Kenji Iwahori

Circularly Polarized Luminescent CdS Quantum Dots Prepared in a Protein Nanocage

Semiconductor quantum dots (QDs) have attracted a great deal of attention because of their optically tunable light emissions, which are derived from the quantum confinement effect. Unlike bulk materials, QDs utilizing these unique optical properties have a wide range of potential applications, especially in light-emitting devices, sensory materials, and bioassays. More recently, several attempts to develop QDs with optical activity have been reported both experimentally and theoretically. AlthoughQDs prepared with chiral stabilizers, such as CdS or CdTe, showed significant circular dichroism (CD), their circularly polarized luminescence (CPL) was inactive. Density functional calculations revealed that the chiral stabilizer distorted the QD surface only, transmitting an enantiomeric structure to the surface layers. However, the QD core with hexagonal phase remained undistorted and achiral. Herein, we hypothesize that if the whole QD crystal adopted a chiral structure, emission from the QD would express CPL activity. Within this context, we focused on a QD prepared in a rhombic dodecahedral protein, horse spleen ferritin, as a hollow chiral template. The ferritin is composed of 24 a-helixrich subunits with exterior and interior diameters of 12 nm and 8 nm, respectively (Figure 1a). The ferritin ubiquitously regulates intracellular iron homeostasis, in which an iron ion is temporally stored as ferrihydride. Recently, sophisticated structural analysis has revealed that the ferrihydride in the ferritin was structurally distorted, reflecting a chiral nanosphere comprised of the a-helix-rich subunits. Along with the ferrihydride, a variety of QDs were prepared within the ferritin, in which the flow of ion precursors in the hollow core took place through specific channels at the interface of the apoferritin subunits. Here 72 glutamate residues on the interior surface of the apoferritin shell are thought to promote the formation of the QDs (Figure 1b). Therefore, we expected that the QD prepared in the hollow chiral template would adopt a chiral crystal structure, resulting in CPL activity. We first demonstrated that a water-soluble CdS QD prepared in ferritin (CdS@ferritin) exhibits significant lefthanded CPL emissions from direct transition and surfacetrapping sites of the CdS QDs. Furthermore, wavelengths of the photoluminescence (PL)/CPL were modulated by laser photoetching. Figure 2a,b shows PL and CPL spectra of the apoferritin and CdS@ferritin with an excitation wavelength at 325 nm. The apoferritin exhibited an intense PL band with lmax at 396 nm, which originated from post-translationally modified dityrosine in the ferritin shell (Figure 2a, blue line; Figure 2c), whereas its CPL signal was negligibly small (Figure 2b, blue line). On the other hand, CdS@ferritin afforded a broad PL band with lmax at 780 nm and a shoulder peak at ca. 498 nm, although the PL signal from ferritin disappeared, which was probably due to intramolecular energy migration from the ferritin shell to the CdS QD (Figure 2a, red line; Figure 2d). Furthermore, the CPL of CdS@ferritin showed an intense band at 498 nm and a broad band at 780 nm (Figure 2b, red line). To quantify these PL/ CPL spectra, Kuhn s anisotropy factor (gLum) was calculated: gLum is defined as gLum= 2(IL IR)/(IL+IR), where IL and IR indicate the signals for leftand right-handed CPL, respectively (Figure 2e). Consequently, the gLum values at 498 nm Figure 1. a) Illustration of apoferritin from a threefold channel (ribbon model) and b) a cross-sectional view (slab 65%). The 72 glutamate residues on the interior surface are depicted as a yellow space-filling model.

Electrostatic placement of single ferritin molecules

We electrostatically placed a single ferritin molecule on a nanometric 3-aminopropyltriethoxysilane (APTES) pattern that was on an oxidized Si substrate. The numerical analysis of the total interaction free energy for ferritin predicted that a quadrilateral array of 15nm diameter APTES nanodisks placed at intervals of 100nm would accommodate a single molecule of ferritin in each disk under a Debye length of 14nm. The experiments we conducted conformed to theoretical predictions and we successfully placed a single ferritin molecule on each ATPES disk without ferritin adsorbing on the SiO2 substrate surface.

Janus-like Protein Cages. Spatially Controlled Dual-Functional Surface Modifications of Protein Cages

Protein cages have been used both as size-constrained reaction vessels for nanomaterials synthesis and as nanoscale building blocks for higher order nanostructures. We generated Janus-like protein cages, which are dual functionalized with a fluorescent and an affinity label, and demonstrated control over both the stoichiometry and spatial distribution of the functional groups. The capability to toposelectively functionalize protein cages has allowed us to manipulate hierarchical assembly using the layer-by-layer assembly process. Janus-like protein cages expand the toolkit of nanoplatforms that can be used for directed assembly of nanostructured materials.

Critical Amino Acid Residues for the Specific Binding of the Ti-Recognizing Recombinant Ferritin with Oxide Surfaces of Titanium and Silicon

The interactions of ferritins fused with a Ti-recognizing peptide (RKLPDA) and their mutants with titanium oxide substrates were explored with an atomic force microscope (AFM). The amino acid sequence of the peptide was systematically modified to elucidate the role of each amino acid residue in the specific interaction. Force measurements revealed a clear correlation among the sequences in the N-terminal domain of ferritin, surface potentials, and long-range electrostatic interactions. Measurements of adhesion forces clearly revealed that hydrogen bonds take part in the specific binding as well as the electrostatic interaction between charged residues and surface charges of Ti oxides. Moreover, our results indicated that not only the charged and polar residues but also a neutral residue (proline) govern the strength of the specific binding, with the order of the residues also being significant. These results demonstrate that the local structure of the peptide governs the special arrangement of charged residues and strongly affects the strength of the bindings.

Bio-templated CdSe nanoparticle synthesis in a cage shaped protein, Listeria-Dps, and their two dimensional ordered array self-assembly

We report here, for the first time, a biotemplated synthesis of uniform CdSe nanoparticle (4.1 ± 0.5 nm) and a fabrication of two-dimensional CdSe nanoparticles (over one micrometre) with nanometric gaps by cage-like protein, Listeria-Dps.

Direct production of a two-dimensional ordered array of ferritin-nanoparticles on a silicon substrate

We have proposed a Bio Nano process that is a bottom-up technique using protein supramolecules for the fabrication of nanoelectronic devices that exceed the limits imposed by conventional lithography. One of its features is a nanostructure, for example, a two-dimensional (2D) ordered array using protein self-assembly. In this study, we show that a 2D ordered array with nanoparticles forms on a thermally-oxidized silicon substrate by using a solution without metals or sodium ions, which cause malfunctions of semiconductor devices. Furthermore, after protein elimination by UV-ozone treatment, a 2D ordered array was confirmed. The clarification of the principle of a 2D ordered array will be promoted in the future.

Electrostatic Placement of Nanodots onto Silicon Substrate Using Ferritin Protein Supramolecules with Control of Electrostatic Interaction in Solution

The behavior of the electrostatic adsorption of a single ferritin protein supramolecule, which formed a nanodot in its inner cavity, on a nanometric 3-aminopropyltriethoxysilane (APTES) pattern made on an oxidized Si substrate was studied using a numerical calculation. The total interaction free energy of the system, which included a ferrin, a substrate with an APTES nanopattern and a buffer solution, was calculated. The obtained distribution of the interaction potential that ferritin experiences can be used to explain theoretically the ferritin adsorption onto a quadrilateral array of 15-nm-diameter APTES nanodisks placed at intervals of 100 nm under a Debye length of 14 nm. This numerical calculation method described here can be applied to the estimation of the electrostatic adsorption behavior of nanometer-sized material as well as proteins.

Solid-phase PEGylation of an immobilized protein cage on polyelectrolyte multilayer.

We used a quartz crystal microbalance (QCM) to quantitatively characterize solid-phase poly(ethylene glycol) modification (PEGylation) of apoferritin that was electrostatically immobilized on the surface of a polyelectrolyte multilayer. The solid-phase PEGylation processes were monitored by analyzing QCM frequency shifts, which showed that the PEG chains were covalently introduced onto the surface of the immobilized apoferritin. We investigated the effect of PEG concentration, PEG molecular weight, and two-dimensional coverage of the immobilized apoferritin on the solid-phase PEGylation process in addition to the surface properties of the PEGylated apoferritin film, such as wettability and protein adsorption capacity. Since the reaction field is more spatially restricted in solid-phase PEGylation than in traditional aqueous-phase PEGylation, this study shows that a ferritin protein cage is potentially useful as a tailored building block, one that has well-defined structures different from the PEGylated ferritin prepared by an aqueous-phase approach.

Dispersed Gold Nanoparticle Array Produced by Apoferritins Utilizing Biomineralization and Chemical Conversion

A new method for producing a dispersed gold nanoparticle (Au NP) array to anchor probe DNAs onto a DNA-sensing electrode has been developed. A homogenous gold sulfide (Au2S) core (precursor of Au NP) was biomineralized in the cavity of a mutant apoferritin (K98E) with enhanced negative outer-surface charges. We employed a slow chemical reaction system utilizing a stable cationic gold complex. K98E could attract the gold complex, and Au2S NPs were synthesized. K98E enabled dispersed placement of the synthesized Au2S core onto a cationic 3-aminopropyltriethoxysilane (APTES) layer on a substrate. UV–ozone treatment eliminated the protein shells and APTES layer. X-ray photoelectron spectroscopy confirmed that the Au2S core was reduced to Au NPs under the same treatment. Atomic force microscopy (AFM) clearly showed that the combination of apoferritin versatility, chemical system design, and UV–ozone treatment successfully produced a dispersed Au NP array on the substrate.

Fabrication of Semiconductor Nano-particles in the Protein Cage of Apoferritin

We specially designed a slow chemical reaction system to synthesize the zinc selenide nanoparticles (ZnSe NPs), in the cavity of the cage-shaped protein, apoferritin. The newly designed chemical synthesis system for ZnSe NPs makes the chemical reaction of compound semiconductor element ions dramatically slow, resulting in that ZnSe NPs can be synthesized in the internal cavity of the apoferritin. The ZnSe NPs synthesized by the optimized reaction parameters are efficiently produced in the aqueous solution. The UVVis spectrum analysis of synthesized ZnSe-ferritin suggests that the formation of ZnSe nuclei in the apoferritin cavity takes about 6 hours by using our slow chemical reaction system. The synthesized ZnSe NPs were characterized by high resolution TEM, X-ray powder diffraction (XRD) and Energy Dispersive Spectrometory (EDS) and it was revealed that the synthesized NPs are a collection of cubic ZnSe crystals.