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The increasing functionality of NPs that form complex with MspA/MspAcys could broaden the area of application of this complex. The complex of MspA/MspAcys with magnetic NPs might be another potentially important area (magnetic hyperthermia). Here preliminary data of the complex formation of MspA/MspAcys with magnetic Fe/Pt NPs are presented. The experimental procedures are similar to the ones described in chapter 5 (A) to produce the complex of MspA/MspAcys with magnetic Fe/Pt NPs with different sizes. Fe/Pt NPs are chosen for this experiment, because it is metallic and magnetic material at the same time. These NPs are attracting a lot of interest for the potential application in the field of biomedical sciences because bimetallic Fe/Pt NPs show extremely stable behavior in presence of oxygen during in vivo experiments.1
Experiment details
Chemicals
Platinum acetylacetonate, Pt(acac)2 , iron acetylacetonate, Fe(acac)2, 1,2-hexadecanediol, octyl ether, and 10% tetramethylammonium hydroxide (TMAOH), oleic acid, oleyl amine are purchased from Sigma-Aldrich and used without purification.
NPs synthesis
Two different sizes Fe/Pt NPs are used in this experiment. Fe/Pt I NPs are smaller than Fe/Pt II NPs. Fe/Pt I is synthesized by chemical reduction method as described by Elkins et al.2 Briefly, 1.5 mmol of 1,2-hexadecanediol is used to reflux 0.5 mmol of iron acetylacetonate and 0.5 mmol of platinum acetylacetonate in 30 mL of dioctyl ether in presence of 0.05 mmol of oley
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lamine and 0.05 mmol of oleic acid at the boiling temperature of octyl ether for 30 minutes. Then, the solution is cooled down to room temperature. The residual NPs are subjected to successive washing/centrifuging cycles to remove excessive surfactant using ethyl alcohol. The remaining dark brown precipitate is redispersed in hexane. Thus prepared NPs are transferred into aqueous medium through phase transfer using 10% TMAOH as described by Salgueirino- Maceira et al.3 Briefly, hexane dispersed NPs are redispersed in 2 mL of 10% TMAOH and 100 mL of deionized water. Sonication and shaking of the mixture transform the NPs from hexane to water. The NPs in water are centrifuged several times to eliminate the excess of surfactants, and the precipitates are redispersed in TMAOH. Thus obtained monodispersed Fe/Pt nanoparticles have average size around 3.5 nm. The representative TEM images and size distributions of both NPs are shown in Figure 5.11.
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Figure 5.11 Representative TEM images of (a) Fe/Pt I & (b) Fe/Pt II NPs and (c) and (d) are their respective size distributions.
PL measurements
Tryptophan molecules are the source of fluorescence in MspA and MspAcys. Every MspA molecule contains eight identical amino acid chains and each chain is composed of 184 amino acid residues with four tryptophan fluorophores at positions 21, 40, 72 and 181. There are altogether 32 tryptophan molecules in each MspA molecule.4 Fluorescence emission spectra, quantum yields, and decay times of amino acid tryptophan has long been known to be highly sensitive of its local environment and presence of nearby quenchers.5, 6 In this experiment,
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tryptophan alone is also used with NPs to see the photoluminescence change due to the addition of magnetic NPs.
Fluorescence data are taken after the addition of every 5 µL of NPs solution to assess the change in PL due to the interactions between the fluorophore and NPs’ surface. The relative change in intensity of the fluorescence is shown in Figure 5.12.
Figure 5.12 Increasing fluorescence intensity of the mixture of (a) Fe/Pt I (small) (b) Fe/Pt II (large) with MspA, MspAcys, and tryptophan.
The increasing intensities of fluorescence of the mixture of NPs and MspA, MspAcys are not similar with the mixture of similar size of gold NPs and MspA, MspAcys. However, the size of Fe/Pt NPs is smaller than the opening pore of MspA and MspAcys. This indicates that the complex formation process of MspA/MspAcys with gold and magnetic Fe/Pt NPs is different. Regardless the size (larger or smaller than the opening of porin) of the magnetic NPs Fe/Pt, the interaction is always the same. Interaction of magnetic Fe/Pt NPs with MspA/MspAcys is similar with that of large gold NPs. It might be because of the magnetic moment of the magnetic NPs
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that forces the NPs come near to each other and make aggregates and behaves like large NPs. Our speculation is that the interaction of magnetic Fe/Pt NPs between MspA/MspAcys takes place only from outside of porion. Magnetic moment of magnetic NPs brings them closer and forms large aggregates which make them unable to enter into the porin channel.
Conclusions
The interaction of magnetic Fe/Pt NPs with MspA and MspAcys is studied using fluorescence spectroscopy. The experiment shows both Fe/Pt NPs could not quench the fluorescence from tryptophan of MspA and MspAcys. While gold NPs are able to quench the fluorescence and form supramolecular assemblies. This result clearly indicates that the interactions of Fe/Pt and gold NPs with Mspa/MspAcys are different though the sizes are comparable. The future studies need to be focused on the aggregation of NPs because magnetic moments drag the particles come closer to each other to form bigger cluster. The larger NP size prevents complex formation with MspA. In the previous chapter on the Faraday rotation of magnetic Fe2O3 NPs, the results indicate formation of large aggregates from single NPs in solution. It is speculated that similar thing could happen in the case of the Fe/Pt NPs. However, no aggregation data are taked for this experiment. Therefore, future experiments need to be focused on the quantifying the magnetic interactions. Controlling these interactions could produce useful inorganic/biological complexes.
References
1. Kim, D. K.; Kan, D.; Veres, T.; Normadin, F.; Liao, J. K.; Kim, H. H.; Lee, S. H.; Zahn, M.; Muhammed, M., J. Appl. Phys. 2005, 97, 10Q918.
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2. Elkins, K. E.; Vedantam, T. S.; Liu, J. P.; Zeng, H.; Sun, S. H., Nano Lett. 2003, 3 (12), 1647-1649.
3. Salgueirino-Maceira, V.; Liz-Marzan, L. M.; Farle, M., Langmuir 2004, 20 (16), 6946- 6950.
4. Dani, R.; Kang, M.; Kalita, M.; Smith, P.; Bossmann, S.; Chikan, V., Nano Lett. 2008, 8 (4), 1229-1236.
5. Vivian, J. T.; Callis, P. R., Biophys. J. 2001, 80, 2093-2109.
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