Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of tripolyphosphate

Gold nanoparticles were prepared by reducing gold salt with a polysaccharide, chitosan, in the absence/presence of tripolyphosphate (TPP). Here, chitosan acted as a reducing/stabilizing agent. The obtained gold nanoparticles were characterized with UV--vis spectroscopy and transmission electron microscopy. The results indicated that the shape and size distribution of gold nanoparticles changed with the molecular weight and concentration of chitosan. More interestingly, the gelation of chitosan upon contacting with polyanion (TPP) can also affect the shape and size distribution of gold nanoparticles. By adding TPP to chitosan solution before the reduction of gold salt, gold nanoparticles have a bimodal size distribution, and at the same time, polygonal gold particles were obtained in addition to spherical gold nanoparticles.     

Functionalization of gold and glass surfaces with magnetic nanoparticles using biomolecular interactions

Advances in nanotechnology have enabled the production and characterization of magnetic particles with nanometer-sized features that can be functionalized with biological recognition elements for numerous applications in biotechnology. In the present study, the synthesis of and interactions between self-assembled monolayers (SAMs) on gold and glass surfaces and functionalized magnetic nanoparticles have been characterized. Immobilization of 10-15 nm streptavidin-functionalized nanoparticles to biotinylated gold and glass surfaces was achieved by the strong interactions between biotin and streptavidin. Fluorescent streptavidin-functionalized nanoparticles, biotinylated surfaces, and combinations of the two were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electron and fluorescent microscopy to confirm that little or no functionalization occurred in nonbiotinylated regions of the gold and glass surfaces compared to the biotinylated sites. Together these techniques have potential use in studying the modification and behavior of functionalized nanoparticles on surfaces in biosensing and other applications.     

Tunable Assembly of Peptide-coated Gold Nanoparticles

The interaction between peptides and gold surfaces has increasingly been of interest for bionanotechnology applications. To more fully understand how to control such interactions, we have studied the optical properties of peptide-modified gold nanoparticles as a function of peptide composition, pH of the surrounding medium, and peptide concentration. We show using localized surface plasmon resonance, transmission electron microscopy, and surface-enhanced Raman scattering (SERS) that selected “gold-binding peptides” (GBPs), similar to those isolated for binding to gold films using yeast display, can bind to gold nanoparticles at a variety of pHs. Peptide modifications of nanoparticles can lead to irreversible particle aggregation when the pH of the solution is kept below the isoelectric point (pI) of the peptide. However, at pHs above the peptide’s pI, particles remain stable in solution, and peptides remain bound to the particles possibly through amine coordination of gold. Additionally, we demonstrate the potential in using SERS for the direct detection of GBPs on gold-silica nanoshells, eliminating the need for indirect labeling methods.     

Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.     

Biological synthesis of gold nanocubes from Bacillus licheniformis

Microorganisms play an important role in the eco-friendly synthesis of metal nanoparticles. This study illustrates the synthesis of gold nanocubes using the bacterium Bacillus licheniformis after 48 h of incubation at room temperature. The morphology of the samples was analyzed using scanning electron microscopy (SEM) and the particles formed were characterized to be nanocubes. The size of gold nanocubes in aqueous solution has been calculated using UV–Vis spectroscopy, XRD and SEM measurements. The nanoparticles are found to be polydisperse nanocubes in the size range 10–100 nm.     

Secondary structure analyses of protein films on gold surfaces by circular dichroism

In order to analyze the secondary structures of protein molecules adsorbed on gold surfaces, circular dichroism (CD) spectra were measured and the secondary structure contents of protein ultra-thin films were estimated quantitatively. A disulfide group was introduced to cytochrome b(562) (cyt.b562), which is a water-soluble b-type heme protein. The cyt.b562 molecules self-assembled to form an ultra-thin protein film both on a gold substrate modified with 2,2(')-dithiodiacetic acid and on a bare gold surface. CD measurements were carried out both in solution and in air, and these results were compared. The protein denaturation was partially prevented, not only in solution but also in air, by both the modification of the substrate and the introduction of the anchor group to the protein molecule. The secondary structure contents of ultra-thin protein films on flat gold surfaces were observed for the first time both in solution and in air by CD spectra.     

SPR-based DNA Detection with Metal Nanoparticles

In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays.     

Gold Nanoring Arrays for Near Infrared Plasmonic Biosensing

Gold nanoring array surfaces that exhibit strong localized surface plasmon resonances (LSPR) at near infrared (NIR) wavelengths from 1.1 to 1.6 μm were used as highly sensitive real-time refractive index biosensors. Arrays of gold nanorings with tunable diameter, width, and spacing were created by the nanoscale electrodeposition of gold nanorings onto lithographically patterned nanohole array conductive surfaces over large areas (square centimeters). The bulk refractive index sensitivity of the gold nanoring arrays was determined to be up to 3,780 cm⁻¹/refractive index unit by monitoring shifts in the LSPR peak by FT-NIR transmittance spectroscopy measurements. As a first application, the surface polymerization reaction of dopamine to form polydopamine thin films on the nanoring sensor surface from aqueous solution was monitored with the real-time LSPR peak shift measurements. To demonstrate the utility of the gold nanoring arrays for LSPR biosensing, the hybridization adsorption of DNA-functionalized gold nanoparticles onto complementary DNA-functionalized gold nanoring arrays was monitored. The adsorption of DNA-modified gold nanoparticles onto nanoring arrays modified with mixed DNA monolayers that contained only 0.5 % complementary DNA was also detected; this relative surface coverage corresponds to the detection of DNA by hybridization adsorption from a 50 pM solution.

     

Polydimethylsiloxane Films Modified with Chitosan/Pectin Multilayers as Scaffolds for Mesenchymal Stem Cells

Solid (smooth) and porous films of polydimethylsiloxane (PDMS) have been obtained; the effect of their structure on the adhesion of mesenchymal stem cells (MSCs) on their surface was found. It is shown that modification of these films with a (chitosan/pectin)₄ multilayer increased the efficiency of viable cell adhesion. A (3-aminopropyl)triethoxysilane–glutaraldehyde system was used to enhance the binding of the polysaccharide layer to the hydrophobic surface of PDMS. It was found that MSCs formed a monolayer culture of the fibroblast-like cells with high viability on porous PDMS modified with (chitosan/pectin)₄.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.     

Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles

The effects of small size (～2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the “free space” between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.     

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

BACKGROUND: Surface bound proteins on colloid particles are widely used in biotechnological applications such as diagnostics or separation. Analysis of colloid surfaces by imaging methods provides information on the structure of these protein films, and an understanding of the functional relationships of biomolecules immobilised on solid surfaces. METHODS: In order to visualise protein molecules organised in films on surfaces of nano-sized gold-particles, an electron-microscopic approach based on the scattering absorption contrast of the specimen was applied. RESULTS: Analysing protein conjugated gold particles with a transmission electron microscope, protein films on gold particle surfaces cause a significant scattering absorption contrast based on the materials' electron density. Thus, the thickness of such films becomes directly measurable in planar projection and the shape of these films are visualised without negative staining methods. The insertion of Ruthenium-labelled antibodies instead of non-labelled antibodies as a marker with increased electron-density in these films yields a contrast enhancement of the whole film. Additional labelling with anti-Mouse IgG Gold conjugates localises the position of the surface bound antibodies in such protein films. CONCLUSIONS: The power of transmission electron microscopy to resolve protein-films on colloid surfaces without staining or labelling as a sample preparation procedure has been demonstrated. Thus, this direct method provides an analytical tool for studying protein films and their structural features on particle surfaces.     

A chemical surface modification of chitosan by glycoconjugates to enhance the cell-biomaterial interaction

The use of wheat germ agglutinin (WGA), a lectin molecule, to modify chitosan and enhance the cell-biomaterial interaction was examined. The percentage of living fibroblast cells on the surfaces of tissue culture polystyrene (TCPS) control, WGA-modified chitosan, and unmodified chitosan films increased to 99%, 99%, and 85%, respectively, after seeding for 48 h. DNA staining revealed that a portion of fibroblasts cultivated on chitosan films( )were undergoing apoptosis. In contrast, fibroblasts growing on WGA-modified chitosan film surfaces did not show any indication of apoptosis. The number of fibroblast cells was the highest on the WGA-modified chitosan surfaces, followed by the TCPS and unmodified chitosan surfaces. This WGA-mediated enhancement on the fibroblast cell-biomaterial interaction was cell type dependent. Other types of cells may need different lectin molecules for enhanced interaction with biomaterials. Further, the evaluation of the heat shock protein (HSP) mRNA expression indicated that HSP 90 expression was increased in the fibroblast cells cultivated on chitosan films and decreased to basal levels on the WGA-modified chitosan films. Taken together, our data suggest that the use of WGA and other lectin molecules to enhance the cell-biomaterial interaction via oligosaccharide-mediated cell adhesion is a promising way to improve cell adhesion and proliferation, the two key issues in tissue engineering.     

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.     

Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRAS) of fibrinogen adsorbed on metal and metal oxide surfaces

We have investigated the possibilities of using Infrared Reflection Absorption Spectroscopy in the study of the interaction of proteins with metal surfaces. Structural information can be obtained since the infrared radiation at the metal surface interacts only with dipole transition moments perpendicular to the metal surface. Fibrinogen spontaneously adsorbed from solution onto gold, titanium and aluminum was used as model systems. The infrared studies were carried out on dried protein films. The amide I bands of fibrinogen adsorbed on the metal surfaces shift towards higher frequencies (ca. 20 cm-1) relative to the same band in buffer solution. The magnitude of these shifts indicates that conformational change of the protein occurs upon adsorption on metal surfaces. The change in conformation of the fibrinogen also can partly be due to one week of drying at room temperature. The amide I and amide II bands show a slightly different behaviour in terms of frequency and intensity for each metal-protein system studied. The side chains appeared to be more substrate sensitive than the peptide group. Orientational effects were observed for a number of side-chain related groups.     

Green synthesis of gold nanoparticles using Nyctanthes arbortristis flower extract

The present study explores the reducing and capping potentials of ethanolic flower extract of the plant Nyctanthes arbortristis for the synthesis of gold nanoparticles. The extract at different volume fractions were stirred with HAuCl₄ aqueous solution at 80 °C for 30 min. The UV–Vis spectroscopic analysis of the reaction products confirmed successful reduction of Au³⁺ ions to gold nanoparticles. Transmission electron microscope (TEM) revealed dominant spherical morphology of the gold nanoparticles with an average diameter of 19.8 ± 5.0 nm. X-ray diffraction (XRD) study confirmed crystalline nature of the synthesized particles. Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) analysis of the purified and lyophilized gold nanoparticles confirmed the surface adsorption of biomolecules during preparation and caused long-term (6 months) stability. Low reaction temperature (25 °C) favored anisotropy. The strong reducing power of the flower extract can also be tested in the green synthesis of other metallic nanoparticles.     

Use of biotinylated chitosan for substrate-mediated gene delivery

To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.     

DNA monolayer on gold substrates characterized by nanoparticle labeling and scanning force microscopy

Monolayers of single-stranded DNA on gold substrates were studied by scanning force microscopy. Complementary DNA probes labeled by gold nanoparticles were applied for contrast enhancement. Substrate regions modified with DNA could be visualized in a highly specific manner. The influence of the solution concentration on the surface density of adsorbed nanoparticles could be visualized. Because individual label particles can be easily detected, this labeling technique opens the way for characterization of DNA monolayers with a lateral resolution in the nanometer range.     

Optical Absorption in Overcoats of Nanoparticle Arrays on a Metallic Substrate

Surface plasma oscillations in metallic particles as well as in thin metallic films have been studied extensively in the past decades. New features regarding surface plasma excitations are, however, constantly discovered, leading, for example, to surface-enhanced Raman scattering studies and enhanced optical transmission though metal films with nanohole arrays. In the present work, the role of a metallic substrate is examined in two cases, one involving an overcoat of dielectric nanoparticles and the other an overcoat of metallic nanoparticles. Theoretical results are obtained by modeling the nanoparticles as forming a two-dimensional, hexagonal lattice of spheres. The scattered electromagnetic field is then calculated using a variant of the Green function method. Comparison with experimental results is made for nanoparticles of tungsten oxide and tin oxide deposited on either gold or silver substrates, giving qualitative agreement on the extra absorption observed when the dielectric nanoparticles are added to the metallic surfaces. Such absorption would be attributed to the mirror image effects between the particles and the substrate. On the other hand, calculations of the optical properties of silver or gold nanoparticle arrays on a gold or a silver substrate demonstrate very interesting features in the spectral region from 400 to 1,000 nm. Interactions between the nanoparticle arrays surface plasmons and their images in the metallic substrate would be responsible for the red shift observed in the absorption resonance. Moreover, effects of particle size and ambient index of refraction are studied, showing a great potential for applications in biosensing with structures consisting of metallic nanoparticle arrays on metallic substrates.     

Preparation and characteristics of novel porous hydrogel films based on chitosan and glycerophosphate

In this study, we prepared thermosensitive hydrogels by adding α-β-glycerophosphate (α-β-GP) to chitosan (CS) solutions. Then the hydrogels were dried to form films at room temperature. Scanning electron microscope (SEM) revealed that the hydrogel films had rough surfaces and porous cross-sections. Compared with pure chitosan films, the CS/GP hydrogel films showed better elasticity and lower tensile strength. Contact angle studies indicated that all these materials have good hydrophilicity. The CS/GP hydrogel films exhibited higher protein adsorption against both negatively charged protein (bovine serum albumin) and positively charged protein (lysozyme) than pure chitosan films. The results of MTT assay performed with the extracts of the CS/GP hydrogel films revealed the films had nontoxicity. The mouse embryonic fibroblast cells cultured on the CS/GP hydrogel films had good spreading and no apparent impairment of cell morphology. The results indicated that the CS/GP hydrogel film could be a promising candidate biomaterial for biomedicine applications.