Improving the sensitivity of immunoassays with PEG-COOH-like film prepared by plasma-based technique.

Herein we report on a preparation and performance of stable, hydrophilic and biocompatible polymeric material suitable for functionalization of disposable substrates used in biosensors. This new material features COOH surface groups cross-linked with ethylene glycol molecules and was prepared in situ on disposable, plastic substrate by high-throughput and environmentally friendly technique called plasma-enhanced chemical vapor deposition (PECVD). The film is grafted to the plasma activated plastic by sequential deposition of tetraethylorthosilicate, forming a bonding layer, and mixed vapors of acrylic acid and diethyleneglycol dimethylether (AA/PEG) that provide the desired functional groups forming a sensing, contact layer.

Nanoparticle-based drug delivery: case studies for cancer and cardiovascular applications.

Nanoparticles (NPs) comprised of nanoengineered complexes are providing new opportunities for enabling targeted delivery of a range of therapeutics and combinations. A range of functionalities can be included within a nanoparticle complex, including surface chemistry that allows attachment of cell-specific ligands for targeted delivery, surface coatings to increase circulation times for enhanced bioavailability, specific materials on the surface or in the nanoparticle core that enable storage of a therapeutic cargo until the target site is reached, and materials sensitive to local or remote actuation cues that allow controlled delivery of therapeutics to the target cells. However, despite the potential benefits of NPs as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of NP materials, as well as their size and shape.

Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay.

Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS).

Novel disposable biochip platform employing supercritical angle fluorescence for enhanced fluorescence collection.

This paper presents an overview of development of a novel disposable plastic biochip for multiplexed clinical diagnostic applications. The disposable biochip is manufactured using a low-cost, rapid turn- around injection moulding process and consists of nine parabolic elements on a planar substrate. The optical elements are based on supercritical angle fluorescence (SAF) which provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the immediate proximity of the biochip surface, thereby having the potential to discriminate against background fluorescence from the analyte solution.

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.

Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays.

This protocol describes an improved and optimized approach to develop rapid and high-sensitivity ELISAs by covalently immobilizing antibody on chemically modified polymeric surfaces. The method involves initial surface activation with KOH and an O(2) plasma, and then amine functionalization with 3-aminopropyltriethoxysilane. The second step requires covalent antibody immobilization on the aminated surface, followed by ELISA.

Shear-mediated platelet adhesion analysis in less than 100 μl of blood: toward a POC platelet diagnostic.

We report a microfluidic chip-based hydrodynamic focusing approach that minimizes sample volume for the analysis of cell-surface interactions under controlled fluid-shear conditions. Assays of statistically meaningful numbers of translocating platelets interacting with immobilized von Willebrand factor at arterial shear rates (∼1500 s(-1)) are demonstrated. By controlling spatial disposition and relative flow rates of two contacting fluid streams, e.

Evaluation of apparent non-specific protein loss due to adsorption on sample tube surfaces and/or altered immunogenicity.

The non-specific loss of protein analytes can have a major effect on assay results particularly where the concentrations of such analytes are extremely low and the matrix is complex. This report assesses how the protein incubated in sample tubes may be lost due to adsorption. Use of proteins, such as bovine serum albumin (BSA), may be used to pre-treat tubes to reduce such losses.

Signal enhancement of surface plasmon-coupled emission (SPCE) with the evanescent field of surface plasmons on a bimetallic paraboloid biochip.

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG.

Novel multiparametric approach to elucidate the surface amine-silanization reaction profile on fluorescent silica nanoparticles.

This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process.

TIRF microscopy as a screening method for non-specific binding on surfaces.

We report a method for studying nanoparticle-biosensor surface interactions based on total internal reflection fluorescence (TIRF) microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor® surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction.

A multiplexed point-of-care assay for C-reactive protein and N-terminal pro-brain natriuretic peptide.

Several new plasma protein biomarkers have been associated with increased risk of cardiovascular events. It would be of great value if sets of these markers could be measured in a multiplexed format at point-of-care settings. A major challenge is the extremely wide concentration range in which different plasma biomarkers are present.

Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach.

A highly sensitive and rapid sandwich enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of human fetuin A/AHSG (alpha2-HS-glycoprotein), a specific biomarker for hepatocellular carcinoma and atherosclerosis. Anti-human fetuin A antibody was immobilized on aminopropyltriethoxysilane-mediated amine-functionalized microtiter plates using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide-based heterobifunctional cross-linking. The analytical sensitivity of the developed assay was 39 pg/mL, compared to 625 pg/mL for the conventional assay.

Fluorescence lifetime analysis and fluorescence correlation spectroscopy elucidate the internal architecture of fluorescent silica nanoparticles.

Fluorescence lifetime (FL) analysis and fluorescence correlation spectroscopy (FCS) have been successfully employed to reveal detailed information about the internal architecture of fluorescent silica nanoparticles (NPs). The dual-component lifetime behavior shows a two-domain dye distribution in the NP as a function of solvent accessibility. The introduction of an undoped silica shell serves to stabilize the outer dye fraction that is manifest as an increase in lifetime.

Demonstration of a surface plasmon-coupled emission (SPCE)-based immunoassay in the absence of a spacer layer.

The technique of surface plasmon-coupled emission (SPCE) involves the coupling of light which is emitted from a fluorophore into the surface plasmon of an adjacent thin metal film, giving rise to highly directional emission. We have combined the advantages of SPCE with the high light collection efficiency of supercritical angle fluorescence by carrying out an immunoassay on a paraboloid array biochip in the absence of the conventional SPCE spacer layer normally used to minimize metal quenching of the fluorescence. In this work, we have successfully demonstrated an SPCE-based assay by utilizing the protein assay layer as the spacer layer.

Microfluidic device to study arterial shear-mediated platelet-surface interactions in whole blood: reduced sample volumes and well-characterised protein surfaces.

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on well-characterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 μL of whole human blood per assay. The device's parallel-plate flow chamber, with 0.

Integrated system investigating shear-mediated platelet interactions with von Willebrand factor using microliters of whole blood.

We report an integrated platelet translocation analysis system that measures complex dynamic platelet-protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics.

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.

Surface plasmon-coupled emission (SPCE)-based immunoassay using a novel paraboloid array biochip.

We have carried out a human IgG immunoassay on a novel disposable optical array biochip using surface plasmon-coupled emission (SPCE) detection. The work successfully combines the advantages of the highly directional SPCE emission profile and enhanced surface plasmon excitation with the high light collection efficiency achieved using supercritical angle fluorescence (SAF). This is achieved using an array of transparent paraboloid polymer elements which have been coated with a thin gold layer to facilitate SPCE.

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.

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.

Surface immobilisation of antibody on cyclic olefin copolymer for sandwich immunoassay.

In this work, the surface functionalisation of the commercially available cyclic olefin copolymer (COC) materials, Zeonor and Zeonex, has been studied. The methodology employed involved oxidation in oxygen plasma, functionalisation of the oxidized surface with aminopropyl triethoxy silane and, finally, attachment of antibody using covalent linker molecules. 1,4-Phenylene diisothiocyanate was selected as the most suitable cross-linker for the attachment of protein, as assessed by fluorescent intensity measurements on immobilised FITC-labelled IgG antibody.

Fabrication of substrate-independent protein microarrays using polyelectrolyte scaffolding.

While significant advances have been made in regard to protein microarray surface chemistries, the surface modification strategies developed are generally substrate-specific and cannot be interchanged between different materials and platforms. This current lack of substrate-independent surface modification strategies makes it difficult to compare or transfer fabrication methodologies between dissimilar substrates. To address this shortcoming, we have developed an interchangeable surface scaffold, which can be utilized to fabricate protein microarrays on a variety of materials with nearly identical results.

Experimental and theoretical evaluation of surface plasmon-coupled emission for sensitive fluorescence detection.

Surface plasmon-coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer, resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. In addition to high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling diminishes with increasing distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed.

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.