User:Sarah Burkhard/Notebook/471 Nano Notebook/2016/09/20

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Fluorescence Round 2

pH : 5

the sample solution was made with : 30 uL HCl, 147.6 uL AuCl, 190.9 uL BSA, 2631 uL H20 --> 3000 uL total Volume

calculations were made with Anneliese's spreadsheet and M1V1 = M2V2 formula, where V2 = 3000 uL, M2 is specified (see below), and M1 depends on Dr. Harting's stock solutions here.

concentrations: BSA Fluorescence Solution

  1. [Au]=0.25 mM
  2. [BSA]=3.125 uM


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.

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

  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

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.)

Image:PH 5 fluor 160920 fluorescence.PNG


Image:Fluor ph5 bsa A PART 1 160920.PNG

The chart above shows the emission maxima over time for the solution of pH 5 BSA with gold nanoparticles. A major increase in maximum emission occurs around ~7 minutes and another smaller increase occurs around ~17 minutes. The emission maximum then plateaus around 420-440 nm.

Image:Fluor ph5 bsa part 2 160920.PNG

Fluorescence vs. Ocean Optics pH 5

Image:Fluor vs ocean optics pH 5 160928.PNG

Notes

we accidentally overrode the data for 0 minutes so we had to restart.

how to save data: 'setup parameters' saves as .sp to save it as text : go to software icon "FL win lab" . you will see an empty graph. click open > sort according to date > choose most recent data

go to save as > switch to ASKII > change to .txt in file name


preparation for tomorrow: UV-Vis solutions with pH 4-12 and Fructose concentrations 0.75 and 1.25 were made



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