Hcp1_cys3 induced assembly of 10 nm silver nanoparticles …

The approach of Hcp1_cys3 induced NP self-assembly with Au-NPs with size of 10.7 nm and Hcp1_cys3 structure with cysteines on the top and bottom rim of the toroid is applied to the silver (Ag)-NP system. For the synthesis of metallic NPs e.g. gold, silver, copper reduction agents such sodium citrate or sodium borohydride are used. But for Ag-NPs with sizes between 1-20 nm, the reproducibility of the synthesis as well as the size control is more difficult to achieve compared to Au-NPs of the same size range¹⁷¹. Anyhow, Ag-NPs of size about 12 nm can be synthesized by the protocol of Wan¹⁴⁸. But it should be mentioned that following this synthesis protocol should give Ag-NPs with a mean size of 4 nm. But here, Ag-NPs with a mean size of 12 nm can be obtained, even after five repetitions of the synthesis. Anyhow, Ag-Hcp1_cys3 samples are prepared by the same protocol as for the Au-Hcp1_cys3 network sample. The Hcp1_cys3 protein concentration is varied between 1 and 20 equivalents (eq.). UV-Vis and TEM are applied to follow the NP assembly process. In Fig. 50 A, the UV-Vis spectra show no shift of the SPR band, but a small shift of the peak maximum from 390 nm to 395 nm, as the protein concentration in the Ag-NP mixture increases. The TEM images of Ag-NP mixtures with 20, 2 eq.

Hcp1_cys3 and 12 mM NaCl in Fig. 50 C-D show similar results as for pure Ag-NPs in Fig. 50 B.

The single NPs do not assemble or aggregate and stay as single particles. Both TEM and UV-Vis results anyhow are consistent indicating separated Ag-NPs. By increasing the NaCl concentration up to 50 mM also does not create any change of the SPR band in the UV-Vis spectra. The reason for this observation can be that the Hcp1_cys3 protein does not bind to Ag-NP, which is unexpected, since the Ag-S binding is in the similar magnitude as the Au-S binding strength¹⁷². Therefore, the sedimentation experiments are conducted and the results are shown in Fig. 51. The sedimentation coefficient (s)-distributions of Ag-NPs and Ag-Hcp1_cys3 mixtures are almost the same. In the case of NP-Hcp1 binding the hybrid structure should show a clear shift of the s-distribution to smaller values, as shown for Au-Hcp1_Q54C in Fig. 45 A. Anyhow, this result explains the nearly unchanged UV-Vis spectra of the Ag-Hcp1_cys3 mixtures and single Ag-NPs in the TEM images. In the attempt to name the reasons for the not-successful binding of Hcp1_cys3 to Ag-NP several factors can be considered. First of all, the antibacterial property of silver NPs is well known, but several explanations are proposed for the same property¹⁷³. But one of them bases on the release of Ag ions on the Ag-NP surface in the presence of oxygen. The Ag ions can interact with the biological systems by forming irreversible bonding and finally lead to bacteria death. This scenario can somehow be excluded for our Ag-NPs. First of all, the Ag-NP solution is always flushed with N2 after all experiments to avoid oxygen in the solution. The solution flask is wrapped in aluminum foil to avoid light and kept in

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the fridge, which should slow down possible oxidation reaction. It may be that in spite of the precautionary measures oxygen is still present in the solution or the mixing step with the aqueous Hcp1_cys3 protein solution can also bring additional oxygen to the mixture leading to Ag ions release. Thus, the ions can saturate the thiol binding site in the Hcp1_cys3 structure causing the impossible binding to the actual Ag-NPs. But the crucial argument against this assumption is the high NaCl concentration (mM) compared to the low Hcp1_cys3 and Ag-NP concentrations in the nanomolar range in the mixture. It means that the released Ag ions will react with the Cl anions forming insoluble AgCl, since the Ag-Cl bond is stronger than the Ag-S bond¹⁷⁴. This should lead to the free thiol binding sites and finally to Ag-Hcp1_cys3 binding.

Unfortunately, the trial to explain the observations in UV-Vis, TEM and AUC fails. Therefore, it can only be stated that for unknown reasons the Ag-Hcp1_cys3 binding is not possible leading to not successful Hcp1_cys3 induced Ag-NP assembly.

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Fig. 50: A) UV-Vis spectra of Ag-NP mixtures with 12 mM NaCl and 2-20 eq. Hcp1_cys3. B-D) The corresponding TEM images of A): pure Ag-NPs, Ag-NPs with 20 eq. Hcp1_cys3 and Ag-NPs with 2 eq. Hcp1_cys3 samples.

A B

C D

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Fig. 51: Sedimentation coefficient distribution (s) of Ag-NP mixtures with increasing Hcp1_cys3 concentration. The NaCl concentration is set to be 15 mM.

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The investigation of gold nanoparticle assembly with sizes between 3.7 and 10.7 nm triggered by Hcp1_cys3 and Hcp1_Q54 toroidal structures is successful. For 10.7 nm Au-NPs chain-like structures of 1D nanoparticle assembly can be obtained with Hcp1_cys3 due to the binding to the cysteines on the top and bottom of the protein ring. Hcp1_cys3 acts as a connector and leads the self-assembly of the NPs to linear structures of limited length until branches are statistically formed by the adsorption of three protein rings on one NP giving a branch.

Therefore, only short linear chains are feasible by this approach using under-stoichiometric Hcp1_cys3 concentrations and all larger structures on the micrometer-scale contain branches.

Nevertheless, the physical property of these structures such as the surface plasmon resonance still corresponds to those of linear structures in a nanoparticle network. The Hcp1_Q54C structure cannot achieve the same Au-protein hybrid architecture due to the absence of the thiol binding sites on both protein surfaces. But in case of 3.7 nm Au-NPs Hcp1_Q54C contributes to the formation of nanostring structures up to 50 nm due to the particle encapsulation inside the Hcp1 cavity. The obtained linear structures are only on the nm-scale.

Therefore, assembly of NPs with defined interparticle distances using the toroidal cysteine modified Hcp1_cys3 protein as a connecting unit is a promising approach towards different NP chains and to explore their physical properties. Unfortunately, for the Ag-NP system with a strong Ag-S bond the Hcp1_cys3 induced NP assembly inexplicably does not occur. Anyhow, the following investigations with CdS- and ZnS-NPs as well as magnetite and cobalt ferrite NPs will focus on the NP size above 4 nm and the Hcp1_cys3 protein. The results for Au-NPs lead to the below scheme, which represents the Au-Hcp1_cys3 chain and nanostring by NPs trapped inside of Hcp1_Q54C.

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103

Chapter 6

Characterization of nanoparticle-protein hybrid structures using

analytical ultracentrifugation and asymmetrical flow-field-flow

fractionation

Chapter 6 – Characterization of nanoparticle-protein hybrid structures using analytical