Today's objective is to perform a UV-Vis and a fluorescence spectroscopy on the three solutions we have been preparing last 2 weeks. The main purpose of these measures is to quantify the amount of dye and Gold NPs in each solution and to assess for the efficiency of the binding reaction.
The UV-Vis spectroscopy is going to allow us to quantify dye in each solution, using the fact that our dye absorb at 602nm. To do so, we are going to make a spectrum of the solution by using a spectrophotometer (UV-1800 Shimadzu) in the wavelength range of 200-800nm. We'll then determine the dye/BSA molecule ratio using the initial amount of BSA (we assume that no BSA has been lost during the experiment). The fluorescence spectroscopy will allow us to compare how efficient the dying process is; to do so we'll compare fluorescence spectrum btw our three different solutions. We are going to use the fact that our dye absorbs at 602nm and emits at 672nm.
- Preparation of the BSA control solution (the protocol is the same as on the 2/8/12):
- 8mL of water + 1mL of HCl (2.84mM) + 1mL of BSA (17.7µM)
- 2 hours in the oven (80°C)
- At the end of the dialysis we obtained as solution:
- For the control solution, a pink solution
- For the 70 ratio solution, a pink solution and lots of small purple fibers
- For the 166 ratio solution, a pink solution and one big purple agregats
- We centrifuged the 70 and 166 solutions (5 minutes at 13200 rpm)
- UV spectroscopy: using UV-2550 Shimadzu Corporation spectrometer, with a wavelength range from 200 to 800nm and measuring the absorbance.
- BSA control with dye
- 70 ratio with dye (cuvette wasn't full)
- 166 ratio with dye (cuvette wasn't full)
- 70 ratio with dye (higher volume)
- 70 ratio without dye (control)
- 166 ratio without dye (control)
- Fluorescence spectroscopy: using LS55 PerkinElmer fluorescence spectrometer, with emission range from 620 to 800nm and excitation at 600nm.
- BSA control with dye (nadh70)
- BSA control with dye (excitation at 575nm)
- BSA control with dye (excitation at 550nm)
- BSA control with dye (excitation at 525nm)
- 70 ratio with dye
- 166 ratio with dye
- Preparing a control solution with the dye only (dye solution control):
- 80µL of DMSO
- 1mg of dye
- 5mL of water
- Centrifugation: 5minutes at 13200rpm
- 70 ratio solution with dye
- dye solution control
- Fluorescence spectroscopy results.