- Analyze solutions of AuHRP and AuADA, both made on 2012/11/14 with UV-vis.
- Note that the solutions of AuADA were made with undialyzed ADA.
- Make solutions of AuADA with dialyzed ADA.
- ADA dialysis began on 2012/11/14 and ended on 2012/11/27.
- Please refer to Keyun Wang's entry for the procedure used to remove the ADA from the dialysis tubing and prepare it for use in AuADA solutions.
- Resuspend AuHRP fibers in Tris buffer.
UV-Vis on AuHRP and AuADA
- Exactly 3 mL of each solution was pipetted into a quartz cuvette for analysis using an automated pipetter (Pipetman).
- Please refer to Keyun Wang's entry for a photograph of the 150 AuADA solution.
- Small pellets of fibers were visible in all AuHRP solutions except 60 AuHRP. The 60 AuHRP solution did not contain a fiber pellet. All AuHRP solutions were purple in color.
- All AuADA solutions were colorless and contained no aggregates. However, it was observed that 130, 140, and 150 AuADA contained small white aggregates. It is speculated that these white aggregates were ADA that precipitated out of solution due to interactions with ions from the elution buffer.
- The spectra for all solutions displayed a peak at around 520 nm, which indicates the presence of GNPs. The general trend was that the absorbance of these peaks increased as the mole ratio of Au:HRP increases. This implies that the amount of GNPs in solution increased in accordance with the amount of gold initially added to each solution.
- However, the peak absorbances for the 130 and 140 AuHRP solutions were almost identical. This is likely because the 130 AuHRP solution contained 1 mL less of H2O due to an error during preparation of the solution.
- The spectra for all solutions did not display any significant peaks, which indicated that GNPs were not formed. This was corroborated by visual examination of the solutions, as described above.
- It was suggested that the elution buffer from FPLC in which the ADA was suspended interfered with the formation of GNPs. Consequently, the protocol was repeated with dialyzed ADA, as described below.
AAS on AuHRP and AuADA
- There were no deviations from the standard protocol for analyzing solutions with AAS. The standard HAuCl4 solutions were made by Dr. Miller on 2012/10/08. Please refer to Keyun Wang's entry for tables of the concentrations and absorbances of the standard HAuCl4 solutions used to calibrate the AAS, and the absorbances and concentrations of the AuHRP and AuADA solutions.
- The general trend for [Au] was that it increased as the mole ratio of Au:HRP increased. It is possible that the increased [Au] in the 130 AuHRP solution is due to resuspension of purple fibers containing Au. Overall, it is likely that the [Au] increase for each solution is due to the increasing amount of Au added to each solution before reaction.
- The general trend for [Au] is the same as with solutions of AuHRP in that [Au] increased as the mole ratio of Au:ADA increased. Again, it is possible that the increased [Au] in the 100 AuADA solution is due to resuspension of fibers during sample collection. Overall, it is likely that the [Au] increase for each solution is due to the increasing amount of Au added to each solution before reaction.
- Please refer to Keyun Wang's entry for analysis of the differences between the mechanism of formation of GNPs between AuADA solutions and AuHRP solutions. Note that since HRP is a larger protein than ADA, it is possible that there are more residues within HRP that react with Au to form GNPs, which explains the increased [Au] values for AuHRP compared to that of AuADA.
Synthesizing AuADA Solutions
- Please refer to the protocol on 2012/11/14. There were no deviations from the protocol and the same stock solution of HAuCl4 was used. However, dialyzed ADA was used instead of ADA in FPLC elution buffer.
- Please refer to Keyun Wang's entry for observations made during the procedure.
Resuspension of AuHRP Fibers in Tris Buffer
- Based on visual observation, it was determined that the 130, 140, and 150 AuHRP solutions contained the most fiber and were used for fiber resuspension.
- Please refer to Keyun Wang's entry for the protocol and concentrations and volumes of Tris buffer used. After Tris buffer was added to the three AuHRP solutions, the solutions were left to sit for 24 hours before being analyzed.
- After 24 hours it was observed that no fibers had formed and that all solutions were translucent purple in color. It was concluded that the AuHRP fibers were successfully resuspended in all concentrations and pH values of Tris buffer. This correlates to literature that states that the optimal pH of HRP is between 6.0 and 6.5; HRP in solutions at higher or lower pH values that its optimal pH must be denatured and therefore not forming fibers with GNPs.