Direct immunoassays and their performance - theoretical modelling of the effects of antibody orientation and associated kinetics.

The orientation and activity of antibodies immobilized on solid surfaces are of direct relevance to many immunosensing applications. We therefore investigate a mathematical model which estimates the fraction of antibodies which are available for reaction in a randomly adsorbed sample. Numerical simulations are presented which highlight the separate effects of antibody orientation, accessibility and loss of binding ability on the amount of captured antigen. The assay response can then be expressed as a function of total antibody density and used for optimizing the surface coverage strategy under various conditions.

Cyanine5-doped silica nanoparticles as ultra-bright immunospecific labels for model circulating tumour cells in flow cytometry and microscopy.

In this work, ultra-bright fluorescent silica nanoparticles (NPs) labels have been shown to selectively bind to a model circulating tumour cell (CTC) line, MCF-7, a metastatic breast cancer by targeting epithelial cellular adhesion molecule (EpCAM) present on the MCF-7 cell membrane. Silica NPs approximately 40nm in diameter were doped with different concentrations of Cyanine5 dye molecules, using the reverse microemulsion method. The NPs were two orders of magnitude brighter than Cyanine5 free dye and the measured fluorescence intensity matched a homo-Förster Resonance Energy Transfer model. NPs were conjugated with anti-EpCAM antibody to the NP surface for immunospecific targeting. In flow cytometry experiments the NPs were twice as bright as two commercial anti-EpCAM red fluorophore conjugates, APC and AlexaFluor®647. This increase is achieved while keeping non-specific binding low as established in control tests with a non-metastatic cancer cell line (HeLa). The NPs were also immunospecific in fluorescence microscopy experiments performed at room temperature on non-fixed cells. Confocal microscopy was used to confirm the NPs were located on the surface of the cells, matching with the location of the EpCAM marker. These NPs labels have excellent potential in biomedical diagnostics, particularly when high signal to noise and good photostability are needed, for example, in point-of-care testing.

Investigating the colloidal stability of fluorescent silica nanoparticles under isotonic conditions for biomedical applications.

Fluorescent silica nanoparticle (NP) labels are of great interest in biomedical diagnostics, however, when used in bioassays under physiological conditions they rapidly agglomerate and precipitate from solution leading to high levels of non-specific binding. In this work, using size and zeta-potential data obtained from Dynamic and Electrophoretic Light Scattering analysis, the improvement in colloidal stability of silica NPs under physiological conditions was correlated with an increase in the concentration of three additives: (1) a protein, bovine serum albumin (BSA); (2) a neutral surfactant, Tween 20®; and (3) a charged surfactant, sodium dodecyl sulfate (SDS). The number of BSA molecules present in the NP corona at each concentration was calculated using UV-Vis spectroscopy and a bicinchoninic acid protein assay (BCA). The optimal concentration of each additive was also effective in stabilizing antibody labeled fluorescent nanoparticles (αNPs) under physiological conditions. Using a fourth additive, trehalose, the colloidal stability of αNPs after freeze-drying and long-term storage also significantly improved. Both as-prepared and freeze-dried αNPs were tested in a standard fluorescence immunoassay for the detection of human IgG. The as-prepared assay showed a higher sensitivity at low concentration and a lower limit of detection when compared to a free dye assay. Assays performed with freeze dried αNPs after 4 and 22 days also showed good reproducibility.

Optimization of size, morphology and colloidal stability of fluorescein dye-doped silica NPs for application in immunoassays.

Fluorescent nanoparticle (NP) labels are of great interest for point-of-care medical diagnostics where high fluorescence signals combined with low limits of detection are required. In this work, hydrophilic and hydrophobic fluorescein dye derivatives were covalently doped into silica NPs. The NPs were prepared in a range of sizes from 16 to 80 nm using both ternary and quaternary microemulsion methods where the diameter varied linearly with changes in the water to surfactant ratio. The morphology and colloidal stability of the NPs were characterised using transmission electron microscopy and photon correlation spectroscopy; NPs doped with hydrophobic fluorescein dye were significantly smaller and more polydispersed. Optical properties including absorption, fluorescence and quantum efficiency were also determined. Representative NPs from each microemulsion method (ternary, Ø = 25 nm and quaternary, Ø = 80 nm) were tested as labels in a fluorescence based immunoassay for the detection of human IgG and human chorionic gonadotropin. Both sets of nanoparticle assays showed lower limits of detection and better coefficients of variance than a free dye label with good day to day reproducibility. The optimal surface coverage of detection antibody was also found to depend on the size of the nanoparticle.

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

BACKGROUND: Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery. PRINCIPAL FINDINGS: Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. SIGNIFICANCE: These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Kinetics of immunoassays with particles as labels: effect of antibody coupling using dendrimers as linkers.

In this article, we report on poly(amidoamine) dendrimers (PAMAM) as coupling agents for recombinant single-chain (ScFv) antibodies to nanoparticle (NP) labels, for use in immunoassay. We present a simple theory for the kinetics of particle capture onto a surface by means of an antibody-antigen reaction, in which the important parameter is the fraction of the particle surface that is active for reaction. We describe how increasing the generation number of the linking dendrimers significantly increased the fraction of the NP surface that is active for antigen binding and consequently also increased the assay kinetic rates. Use of dendrimers for conjugation of the NP to the antibody resulted in a significantly higher surface coverage of active antibody, in comparison with mono-valent linker chemistry. As a direct consequence, the increase in effective avidity significantly out-weighed any effect of a decreased diffusion coefficient due to the NP, when compared to that of a molecular dye-labelled antibody. The signal to noise ratio of the G4.5 dendrimer-sensitised nanoparticles out-performed the dye-labelled antibody by approximately four-fold. Particle aggregation experiments with the multi-valent antigen CRP demonstrated reaction-limited aggregation whose rate increased significantly with increasing generation number of the dendrimer linker.

Optical properties of micro-patterned silver nanoparticle substrates.

In this paper, we describe a novel technique for depositing metal nanoparticles (NPs) on a planar substrate whereby the NPs are micro-patterned on the surface by a simple stamp-printing procedure. The method exploits the attractive force between negatively charged colloidal metal NPs and positively-charged polyelectrolyte layers which have been selectively deposited on the surface. Using this technique, large uniform areas of patterned metal NPs, with different plasmonic properties, were achieved by optimisation of the stamping process. We report the observation of unusual fluorescence emission from these structures. The emission was measured using epifluorescence microscopy. Fluorescence lifetime behaviour was also measured. Furthermore, the mu-patterned NPs exhibited blinking behaviour under 469 nm excitation and the fluorescence spectrum was multi-peaked. It has been established that the fluorescence is independent of the plasmon resonance properties of the NPs. As well as optimising the novel NP mu-patterning technique, this work discusses the origin and characteristics of the anomalous fluorescence behaviour in order to characterise and minimise this unwanted background contribution in the use of metal NPs for plasmonic enhancement of fluorescence for optical biochip applications.

Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence.

In this work, we used a model assay system (polyclonal human IgG-goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 +/- 10 and 149 +/- 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of approximately 66x in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.

Nanoparticle strategies for enhancing the sensitivity of fluorescence-based biochips.

This article describes strategies for achieving fluorescence enhancement in optical biochips. Two strategies are discussed: plasmonic enhancement, which is due to the localized surface plasmon resonance of metal nanostructures that are adjacent to the fluorescent labels in optical immunoassays; and the use of high-brightness silica nanoparticles as enhanced labels. We present a review of the state-of-the-art in both areas, including synthesis techniques for the metal and silica nanoparticles and the use of the nanoparticles in optical immunoassays. Data are presented that highlight the key design parameters which influence the level of enhancement and model assay data are presented that illustrate potential enhancements in assay performance.

Experimental and theoretical studies of the optimisation of fluorescence from near-infrared dye-doped silica nanoparticles.

There is substantial interest in the development of near-infrared dye-doped nanoparticles (NPs) for a range of applications including immunocytochemistry, immunosorbent assays, flow cytometry, and DNA/protein microarray analysis. The main motivation for this work is the significant increase in NP fluorescence that may be obtained compared with a single dye label, for example Cy5. Dye-doped NPs were synthesised and a reduction in fluorescence as a function of dye concentration was correlated with the occurrence of homo-Förster resonance energy transfer (HFRET) in the NP. Using standard analytical expressions describing HFRET, we modelled the fluorescence of NPs as a function of dye loading. The results confirmed the occurrence of HFRET which arises from the small Stokes shift of near-infrared dyes and provided a simple method for predicting the optimum dye loading in NPs for maximum fluorescence. We used the inverse micelle method to prepare monodispersed silica NPs. The NPs were characterised using dynamic light scattering, UV spectroscopy, and transmission electron microscopy (TEM). The quantum efficiency of the dye inside the NPs, as a function of dye loading, was also determined. The fluorescent NPs were measured to be approximately 165 times brighter than the free dye, at an optimal loading of 2% (w/w). These experimental results were in good agreement with model predictions.

Optimization of plasmonic enhancement of fluorescence on plastic substrates.

In this work, we report on the uniform deposition of tailored plasmonic coatings on polymer substrates and on the distance dependence of the plasmonic enhancement of a fluorescent dye. Silver, gold, and silver/gold alloy nanoparticles (NPs) with a range of diameters were synthesized using chemical techniques and characterized using UV-vis absorption spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Reproducible polyelectrolyte (PEL) layers, which were deposited on plastic microwell plates using a layer-by-layer technique, served as both a stable and uniform substrate for deposition of the NPs as well as providing spacer layers of known thickness between the NPs and the fluorescent dye. A maximum enhancement factor of approximately 11 was measured for 60 nm diameter pure silver NPs, for a dye-NP separation of approximately 3 nm. A shift in the localized surface plasmon resonance (LSPR) wavelength as a function of the effective refractive index of the PEL layers was also observed, and the measured shifts show a similar trend with theoretical predictions. This work will contribute toward the rational design of optical biochip platforms based on plasmon-enhanced fluorescence.