IGEM:American University/2009/Notebook/BCHM 2/2017/04/04
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Results and Discussion
Anneliese Faustino CHEM 672 Optimizing a Method for Protein Templated CdS Quantum Dot Synthesis
Fluorescence spectroscopy was used to analyze the synthesized CdS QDs. The excitation wavelength for each of the CdS experiments was 341 nm as per the literature and a pre-run fluorescence experiment to determine the excitation wavelength.1 The pilot experiment analyzed myoglobin synthesized CdS QDs after one day of stirring. The fluorescence spectra of the QDs at various pH are shown in Figure 1. The formation of CdS QDs after one day of stirring was poor. Samples with pH 4 and 5 showed some formation of QDs with broad peaks around 500 nm. CdS QDs are known to fluoresce around 498 nm, confirming a successful synthesis.1 Another smaller peak was visible for pH 4 and 5 around 535 nm; this peak was not reported in the literature. The fluorescence spectra for pH 7 through 10 did not present any visible fluorescence peaks. The myoglobin synthesized QDs were analyzed again after seven days of stirring to achieve better QD yield and better fluorescence spectra resolution.2 The excitation wavelength remained the same at 341 nm. The results of this experiment are shown in Figure 2. Seven days of stirring yielded much more interesting fluorescence spectra. Broad peaks were visible between 490 nm and 535 nm. There was a small decrease in intensity between pH 4 and pH 8, then a larger decrease for pH 9. The sample at pH 10 had an extremely broad and low intensity peak.
BSA templated CdS QDs were prepared and stirred for seven days. The synthesized QDs were analyzed via fluorescence spectroscopy at an excitation wavelength of 341 nm. The results of this experiment are shown in Figure 3. The BSA templated QD spectra are different than that of myoglobin for certain pH samples. pH 4 shows a high intensity peak around 430 nm; this may be a protein CdS complex. It also has the usual peaks around 490 nm and 535 nm. pH 5 and 6 have slightly lower intensity than pH 4 in the 490-535 nm region. pH 7 and 8 have even lower fluorescence intensity than the lower pH samples, and they also exhibit a very broad peak in the 425-535 nm region. pH 9 and 10 exhibit very low intensity peaks in the 490-535 nm region.
Lysozyme templated CdS QDs were prepared and stirred for seven days. The synthesized QDs were analyzed via fluorescence spectroscopy at an excitation wavelength of 341 nm. The results of this experiment are shown in Figure 4. Lysozyme templated QDs followed the general trend of the fluorescence intensity decreasing with increasing pH except in the case of the pH 10 sample. Samples with pH 4 through 9 all had broad peaks between 490 and 535 nm, but pH 10 had a fluorescence intensity 2-3 times greater than any other pH in the 400-490 nm region. Furthermore, there is no visible peak in the 490-535 nm region for the sample at pH 10. This could be due to a CdS lysozyme complex or from the protein unfolding due to the high pH, revealing fluorescent amino acid residues.3
Myoglobin with Ammonium
A 2007 study found that synthesis of CdS QDs could be improved by having a high concentration of ammonium ions as compared to cadmium concentration. This is because ammonium ions may stabilize the cadmium ions as tetraaminecadmium ions. This in turn slows the reaction, preventing the bulk sulfur from precipitating out of the solution.4 The previous experiments had problems with sulfur precipitate, particularly in samples with acidic pH. To analyze the effect of ammonium on CdS QD formation, myoglobin templated CdS QDs were prepared with a 40 mM ammonia acetate additive and stirred for seven days. The synthesized QDs were analyzed via fluorescence spectroscopy at an excitation wavelength of 341 nm. The results of this experiment are shown in Figure 5. Fluorescence intensity for this experiment was much higher than that of the CdS myoglobin experiment without the ammonium additive, demonstrating the synthesis of more or more fluorescent quantum dots. The broad fluorescence peaks were in the wavelength range of 490-535 nm. Again, the general trend of increasing fluorescence intensity with decreasing pH was apparent. pH 9 and 10 had extremely low fluorescence intensities.
Myoglobin with Ammonium and a Na2S Sulfur Source
To better understand the effect of the sulfur source myoglobin templated CdS QDs were prepared with a Na2S sulfur source (instead of the potassium thioacetate sulfur source) as well as a 40 mM ammonia acetate additive and stirred for seven days. The synthesized QDs were analyzed via fluorescence spectroscopy at an excitation wavelength of 341 nm. The results of this experiment are shown in Figure 6. The resulting fluorescence spectra were different than those of any of the previous experiments. Broad peaks around 610 nm were formed at pH 7-10 with pH 10 having the most intense peak.
Discussion Among the three protein scaffolds that were studied, myoglobin showed the most consistent production of CdS QDs, which is why it was chosen for the ammonium acetate and Na2S studies. The most distinct difference between myoglobin and the other two proteins is its heme prosthetic group. Heme groups are known for attracting heavy metals such as cadmium, which is often a problem in heavy metal poisoning patients.5 This heme group-cadmium interaction may be the cause of the more optimized synthesis of CdS QDs. The addition of the ammonium acetate was meant to prevent the formation of sulfur precipitate by stabilizing the cadmium ions as tetraaminecadmium ions and allowing for slower formation of the QDs. Sulfur precipitate can disrupt fluorescence readings due to the inner filter effect of the precipitated sulfur absorbing light.6 To prevent the inner filter effect from affecting the fluorescence measurements, ammonium acetate was added to the reaction mixture. Fluorescence intensity readings increased after the addition of ammonium acetate. Furthermore, fluorescence peaks were easier to identify in the spectra. The addition of stabilizing ammonium ions is an important step in optimizing CdS QD synthesis.4 The sulfur source may have a major impact on the formation of CdS quantum dots. Potassium thioacetate was the sulfur source used in the majority of the experiments as a less toxic version of thioacetic acid, which was described as the most effective sulfur source for synthesis in a 2010 study of CdS QDs.1 Thioacetic acid degrades to H2S, HS- or S2- based on the solution pH as shown below:
CH3COSH + H2O CH3COOH +H2S H2S HS- + H+ pKa = 7.0 HS- S2- + H+ pKa = 13.9
Acidic conditions cause H2S to be the sulfur source in the reaction mixture; however, basic conditions will have HS- or S2- species based on the pH of the reaction mixture. For the purposes of this experiment, HS- was the sulfur source in the basic solutions. Negatively charged amino acids will repel the negatively charged sulfur sources. The RSCB Protein Data Bank was used to check the primary sequences of myoglobin, BSA, and lysozyme. Each of these proteins was found to contain negatively charged amino acid residues.7 The decrease in fluorescence intensity with increasing pH may therefore be related to the negatively charged amino acids and sulfur source speciation.4 The experiment that used Na2S as the sulfur source further demonstrated that there can be major effects on the QD synthesis based on the sulfur source speciation. Na2S will only produce S2- ions in samples of every pH.8 The negatively charged S2- species will enter the protein scaffold slowly because of electrostatic repulsion of the negatively charged amino acid residues. In this experiment, pH 8-10 showed a fluorescence peak at 610 nm. This may be because of a complex forming between the protein, sulfur source, and QDs. It may also be because the QDs are forming at a different size. To better understand the process of sulfur source speciation and QD formation, future study will involve control experiments without the protein in order to rule out a protein, sulfur source, QD complex and to better understand how the QDs form without the protein scaffold.9
Figure 2: Fluorescence spectra of myoglobin templated CdS QDs at various pH. These spectra were analyzed after seven days of stirring.
Figure 3: Fluorescence spectra of BSA templated CdS QDs at various pH. These spectra were analyzed after seven days of stirring.
Figure 4: Fluorescence spectra of lysozyme templated CdS QDs at various pH. These spectra were analyzed after seven days of stirring.
Figure 5: Fluorescence spectra of myoglobin templated CdS QDs with ammonium at various pH. These spectra were analyzed after seven days of stirring.
Figure 6: Fluorescence spectra of myoglobin templated CdS QDs with an ammonium additive and a Na2S sulfur source at various pH. These spectra were analyzed after seven days of stirring. References 1. Naito, M.; Iwahori, K.; Miura, A.; Yamane, M.; Yamashita, I., Circularly Polarized Luminescent CdS Quantum Dots Prepared in a Protein Nanocage. Angewandte Chemie International Edition 2010, 49 (39), 7006-7009. 2. Iwahori, K.; Yoshizawa, K.; Muraoka, M.; Yamashita, I., Fabrication of ZnSe Nanoparticles in the Apoferritin Cavity by Designing a Slow Chemical Reaction System. Inorganic Chemistry 2005, 44 (18), 6393-6400. 3. Imoto, T.; Forster, L. S.; Rupley, J. A.; Tanaka, F., Fluorescence of Lysozyme: Emissions from Tryptophan Residues 62 and 108 and Energy Migration. Proceedings of the National Academy of Sciences of the United States of America 1972, 69 (5), 1151-1155. 4. Iwahori, K.; Enomoto, T.; Furusho, H.; Miura, A.; Nishio, K.; Mishima, Y.; Yamashita, I., Cadmium Sulfide Nanoparticle Synthesis in Dps Protein from Listeria innocua. Chemistry of Materials 2007, 19 (13), 3105-3111. 5. Schauder, A.; Avital, A.; Malik, Z., Regulation and gene expression of heme synthesis under heavy metal exposure--review. Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer 2010, 29 (2), 137-58. 6. Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B., Experimental correction for the inner-filter effect in fluorescence spectra. Analyst 1994, 119 (3), 417-419. 7. H.M. Berman; J. Westbrook; Z. Feng, G. G.; T.N. Bhat, H. W.; I.N. Shindyalov; Bourne, P. E., The Protein Data Bank. Nucleic Acids Research 2000, 28, 238-232. 8. Zhou, W.; Schwartz, D. T.; Baneyx, F., Single-Pot Biofabrication of Zinc Sulfide Immuno-Quantum Dots. Journal of the American Chemical Society 2010, 132 (13), 4731-4738. 9. Yanhong, L.; Dejun, W.; Qidong, Z.; Min, Y.; Qinglin, Z., A study of quantum confinement properties of photogenerated charges in ZnO nanoparticles by surface photovoltage spectroscopy. The Journal of Physical Chemistry B 2004, 108 (10), 3202-3206.