User:Sanuja Mohanaraj/Notebook/Experimental Biochemistry 1 Fall 2017/2017/09/20

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Fluorescence

An absorbance ranging from 515nm to 530nm is observed for stabilized gold nanoparticle colloids as a result of plasmon resonance. As suggested by some literature, a photoluminescence band can be observed between 420nm and 450nm as a result of the excited gold nanoparticles at 308nm. The nature of this phenomenon is used to analyze electron transferring abilities, such as FRET.

Two solutions of 10μM acridine orange and 10μM fluorescein were prepared by diluting ~1.51e-4 grams of acridine orange and ~1.66e-4 grams of fluorescein, into separate 50mL volumetric flasks. Through serial dilutions, solutions ranging from 1μM-9μM were made using the prepared acridine orange and fluorescein solutions. The fluorescence of each stock solution was measured using excitation wavelengths of 308nm, 440nm, and 490nm using the Perkin Elmer LS55 fluorescence spectrometer; with parameters of 8nm excitation and emission windows and a scan speed of 50nm/min. The emission scan was started at (emission wavelength + 10nm), and was scanned at a range of 200nm. Next, the fluorescence of the gold nanoparticle solutions using each amino acid and BSA were measured at all three excitation wavelengths as well.

Calculations

10μM Acridine Orange Solution
10μM = moles / 0.05L
moles = 5e-07 moles
(5e-07 moles)(301.81g/mol) = 1.509e-04 grams

10μM Fluorescein Solution
10μM = moles / 0.05L
moles = 5e-07 moles
(5e-07 moles)(332.31g/mol) = 1.6615e-04 grams

Acridine Orange & Fluorescein Solutions
M1V1=M2V2

(1μM)(5mL)=(10μM)(V2)
V2=500μL

(2μM)(5mL)=(10μM)(V2)
V2=1000μL

(3μM)(5mL)=(10μM)(V2)
V2=1500μL

(4μM)(5mL)=(10μM)(V2)
V2=2000μL

(5μM)(5mL)=(10μM)(V2)
V2=2500μL

(6μM)(5mL)=(10μM)(V2)
V2=3000μL

(7μM)(5mL)=(10μM)(V2)
V2=3500μL

(8μM)(5mL)=(10μM)(V2)
V2=4000μL

(9μM)(5mL)=(10μM)(V2)
V2=4500μL

Results

Screen Shot 2017-11-22 at 2.01.53 PM.png
The above spectrum depicts the measured fluorescence of the 1μM acridine orange solution, at an excitation wavelength of 490nm. There is an observed peak at approximately 525nm.

Screen Shot 2017-11-22 at 2.02.09 PM.png
The above spectrum displays the measured fluorescence of the 3μM acridine orange solution, at an excitation wavelength of 308nm. There is an observed peak at ~345nm.

Screen Shot 2017-11-22 at 2.03.14 PM.png
The above spectrum depicts the measured fluorescence of the 1μM fluorescein solution, at an excitation wavelength of 490nm. There is an observed peak at approximately 500nm.

Screen Shot 2017-11-22 at 2.03.04 PM.png
The above spectrum displays the measured fluorescence of the 3μM fluorescein solution, at an excitation wavelength of 308nm. There is an observed peak at ~512nm.

Screen Shot 2017-11-22 at 2.02.26 PM.png
The above spectrum displays the measured fluorescence of the phenylalanine-stabilized gold nanoparticle solution, at an excitation wavelength of 308nm. There is an observed peak at ~415nm.

Screen Shot 2017-11-22 at 2.02.37 PM.png
The above spectrum displays the measured fluorescence of the phenylalanine-stabilized gold nanoparticle solution, at an excitation wavelength of 440nm. There is a slight observed peak at ~520nm.

Screen Shot 2017-11-22 at 2.02.50 PM.png
The above spectrum displays the measured fluorescence of the phenylalanine-stabilized gold nanoparticle solution, at an excitation wavelength of 490nm. There is no apparent peak in the above spectrum.