User:Sarah Burkhard/Notebook/471 Nano Notebook/2016/09/06

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Ocean Optics

Objective

Draw conclusions on reaction speed of nanoparticle formation by measuring absorbance of light, every 2 minutes over a period of 3 hours.


Protocol

samples

  1. BSA Ocean Optics
    1. 3 mL total
      1. 0.25 mM Au
      2. 3.125 μM BSA
      3. HCl or NaOH appropriate for your pH -- we used pH 4
      4. water

convert 3.125 micromolar into volume needed in microliter: M1V1 = M2V2 | M1 = 34 micromolar , V1 = x , M2 = 3.125 micromolar, V2 = 3000 microliter

V1 = 3.125 * 3000 / 34 = .27573 ml

pH 4 means proton concentration = 1*10^-4 mol/L. one tenth needs to be proton concentration, so 300 microliter acid.

147 microliter AuCl (same set up of M1V1 = M2V2, calculations made in Anneliese's spreadsheet)

water= 3000 - 275 - 300 = 147 microliter


The Protocol is copied and pasted from the AU Design Lab

  1. Extra Steps
    1. Do you need temperature control or need stirring?
      1. See the protocol for controlling the cuvette controller Quantum Northwest Cuvette Holder
  2. Turn on the jaz spectrophotometer (red on-off button)
  3. Turn on the Lamps (Ocean Optics DH-2000)
    1. Turn on the switch in the back of the controller box
    2. Press the blue "Deuterium" button and wait for the light to stay on
    3. Press the red "Halogen" button
    4. The lamps need 15 minutes to warm up before you are ready to proceed to the next step.
  4. Starting the software
    1. Open the OceanView software.
      1. Click on the "Create New Spectroscopy Application" icon (looks like a bunch of shark fins stacked on one another)
        1. Click on "Spectroscopy"
        2. Select JAZA1666 and click "Next"
        3. Select "Absorbance" and click "Next"
        4. Set Acquisition Parameters
          1. Set "Integration Time" to 1 ms
          2. Set "Scans to Average" to 100
          3. Click "Next"
        5. Store Reference Spectrum
          1. Make sure that the toggle on the light source is switched to "Open"
          2. Click on the light bulb icon
            1. A spectrum should appear in the middle display
          3. Click "Next"
        6. Store Background Spectrum
          1. Make sure that the toggle on the light source is switched to "Closed"
          2. Click on the light bulb icon
            1. A spectrum should appear in the middle display
          3. Click "Finish"
        7. Make sure that the toggle on the light source is switched to "On"
  5. Set up your data acquisition schedule
    1. Click on the "Save graph to file/files" icon (the floppy disc icon on the lower icon panel)
    2. Click "Yes" for "You must configure save parameters first. Do you want to configure now?"
    3. Set Save Options
      1. Choose the "Between saved scans, wait at least" option
      2. Set the time to whatever the experiment requires
        1. For most nanoparticle syntheses, this will be 2 minutes
      3. Choose the "Stop after this amount of time" option
        1. For most nanoparticle syntheses, this will be 3 hours
    4. Set File Options
      1. Save to Directory: C:\Users\Lab Admin\DropBox\CHEM471 2016\Ocean Optics\Year\Month\Date
      2. Select Open
      3. Set Basename to something that will be descriptive of your data for example Lysozyme_AuNP_Ratio45_80C
      4. Click "Apply"
      5. Click "Exit"
  6. Start data collection
    1. After you have your sample, cuvette, and cuvette holder properly prepared perform the following:
      1. Click the "Save graph to file/files" icon (the floppy disc icon on the lower icon panel)
      2. Data files should be showing up in your selected folder. If it isn't, you have done something wrong.
  7. Shutting down
    1. When your data collection is finished, perform the following steps
      1. Shut down the OceanView software
      2. Turn off the jaz spectrophotometer
      3. Turn off the lamps (main switch in the back)
      4. Shut down the Quantum Northwest Cuvette Holder if necessary
  8. Analyzing data
    1. Open data compilation folder. (desktop > data compilation)
    2. Open copy command.txt document.
    3. In data compilation folder shift right click and choose open command window here.
    4. Copy the first line from the copy command.txt document and paste into the command window. Hit enter.
    5. It will prompt “location?” Copy the location of the saved scans, paste it into the command window, and hit enter.
    6. Then copy the second line from the copy command.txt document, paste into the command window, and hit enter.
    7. It will prompt “number of files.” Enter the number of files that it has copied which will be immediately above the prompt.
    8. It will prompt “number of lines per file.” Enter 2048.
    9. It will prompt “number of lines before data.” Enter 17.
    10. It will prompt “Are you sure (Y/N)?” Enter Y.
    11. In the data compilation there will a document titled out.txt. Open out.txt and save as an excel file in the folder with the saved scans and rename to match the folder.
    12. Delete out.txt if still saved in data compilation folder.

Data

How to use data from Ocean Optics:

  • Copy data in DAR desktop data compilation
  • Shift right click open a command window
  • Location = paste file
  • Copy location
  • Paste
  • Run java / jar :90 files
  • Number lines per file: 2048
  • 17 lines before data
  • Copy "out.txt"
  • Rename
  • Open with excel
  • Only absorbance, y-data
  • Enter wavelength from individual file
  • Time in seconds start w 0, 120, 240
  • Absorbance max = max between 450 - 700 so find those rows
  • Select all rows and columns
  • Wavelength: use function to go through data and find cells with max
  • Add 709 to exclude 710

Excel tip: Use x function

  • Highlight and copy through columns
  • Value special transpose
  • Make graph :
  1. absorbance vs time
  2. wavelength vs time
  3. Absorbance at max wavelength vs time

Excel tip : Paste values transpose

  1. Compilation of spectra in 30 minute interval

directory: type DEL "dir" > Y

Data Analysis

The graphs below visualize our most important findings:

1) Graph 1 shows that absorbance increases almost linearly with time; this is an important finding within analysis of the kinetics of a reaction. It most certainly fits a model of kinetics which would allow us to raw further conclusions on concentration of the analyte (in our case gold nanoparticles.) It means that our gold nano particle forms steadily over time. Max abs over time.PNG

2) Graph 2 shows a clear shift in energy absorbance. At the beginning, light at smaller wavelengths is absorbed, corresponding to higher energy values. After approximately 3000 seconds we see the jump, and a higher wavelength (lower energy) is absorbed. This shift most likely indicates the time of protein unfolding. We can conclude that after around 50 minutes our protein unfolding process is in the final stage. Max wavelength over time.PNG

3) Graph 3 shows the beginning of the protein unfolding. Absorbance at wavelength 535 increases with increased concentration of unfolded protein and formed gold nanoparticle. A353 over time.PNG

Notes

keep cuvette !

waste from sample goes in liquid heavy metal waste. to clean, add 3 drops HCl , 1 drop nitric acid to make "royal water" (as it dissolves gold) to clean heavy metal from cuvette, soak over night