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.

Measuring protein-protein interactions using Biacore.

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1,179 papers that involved the application of biosensor data were identified for the year 2007 alone (Rich and Myszka, J Mol Recognit 21:355-400, 2008), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid.

New trends in bioanalytical microdevices to assess platelet function.

Cardiovascular disease is the major cause of mortality globally. The role of platelets and antiplatelet drugs in the treatment of cardiovascular disease is widely appreciated. Platelets have a less well-known role in cancer and inflammation and as the role of platelets in cancer and inflammation is increasingly understood, there is a compelling need to develop diagnostic assays of platelet function to guide clinical management.

A high-affinity recombinant antibody permits rapid and sensitive direct detection of myeloperoxidase.

Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display.

Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk.

Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis.

Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer.

We report a label-free optical detection technique, called total internal reflection ellipsometry (TIRE), which can be applied to study the interactions between biomolecules and a functionalized polymer surface. Zeonor (ZR), a cycloolefin polymer with low autofluorescence, high optical transmittance and excellent chemical resistance, is a highly suitable material for optical biosensor platforms owing to the ease of fabrication. It can also be modified with a range of reactive chemical groups for surface functionalization.

Optically addressable single-use microfluidic valves by laser printer lithography.

We report the design, fabrication, and characterization of practical microfluidic valves fabricated using laser printer lithography. These optofluidic valves are opened by directing optical energy from a solid-state laser, with similar power characteristics to those used in CD/DVD drives, to a spot of printed toner where localized heating melts an orifice in the polymer layer in as little as 500 ms, connecting previously isolated fluidic components or compartments. Valve functionality, response time, and laser input energy dependence of orifice size are reported for cyclo-olefin polymer (COP) and polyethylene terephthalate (PET) films.

Functionalization of cyclo-olefin polymer substrates by plasma oxidation: stable film containing carboxylic acid groups for capturing biorecognition elements.

Many current designs in biomedical diagnostics devices are based on the use of low cost, disposable, easy-to-fabricate chips made of plastic material, typically a cyclo-olefin polymer (COP). Low autofluorescence properties of such material, among others, make it ideal substrate for fluorescence-based applications. Functionalization of this plastic substrate for biomolecule attachment is therefore of great importance and the quality of films produced on such surface have often a significant influence on the performance of the device.

Highly sensitive recombinant antibodies capable of reliably differentiating heart-type fatty acid binding protein from noncardiac isoforms.

During recent times, heart-type fatty acid binding protein (hFABP) has gained increasing credence as a promising cardiac biomarker. This is largely due to its rapid myocardial release and subsequent clearance kinetics, which are superior to those of myoglobin and offer an earlier diagnostic window than the troponins. Realization of its full diagnostic and prognostic potential is dependent on accessibility to robust hFABP-specific assays.

Formation and growth of oxide layers at platinum and gold nano- and microelectrodes.

The construction and characterization of platinum and gold disk electrodes with minimum radii of 7 nm (platinum) and 500 nm (gold) is reported. The electrodes were prepared with a micropipet puller using a two step procedure and have been characterized using scanning electron microscopy, scanning electrochemical microscopy, high speed chronoamperometry, and cyclic voltammetry. The formation and growth of platinum and gold oxide layers, on the electrodes at time scales from microseconds to seconds, is reported.

Microfluidic sedimentation cytometer for milk quality and bovine mastitis monitoring.

We report a rapid, low-cost, portable microfluidic sedimentation cytometer (SeCy) for assessing the somatic cell count and fat content of milk in 15 min using a "sample-in, answer-out" approach. The system consists of 12 independent microfluidic devices, essentially flattened funnel structures, fabricated on the footprint of a single plastic compact disc (CD). Each funnel structure holds 150 μL of milk, has an inlet for milk filling and an outlet for air to escape, and ends in a narrow, closed-end microfluidic channel that facilitates packing of the cells into a column whose length is proportional to cell count.

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.

Electrogenerated chemiluminescence.

In electrogenerated chemiluminescence, also known as electrochemiluminescence (ECL), electrochemically generated intermediates undergo a highly exergonic reaction to produce an electronically excited state that then emits light. These electron-transfer reactions are sufficiently exergonic to allow the excited states of luminophores, including polycyclic aromatic hydrocarbons and metal complexes, to be created without photoexcitation. For example, oxidation of [Ru(bpy)(3)](2+) in the presence of tripropylamine results in light emission that is analogous to the emission produced by photoexcitation.

Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends.

The lack of a clear correlation between design and protection continues to present a barrier to progress in vaccine research. In this article, we outline how surface plasmon resonance (SPR) biosensors are emerging as tools to help resolve some of the key biophysical determinants of protection and, thereby, facilitate more rational vaccine design campaigns. SPR technology has contributed significantly to our understanding of the complex biophysical determinants of HIV neutralization and offers a platform for preclinical evaluation of vaccine candidates.

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.

Plasma functionalization of AFM tips for measurement of chemical interactions.

In this paper, a new, fast, reproducible technique for atomic force microscopy (AFM) tips functionalization used for chemical interaction measurements is described. Precisely, the deposition of an aminated precursor is performed through plasma-enhanced chemical vapor deposition (PECVD) in order to create amine functional groups on the AFM tip and cantilever. The advantages of the precursor, aminopropyltriethoxysilane (APTES), were recently demonstrated for amine layer formation through PECVD deposition on polymeric surfaces.

Simple method of 'on-plate' growth for improved antibody screening.

Antibody phage display is a powerful biomolecular selection technology now routinely used for refining antibody diversity towards analytes of both therapeutic and diagnostic interest. Post selection, automated robotic systems can be utilised to pick, express and analyse large numbers of putative analyte-specific clones allowing the parallel screening of thousands of antibodies in less time. Most screening techniques involve a spatial addressing process whereby the selected antibodies are extracted from the cells and analysed to verify specificity.

Hybrid integrated PDMS microfluidics with a silica capillary.

To harness the properties of both PDMS and silica, we have demonstrated hybrid integrated PDMS microfluidic systems with fused silica capillaries. The hybrid integrated PDMS microfluidics and silica capillary (iPSC) modules exhibit a novel architecture and method for leakage free CE sample injection merely requiring a single high voltage source and one pair of electrodes. The use of the iPSC device is based on a modular approach which allows the capillary to be reused extensively whilst replacing the attached fluidic module for different experiments.

Photochromic imidazolium based ionic liquids based on spiropyran.

We investigate the physicochemical properties of a novel imidazolium benzospiropyran derivative, SP(Im), in imidazolium based ionic liquids (ILs). SP(Im) was prepared through alkylation of an imidazole to the photoswitchable compound and this derivative was characterised in imidazolium based ILs with increasing chain length to examine the stability of its merocyanine (MC) and spiropyran (SP) forms and compared to standard spiropyran, BSP. The rate of thermal relaxation of the new derivative is found to be about ten times faster than that of BSP as reflected in rates of 13.

Deposition of chemically reactive and repellent sites on biosensor chips for reduced non-specific binding.

The performances of new polymeric materials with excellent optical properties and good machinability have led the biomedical diagnostics industry to develop cheap disposable biosensor platforms appropriate for point of care applications. Zeonor, a type of cycloolefin polymer (COP), is one such polymer that presents an excellent platform for biosensor chips. These polymer substrates have to be modified to have suitable physico-chemical properties for immobilizing proteins.

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance.

When designing devices for biomedical diagnostics, increasing the signal to noise ratio is often critical for achieving clinically relevant sensitivity and limits of detection (LOD). In antibody-based assays, the measured signal can be amplified through the replacement of molecular fluorophores with doped nanoparticles (NP). However, the benefits of using NPs can only be realized if the NPs are coated efficiently with detection antibody, have good colloidal stability and the ratio of specific to non-specific binding (NSB) is high enough.

Development of a fluorescent anti-factor Xa assay to monitor unfractionated and low molecular weight heparins.

Fluorogenic assays have many potential advantages over traditional clot-based and chromogenic assays such as the absence of interference from a range of factor deficiencies as well as offering the possibility of assays in platelet rich plasma or whole blood. A fluorogenic anti-factor Xa (anti-FXa) assay has been developed for the determination of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), namely enoxaparin and tinzaparin, and the synthetic heparinoid danaparoid, in commercial human pooled plasma. The assay was based on the complexation of heparin-spiked plasmas with exogenous FXa at a concentration of 4nM in the presence of 0.

Functionalization of cycloolefin polymer surfaces by plasma-enhanced chemical vapour deposition: comprehensive characterization and analysis of the contact surface and the bulk of aminosiloxane coatings.

The surface science of bioassay devices is of great importance in the development of modern diagnostic platforms. The quality of surface is one of the most important elements of the device, often governing the background response, hence controlling the sensitivity of an assay. Detailed surface characterization and analysis are imperative for the preparation of reproducible coatings with desired properties.