User:Sarah Burkhard/Notebook/471 Nano Notebook/2016/11/01

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Fluorescence pH 9

  • Sample preparation:

BSA Fluorescence Solution

  1. pH 9
  2. [Au]=0.25 mM
  3. [BSA]=3.125 uM
  4. Vf=3000 uL

Stock Concentrations 160920

  1. [BSA]= 52.8 uM
  2. [Au]= 2.64 mM
  3. [NaOH]= 1 mM

Volumes for making the solution:

  1. 177.556 uL BSA
  2. 284.09 uL Au
  3. 2508.35 uL HOH
  4. 30 uL NaOH

Data Analysis

AnnelieseFaustino fluorescencedataanalysis pH9BSAAUNP 161101 Part1.PNG

The chart above shows wavelength of the emission maximums for 3.125 uM BSA, 0.25 mM Au at pH 9 over time. The emission maximum holds relative steady, fluctuating by 5 nm above and below 420 nm for the entire length of the run.

AnnelieseFaustino fluorescencedataanalysis pH9BSAAUNP 161101 Part2.PNG

The chart above shows the integrated emission for 3.125 uM BSA, 0.25 mM Au at pH 9 over time. The integrated emission intensity begins at about 8000 at the start of the run. The integrated intensity increases to 11,500 at around 1200 s (forming a peak). The integrated emission intensity then decreases until about 5500 s (to a value of about 8500 intensity units), when it slowly begins to increase up to 11,000 intensity units until the end of the run at 12,000 s.

AnnelieseFaustino fluorescencedataanalysis pH9BSAAUNP 161101 Part3.PNG

The above chart shows the first 1000 s of emission intensity vs. wavelength for 3.125 uM BSA, 0.25 mM Au at pH 9. The water raman peak is pronounced in all scans. The emission intensity is seen to increase rapidly at first, then less slowly over time.

AnnelieseFaustino fluorescencedataanalysis pH9BSAAUNP 161101 Part4.PNG

The above chart shows 1000 s.to 4500 s. of emission intensity vs. wavelength for 3.125 uM BSA, 0.25 mM Au at pH 9. The water raman peak is pronounced in all scans. The emission intensity is seen to not increase much more after 1000 s.

Notes

  • Making gold chloride solution

parafilm the spatula for gold (otherwise it will corrode) keep in dust cater . slide off lid 27.8 mg rinse with water 27.8 mg, fill up to 25 ml

0.0278 g / (303.8 g / mol AuCl) / 0.025 l = 0.00366 M AuCl = 3.66 mM


We prepared 45 UV Vis solutions for 0 M BSA concentration and Fructose concentrations 0.0625, 0.125, 0.25, 0.75, and 1.25 mM, for pH 4 - 12.

Data Analysis

- open MATlab - open fluorescence data in txt file

want to fit dataset and keep peaks at same place. water ramen peak alone.

Cphysicsadmin/desktop command window >>M=dlmread('filename here.txt');

open M

>>X= M(:,1) >>Y= M(:,2) ;

run curve fitting tool

>>cftool

opens new window

Xdata = X Ydata = Y


choose Gaussian number of terms 4 note location of peaks via a1, b1 etc

>>Myo=dmlread('filename'); >> Xm = Myo (:,1)

>>t0 = Myo (;,2)

fit options define limits of centroid (b peak found before)

note peaks you want. 353 . 437.

>>t29 = Myo (;,31)

430 set startpoint for 430 to 328 and 327.5 upper 328.5 a1 between 1 and 10 (this is the Raman so set small)

peak at 437 should not be broader than 37.5

keep all peaks. raman 5 - 10, others 30 - 50 identify protein peak and others end up fitting w 5 peaks


may miss one peak. so go to 5. change fit options. keep at 5 peaks : c 35 -40 ; b +- 5 . water raman +- 0.5 c is width, a is intensity, b is shift (from function). b values are wavelengths at peak +- 10

peaks: 328 , 353, 385, 438, 465, 488 . remove 465.

copy and paste results.

look at area vs time. MATlab fitting to force fit options always be the same. copy values into spreadsheet t0 as column a1 b1 c1 as rows