Gold nanoclusters are known for their unique size and surface quality and do not have the same metallic properties as nanoparticles or their corresponding bulk material. The formation of nanoclusters depends on many variables, including temperature, pH, and type of reductant. This experiment was done to begin to understand how these variables can affect synthesis and what exactly they are.
Data were collected showing that at a neutral pH, using a solution of 50mM Tris and 150mM NaCl, highly fluorescent gold nanoclusters were formed. These nanoclusters synthesized using 1M sodium hydroxide reducing agent showed the brightest fluorescence, a bright red-orange color. It was found that in samples showing fluorescence, gold nanoclusters with a mass of 4 kDa could be observed.
Introduction
Nanoparticles & Nanoclusters
Protein Expression and Purification
Materials and Methods
This thawed the cells until they were ready to be diluted in 40 µL of distilled water. This culture was grown for four hours until the OD600 (optical density measured at 600 nm) was found to be between 0.4 and 0.6. At this point, 5 mL of cells were kept in a culture tube to serve as a control.
This is the uninduced culture and was later saved for running in the gel. After this time, the cells were centrifuged and harvested for ten minutes at 4°C and the pellet was then stored at -20°C. The cells were then sonicated with a sonication buffer to lyse the cells and release the protein.
This mixture was sonicated again and the supernatant containing the protein was retained to be passed through a StrepTrap column for the final purification step;. A StrepTrap column is used because of its high affinity for CSP-1, allowing impurities and other material to be separated. Protein was loaded onto the column at a flow rate of 4mL/min and the flow through was collected; this was labeled Flow Through 1.
The column was then washed with Buffer W for 6 minutes and the flow-through was collected (flow-through 2). The column was then washed with NaOH, water and Buffer W to be stored at 4°C. However, the pellet is resuspended in SDS to run through the gel along with the purified protein.
StrepTrap has a high affinity for proteins and keeps them bound while impurities flow through. This figure shows the flow-throughs collected from the column and the purified WT CSP-1 in the final step. The protein was first denatured in a hot bath at 90 °C before being placed in a gel to resolve it into its monomeric form.
Results and Discussion
The purified protein was then dialyzed in dialysis buffer (50 mM Tris, 150 mM NaCl, pH 7.8). This is done because of the high possibility of metal ions being present in the DI water used to make the dialysis buffer. Flow Through 1, Flow Through 2, the uninduced sample, the post-sonication pellet, and the pre-purification protein sample are all on the gel as well.
From left to right, the wells are filled with high molecular weight scale, pellet, protein before purification, protein after purification, flow 1 and flow 2. The numbers on the far left represent the mass in kDa of the corresponding band of the high molecular weight scale. In Figure 8, in the fourth well from the left, which is the protein after purification, a band at about 13 kDa is clearly visible.
The protein exists as a tetramer, but is seen in the gel in its 13 kDa monomeric form, due to denaturation. This protein pool was found to be 39 μM via its UV-Vis spectrum, as seen in Figure 9. Using Beer's law and an extinction coefficient of 8480, the concentration was determined to be 39 μM, shown in Equation 1.
Gold and Platinum Nanocluster Synthesis
Materials and Methods
The UV-Vis spectrophotometer will be blanked with either 50/50 5mM Tris and water or 50/50 dialysis buffer and water, depending on what was used in the sample. Instead of being in pH 7.8 dialysis buffer, the protein was diluted in 5 mM Tris buffer at different pHs. The protein was placed in a 10K filter with 5mM solutions of Tris buffer with pH values of and 12 for centrifugal filtration.
After washing with Tris buffer, the protein was then diluted back to 50 mM in Tris buffer at the appropriate pH. Conventional synthesis was performed on each of the samples and data were collected the next day. The second set proceeded similarly using the same method for diluting the protein in 5 mM Tris buffer, but pH 1 and 13 buffers were used instead.
Instead of the samples being half protein and half water, more Tris buffer was used to replace the water. A normal synthesis was made with 9 equivalents of platinum, instead of gold, and another sample of 8 eq. NaOH was the reducing agent in both of these samples and was added at 5% by volume.
More experiments with platinum were then carried out, using NaBH4 as the reducing agent in all of them. A MALDI was performed on the samples of 9 equivalents of Au, 9 equivalents of Pt, and the mixture of 8 and 1 equivalents to check the mass of the protein together with the mass of the equivalents of Pt and Au. Matrices of CHCA and sinapinic acid were used with the MALDI to determine the mass of the components of the samples.
The samples were also run into a native gel along with the protein itself to further check molecular mass. The samples did not require heating/denaturing because they were run in a native gel. This is a reference table showing all samples run and the different variables in each sample.
Results and Discussion
The fluorescence of these samples appears to be similar, with the 15% NaOH slightly stronger and although a little stronger than the fluorescence of the samples with 10% or less additions of NaOH. The reduction of the sulfhydryl group of cysteine comes from the NaOH added through an endogenous reaction. This is the only method that worked according to the results, as none of the samples reduced with NaBH4 showed significant fluorescence.
This figure shows the UV-Vis spectrum of gold clusters synthesized with 5 mM Tris at the respective pHs shown. This figure shows the UV-Vis spectrum of gold clusters synthesized with 5 mM Tris at the respective pH and temperatures shown. However, no precipitate was visually observed and no fluorescence was observed in any of the samples in Figure 16 .
This figure shows the UV-Vis spectrum of normally synthesized gold clusters at pH 7.8 and 37°C. A slight absorption peak at about 280 nm can be seen in all of the above graphs, which represents the protein peak observed in Figure 9. However, it is not as clearly defined in any of the above spectra as in Figure 9.
This could be due to the formation of the nanoclusters, or just the metal and reducing agent themselves, disrupting the structure of the protein. Another spectrum of the samples of 9 equivalents of Au, 9 equivalents of Pt and 8/1 equivalents of Au and Pt were retaken for each after being stored in a refrigerator at 4°C for two weeks. The spectra were almost identical, demonstrating the strong stability of the protein and the Au nanoclusters.
A matrix-assisted laser desorption/ionization (MALDI) was also taken from the normal sample of 9 equivalents of gold, the 9 equivalents of platinum sample, and the sample with 8 equivalents of gold and 1 equivalent of platinum added. A sharp, distinct peak can be seen at approximately 13800 m/z representing the monomeric form of the protein. This figure shows the MALDI of the sample of 8 equivalent Au and 1 equivalent Pt compared to the 9 equivalent Au sample.
The sinapic acid matrix worked best for all samples and the peak intensities were most clearly displayed. This figure shows the emission and excitation spectra of the samples normally synthesized with varying percentages of NaOH reducing agent.
Conclusion
Further extensive research into Au nanoclusters and nanoparticles could lead to applications in biological sensing and catalysis. In the field of electrocatalysis, Au nanoclusters can also play an important role as they are excellent reducing agents in the electroreduction of oxygen. Jin, Rongchao; Zeng, Chenjie; Zhou, Meng; Chen, Yuxiang; “Atomically precise colloidal metal nanoclusters and nanoparticles: fundamentals and.
Kawasaki, Hideya, ken dagiti dadduma pay; Hamaguchi, ken ni Kenji; Osaka, Issey, ken ni Issey; Arakawa, Ryuichi, ken dagiti dadduma pay; "pH-a nakadepende a sintesis dagiti pepsin-a naipamaysa a balitok a nanocluster nga addaan iti asul-berde ken nalabaga a fluorescente a panagibuyat." Wiley-VCH Balay ti Panagpablaak & Co.
