Alastair W. Wark

Nanoparticle-enhanced surface plasmon resonance detection of proteins at attomolar concentrations: comparing different nanoparticle shapes and sizes.

The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40-50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NP-antibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (>10(3)) on previous NP-enhanced SPR studies utilizing smaller (~15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.

Microarray methods for protein biomarker detection

The application of protein biomarkers as an aid for the detection and treatment of diseases has been subject to intensified interest in recent years. The quantitative assaying of protein biomarkers in easily obtainable biological fluids such as serum and urine offers the opportunity to improve patient care via earlier and more accurate diagnoses in a convenient, non-invasive manner as well as providing a potential route towards more individually targeted treatment. Essential to achieving progress in biomarker technology is the ability to screen large numbers of proteins simultaneously in a single experiment with high sensitivity and selectivity. In this article, we highlight recent progress in the use of microarrays for high-throughput biomarker profiling and discuss some of the challenges associated with these efforts.

Controlled side-by-side assembly of gold nanorods and dye molecules into polymer-wrapped SERRS-active clusters

The controlled side-by-side assembly of gold nanorods in solution together with Raman reporter dye molecules to create small SERRS-active clusters stabilised by a surrounding polymer layer is demonstrated. This promising new class of nanotags offers several advantages over spherical nanoparticles for bioimaging and is of potential importance for a wide range of plasmon-enhanced spectroscopies and can also serve as building blocks for more complex solution-phase nanostructures.

Ultrasensitive and ultrawide range detection of a cardiac biomarker on a surface plasmon resonance platform

One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.

Tuning the interparticle distance in nanoparticle assemblies in suspension via DNA-triplex formation : correlation between plasmonic and surface-enhanced Raman scattering responses

The understanding of the relationship between plasmonic and surface-enhanced Raman scattering (SERS) properties of dynamic nanoparticle assemblies is of paramount importance for the optimal design of related plasmonic nanostructures, especially for SERS applications. In this regard, recent studies have provided new important insights for well-ordered nanoparticle assemblies but little is known about the relationship between the physical and optical properties for large ensembles of randomly aggregated metal nanoparticles in suspension, which still represents the simplest and most common route to obtain highly effective SERS substrates. Here we exploit the triplex-assembling ability of DNA-conjugated silver nanoparticles to engineer interparticle junctions with controlled interparticle distance and tune the aggregation rate to allow accurate investigation into the correlation between the averaged time-dependent plasmonic and SERS responses within a complex ensemble of nanoparticles in suspension. Solution-based single particle tracking was used to characterize the heterogeneity of the nanoparticle assembly with statistical reliability, acting as a key tool to unravel the connection between these two bulk responses. To achieve this, we report the first example of the parallel hybridization of dye-labeled locked nucleic acid (LNA) silver nanoparticle probes to double stranded DNA bridges of different lengths to form a triplex assembly, that provides SERS enhancements directly related to the interparticle distance imposed by the high structural rigidity of the double stranded linker. This is also a crucial step towards utilising SERS for the study of DNA in its natural double stranded state and, ultimately, to obtain nanoscale distance-dependent information in challenging biological environments using specially designed nanoparticles.

Dual Nanoparticle Amplified Surface Plasmon Resonance Detection of Thrombin at Subattomolar Concentrations

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.

Highly sensitive electrochemical detection of proteins using aptamer-coated gold nanoparticles and surface enzyme reactions

A novel electrochemical detection methodology is described for the femtomolar detection of proteins which utilizes both DNA aptamer-functionalized nanoparticles and a surface enzymatic reaction. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was pre-modified with a monolayer of aptamer-nanoparticle conjugates followed by the specific interaction of alkaline phosphatase (ALP) conjugated anti-IgE. To clearly demonstrate the signal enhancement associated with nanoparticle use, anodic current measurements of the ALP catalyzed oxidation of the enzyme substrate 4-aminophenylphosphate (APP) were also compared with electrode surfaces upon which the aptamer was directly attached. The detection of an unlabelled protein at concentrations as low as 5 fM is a significant improvement compared to conventional electrochemical-based immunoassay approaches and provides a foundation for the practical use and incorporation of nanoparticle-enhanced detection into electrochemical biosensing technologies.

Universal Surface-Enhanced Raman Tags: Individual Nanorods for Measurements from the Visible to the Infrared (514–1064 nm)

Surface-enhanced Raman scattering (SERS) is a promising imaging modality for use in a variety of multiplexed tracking and sensing applications in biological environments. However, the uniform production of SERS nanoparticle tags with high yield and brightness still remains a significant challenge. Here, we describe an approach based on the controlled coadsorption of multiple dye species onto gold nanorods to create tags that can be detected across a much wider range of excitation wavelengths (514-1064 nm) compared to conventional approaches that typically focus on a single wavelength. This was achieved without the added complexity of nanoparticle aggregation or growing surrounding metallic shells to further enhance the surface-enhanced resonance Raman scattering (SERRS) signal. Correlated Raman and scanning electron microscopy mapping measurements of individual tags were used to clearly demonstrate that strong and reproducible SERRS signals at high particle yields (>92%) were readily achievable. The polyelectrolyte-wrapped nanorod-dye conjugates were also found to be highly stable as well as noncytotoxic. To demonstrate the use of these universal tags for the multimodal optical imaging of biological specimens, confocal Raman and fluorescence maps of stained immune cells following nanoparticle uptake were acquired at several excitation wavelengths and compared with dark-field images. The ability to colocalize and track individual optically encoded nanoparticles across a wide range of wavelengths simultaneously will enable the use of SERS alongside other imaging techniques for the real-time monitoring of cell-nanoparticle interactions.

Enhanced bioaffinity sensing using surface plasmons, surface enzyme reactions, nanoparticles and diffraction gratings

This paper introduces a novel approach to surface bioaffinity sensing based on the adsorption of nanoparticles onto a gold diffraction grating that supports the excitation of planar surface plasmons. A surface enzymatic amplification reaction is also incorporated into the detection scheme to enhance the sensitivity and utility of the nanoparticle-enhanced diffraction grating (NEDG) sensors. As a demonstration, the detection of microRNA is described where a combination of a surface polymerase reaction and DNA-modified nanoparticles is used to detect the bioaffinity adsorption of the target onto the probe-functionalized gold grating surface. The enzymatically-amplified NEDG sensors possess a great potential for a wide range of applications including the detection of biosecurity agents, DNA and RNA viruses, biomarkers, and proteins.

Bioaffinity detection of pathogens on surfaces

Abstract The demand for improved technologies capable of rapidly detecting pathogens with high sensitivity and selectivity in complex environments continues to be a significant challenge that helps drive the development of new analytical techniques. Surface-based detection platforms are particularly attractive as multiple bioaffinity interactions between different targets and corresponding probe molecules can be monitored simultaneously in a single measurement. Furthermore, the possibilities for developing new signal transduction mechanisms alongside novel signal amplification strategies are much more varied. In this article, we describe some of the latest advances in the use of surface bioaffinity detection of pathogens. Three major sections will be discussed: (i) a brief overview on the choice of probe molecules such as antibodies, proteins and aptamers specific to pathogens and surface attachment chemistries to immobilize those probes onto various substrates, (ii) highlighting examples among the current generation of surface biosensors, and (iii) exploring emerging technologies that are highly promising and likely to form the basis of the next generation of pathogenic sensors.