December 4, 2013
This semester I’ve been working on replicating the fluorescence observed for the combination of ZnPPIX, DNA, and AuNPs, excited at 420nm, on [06/05/2013|User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05]. In this trial, the concentrations were: DNA- 0.94μM, AuNP- 12.55nM, ZnPPIX- 1.9364μM.
[[Image:Pic of ZnPPIX abs.png]
In my first attempt at replication on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/09/04|09/04/2013], I ran several different combinations of ZnPPIX, DNA, and AuNPs at the concentrations used on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013], in addition to modified concentrations for the 4 and 8-strand DNA, and concentrations adapted from a published procedure(1). All samples were excited at 420nm again, though no signal was observed for any of them.
I remade the samples from [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013] again on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/09/11|09/11/2013], taking absorbance measurements this time to confirm the presence of ZnPPIX, DNA, and AuNPs in each sample. Samples were excited at 440nm, from the published procedure, and 420nm, from [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013], but again no fluorescence was observed.
On [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/09/18|09/18/2013], I remade the samples from [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013], using PBS instead of water to dilute the solutions in case the water had caused the DNA to degrade rather than react with the ZnPPIX. Absorbance measurements again confirmed the presence of compounds, but no fluorescence signal was observed.
Since I was unable to see any fluorescence signal with any of the above attempts, I checked the concentration of the ZnPPIX by taking absorbance measurements and performing Beer’s law calculations. On [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/10/02|10/02/2013], I determined the concentrations were about an order of magnitude less than what I’d assumed them to be (based off of the mass I dissolved when I created the solution about a year ago).
Beer's Law caluclations:
I synthesized and filtered new ZnPPIX with Dr. Hartings, calculating the concentration from absorbance measurements using Beer’s law. On [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/10/30|10/30/2013], samples from [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013] were remade with the new ZnPPIX, modified for the 4 and 8-strand DNA rather than thiol DNA. All samples were excited at 420nm, and some fluorescence signal was observed for all samples. However, the signal was minimal, did not align with what was previously observed, and may have been due to instrumentation rather than the samples. I attempted to further purify the ZnPPIX on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/11/06|11/06/2013], however, I was unable to get the ZnPPIX to precipitate out of solution to filter it. On [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/11/13|11/13/2013] I made ZnPPIX using Dr. Miller’s porphyrin instead, determined the concentration with absorbance measurements and Beer’s law, and remade the samples modified for 4 and 8-strand DNA from [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013]. There was some very minimal fluorescence signal, though again the differences in intensity did not align with what was expected.
Beer's Law calculations:
My attempt to replicate the fluorescence signal from the combination of ZnPPIX, DNA, and AuNPs that was observed on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013] was unsuccessful. I was unable to use the same AuNPs used in the original solution because we ran out over the summer and made new ones of a similar size, which may have been a reason why the signal could not be replicated. Since some signal was observed in the last two trials, perhaps the concentrations of the solution components could be increased to increase and better distinguish the observed signals and potentially determine why the produced signal is not the same as it was on [User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/05|06/05/2013].
(1). Zhang, Z.; Sharon, E.; Freeman, R.; Liu, X.; Willner, I., Fluorescence Detection of DNA, Adenosine-5′-Triphosphate (ATP), and Telomerase Activity by Zinc(II)-Protoporphyrin IX/G-Quadruplex Labels. Analytical Chemistry 2012, 84 (11), 4789-4797.