Shape control of Au nanostructures using peptides for biotechnological applications.

Metallic gold (Au) nanostructures have attracted attentions in various fields of materials science and electrical science in terms of catalysts, sensing systems, photonic devices, and drug delivery systems because of their characteristic physical, chemical, and biocompatible properties. Recently, Au nanostructures with near-infrared light absorbing properties have shown potential for applications such as biological imaging and thermotherapy in biotechnological fields. However, fabrication of Au nanostructures with complex shapes often requires the use of highly biotoxic substances such as surfactants and reducing agents. Peptides are promising compounds for controlling the shape of Au nanostructures by mineralization with several advantages for this purpose. In this highlight, we focus on the shapes with respect to the fabrication of Au nanostructures using biocompatible peptides. We classify the peptides that form Au nanostructures into three broad categories: those that bind Au ions, those that reduce Au ions, and those that control the direction of Au crystal growth. Then, we briefly summarize the correlations between peptide sequences and their roles, and propose future strategies for fabricating Au nanostructures using peptides for biotechnological applications.

Effect of linearly polarized microwaves on nanomorphology of calcium carbonate mineralization using peptides.

Microwaves are used for diverse applications such as mobile phones, ovens, and therapy devices. However, there are few reports on the effects of microwaves on diseases other than cancer, and on physiological processes. Here, we focused on CaCO3 mineralization as a model of biomineralization and attempted to elucidate the effect of microwaves on CaCO3 mineralization using peptides. We conducted AFM, ζ potential, HPLC, ICP-AES, and relative permittivity measurements. Our findings show that microwaves alter the nanomorphology of the CaCO3 precipitate, from sphere-like particles to string-like structures. Furthermore, microwaves have little effect on the mineralization when the mineralization ability of a peptide is high, but a large effect when the precipitation ability is low. Our findings may be applicable to not only the treatment of teeth and bones but also the development of organic-inorganic nanobiomaterials. This methodology can be expanded to other molecular/atomic reactions under various microwave conditions to alter reaction activity parameters.

Elemental composition control of gold-titania nanocomposites by site-specific mineralization using artificial peptides and DNA.

Biomineralization, the precipitation of various inorganic compounds in biological systems, can be regulated in terms of the size, morphology, and crystal structure of these compounds by biomolecules such as proteins and peptides. However, it is difficult to construct complex inorganic nanostructures because they precipitate randomly in solution. Here, we report that the elemental composition of inorganic nanocomposites can be controlled by site-specific mineralization by changing the number of two inorganic-precipitating peptides bound to DNA. With a focus on gold and titania, we constructed a gold-titania photocatalyst that responds to visible light excitation. Both microscale and macroscale observations revealed that the elemental composition of this gold-titania nanocomposite can be controlled in several ten nm by changing the DNA length and the number of peptide binding sites on the DNA. Furthermore, photocatalytic activity and cell death induction effect under visible light (>450 nm) irradiation of the manufactured gold-titania nanocomposite was higher than that of commercial gold-titania and titania. Thus, we have succeeded in forming titania precipitates on a DNA terminus and gold precipitates site-specifically on double-stranded DNA as intended. Such nanometer-scale control of biomineralization represent a powerful and efficient tool for use in nanotechnology, electronics, ecology, medical science, and biotechnology.

Selective Gold Recovery from Homogenous Aqueous Solutions Containing Gold and Platinum Ions by Aromatic Amino Acid-Containing Peptides.

There is increasing interest in the development of noble metal separation/recovery processes, especially for applications to "urban mining". Common separation/recovery processes for noble metals use a solvent (liquid-liquid) extraction technique in hydrometallurgy. However, these processes are time-consuming and not environmentally friendly, because they use organic solvents for sequential metal ion extractions. Electrowinning is an alternative approach for selective metal precipitation that involves controlling the redox potentials of electrodes but requires specialized equipment and generates hydrogen as a byproduct at the cathode surface under dilute conditions. In the present study, we investigated selective gold recovery from a homogenous aqueous solution containing a mixture of dilute HAuCl4 and H2PtCl6 (5.0 × 10-5 M each) and aromatic amino acid-containing peptides (2.0 × 10-4 M each). Gold selectivity was determined by analyzing the compositions of the solids and supernatants obtained from the reaction mixtures. A much higher gold selectivity (gold/platinum (Au/Pt) atomic ratio = 7.5) was obtained using an anthracene-containing peptide compared to peptides containing one or two naphthalene ring(s). Our proposed approach is applicable to the sequential separation of several noble metal ions, such as Au, palladium (Pd), Pt, iridium (Ir) and rhodium (Rh), and simply requires developing aromatics suitable for each noble metal of interest.

Incorporation of an N-methyl Amino Acid into a Template Peptide Alters Anisotropy in the Crystal Growth of Gold Nanoparticles Synthesized by the Peptide Template Method.

BACKGROUND: Gold nanocrystals have unique physicochemical and biocompatible properties, and hold promise for use as catalysts and in the fields of electronics, photonic and/or plasmonic devices, sensing and/or imaging systems, targeted drug delivery, and photothermal therapies. A variety of organic templates have been used to control the size, shape, and structure of gold nanocrystals, and to modify their surfaces. For the control of the shape of gold nanocrystals, we previously designed and synthesized a ß-sheet-forming nonapeptide (RU006: Ac- AIAKAXKIA-NH2, X = L-2-naphthylalanine, Nal). A mixture of RU006 and HAuCl4 in water produced ultrathin gold nanoribbons with 50-100 nm wide, several nanometers high, and microns long. OBJECTIVES: The main objective of this study is the control of the nanoribbon crystal growth by designing and synthesizing RU006 analogs containing an N-methyl-L-alanine residue. METHODS: We report (i) the design and synthesis of four RU006 analogs in which an L-alanine (Ala) at four positions in the RU006 sequence (N-methylated RU006 analogs) is replaced with an N-methyl alanine, (ii) conformational and morphological analyses of the self-assembled Nmethylated RU006 analogs, (iii) gold nanocrystal synthesis by the peptide templating method with N-methylated RU006 analogs, and (iv) the roles of peptide self-assembly in anisotropic gold crystal growth. RESULTS: RU006 with an N-methyl moiety at the center position resulted in flattened/platelet gold nanocrystals. It was also found that decreasing the mole fraction of RU006 in mixtures with Nmethylated RU006 analogs afforded significantly different absorption spectra compared to that obtained using RU006 alone under gold nanocrystal synthesis conditions. CONCLUSION: We found that morphology of gdd nanocrystals is significantly affected by electron transfer from the naphthalene rings to HAuCl4, accompanied by cross-linking reactions between spatially adjacent naphthalene rings within the hydrophobic cavity of a template assembly.

α-Helical Peptide-Gold Nanoparticle Hybrids: Synthesis, Characterization, and Catalytic Activity.

BACKGROUND: Gold nanoparticles are promising nanomaterials for catalytic reactions, sensing/imaging systems, photonic/plasmonic devices, and electronics because of their unique physical and chemical properties. To date, significant catalytic activities of gold nanoparticles have been reported for reactions such as carbon monooxide oxidation and 4-nitrophenol reduction, and diverse gold nanoparticle morphologies such as nanospheres, wires, rods, and cubes have been achieved using a variety of capping/stabilizing organic molecules. However, there are few reports on the simultaneous assembly of peptides forming secondary structures and metallic nanoparticles into peptide-metallic particle hybrids under mild aqueous conditions and demonstration of their use as catalysts. Furthermore, the gold nanoribbon surfaces are covered with ß-sheet structures, disrupting the access of substrates to the active sites, thereby possibly inhibiting their catalytic activity. OBJECTIVES: The main objective of this study is design, synthesis, and characterization of peptidegold nanoparticle hybrids that are prepared by an α-helical conformation of a template and examination of the catalytic activities of the hybrids. METHODS: We here report (i) the design, synthesis, and characterization of a new template peptide, RU025, that tends to form an α-helical conformation and self-assembles into network nanoarchitectures in aqueous solution through possibly hydrophobic and electrostatic interactions, (ii) the characterization of gold seed crystals synthesized by mixing RU025 and HAuCl4, (iii) the characterization of peptide-gold nanoparticle hybrids directed by crystal growth with NaBH4 and the dependence on the conditions used for nucleation, and (iv) the catalytic activities of the hybrids towards the reduction of 4-nitrophenol to 4-aminophenol in the presence of excess NaBH4. RESULTS: We demonstrated the design, synthesis, and characterization of a new template peptide, RU025, that tends to form an α-helical conformation and self-assembles into network nanoarchitectures in aqueous solution. Gold seed crystals were synthesized by mixing RU025 and HAuCl4 in a 1:2 molar ratio, followed by further reduction of the gold seed crystals with NaBH4. This reaction afforded worm-like gold nanoparticles embedded in the peptide self-assemblies. The peptide-gold nanoparticle hybrids exhibited catalytic activities for the Langmuir-Hinshelwood type reduction of 4-nitrophenol to 4-aminophenol in the presence of excess NaBH4, with an activation energy of 33 kJ mol-1. CONCLUSION: The size and morphology of gold nanoparticles can be tuned in the nanometer range by altering the peptide concentration relative to HAuCl4 and by changing the nucleation time. This method for constructing peptide-metallic nanoparticle hybrids, in which metallic nanoparticles are dispersed in the peptide self-assemblies, provides highly reactive catalysts.

Modification of the N-Terminus of a Calcium Carbonate Precipitating Peptide Affects Calcium Carbonate Mineralization.

BACKGROUND: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP- 1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. OBJECTIVES: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. METHODS: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). RESULTS: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro- and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. CONCLUSION: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research.

Non-Covalent Loading of Anti-Cancer Doxorubicin by Modularizable Peptide Self-Assemblies for a Nanoscale Drug Carrier.

We prepared nanoscale, modularizable, self-assembled peptide nanoarchitectures with diameters less of than 20 nm by combining ß-sheet-forming peptides tethering a cell-penetrating peptide or a nuclear localization signal sequence. We also found that doxorubicin (Dox), an anti-cancer drug, was non-covalently accommodated by the assemblies at a ratio of one Dox molecule per ten peptides. The Dox-loaded peptide assemblies facilitated cellular uptake and subsequent nuclear localization in HeLa cells, and induced cell death even at low Dox concentrations. This peptide nanocarrier motif is a promising platform for a biocompatible drug delivery system by altering the targeting head groups of the carrier peptides.

Site-specific control of multiple mineralizations using a designed peptide and DNA.

We have developed a site-specific method for precipitating multiple inorganic compounds using target DNA and a designed peptide consisting of a peptide nucleic acid (PNA) sequence and an inorganic compound-precipitating sequence. This system for controlled site-specific precipitation represents a powerful tool for use in nanobiotechnology and materials science.

The alkyl linkers in tandem-homodimers of a ß-sheet-forming nonapeptide affect the self-assembled nanostructures.

There is increasing interest in designing smart biomaterials by employing the self-assembly characteristics of synthetic peptides. The use of amyloid-like fibrils is one approach to nanometer- and micrometer-sized supramolecular structures. However, it is generally difficult to predict and/or analyze peptide conformations in nanostructures generated by the self-assembly of ß-sheet-forming peptides such as amyloid-ß peptide because each peptide experiences a slightly different environment. Therefore, a methodology for rationally designing peptide-based smart materials is required. In this study, we demonstrate the design and synthesis of tandem-homodimers of a ß-sheet-forming peptide where the amino acid sequence is duplicated in series and joined via alkyl linkers of different chain length. The conformations of these tandem-homodimers within the self-assembled nanoarchitectures in aqueous solution were characterized. Our findings demonstrate that the hydrophobicity and/or flexibility of the alkyl linkers significantly affect the peptide conformation (extended or bent) of the self-assembled peptide nanostructures. We believe that the present tandem-homodimerization method represents a new direction for the rational design of peptide-based smart biomaterials.

Anomalous Reflection of Gold: A Novel Platform for Biochips.

The importance of protein detection system for protein functions analyses in recent post-genomic era is rising with the emergence of label-free protein detection methods. We are focusing on a simple and practical label-free optical-detection method called anomalous reflection (AR) of gold. When a molecular layer forms on the gold surface, significant reduction in reflectivity can be observed at wavelengths of 400-500 nm. This allows the detection of molecular interactions by monitoring changes in reflectivity. In this chapter, we describe the AR method with three different application platforms: (1) gold, (2) gold containing alloy/composite (AuAg2O), and (3) metal-insulator-metal (MIM) thin layers. The AuAg2O composite and MIM are implemented as important concepts for signal enhancement process for the AR technique. Moreover, the observed molecular adsorption and activity is aided by a three-dimensional surface geometry, performed using poly(amidoamine) or PAMAM dendrimer modification. The described system is suitable to be used as a platform for high-throughput detection system in a chip format.

A Cell Microarray Format: A Peptide Release System Using a Photo-Cleavable Linker for Cell Toxicity and Cell Uptake Analysis.

There has been increasing interest in the potential use of microarray technologies to perform systematic and high-throughput cell-based assays. We are currently focused on developing more practical array formats and detection methods that will enable researchers to conduct more detailed analyses in cell microarray studies. In this chapter, we describe the construction of a novel peptide-array format system for analyzing cellular toxicity and cellular uptake. In this system, a peptide is immobilized at the bottom of a conventional 96-well plate using a photo-cleavable linker. The peptide can then be released from the bottom by irradiating the desired wells with UV light, thus allowing the cytotoxicity or cellular uptake of the peptide to be monitored. This system will facilitate the realization of high-throughput cell arrays for cellomics analyses and cell-based phenotypic drug screens.

Site-specific control of silica mineralization on DNA using a designed peptide.

We developed a site-specific method for precipitating inorganic compounds using organic compounds, DNA, and designed peptides with peptide nucleic acids (PNAs). Such a system for site-specific precipitation represents a powerful tool for use in nanobiochemistry and materials chemistry.

Roles of aromatic side chains and template effects of the hydrophobic cavity of a self-assembled peptide nanoarchitecture for anisotropic growth of gold nanocrystals.

Gold nanocrystals are promising as catalysts and for use in sensing/imaging systems, photonic/plasmonic devices, electronics, drug delivery systems, and for photothermal therapy due to their unique physical, chemical, and biocompatible properties. The use of various organic templates allows control of the size, shape, structure, surface modification and topology of gold nanocrystals; in particular, currently the synthesis of gold nanorods requires a cytotoxic surfactant to control morphology. To control the shape of gold nanocrystals, we previously demonstrated the de novo design and synthesis of a ß-sheet-forming nonapeptide (RU006: Ac-AIAKAXKIA-NH2, X=L-2-naphthylalanine, Nal) and the fabrication of gold nanocrystals by mixing RU006 and HAuCl4 in water. The reaction afforded ultrathin gold nanoribbons 50-100 nm wide, several nanometers high, and microns long. To understand the mechanism underlying gold nanoribbon formation by the RU006 system, we here report (i) the effects of replacement of the Nal aromatic side chain in the RU006 sequence with other aromatic moieties, (ii) the electrochemical properties of aromatic side chains in the de novo designed template peptides to estimate the redox potential and number of electrons participating in the gold crystallization process, and (iii) the stoichiometry of the RU006 system for gold nanoribbon synthesis. Interestingly, RU006 bearing a naphthalene moiety (oxidation peak potential of 1.50 V vs Ag/Ag(+)) and an analog [Ant(6)]-RU006 bearing a bulky anthracene moiety (oxidation peak potential of 1.05 V vs Ag/Ag(+)) allowed the growth of anisotropic (ribbon-like) and isotropic (round) gold nanocrystals, respectively. This trend in morphology of gold nanocrystals was attributed to spatially-arranged hydrophobic cavities sufficiently large to accommodate the gold precursor and to allow directed crystal growth driven by cross-linking reactions among the naphthalene rings. Support for this mechanism was obtained by decreasing the mole fraction of [Ant(6)]-RU006 against the total concentration of [Ant(6)]-RU006 and [Phe(6)]-RU006: absorption spectra similar to that for RU006 were obtained. Differences in the redox properties of the anthracene and naphthalene moieties scarcely affected morphology. We propose that construction of an appropriate hydrophobic cavity is important for templating gold nanocrystal architectures by peptide self-assembly. This mechanism would be applicable for developing simple, low toxicity, mild synthetic methods for constructing metallic nanomaterials for therapeutic use.

Label and Label-Free Detection Techniques for Protein Microarrays.

Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano­biological events.

Ultrathin gold nanoribbons synthesized within the interior cavity of a self-assembled peptide nanoarchitecture.

There is increasing interest in gold nanocrystals due to their unique physical, chemical, and biocompatible properties. In order to develop a template-assisted method for the fabrication of gold nanocrystals, we demonstrate here the de novo design and synthesis of a ß-sheet-forming nonapeptide (RU006: Ac-AIAKAXKIA-NH2, X = L-2-naphthylalanine) which undergoes self-assembly to form disk-like nanoarchitectures approximately 100 nm wide and 2.5 nm high. These self-assemblies tend to form a network of higher-order assemblies in ultrapure water. Using RU006 as a template molecule, we fabricated ultrathin gold nanoribbons 50-100 nm wide, 2.5 nm high, and micrometers long without external reductants. Furthermore, in order to determine the mechanism of ultrathin gold nanoribbon formation, we synthesized four different RU006 analogues. On the basis of the results obtained using RU006 and these analogues, we propose the following mechanism for the self-assembly of RU006. First, RU006 forms a network by the cooperative association of disk-like assemblies in the presence of AuCl4(-) ions that are encapsulated and concentrated within the interior cavity of the network architectures. This is followed by electron transfer from the naphthalene rings to Au(III), resulting in slow growth to form ultrathin gold nanoribbons along the template network architectures under ambient conditions. The resulting ribbons retain the dimensions of the cavity of the template architecture. Our approach will allow the construction of diverse template architectural morphologies and will find applications in the construction of a variety of metallic nanoarchitectures.

A novel array format for monitoring cellular uptake using a photo-cleavable linker for peptide release.

We developed a novel peptide array format incorporating a photo-cleavable linker for monitoring cellular uptake. Model peptides were successfully immobilised via the photo-cleavable linker onto conventional plates and could be released spatiotemporally using UV irradiation. Incorporation of confocal microscopy allowed for detailed real-time monitoring of cellular internalisation of peptides.

Biomimetic alignment of zinc oxide nanoparticles along a peptide nanofiber.

Zinc oxide (ZnO) has potential applications in solar cells, chemical sensors, and piezoelectronic and optoelectronic devices due to its attractive physical and chemical properties. Recently, a solution-phase method has been used to synthesize ZnO crystals with diverse (from simple to hierarchical) nanostructures that is simple, of low cost, and scalable. This method requires template molecules to control the morphology of the ZnO crystals. In this paper, we describe the design and synthesis of two short peptides (RU-003,Ac-AIEKAXEIA-NH(2); RU-027, EAHVMHKVAPRPGGGAIEKAXEIA-NH(2); X = l-2-naphthylalanine) and the characterization of their self-assembled nanostructures. We also report their potential for ZnO mineralization and the alignment of ZnO nanoparticles along peptide nanostructures at room temperature. Interestingly, nonapeptide RU-003 predominantly formed a straight fibrous structure and induced the nucleation of ZnO at its surface, leading to an alignment of ZnO nanoparticles along a peptide nanofiber. This novel method holds promise for the room-temperature fabrication of ZnO catalysts with increased specific surface area, ZnO-gated transistors, and ZnO-based nanomaterials for optical applications.

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity.

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.