7. Hcp1 induced cadmium- and zinc sulfide nanoparticle assembly
7.2.5 In-situ synthesis of nanoparticles inside of the Hcp1_Q54C
The in-situ synthesis of CdS-QDs was performed by the protocols of Wong206 and Iwahori207. In these protocols the proteins such as apo-ferritin or Dps cage-shaped protein from Listeria innocua (riLiDps) were added to a buffer mixture such as Tris-NaCl or TES-NaCl with a pH of 7.5 or 8. But here water was used as solvent for the synthesis.
In the protocol of Wong a 2 mM CdAc solution was carefully deareated with nitrogen for 1 h.
The synthesis contains a step-wise addition of the Cd and S precursors into the protein solution in four cycles. In each cycle 55 equivalents (eq.) of Cd2+ per one Hcp1 hexamer protein was added to the Hcp1_Q54C solution. The mixture was continuously stirred under nitrogen flow for another 1 h. After approximately 1 h, 2.5 eq. of an aqueous solution of Na2S based on the Cd2+
concentration was added to the mixture. After four cycles the mixture has a molar ratio of 1:220:1100 for Hcp1_Q54C to Cd to S. The sample was dialyzed after the reaction. In our synthesis, 220 eq. Cd2+ were directly added to the Hcp1_Q54C solution and then two increments of Na2S of each 550 eq. in a time distance of 1 h were added to the mixture. The sample was dialyzed against water after the reaction.
In the protocol of Iwahori a mixture containing Hcp1_Q54C, 3.6 M NH3COOH, 700 eq. CdAc based on the Hcp1_Q54C concentration was prepared. The pH of the mixture was adjusted to be 8.6 with a NH3 solution. After that 5 eq. (based on the Cd concentration) of an aqueous thioacetic acid solution was added to the mixture. The reaction solution was shaken at room temperature for 24 h. At the end the solution was centrifuged at 9000 rpm for 10 min to remove bulk precipitates and the supernatant was dialyzed in water for 24 h.
7.2.6 Analytical methods
The absorption spectra were recorded on a UV-Vis Cary 50 Probe from Varian. The protein concentration was calculated by measuring the absorbance at 280 nm with a NanoDrop®ND-1000 from PEQLAB. The fluorescence spectra were recorded on a Luminescence spectrometer LS50 B from Perkin Elmer. TEM images were collected using a Zeiss Libra120 TEM operated at 120 keV. A sample volume of 10 µl was transferred onto a glow discharged carbon coated copper grid. After 10 min the drop was removed by a filter paper. 310 particles were analyzed to determine the mean size of the particles in the sample.
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The CdS- and ZnS-NPs are synthesized by the protocol of Joo et al.205. The obtained nanoparticles have a mean size of 5 and 9 nm for the CdS and ZnS systems, as shown in TEM images of Fig. 57 A-C. The particle shape is quite irregular, but tends to be spherical. Anyhow, the UV-Vis spectra of the NPs in Fig. 57 B-D show the typical exciton absorption bands around 476 nm and 325 nm indicating a blue-shift of the bandgap in respect to the bandgap of the CdS (2.38 eV/520 nm) and ZnS (3.64 eV/340 nm) bulk materials, as observed in the original protocol205. Therefore, the obtained NPs are in the size-confinement regime of the semiconductor nanostructures. A ligand exchange with phosphonoacetic acid, which was already successfully performed for CdSe-QDs117, was conducted for CdS- and ZnS-NPs. The absorption property and the morphology remain the same as shown for the CdS- and ZnS-NPs before the ligand exchange in Fig. 57 A-D.
Fig. 57: TEM image and UV-Vis spectrum of CdS-NPs (A-B) and ZnS-NPs (C-D). The NPs show a mean size of 5 nm and 9 nm, as well as a exciton absorption band at 476 nm and 325 nm for CdS- and ZnS-NPs.
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The phosphonoacetic acid stabilized and ZnS-NPs are dispersible in water and thus, CdS-Hcp1_cys3 and ZnS-CdS-Hcp1_cys3 samples are prepared by the mixing protocol of Au-CdS-Hcp1_cys3 network sample. Since the Hcp1_cys3 concentration is fixed to 3 equivalents for all samples, the effect of salt concentration on the assembly structure is investigated. The TEM result of a sample with 12 mM NaCl in Fig. 58 A shows CdS-NPs assembled in a randomly aggregated manner. As the salt concentration is reduced to be around 1 mM, the CdS-NPs assemble into a more defined structure with linear areas on the nm-scale, as shown in Fig. 58 B. However, the ZnS-NPs show similar assembly structures in the samples with 12 mM and with 1 mM NaCl concentration, as shown in Fig. 59 A-B. It can be noted that the overall-shape of the aggregated NPs in both samples is very similar to the Au-Hcp1_cys3 sample in Fig. 32 D showing elongated particle aggregates containing 6-60 Au-NPs. The linear NP assembly structures are only limited to the nanoscale containing around 10 CdS-NPs. For the ZnS system, the NP-Hcp1_cys3 samples contain equimolar protein concentration. The salt concentration is varied between 12 and 1 mM NaCl (Fig. 59 A-B). The TEM images in both cases show a ZnS-NP assembly structure of a size about 200 nm, but in a random fashion, indicating the minor role of salt concentration in the NP assembly. This can also be observed for the Fe3O4-Hcp1_cys3 samples with low and high NaCl concentration in Fig. 63 C and 65 B as well as CoFe2O4-Hcp1_cys3 sample with zero NaCl concentration in Fig. 74 B of
chapter 8
. The CdS- and ZnS-NP assembly structures prove the general ability of Hcp1_cys3 structure to trigger nanoparticle assembly. The overall shape of the aggregate is elongated, which is similar to the TEM result of the Au-Hcp1_cys3 sample in Fig.32 D. This effect can be related to the tendency of single Hcp1 toroids to linearly assemble on the nm-scale, as described in the Fundamentals chapter. But the desired linear structure on the micrometer scale length, as shown for the Au-Hcp1_cys3 network, cannot be achieved with the CdS- and ZnS-NPs. As a summary, the Hcp1_cys3 can obviously trigger a CdS-NP assembly by comparing the TEM images before in Fig. 57 and after the protein addition in Fig. 58 and 59.
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Fig. 58: TEM image of CdS-NP solution with 3 eq. Hcp1_cys3 containing 12 mM (A) and 1 mM NaCl (B).
Fig. 59: TEM image of ZnS-NP solution with 3 eq. Hcp1_cys3 containing 12 mM (A) and 1 mM NaCl (B).
The next approach is to use the Hcp1_Q54C protein cavity and Hcp1_Q54C fiber, which are spontaneously formed in water, as a nanoreactor and template for the mineralization of CdS-NPs inside of the Hcp1_Q54C fiber. The literature search reveals some interesting biomineralization protocol of NPs inside of the protein cavities. The in-situ synthesis of CdS-QDs inside of the apo-ferritin or Dps cage-shaped protein from Listeria innocua (riLiDps) is performed by Wong206 and Iwahori207. The described syntheses take place in the buffer mixture, but the linear Hcp1_Q54C structures on the nm- and m-scale are observed at a salt concentration below 3 mM. Thus, the synthesis of CdS-QDs following both protocols of Wong
A B
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and Iwahori is accomplished in water. The UV-Vis and photoluminescence investigations are conducted to determine the optical features of the nanosized CdS-QDs. The exemplary UV-Vis spectrum in Fig. 60 A shows an absorption edge at 525 nm, which is the bandgap of the bulk material. According to the published result this absorption edge should be located at 470 nm for CdS-QDs with a size of 4.5 nm. This result hints at CdS material, which does not belong to the size-confinement regime and thus, does not show a blue-shift of the absorption band, as observed for the synthesized CdS-NPs in Fig. 57 B. The photoluminescence spectrum in Fig. 60 B recorded at an excitation wavelength of 360 nm shows no emission signal around 590 nm as expected for 4.5 nm CdS-QDs. The only emission signal is observed at 340 nm in Fig. 60 C, which is typical for the tryptophan residues in the Hcp1 hexamers, as shown in Fig. 52 C. The UV-Vis and photoluminescence results are consistent and point out that the synthesized CdS solution contain semiconductor material with size larger than 4.5 nm and outside of the size-confinement regime. The mineralization approach of CdS-QDs inside of the 4 nm Hcp1_Q54C cavity leading to a fibrous structure with inorganic core as demonstrated for the Au-Hcp1_Q54C nanostrings in chapter 3, unfortunately fails.
Fig. 60: A) UV-Vis spectrum of the in-situ synthesized CdS-NP solution. Photoluminescence spectra of this CdS-NP solution at excitation wavelength of 360 nm (B) and 280 nm (C).
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As a summary, the 1D structures of CdS- and ZnS-NPs prepared by the mixing protocol of Au-Hcp1_cys3 network are limited to the nm-scale. But on larger scale length the obtained structures have the characteristic of a random NP aggregation, as shown in the TEM results.
Therefore, we conclude that for both semiconductor systems the Hcp1_cys3 structure causes NP assembly in a random manner. As a small outlook for this chapter, it can be useful to align the NPs and apply the Hcp1_cys3 as a nanoparticle connecting unit. The resulting hybrid structure can show NP ordering on a larger scale length. Successful example of CdS nanorods alignment in an electric field gives rise to the formation of a 2D array structure on the micrometer scale208. But the applied electric current of around 1000 V/cm is a very high value, which can certainly destroy the Hcp1 protein structure, since proteins in general carry a large amount of charges. Therefore, the utilization of an external electric field seems not to be a promising approach. Thus, in the next chapter magnetic nanoparticles such as magnetite and cobalt ferrite are used in a magnetic field assisted approach for the linear hybrid structure formation. Here, the NPs can be aligned in a magnetic field. After the protein addition, the Hcp1_cys3 structure functions as a connecting unit and leads to hybrid structures on the micrometer scale. The advantage here is that the magnetic field is not destructive towards the protein structure.
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Hcp1_cys3 induced magnetite and cobalt ferrite nanoparticle
assembly and nanocomposite materials
Chapter 8 – Hcp1_cys3 induced magnetite and cobalt ferrite nanoparticle assembly and nanocomposite