Exosomal Annexin II Promotes Angiogenesis and Breast Cancer Metastasis.

Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin II as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal Annexin II (exo-Anx II) are still lacking. In this study, mechanistic insight was sought regarding exo-Anx II and its function in angiogenesis and breast cancer metastasis. Multiple in vitro and in vivo techniques were used to study the role of exo-Anx II in angiogenesis. Using atomic force microscopy and Western blotting, exo-Anx II expression was characterized in normal and breast cancer cells. In addition, organ-specific metastatic breast cancer cells and animal models were used to define the role exo-Anx II in breast cancer metastasis. Results revealed that exo-Anx II expression is significantly higher in malignant cells than normal and premetastatic breast cancer cells. In vitro and in vivo studies demonstrated that exo-Anx II promotes tPA-dependent angiogenesis. Furthermore, in vivo analysis indicated that metastatic exosomes create a favorable microenvironment for metastasis, and exo-Anx II plays an important role in this process, as priming with Anx II-depleted exosomes reduces brain (∼4-fold) and lung (∼2-fold) metastasis. Upon delineating the mechanism, it was discovered that exo-Anx II causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 pathways and increased secretion of IL6 and TNFα. These data demonstrate an important role for exo-Anx II in breast cancer pathogenesis. IMPLICATIONS: Exosome-associated Annexin II plays an important role in angiogenesis and breast cancer metastasis, which can be exploited as a potential biomarker as well as a therapeutic target for diagnosis and treatment of metastatic breast cancer. Mol Cancer Res; 15(1); 93-105. ©2016 AACR.

Steady State and Time Resolved Fluorescence Studies of Azadioxatriangulenium (ADOTA) Fluorophore in Silica and PVA Thin Films.

A cationic azadioxatriangulenium (ADOTA) dye was entrapped in silica thin films obtained by the sol-gel process and in poly (vinyl) alcohol (PVA) thin films. Azadioxatriangulenium is a red emitting fluorophore with a long fluorescence lifetime of ~20 ns. The fluorescent properties of azadioxatriangulenium in silica thin films and PVA films were studied by means of steady-state and time resolved fluorescence techniques. We have found that the azadioxatriangulenium entrapped in silica thin film has a wider fluorescence lifetime distribution (Lorentzian distribution), lower fluorescence efficiencies, shorter lifetimes compared to Azadioxatriangulenium in a PVA film. The local environment of azadioxatriangulenium molecules in the silica thin film is rich with water and ethanol, which creates the possibility of forming excited state aggregates due to high concentration of dye within a small confined area. In contrast to the PVA matrices, the porous silica films allow restricted rotations of Azadioxatriangulenium molecules, which result in faster and complex fluorescence anisotropy decays suggesting energy migration among dye molecules.

Preparation of plasmonic platforms of silver wires on gold mirrors and their application to surface enhanced fluorescence.

In this report we describe a preparation of silver wires (SWs) on gold mirrors and its application to surface enhanced fluorescence (SEF) using a new methodology. Silica protected gold mirrors were drop-coated with a solution of silver triangular nanoprisms. The triangular nanoprisms were slowly air-dried to get silver wires that self-assembled on the gold mirrors. Fluorescence enhancement was studied using methyl azadioxatriangulenium chloride (Me-ADOTA · Cl) dye in PVA spin-coated on a clean glass coverslip. New Plasmonic Platforms (PPs) were assembled by placing a mirror with SWs in contact with a glass coverslip spin-coated with a uniform Me-ADOTA · Cl film. It was shown that surface enhanced fluorescence is a real phenomenon, not just an enhancement of the fluorescence signal due to an accumulation of the fluorophore on rough nanostructure surfaces. The average fluorescence enhancement was found to be about 15-fold. The lifetime of Me-ADOTA · Cl dye was significantly reduced (∼ 4 times) in the presence of SWs. Moreover, fluorescence enhancement and lifetime did not show any dependence on the excitation light polarization.

Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange.

Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 µM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.

FRET enhanced fluorescent nanodiamonds.

Fluorescent nanodiamonds (FNDs) are one of the new and very promising biocompatible nanomaterials that can be used both as a fluorescence imaging agent and a highly versatile platform for controlled functionalization to target and deliver a wide spectrum of therapeutic agents. Among the remarkable fluorescence properties are excellent photostability, emission between 600-700nm, quantum yield of 1 and moderately long fluorescence lifetimes. However the low absorption cross section of fluorescent (N-V)(-) centers limits FNDs' brightness. In this work we show that an approach based on the Forster resonance energy transfer (FRET) may significantly enhance the fluorescence signal observed from a single ND. We demonstrate that organic dyes (fluorophores) attached to the FND surface can efficiently transfer the excitation energy to (N-V)(-) centers. Multiple dyes positioned in close proximity to the ND facile surface may serve as harvesting antennas transferring excitation energy to the fluorescent centers. We propose that, with the help of some of the functional groups present on the FND surface, we can either directly link flurophores or use scalable dendrimer chemistry to position many organic dyes at a calibrated distance. Also, the remaining multiple functional groups will be still available for particle targeting and drug delivery. This opens a new way for designing a new type of theranostics particles of ultrahigh brightness, high photostability, specific targeting, and high capacity for drug delivery.

YY1 and a unique DNA repeat element regulates the transcription of mouse CS1 (CD319, SLAMF7) gene.

CS1 (CD319, CRACC, SLAMF7, novel Ly9) activates NK cell-mediated cytotoxicity and proliferation of B lymphocytes during immune responses. The expression of CS1 is up regulated on B cells in multiple myeloma and systemic lupus erythematosus. In this study we describe the transcriptional regulation of mouse CS1 (mCS1) gene. We show that mCS1 gene transcription is regulated by YY1 (Ying Yang 1) and a unique (AG)n=36 DNA repeat element. YY1 is known to play a significant role in B cell development by regulating the pro B cell to pre B cell transition. The consensus DNA binding site for YY1 was detected using TRANSFAQ on the mCS1 promoter region. Mutations in the YY1 site led to a significant increase in mCS1 promoter activity indicating that YY1 represses mCS1 transcription. YY1 binds to the mCS1 promoter at the expected site in vivo and in vitro as tested by chromatin immunoprecipitation assays and super-shift EMSA assays respectively. Unique (CT)n=24 and (AG)n=36 DNA repeat elements are present on mCS1 promoter that are sensitive to S1 nuclease and engage in DNA triplex structure as confirmed by AFM (atomic force microscopy) imaging. Interestingly, the (AG)n=36 repeat element enhances mCS1 promoter activity.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Pre-Clinical Evaluation of rHDL Encapsulated Retinoids for the Treatment of Neuroblastoma.

Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (NB) (HRNB). The long term survival rates of HRNB in the United States are still only 30-50%. Due to resistance that often develops during therapy, development of new effective strategies is essential to improve the survival and overcome the tendency of HRNB patients to relapse subsequent to initial treatment. Current chemotherapy regimens also have a serious limitation due to off target toxicity. In the present work, we evaluated the potential application of reconstituted high density lipoprotein (rHDL) containing fenretinide (FR) nanoparticles as a novel approach to current NB therapeutics. The characterization and stability studies of rHDL-FR nanoparticles showed small size (<40 nm) and high encapsulation efficiency. The cytotoxicity studies of free FR vs. rHDL/FR toward the NB cell lines SK-N-SH and SMS-KCNR showed 2.8- and 2-fold lower IC50 values for the rHDL encapsulated FR vs. free FR. More importantly, the IC50 value for retinal pigment epithelial cells (ARPE-19), a recipient of off target toxicity during FR therapy, was over 40 times higher for the rHDL/FR as compared to that of free FR. The overall improvement in in vitro selective therapeutic efficiency was thus about 100-fold upon encapsulation of the drug into the rHDL nanoparticles. These studies support the potential value of this novel drug delivery platform for treating pediatric cancers in general, and NB in particular.

Metal-enhanced immunoassays.

The surface-confined assay format is one of the most convenient detection formats used in many immunoassays. Fluorescence emission from monolayers of dyes requires a strong excitation and good detection system. Such samples are susceptible to artifacts due to background fluorescence from substrates. We demonstrate that using silver nanostructures deposited on the slide substrate can significantly enhance measured fluorescence, reduce unwanted background and increase photostability of the used probes. Using thin layers of polymer doped with fluorescein, we tested two nanostructures--silver island films (SIFs) deposited on glass slides and self-assembled colloidal structures (SACS) deposited on thin silver film. The SACS surfaces show extraordinary fluorescence enhancements: over 100-folds in hot spots. We applied these surfaces for enhanced Alexa488 model immunoassay.

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

Fluorescent Polyelectrolyte Capped Silver Nanoclusters: Optimization and Spectroscopic Evaluation.

In the present work, we have synthesized water soluble Ag nanoclusters using PMAA as a template with different Ag+: COO-ratios, to optimize it for highest brightness using less UV exposure time. Fluorescence polarization was 0.30 for and was found to vary with excitation and emission wavelength with few hundred picoseconds average fluorescence lifetime. Fluorescence Correlation Spectroscopy data depicts slower diffusion at red excitation compared to blue excitation in confocal volume than conventionally synthesized colloids proving presence of multiple sizes. The optical properties of the particles are dependent upon the excitation wavelength used and the emission wavelength collected.

Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone.

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca(2+) entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca(2+) entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca(2+) entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H(2)O(2) itself evoked Ca(2+) influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H(2)O(2) also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H(2)O(2) stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H(2)O(2) triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.

Förster resonance energy transfer evidence for lysozyme oligomerization in lipid environment.

Intermolecular time-resolved and single-molecule Förster resonance energy transfer (FRET) have been applied to detect quantitatively the aggregation of polycationic protein lysozyme (Lz) in the presence of lipid vesicles composed of phosphatidylcholine (PC) and its mixture with 5, 10, 20, or 40 mol % of phosphatidylglycerol (PG) (PG5, PG10, PG20, or PG40, respectively). Upon binding to PC, PG5, or PG10 model membranes, Lz was found to retain its native monomeric conformation, while increasing content of anionic lipid up to 20 or 40 mol % resulted in the formation of Lz aggregates. The structural parameters of protein self-association (the degree of oligomerization, the distance between the monomers in protein assembly, and the fraction of donors present in oligomers) have been derived. The crucial role of the factors such as lateral density of the adsorbed protein and electrostatic and hydrophobic Lz-lipid interactions in controlling the protein self-association behavior has been proposed.

Morphological changes of supported lipid bilayers induced by lysozyme: planar domain formation vs. multilayer stacking.

Total internal reflection fluorescence microscopy (TIRFM) has been utilized to explore the effect of cationic protein lysozyme (Lz) on the morphology of solid-supported lipid bilayers (SLBs) comprised of zwitterionic lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL). Kinetic TIRFM imaging of different systems revealed subtle interplay between lipid lateral segregation accompanied by exchange of neutral and acidic lipids in the protein-lipid interaction zone, and the formation of lipid multilayer stacks. The switch between these states was shown to be controlled by CL content. In weakly charged SLBs containing 5 mol% CL, assembling of CL molecules into planar domains upon Lz adsorption has been observed while at higher content of anionic lipid (25 mol%) in-plane domains tend to transform into multilayer stacks, thereby ensuring the most thermodynamically-favorable membrane conformation.

Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally.

Studies on mice showed that chitosan as an adjuvant for H5 inactivated influenza vaccines administered intramuscularly enhances significantly antibody titers and protective efficiency not only against homologous influenza viruses, but also against drift variants. Chitosan adjuvanted vaccines induced high antibody titers after a single immunization and with a low dose of antigen. High antibody titers remained for at least 6 months. Chitosan adjuvanted vaccine stored at 4 degrees C preserves its adjuvant properties for at least 8 months. Chitosan stimulates proliferative and cytotoxic activity of splenic mononuclear leukocytes in mice and promotes an increase in the numbers of CD3, CD3/NK, I-AK (MHC II), and H-2Db (MHC I) cells. After intramuscular immunization, chitosan did not induce IgE antibodies and antibodies against chitosan itself. Chitosan is a promising adjuvant candidate for inactivated influenza vaccines administered parenterally.

Enhanced Fluorescence Emission of Me-ADOTA by Self-Assembled Silver Nanoparticles on a Gold Film.

We report a multi-fold enhancement of the fluorescence of methyl-azadioxatriangulenium chloride (Me-ADOTA*Cl) in PVA deposited on a 50 nm thick gold mirror carrying an evaporation induced self-assembly of colloidal silver nanoparticles (Ag-SACs). The average measured increase in fluorescence emission of about 50-fold is accompanied by hot spots with a local enhancement in brigthness close to 200. The long lifetime of the dye allows for the first direct determination of the correlation between the enhancement of emission intensity and the decrease in fluorescence lifetime. The Ag-SACs surface preparation and observed enhancements are highly reproducible. We believe that these robust plasmonic surfaces will find use in sensing platforms for ultrasensitive detection.

Nanostructured silver-based surfaces: new emergent methodologies for an easy detection of analytes.

In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore-labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes.

Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.

Decreasing photobleaching by silver nanoparticles on metal surfaces: application to muscle myofibrils.

Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching. We now investigate whether the photobleaching could be further reduced when the excited fluorophore interacts with the LSP excited in the metallic nanoparticles resident on mirrored surface. As an example we use myofibrils, subcellular structures within skeletal muscle. We compare nanoparticle-enhanced fluorescence of myofibrils in the presence and in the absence of a mirrored surface. The proximity of the mirrored surface led to enhancement of fluorescence and to a decrease in fluorescent lifetime, much greater than that observed in the presence of nanoparticles alone. We think that the effect is caused by the near-field interactions between fluorophores and LSP, and between fluorophores and propagating surface plasmons (PSPs) produced in the metallic surface by the nanoparticles. Photobleaching is decreased because fluorescence enhancement enables illumination with a weaker laser beam and because the decrease in fluorescence lifetime minimizes the probability of oxygen attack during the time a molecule is in the exited state.

Fluorescence Quenching/Enhancement Surface Assays: Signal Manipulation Using Silver-coated Gold Nanoparticles.

Gold nanoparticles covalently attached to the indium tin oxide coated glass slide drastically quench fluorescence of a surface immunoassay (approximately 5-fold). Silver electrochemically deposited over the gold particles leads to fluorescence amplification: signal increases approximately 7-8 times if compared to the signal on gold particles not covered with silver. This phenomenon allows enhancing of the surface immunoassays utilizing both types of nanoparticles.