We describe a novel camera phone-based optical sensing platform capable of real-time quantitative fluorescence-based measurements. The platform employs the phone's camera as a photodetector in a non-imaging optical configuration that simultaneously detects fluorescence from a number of sensor spots on a disposable optical sensor chip. The platform combines the functionality of the camera with the phone's data-processing capabilities to facilitate on-phone analysis of detected fluorescence intensity. It is envisaged that such a platform will have significant potential in application areas such as environmental monitoring and healthcare where there is a growing demand for decentralized diagnostics using low-cost, portable devices, particularly in remote or low-resource environments.

In this letter, we report a simple optical strategy designed to significantly enhance the fluorescence collection efficiency of planar substrate-based optical sensors. The strategy involves the use of refractive and reflective optical elements that permit supercritical angle fluorescence to be transmitted through the lower surface of the planar substrate and redirected toward an optical detector. Importantly, this approach facilitates simple integration with existing multianalyte sensor systems and microfluidic platforms by leaving the sensor's upper planar surface unstructured. In comparison to a conventional planar substrate configuration, the proposed approach can provide better than an order of magnitude (factor of 20) enhancement in the fluorescence intensity detected. It is envisaged that this approach will have significant potential in a range of application areas where there is need for low-cost optical sensor technologies.

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. The need to validate each NP for safety and efficacy with each therapeutic compound or combination of therapeutics is an enormous challenge, which forces industry to focus mainly on those nanoparticle materials where data on safety and efficacy already exists, i.e., predominantly polymer NPs. However, the enhanced functionality affordable by inclusion of metallic materials as part of nanoengineered particles provides a wealth of new opportunity for innovation and new, more effective, and safer therapeutics for applications such as cancer and cardiovascular diseases, which require selective targeting of the therapeutic to maximize effectiveness while avoiding adverse effects on non-target tissues.

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.

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. A superior performance of the AA/PEG coating as suitable material for substrates in biomedical devices was demonstrated in a model fluorescence linked immunosorbent assay. The results were compared with other commonly used surface materials prepared by wet chemistry methods. The unique characteristic of the AA/PEG film is that the immunoassay can be executed without the need for a blocking step, typically using albumins, without negative consequences on the bioassay results. In fact, the superior quality of the materials modified with AA/PEG film was highlighted by improving the sensitivity of an immunoassay by two orders of magnitude when compared with substrates prepared by standard surface chemistry methods.

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. Two promising biomarkers for cardiac risk prediction are C-reactive protein (CRP) and N-terminal pro-brain natriuretic peptide (NTproBNP). The concentrations of these markers can differ by more than six orders of magnitude. Here we present a chip-based multiplexed assay for CRP and NTproBNP. The high-concentration analyte, CRP, is analyzed in a competitive format, whereas the low-concentration analyte, NTproBNP, is analyzed in a sandwich format. This allows concurrent measurement of the two analytes in a single multiplexed assay. The dynamic ranges for the two assays were optimized to match the relevant serum concentration ranges; thus, no dilutions were needed. Both assays exhibit good precision (5-15% in the clinically relevant concentration ranges), and the limit of detection for the NTproBNP assay was 5 ng/L. Patient plasma samples were used for comparison with clinical methods, resulting in coefficients of determination (R(2)) of 0.9762 and 0.9606 for NTproBNP and CRP, respectively.

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. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ∼3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions.

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. The ELISA procedure developed is 16-fold more sensitive than established methods. This protocol could be used generally as a quantitative analytical approach to perform high-sensitivity and rapid assays in clinical situations, and would provide a faster approach to screen phage-displayed libraries in antibody development facilities. The antibody immobilization procedure is of ∼3 h duration and facilitates rapid ELISAs. This method can be used to perform assays on a wide range of commercially relevant solid support matrices (including those that are chemically inert) with various biosensor formats.

This paper focuses on characterization of the sensing performance of a refractometric sensing platform based on multimode interference couplers (MMICs). Platform fabrication exploited a low-cost process using photocurable organic-inorganic hybrid sol-gel materials which were structured to form optical waveguides by direct UV laser writing. The sensing principle is based upon the high sensitivity of the optical field distribution formed in the MMIC toward changes in the refractive index of its environment. Simulations demonstrated the importance of correctly specifying the length of the MMIC section and illustrated that longer platforms are more sensitive due to a greater shift in self-image position. To characterize sensing performance, a porous sol-gel humidity sensing enrichment layer was coated on the MMIC. Relative humidity was detected by the system with a resolution of 0.097%. Refractive index resolution of the platform was determined to be similar to 2 x 10(-6) RIU, which is an analyte-independent value and illustrates the generic nature of this platform. As such, this platform has immense potential for future applications as a label-free and real-time biosensor platform.

We have presented novel surface plasmon-coupled emission (SPCE) based on solid-state electrochemiluminescence (ECL) of Nafion films containing tris(2,2′-bipyridyl)ruthenium(II). This approach combines the advantages of ECL, efficient emission in the absence of an external light source, with the highly directional emission of SPCE. We described theoretical calculations and optimal Nafion film thickness to get SPCE based on solid-state ECL. We confirmed the SPCE and dose-dependent SPCE response from the concentration range of 0.05–0.5% (w/v) [Ru(bpy)3]2+ in the Nafion film. SPCE based on solid-state ECL can be used as a useful platform for the analysis of chemical and biomolecular interactions.

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). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.

Recently, multifunctional silica nanoparticles have been investigated extensively for their potential use in biomedical applications. We have prepared sub-micron monodisperse and stable multifunctional mesoporous silica particles with a high level of magnetization and fluorescence in the near infrared region using an one-pot synthesis technique. Commercial magnetite nanocrystals and a conjugated-NIR-dye were incorporated inside the particles during the silica condensation reaction. The particles were then coated with polyethyleneglycol to stop aggregation. X-ray diffraction, N2 adsorption analysis, TEM, fluorescence and absorbance measurements were used to structurally characterize the particles. These mesoporous silica spheres have a large surface area (1978m2/g) with 3.40nm pore diameter and a high fluorescence in the near infrared region at λ=700nm. To explore the potential of these particles for drug delivery applications, the pore accessibility to hydrophobic drugs was simulated by successfully trapping a hydrophobic ruthenium dye complex inside the particle with an estimated concentration of 3wt%. Fluorescence imaging confirmed the presence of both NIR dye and the post-grafted ruthenium dye complex inside the particles. These particles moved at approximately 150μm/s under the influence of a magnetic field, hence demonstrating the multifunctionality and potential for biomedical applications in targeting and imaging.

A novel hybrid organic-inorganic photocurable sol-gel material based on tantalum ethoxide and 3-trimethoxysilylpropylmethacrylate has been developed, characterised and used in the fabrication of optical waveguides and three-dimensional woodpile structures employing the single and two-photon polymerisation techniques, respectively. Single mode waveguides operating at 1310 and 1550 nm have been fabricated, optically characterised and their performances correlated to the material formulation. Three-dimensional woodpile structures exhibiting negligible shrinkage have been developed and their remarkable mechanical stability correlated to the molecular structure of the hybrid material. The overall fabrication process of these devices is described and it is shown that the refractive indices of the materials can be tailored by a precise control of the material composition allowing the successful fabrication of performing single mode waveguides. (C) 2010 Elsevier B.V. All rights reserved.

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. An optical reader is also described that enables interrogation and fluorescence collection from the nine optical elements on the chip. The sensitivity of the system was determined with a biotin-avidin assay while its clinical utility was demonstrated in an assay for C-reactive protein (CRP), an inflammation marker.

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. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal –NH2 groups, a second film with a high density of –COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order –NH2>-branched PEG>–COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH.

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. However, such losses may also be associated with structural perturbations leading to changes in immunogenicity (as a result of alterations in specific epitope-related conformations). This can lead to erroneous results or lack of comparability with a range of methodologies such as the bicinchoninic protein assay and immunoassays or when surface plasmon resonance (SPR)-based approaches are used. A model system to evaluate these phenomena is proposed.

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 0.1 mm2 cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm2 cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.