Revisiting the conformational state of albumin conjugated to gold nanoclusters: A self-assembly pathway to giant superstructures unraveled.

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native ß-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.

New strategy for the gene mutation identification using surface enhanced Raman spectroscopy (SERS).

An early and accurate diagnosis of a specific DNA mutations has a decisive role for effective treatment. Especially, when an immediate decision on treatment most needs to be made, the rapid and precise confirmation of clinical findings is vital. Herein, we show a new strategy for the gene mutation (BRAF c.1799T>A; p. V600E) identification using highly SERS-active and reproducible SERS substrate (photo-etched GaN covered with a thin layer of sputtered gold) and surface enhanced Raman scattering (SERS) spectroscopy. The detection is based on the conformation change (gauche → trans) of the alkanethiol linker modifying the capture DNA during the hybridization process. The value of the intensity ratio of the ν(C-S) bands of the trans and gauche conformer higher than 1.0 indicated the presence of mutation. The demonstrated new DNA SERS (bio)sensor is characterized by the low detection limit at the level of pg µL-1, wide analytical range from 6.75 pg µL-1 to 67.5 ng µL-1 and high selectivity. The proposed bioactive platforms, based on nanostructured GaN substrates modified with thiolated ssDNA (single stranded DNA) can be successfully used in the analysis of clinical samples.

Towards Organized Hybrid Nanomaterials at the Air/Water Interface Based on Liquid-Crystal/ZnO Nanocrystals.

The ability to self-assemble nanosized ligand-stabilized metal oxide or semiconductor materials offers an intriguing route to engineer nanomaterials with new tailored properties from the disparate components. We describe a novel one-pot two-step organometallic approach to prepare ZnO nanocrystals (NCs) coated with deprotonated 4-(dodecyloxy)benzoic acid (i.e., an X-type liquid-crystalline ligand) as a model LC system (termed ZnO-LC1 NCs). Langmuir and Langmuir-Blodgett films of the resulting hybrids are investigated. The observed behavior of the ZnO NCs at the air/water interface is rationalized by invoking a ZnO-interdigitation process mediated by the anchored liquid-crystalline shell. The ordered superstructures form according to mechanism based on a ZnO-interdigitation process mediated by liquid crystals (termed ZIP-LC). The external and directed force applied upon compression at the air/water interface and the packing of the ligands that stabilize the ZnO cores drives the formation of nanorods of ordered internal structure. To study the process in detail, we follow a nontraditional protocol of thin-film investigation. We collect the films from the air/water interface in powder form (ZnO-LC1 LB), resuspend the powder in organic solvents and utilize otherwise unavailable experimental techniques. The structural and physical properties of the resulting superlattices were studied by using electron microscopy, atomic force microscopy, X-ray studies, dynamic light scattering, thermogravimetric analysis, UV/Vis absorption, and photoluminescence spectroscopy.

Gold-oxoborate nanocomposites and their biomedical applications.

A novel inorganic nanocomposite material, called BOA, which has the form of small building blocks composed of gold nanoparticles embedded in a polyoxoborate matrix, is presented. It is demonstrated that cotton wool decorated with the BOA nanocomposite displays strong antibacterial activity toward both Gram-positive and -negative bacteria strains. Importantly, the modified cotton does not release any toxic substances, and the bacteria are killed upon contact with the fibers coated with the BOA. Toxicity tests show that the nanocomposite--in spite of its antiseptic properties--is harmless for mammalian cells. The presented method of surface modification utilizes mild, environmentally friendly fabrication conditions. Thus, it offers a facile approach to obtain durable nontoxic antiseptic coatings for biomedical applications.

Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system.

A highly sensitive immunoassay utilizing surface-enhanced Raman scattering (SERS) has been developed with a new Raman reporter and a unique SERS-active substrate incorporated into a microfluidic device. An appropriately designed Raman reporter, basic fuchsin (FC), gives strong SERS enhancement and has the ability to bind both the antibody and gold nanostructures. The fuchsin-labeled immuno-Au nanoflowers can form a sandwich structure with the antigen and the antibody immobilized on the SERS-active substrate based on Au-Ag coated GaN. Our experimental results indicate that this SERS-active substrate with its strong surface-enhancement factor, high stability and reproducibility plays a crucial role in improving the efficiency of SERS immunoassay. This SERS assay was applied to the detection of Hepatitis B virus antigen (HBsAg) in human blood plasma. A calibration curve was obtained by plotting the intensity of SERS signal of FC band at 1178cm(-1) versus the concentration of antigen. The low detection limit for Hepatitis B virus antigen was estimated to be 0.01IU/mL. The average relative standard deviation (RSD) of this method is less than 10%. This SERS immunoassay gives exact results over a broad linear range, reflecting clinically relevant HBsAg concentrations. It also exhibits high biological specificity for the detection of Hepatitis B virus antigen.

Activation of CO2 by tBuZnOH species: efficient routes to novel nanomaterials based on zinc carbonates.

We report on the activation of CO2 by the well-defined alkylzinc hydroxide (tBuZnOH)6 in the absence and presence of tBu2Zn as an external proton acceptor. The slight modifications in reaction systems involving organozinc precursors enable control of the reaction products with high selectivity leading to the isolation of the mesoporous solid based on ZnCO3 nanoparticles or an unprecedented discrete alkylzinc carbonate [(tBuZn)2(µ5-CO3)]6 cluster with the Zn-C bond intact, respectively.

Formation of net-like patterns of gold nanoparticles in liquid crystal matrix at the air-water interface.

Controlled patterning and formation of nanostructures on surfaces based on self-assembly is a promising area in the field of "bottom-up" nanomaterial engineering. We report formation of net-like structures of gold nanoparticles (Au NPs) in a matrix of liquid crystalline amphiphile 4'-n-octyl-4-cyanobiphenyl at the air-water interface. After initial compression to at least 18 mN m(-1), decompression of a Langmuir film of a mixture containing both components results in formation of net-like structures. The average size of a unit cell of the net is easily adjustable by changing the surface pressure during the decompression of the film. The net-like patterns of different, desired average unit cell areas were transferred onto solid substrates (Langmuir-Blodgett method) and investigated with scanning electron microscopy and X-ray reflectivity (XRR). Uniform coverage over large areas was proved. XRR data revealed lifting of the Au NPs from the surface during the formation of the film. A molecular mechanism of formation of the net-like structures is discussed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11051-012-0826-4) contains supplementary material, which is available to authorized users.