Fabrication of fluorescence tunable electrospun conjugated polycarbazole fibers containing gold nanoparticles

In this report, we demonstrate a novel way to tune the fluorescence property of electrospun conjugated polymer fibers. The basic strategy is to use a soluble precursor polymer with gold nanoparticles for electrospinning, which is then cross-linked by applying potential cycles in an electrochemical cell. Electroactive carbazole units in electrospun precursor polymer fibers were converted to conjugated polymer fibers. Since the conjugated polymer fibers can be formed, the fluorescence from the conjugated polymer fibers can be tuned by the rate of the conversion and doping of the fibers. Furthermore, the quenching of the fluorescence, which overlaps with the plasmon band of the gold nanoparticles, was observed. The quenching of the fluorescence properties of the fibers was dependent on the amount of gold nanoparticles inside the fibers.     

Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo

Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.     

Fluorescence enhancement in hot spots of AFM-designed gold nanoparticle sandwiches

We observe an enhancement of fluorescence from a single fluorescent sphere, which is sandwiched between two individual gold nanoparticles, forming a hot spot of strong field enhancement. The fluorescence enhancing hot spot is custom-designed by the deliberate assembly of gold nanoparticles with an atomic force microscope cantilever. The fluorescence intensity is monitored while the separation between the two gold nanoparticles is reduced by gradually pushing the gold nanoparticles closer to the fluorescent sphere. The fluorescence enhancement is maximal when the distance between the two gold nanoparticles is smallest, when the excitation polarization is parallel to the axis of the sandwich, and when the fluorescent sphere is positioned exactly on the axis connecting the two gold nanoparticles.     

Electrochemical synthesis of gold nanoparticles onto indium tin oxide glass and application in biosensors

A simple one-step method for the electrochemical deposition of gold nanoparticles (GNPs) onto bare indium tin oxide film coated glass substrate without any template or surfactant was investigated. The effect of electrolysis conditions such as potential range, temperature, concentration and deposition cycles were examined. The connectivity of GNPs was analyzed by UV-Vis absorption spectroscopy and scanning electron microscopy. The nanoparticles were found to connect in pairs or to coalesce in larger numbers. The twin GNPs display a transverse and a longitudinal localized surface plasmon resonance (LSPR) band, which is similar to that of gold nanorods. The presence of longitudinal LSPR band correlates with high refractive index sensitivity. Conjugation of the twin-linked GNPs with albumin bovine serum-biotin was employed for the detection of streptavidin as a model based on the specific binding affinity in biotin/streptavidin pairs. The spectrophotometric sensor showed concentration-dependent binding for streptavidin.     

Probing the interaction of bovine haemoglobin with gold nanoparticles

The interaction between gold nanoparticles (GNPs) and bovine haemoglobin (BHb) was studied by ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) and fluorescence spectroscopic techniques. The UV-Vis absorption spectrum demonstrated that there was interaction between GNPs and BHb, but no direct interaction between GNPs and haem groups of BHb. The fluorescence data revealed that GNPs effectively quenched the intrinsic fluorescence of BHb via static quenching. The binding of GNPs to BHb occurred at a single site. The binding process was a spontaneous molecular interaction procedure, in which hydrophobic force and hydrogen bonds played a major role. The alternations of protein secondary structure in the presence of GNPs were also determined by CD spectroscopy. This work is helpful to understand the interaction mechanism of GNPs with haemoglobin, which can guide the applications of GNPs in biomedicine.     

The effect of arginine on gold nanoparticles in colloidal solutions and in thin films

Gold nanoparticles were prepared in aqueous colloidal solutions and their interaction with L-arginine solutions at different concentrations was investigated by UV-vis spectroscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). The shift towards red of the absorption maximum of gold nanoparticles with increasing L-arginine concentration and in time, and the apparition of a new large band at higher wavelength evidence the formation of assemblies of gold nanoparticles, mediated by the amino acid. TEM images present the progress in the building process of supermolecular structures. Further, the AFM images show the self assemblies of gold nanoparticles capped with L-arginine well ordered in large domains on silanized glass. As a model for the process, we suggest that the positively charged guanidinium group of L-arginine is anchored on the negative citrate capped gold nanoparticles, while the other two functionalities of L-arginine are involved in the bonding between gold nanoparticles. The ability of arginine to specifically bind gold nanoparticles could lead to an increased ability of proteins, containing arginine, to specifically bind to nanogold. Then, they bind other target proteins or different ligands underlying numerous biological and medical applications that range from nanoscale biosensors, cell-cell communications to targeted delivery of drugs to cancer cells.     

Critical flocculation concentrations, binding isotherms, and ligand exchange properties of peptide-modified gold nanoparticles studied by UV-visible, fluorescence, and time-correlated single photon counting spectroscopies

Protocols for modifying gold nanoparticles with peptide-bovine serum albumin (BSA) conjugates are described within. The resulting constructs were characterized using a number of techniques including static fluorescence spectroscopy and time-correlated single photon counting spectroscopy (TCSPC) in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. TCSPC has proven to be a powerful technique for observing the microenvironment of protein-gold nanoparticle conjugates because it can distinguish between surface-bound and solution-phase species without the need for separation steps. Full characterization of the composition and stability of peptide-modified metal nanoparticles is an important step in their use as intracellular delivery vectors and imaging agents.     

Kinetic studies on water-soluble gold nanoparticles coordinated to poly(vinylpyrrolidone): isotropic to anisotropic transformation and morphology

The growth kinetics, isotropic-to-anisotropic transformation, structural properties and surface morphology of polyvinylpyrrolidone (PVP)-coordinated gold nanoparticles are reported in this work. The reduction of gold ions, kinetics, and growth mechanism of gold nanoparticles, and the coordination between PVP and gold are explored for the first time in this single report. The layer-by-layer growth mechanism (adsorption of gold ions to the nuclei and their subsequent reduction) was observed in the growth of isotropic nanoparticles during the initial stage of the reaction, whereas the Ostwald ripening mechanism (growth of larger particles at the expense of smaller particles) was observed in the growth of the anisotropic nanoparticles in the later stage of the reaction. The surface plasmon resonance band for the anisotropic nanoparticles (average size for a typical sample was ca. 9 nm) was blue-shifted (20 nm) toward that of the isotropic nanoparticles (whose average size is much smaller than that of the anisotropic nanoparticles). The increased effective electron density on the surface of anisotropic particles was the cause of this blue shift. The resultant gold colloids were very stable because the PVP molecules were coordinated through both the C–N and C=O groups, instead of the C=O group alone. The positions of the surface plasmon band and morphology of the gold products were strongly dependent on the amount of PVP.     

Enhancement of luminescence of Rhodamine B by gold nanoparticles in thin films on glass for active optical materials applications

Fluorescent dyes in solid matrices have many potential applications provided that their high optical efficiencies are achieved. We present here gold nanoparticles formed and incorporated together with fluorescent dye Rhodamine B into a film of polyvinyl alcohol (PVA). The increase of fluorescence of the dye results from its interaction with surface plasmons. The electric charge on the gold nanoparticles and the distance between them and the dye molecules has a significant effect on the fluorescence intensity. Fluorescence enhancement of 74% was achieved for the negatively charged particles. Dynamic measurements reveal decrease of fluorescent lifetimes of the dye in presence of gold nanoparticles. Our findings enable utilization of films with enhanced fluorescence in optical materials such as luminescence solar concentrators, solid state tunable laser and active waveguides.     

Shape-controlled synthesis of NIR absorbing branched gold nanoparticles and morphology stabilization with alkanethiols

Gold nanoparticles are ideal candidates for clinical applications if their plasmon absorption band is situated in the near infrared region (NIR) of the electromagnetic spectrum. Various parameters, including the nanoparticle shape, strongly influence the position of this absorption band. The aim of this study is to produce stabilized NIR absorbing branched gold nanoparticles with potential for biomedical applications. Hereto, the synthesis procedure for branched gold nanoparticles is optimized varying the different synthesis parameters. By subsequent electroless gold plating the plasmon absorption band is shifted to 747.2 nm. The intrinsic unstable nature of the nanoparticles' morphology can be clearly observed by a spectral shift and limits their use in real applications. However, in this article we show how the stabilization of the branched structure can be successfully achieved by exchanging the initial capping agent for different alkanethiols and disulfides. Furthermore, when using alkanethiols/disulfides with poly(ethylene oxide) units incorporated, an increased stability of the gold nanoparticles is achieved in high salt concentrations up to 1 M and in a cell culture medium. These achievements open a plethora of opportunities for these stabilized branched gold nanoparticles in nanomedicine.     

Controlling the aggregation behavior of gold nanoparticles

The aggregation of Au nanoparticles (NPs) in solution is influenced by cationic and oligocationic species. The polarization of the conduction electron oscillations in adjacent gold nanoparticles causes a new red-shifted plasmon absorbance attributed to the coupling of the plasmon absorbance of the particles. This appearance of an additional plasmon band is of particular interest to the field of SERS and has led to research works directed at the stabilization of small colloid aggregates in solution. The surface plasmon coupling can be tuned by controlling the aggregation of gold nanoparticles by the addition of some “cross-linking” agent. Here we develop a simple method to fabricate linear-chainlike aggregates of gold nanoparticles (so-called nanochains), tuning the linear optical properties in a wide wavelength range from visible to the near-infrared. The aggregation behavior and linear self-assembly mechanism of citrate-stabilized gold colloids as provoked by the addition of cetyltrimethylammonium bromide (CTAB) and 11-mercaptoundecanoic acid (MUA) are also analyzed. The line-assembly mechanism of gold nanochain is attributed to the preferential binding of CTAB molecules on a certain facet of gold NPs and the Au NP electrostatic interactions. We also found that the 11-mercaptoundecanoic acid was effective to prevent the further aggregation of CTAB-modified gold colloids.     

Detection of phosphopeptides by localized surface plasma resonance of titania-coated gold nanoparticles immobilized on glass substrates

We herein demonstrate a new sensing method for phosphopeptides by localized surface plasmon resonance (LSPR) using titania-coated gold nanoparticles immobilized on the surface of a glass slide as the sensing substrate and using UV-visible spectrophotometry as the detection tool. Titania has been known to be an effective substrate for binding with phosphorylated species. The detection principle is the shift of wavelength of optical absorption due to SPR of the gold nanoparticles induced by binding of phosphorylated species with titania on the surface of the gold nanoparticles. The feasibility of the approach is demonstrated by detection of tryptic digest products of beta-casein and milk. Gold nanoparticles coated with thin films of titania, immobilized on a glass slide, can selectively bind traces of phosphopeptides from complex samples, resulting in a wavelength shift of the absorption band in the SPR spectrum with good reproducibility. The LSPR results are confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The detection limit for the tryptic digest product of beta-casein is 50 nM.     

MspA Porin-Gold Nanoparticle Assemblies: Enhanced Binding through a Controlled Cysteine Mutation

In this study, the interactions of two gold nanoparticles of different sizes (average diameters of 3.7 +/- 2.6 and 17 +/- 3 nm) with octameric mycobacterial porin A from Mycobacterium smegmatis (MspA) and a mutant of MspA featuring a cysteine mutation in position 126 (Q126C) are investigated. From the observation of enhanced photoluminescence quenching, it is inferred that the presence of eight cysteines in the MspA Q126C mutant significantly enhances the binding of selected small gold nanoparticles within the inner pore of MspA. The large gold nanoparticle/porin complex shows photoluminescence enhancement, which is expected since the larger nanoparticles cannot dock within the homopore of MspA due to size exclusion. In addition to the fluorescence experiments, observation of energy transfer from the small gold nanoparticles to the MspA shows the close proximity of the small gold nanoparticles with the porin. Interestingly, the energy transfer of the large nanoparticle/MspA complex is completely missing. From high-performance liquid chromatography data, the estimated binding constants for small Au@MspA, large Au@MspA, small Au@MspAcys, and large Au@MspAcys are 1.3 x 10 (9), 2.22 x 10 (10), > 10 (12) (irreversible), and 1.7 x 10 (10), respectively.     

Optical organophosphate sensor based upon gold nanoparticle functionalized fumed silica gel

We report on the creation of a high surface area, chemically selective material for the efficient adsorption of organophosphate and organophosphonate species. Using silica microparticles in conjunction with gold nanoparticles and surface modification chemistry, we have demonstrated a material with a binding constant for organophosphonates and organophosphates (OPPs) of K=2x10(6) M-1. The binding of OPPs to the modified gold nanoparticles appears as a spectral shift in the gold nanoparticle resonance. The sensitivity of this technique is limited by scattering losses of suspensions of the particles, and we report on how this sensitivity can be recovered to a significant extent by the use of solvents with a refractive index close to that of the silica particles.     

Plasmon-enhanced fluorescence in heterochlorophyllous peridinin-chlorophyll-protein photosynthetic complex

We study the fluorescence properties of peridinin-chlorophyll-protein light-harvesting complex reconstituted with both chlorophyll a and chlorophyll b coupled to spherical gold nanoparticles. Since there is a bidirectional energy transfer between the chlorophylls, the impact of plasmon excitations upon the energy transfer dynamics can be studied in such a hybrid nanostructure. The results show that the emission of both chlorophyll a and chlorophyll b in a reconstituted PCP is strongly enhanced when the light-harvesting complexes are separated from the metallic nanoparticles by a 12-nm-thick SiO₂ layer, the average enhancement reaches a factor of 8. The analysis of fluorescence decay curves indicates that the energy transfer between chlorophyll a and chlorophyll b is also influenced by plasmon excitations in gold nanoparticles.     

Thermal Diffusivity Determination of Protoporphyrin IX Solution Mixed with Gold Metallic Nanoparticles

Nanoparticles appear to be ideally suited for applications in targeted thermal effects in medical therapies and photothermally activated drug delivery; all depend critically on the thermal transport between the nanoparticles and the surrounding liquid. In this work thermal lens spectroscopy (TLS) was used to determine the thermal diffusivity of protoporphyrin IX (PpIX) solutions mixed with gold metallic nanoparticles. PpIX disodium salt (DS) was used in a HCl solution at 25%. Fluids containing gold (Au) nanoparticles at different concentrations were prepared and added to the PpIX solutions. For each solution, UV–Vis spectroscopy was used to obtain the optical absorption spectrum, and transmission electron microscopy (TEM) was used to obtain the gold nanoparticle size. From the TLS signal intensity, it was possible to determine the characteristic time constant of the transient thermal by fitting the theoretical expression to the experimental data. From this characteristic time, the thermal diffusivity was obtained for each solution. The results show that the thermal diffusivity of PpIX mixed with gold nanoparticles increases with an increase of the nanoparticle metallic concentration.     

Finite frequency f-sum rule for assessment of number density of gold nanoparticles (AuNPs) and Kramers-Kronig relation for refractive index of colloidal gold

Absorption spectra from colloids containing different concentrations of spherical gold nanoparticles in water were measured with a spectrophotometer. The absorption spectra were used to calculate the number density of nanoparticles (NPs) with the aid of an unconventional finite spectral band f-sum rule applied for gold colloid. Good correlation between the number density of dispersion electrons, obtained from the f-sum rule, and the number density of nanoparticles was found. The effective absolute refractive index of the gold colloid was obtained with the aid of a singly subtractive Kramers-Kronig relation, and in addition the refractive index change due to the nanoparticles was obtained with the aid of a conventional Kramers-Kronig relation. Such optical properties are valuable in studies of light interaction with nanoparticles.     

Sensitive fluorometric nanoparticle assays for cell counting and viability

We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.     

Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles

A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and magnetic nanoparticles has been developed. The assay is based on the fluorescence quenching of fluorescein isothiocyanate caused by gold nanoparticles coated with monoclonal antibody. To demonstrate its analytical capabilities, the magnetic nanoparticles were coated with anti-alpha-fetoprotein polyclonal antibodies, which specifically bound with alpha-fetoprotein. Gold nanoparticles coated with anti-alpha-fetoprotein monoclonal antibodies could sandwich the alpha-fetoprotein captured by the magnetic nanoparticle probes. The sandwich-type immunocomplex was formed on the surface of magnetic nanoparticles and could be separated by a magnetic field. The supernatant liquid, which contained the unbound gold nanoparticle probes, was used to quench the fluorescence, and the fluorescence intensity of fluorescein isothiocyanate at 516 nm was proportional to the alpha-fetoprotein concentration. The result showed that the limit of detection of alpha-fetoprotein was 0.17 nM. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.     

Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer

Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique, we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.