol for Fluorescence:

  1. Starting the Peltier controller
    1. Make sure that the ribbon cord connects the peltier controller and the cuvette holder
    2. Fill the chiller bucket with water
    3. Place the aquarium pump (with tubes attached to the Peltier heat exchanger) into the bucket and plug in the pump so that water flows
    4. Turn on the peltier controller (switch in the back of the instrument)
    5. Use the arrow buttons to set the temperature.
  2. Turning off the Peltier controller
    1. Turn the controller power off
    2. Unplug the aquarium pump
    3. Dump the water from the bucket into the sink

Preparation of stock solutions for UV-Vis:

    1. 5 mL total
      1. 0.25 mM Au
      2. 3.125 μM BSA
      3. HCl or NaOH appropriate for your pH (pH 4 - 12)
      4. Appropriate amount of fructose (0.125 mM, 0.25 mM)
      5. water
  • Matt Hartings 14:02, 9 September 2016 (EDT): I remade your samples because you got strange results. Here's my description


Data Analysis

The emission peak will shift to higher wavelengths, due to protein unfolding. The tryptophan is fluorescent with an emission peak between 300 and 350 nm. Gold particles are fluorescent as well, but differently as long as they are folded in the Protein (related to the low dielectric constant within the protein.)


Graph 1 shows the shift in intensity vs wavelength over a time period of three hours. With time, intensity increased.

Fluorescence summary 160909.PNG

One can see that BSA unfolds and forms gold nano particles rapidly at pH 4 because emission at the relevant wavelength (~435 nm) increases drastically within the first 15 minutes.

The emission maximum graph also shows us at what time the emission shifts which indicates the time the majority of protein is unfolded. The jump occurs between 5 and 10 minutes.

Emission maximum 160909.PNG

A more detailed graph showing what happens in the first 40 minutes is here, a graph of intensity of emission vs time shows the emission peak and the following decrease:


In another intensity vs time graph you can see how it smoothly increases, representing a smooth, continuous reaction.

Fluorescence vs ocean optics ph4.PNG The integrated intensity versus time (fluorescence) and the absorbance versus time (Ocean Optics) for BSA pH 4 with 0.25 mM and 3.125 mM BSA were plotted in the same chart to draw a further conclusion on the time at which the gold nano particle formation begins and peaks.

Notes

BSA stock solutions go 4 hours in oven, up to 80 C. menu > hill icon > menu > choose program 1 > 1 loop