Nitric oxide (NO) plays an essential role within the immune system since it is involved in the break-down of infectious agents such as viruses and bacteria. The ability to measure the presence of NO in the intracellular environment would provide a greater understanding of the pathophysiological mechanism of this important molecule. Here we report the detection of NO from the intracellular phagolysosome using a fluorescently tagged metalloprotein–gold nanoparticle conjugate. The metalloprotein cytochrome c, fluorescently tagged with an Alexa Fluor dye, was self-assembled onto gold nanoparticles to produce a NO specific nanobiosensor. Upon binding of NO, the cytochrome c protein changes conformation which induces an increase of fluorescence intensity of the tagged protein proportional to the NO concentration. The nanobiosensor was sensitive to NO in a reversible and selective manner, and exhibited a linear response at NO concentrations between 1 and 300 μM. In RAW264.7γ NO− macrophage cells, the nanobiosensor was used to detect the presence of NO that had been endogenously generated upon stimulation of the cells with interferon-γ and lipopolysaccharide, or spontaneously released following treatment of the cells with a NO donor. Significantly, the nanobiosensor was shown to be taken up by the macrophages within phagolysosomes, i.e., the precise location where the NO, together with other species, destroys bacterial infection. The nanobiosensor measured, for the first time, increasing concentrations of NO produced during combined stimulation and phagocytosis of Escherichia coli bacteria from within localised intracellular phagolysosomes, a key part of the immune system.

The aim of this investigation was to define the optimum method of binding antibodies to the surface of gold nanoparticles (AuNPs) and then to apply the optimised antibody-functionalised AuNPs for the detection of a target antigen. A detailed investigation of three different techniques for the functionalisation of AuNPs with anti-cocaine antibody and methods for the subsequent characterisation of the antibody-functionalised AuNP are reported. The addition of anti-cocaine antibody onto the AuNP surface was facilitated by either: a polyethylene glycol (PEG) linker with a COOH terminal functional group; an aminated PEG ligand; or an N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-Protein A/G intermediate. Characterisation of the functionalised particles was performed using transmission electron microscopy, UV–Visible spectrophotometry and by agarose gel electrophoresis. In addition, the cocaine binding efficacy of the resultant AuNPs and their cocaine-binding capacity was determined using a cocaine-horseradish peroxidase conjugate, and by the application of a microtiter plate-based immunoassay. The results showed that the number of antibody per particle was the highest when the AuNP were functionalised with the Protein A/G intermediate. As compared to free antibody, the cocaine binding efficacy was significantly enhanced using the AuNP-Protein A/G-antibody complex. This optimal antibody-antigen binding efficacy is thought to be the result of the large number of antibody per particle and the oriented binding of the antibody to the Protein A/G on the AuNP surface. These results highlight the ideal immuno-gold nanoparticle characteristics for the detection of target antigens such as cocaine.

Photodynamic therapy (PDT) is a treatment of cancer whereby tumours are destroyed by reactive oxygen species generated upon photoactivation of a photosensitizer drug. Hydrophobic photosensitizers are known to be ideal for PDT; however, their hydrophobicity necessitates that they are typically administered using emulsions. Here, a delivery vehicle for photodynamic therapy based on the co-self-assembly of both a Zn(II)-phthalocyanine derivative photosensitizer and a polyethylene glycol (PEG) derivative onto gold nanoparticles is reported. The PEG on the particle surface ensured that the conjugates were water soluble and enhanced their retention in the serum, improving the efficiency of PDT in vivo. The pharmacokinetic behaviour of the nanoparticle conjugates following intravenous injection into C57/BL6 mice bearing a subcutaneous transplanted B78H1 amelanotic melanoma showed a significant increase of retention of the nanoparticles in the tumour. PDT tumour destruction was achieved 3 h following injection of the nanoparticle conjugates leading to a remarkable 40% of the treated mice showing no tumour regrowth and complete survival. These results highlight that dual functionalised nanoparticles exhibit significant potential in PDT of cancer especially for difficult to treat cancers such as amelanotic melanoma.

Carbohydrate molecules are involved in many of the cellular processes that are important for life. By combining the specific analyte targeting of carbohydrates with the multivalent structure and change of solution colour as a consequence of plasmonic interactions with the aggregation of metal nanoparticles, glyconanoparticles have been used extensively for the development of bioanalytical assays. The noble metals used to create the nanocore, the methodologies used to assemble the carbohydrates on the nanoparticle surface, the carbohydrate chosen for each specific target, the length of the tether that separates the carbohydrate from the nanocore and the density of carbohydrates on the surface all impact on the structural formation of metal based glyconanoparticles. This tutorial review highlights these key components, which directly impact on the selectivity and sensitivity of the developed bioassay, for the colorimetric detection of lectins, toxins and viruses.

The functionalisation of therapeutic nanoparticle constructs with cancer-specific biomolecules can enable selective tumour accumulation and targeted treatment. Water soluble gold nanoparticles (ca. 4 nm) stabilised by a mixed monolayer of a hydrophobic zinc phthalocyanine photosensitiser (C11Pc) and hydrophilic polyethylene glycol (PEG) have been prepared. The C11Pc-PEG gold nanoparticle constructs were further functionalised with jacalin, a lectin specific for the cancer-associated Thomsen–Friedenreich (T) carbohydrate antigen, or with monoclonal antibodies specific for the human epidermal growth factor receptor-2 (HER-2). The two biofunctionalised nanoparticle conjugates produced similar levels of singlet oxygen upon irradiation at 633 nm. Importantly, both nanoparticle conjugates demonstrated extensive, yet comparable, phototoxicity in HT-29 colorectal adenocarcinoma cells (80–90%) and in SK-BR-3 breast adenocarcinoma cells (>99%). Non-conjugated C11Pc-PEG gold nanoparticles were only minimally phototoxic. Lysosomal colocalisation studies performed with the HT-29 colon cancer cells and the SK-BR-3 breast cancer cells revealed that both nanoparticle conjugates were partially localised within acidic organelles, which is typical of receptor-mediated endocytosis. The similarity of the targeted PDT efficacy of the two biofunctionalised C11Pc-PEG gold nanoparticles is discussed with respect to targeting ligand binding affinity and cell surface antigen density as key determinants of targeting efficiency. This study highlights how targeting small cell-surface molecules, such as the T antigen, can mediate a selective photodynamic treatment response which is similar to that achieved when targeting overexpressed protein receptors, such as HER-2. The high prevalence of the T antigen present on the cellular surface of primary tumours emphasises the broad potential applications for lectin-targeted therapies.

We describe a gold nanoparticle based assay that can rapidly determine the crosslinking of DNA duplexes by ligands. Such compounds have potential in targeting highly compacted DNA such as that found in the nucleosome.

A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent α2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25:75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.

A tetracationic meso-substituted amphiphilic porphyrin (abbreviated as C14) was encapsulated within silica microparticles to yield a conjugate with a mean particle diameter of ca. 0.9 μm. The conjugate displayed a complete stability for at least 3 months when suspended in a neutral aqueous medium. The encapsulated C14 underwent a limited photobleaching when the conjugate was exposed to full spectrum visible light. Illumination of the silica microparticle-bound C14 by visible light resulted in the generation of singlet oxygen and induced a decrease in the survival of 4 log for a 20 min irradiation of the Gram-positive bacterium meticillin-resistant Staphylococcus aureus (MRSA) and a 30 min irradiation of the Gram-negative bacterium Escherichia coli (E. coli). Under identical experimental conditions photoexcited free C14 caused a decrease in viability of 5 log for MRSA and 6 log for E. coli. When the conjugate loaded with 12 μM C14 was added to a water sample contaminated with MRSA (108 cells per ml) a tight association of the bacterial cells with the silica microparticle–porphyrin system was achieved. Subsequent illumination of the conjugate with visible light (30 min, 100 mW cm−2) caused a 3 log reduction in the population of MRSA cells in the water sample. Importantly, the conjugate was readily recovered by filtration of the aqueous suspension and shown to maintain a high antibacterial photoactivity when introduced into a new MRSA-contaminated medium and irradiated.

Aged fingermarks are difficult to develop using traditional fingermark methods. Here we use anti-cotinine magnetic particle conjugates combined with fluorescently tagged secondary antibody fragments to develop fingermarks, deposited on glass by a smoker volunteer, that have been aged under four different conditions: at room temperature in the light; room temperature in the dark; at an elevated temperature in the dark (55 °C); and stored within a freezer (−20 °C) in the dark. The anti-cotinine magnetic particle conjugates bound to the ridges of the latent fingermarks of the smoker to produce high resolution brightfield and fluorescence images that were suitable for identification purposes. The antibody-particle conjugates were used to develop fingermarks aged for up to 4 weeks stored under the 4 environmental conditions. The best quality images were obtained when the fingermarks were aged at an elevated temperature of 55 °C. This successful development of aged fingermarks using anti-cotinine magnetic particle conjugates suggests that this method is an excellent alternative to powders or cyanoacrylate (superglue) fuming that are typically used for the development of aged fingermarks.

Semiconductor nanoparticles or quantum dots (QDs) have been proposed as potential vehicles for photodynamic therapy (PDT) since 2003. Some studies using cadmium-based QDs have shown promising results when coupled to molecular photosensitizers. However, the toxicity of such QDs and the low overall efficiency of these hybrids are still problematic. We have coupled two types (sizes) of less-toxic InP/ZnS QDs to the photosensitizer chlorin e6. The spectroscopic properties of these hybrids have been studied in detail. Spectroscopic methods have been applied to elucidate the energy transfer pathways and kinetics and the rate of singlet oxygen production of all components. Additionally, the PDT efficacy of the QD/chlorin e6 hybrids has been assessed against a breast cancer (MDA-MB-231) cell line using a colorimetric 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We have found that the energy transfer between QDs and the molecular photosensitizer is the rate-determining step for the production of singlet oxygen and that the cell viabilities of the hybrid and free photosensitizer are comparable. These systematic findings show that the energy transfer between QDs and photosensitizers is a “bottleneck”, which suggests that a better chemical design of the QD/photosensitizer hybrids in future embodiments is essential.

A 4-component antibody–phthalocyanine–polyethylene glycol–gold nanoparticle conjugate is described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles (4 nm) were stabilised with a self-assembled layer of a zinc–phthalocyanine derivative (photosensitiser) and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently bound to the nanoparticlesvia a terminal carboxy moiety on the polyethylene glycol. The nanoparticle conjugates were stable towards aggregation, and under irradiation with visible red light efficiently produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.

Latent fingermarks can be particularly difficult to visualise on highly reflective surfaces such as white porcelain. Here we use a combination of anti-cotinine magnetic particle conjugates and fluorescently tagged secondary antibody fragments in order to develop latent fingermarks on porcelain. Further, the anti-cotinine magnetic particle conjugates were used to distinguish between fingermarks from smoker and non-smoker volunteers by detecting the presence of cotinine, a metabolite of nicotine, within the fingermarks of smokers. For both sets of fingermarks, the anti-cotinine magnetic particle conjugates enabled the development of the fingermarks. Importantly, the brightfield and fluorescence microscopy images of the developed fingermarks were of high resolution and suitable for identification purposes. The anti-cotinine particle conjugates specifically bound to the cotinine antigen present in the sweat within the ridges of the fingermarks of the smoker volunteers as shown by the presence of ridges, minutiae, ridge shape and pores. However, with the absence of the cotinine antigen in the non-smoker fingermarks, the anti-cotinine magnetic particle conjugates non-specifically bound to the porcelain surface to produce a ‘negative’, or inverse, image of the fingermark. The difference in specific and non-specific binding of the antibody–particle conjugates on the porcelain surface enables the high quality images to be used to identify an individual and distinguish between smokers and non-smokers.

An immunoassay based technique is used for the detection of psychoactive substances in the sweat deposited within fingermarks of a narcotic drug user. Magnetic particles functionalized with antimorphine and antibenzoylecgonine antibodies were used for the detection of a metabolite of heroin (morphine) and a metabolite of cocaine (benzoylecgonine), respectively. The drug metabolites were detected individually as well as simultaneously from a single fingermark. The images of the fingermarks obtained using brightfield and fluorescence microscopy were of high evidential quality with resolution to enable identification of an individual in addition to providing information on drug usage.

With the commercial availability of recombinant human erythropoietin (rHuEPO), there is significant scope for athletes, especially those competing in endurance sports, to illicitly enhance their performance by increasing their aerobic capacity through enhanced erythrocyte production and hence oxygen transport. While such abuse has been confirmed in a number of human sports, there is also the possibility that rHuEPO can be abused in animal based sports such as thoroughbred horseracing. The direct detection of rHuEPO abuse, using either urine or blood samples, is challenging as the recombinant glycoprotein is similar to that produced endogenously and typically can only be measured above background levels within 4 days of administration. However, it is known that an immune response occurs when horses are doped with rHuEPO. The production of a specific antibody in response to doping with rHuEPO provides a target analyte that is not only different to endogenous species but one which resides in the body for considerably longer than the glycoprotein itself, significantly extending the measurement window. Here we have developed a glycoprotein microarray which exploits the antibody–antigen interaction to provide a means of detecting rHuEPO abuse in animals through the measurement of erythropoietin (EPO) antibodies (anti-HuEPO antibodies). Three commercially available isoforms of rHuEPO (Eprex®, Aranesp® and NeoRecormon®) were arrayed onto the planar surface of a nitrocellulose-coated microarray slide to act as the capture molecule in the assay. The assay was achieved by incubation of the microarray with solutions containing the anti-HuEPO antibody, followed by incubation with a fluorescently tagged secondary antibody. This ‘sandwich’ based assay enabled the fluorescent based detection of anti-HuEPO antibodies using an array-scanner. The EPO glycoprotein microarray was shown to be specific for anti-HuEPO antibodies. To detect anti-HuEPO antibodies in spiked serum samples an optimal dilution of the serum with buffer of 1:4 was established. Using Eprex®-10,000 IU as the capture molecule, the lowest concentration of anti-HuEPO antibody which was detected using the microarray was 148 pM, suggesting that the developed microarray platform could be used as a screen of EPO abuse.

Magnetic particles functionalised with anti-cotinine antibody have been used to image latent fingermarks through the detection of the cotinine antigen in the sweat deposited within the fingerprints of smokers. The antibody–magnetic particle conjugates are readily applied to latent fingerprints while excess reagents are removed through the use of a magnetic wand. The results have shown that drug metabolites, such as cotinine, can be detected and used to image the fingermark to establish the identity of an individual within 15 minutes.

Photogeneration of singlet oxygen (1O2) by rose bengal is improved through the use of a porous monolithic polymer (PMP) as a support, as compared to a classic gel-type resin matrix. This type of monolithic polymeric matrix can be made at a multigram scale in quantitative yields enabling the preparation of large amounts of supported photosensitizer at low cost. The singlet oxygen induced oxidation of 9,10-diphenylanthracene has been used as a benchmark reaction, and a comparative study using rose bengal in solution, entrapped within gel-type derived polymer and entrapped within a porous monolithic polymer (PMP) has been performed. The enhanced photoreactivity of the PMP-rose bengal conjugates has been utilised for the successful photodynamic therapy (PDT) of melanoma cells.

Ricin is a toxic lectin which presents a potential security threat. Its rapid detection is highly desirable. Here we present a colorimetric bioassay based on the aggregation of carbohydrate-stabilised gold nanoparticles which has been used to detect Ricinus communis Agglutinin 120 (RCA120) – a ricin surrogate. To achieve a stable and robust sensing system the anchor chain length and the density of the assembled carbohydrates on the gold particle surface has been examined to determine the optimal coverage for maximal aggregation with both RCA120 and Concanavalin A (Con A) lectins. Gold nanoparticles were stabilised with either a thiolated galactose derivative (9-mercapto-3,6-diaoxaoctyl-β-D-galactoside) or a thiolated mannose derivative (9-merapto-3,6-dioxaoctyl-α-D-mannoside), for RCA120 and Con A respectively, diluted in each instance with varying ratios of a thiolated triethylene glycol derivative. Aggregation was induced with the respective cognate lectin with the reaction monitored by UV-visible spectrophotometry. The results obtained show that a particle surface with at least 7.5% galactose is required for aggregation with RCA120 and 6% mannose coverage is required for aggregation with Con A. For each lectin the sensitivity of the assay could be controlled by adjustment of the carbohydrate density on the gold nanoparticles, but with differing results. Maximal aggregation with Con A was achieved with a monolayer consisting of 100% mannose, whereas for RCA120 maximal aggregation occurred with 70% coverage of galactose. The limit of detection for RCA120 using the optimally presented galactose-stabilised nanoparticles was 9 nM.

Phthalocyanine-nanoparticle conjugates have been designed and synthesised for the delivery of hydrophobic photosensitizers for photodynamic therapy (PDT) of cancer. The phthalocyanine photosensitizer stabilized gold nanoparticles have an average diameter of 2–4 nm. The synthetic strategy interdigitates a phase transfer reagent between phthalocyanine molecules on the particle surface that solubilises the hydrophobic photosensitizer in polar solvents enabling delivery of the nanoparticle conjugates to cells. The phthalocyanine is present in the monomeric form on the nanoparticle surface, absorbs radiation maximally at 695 nm and catalytically produces the cytotoxic species singlet oxygen with high efficiency. These properties suggest that the phthalocyanine-nanoparticle conjugates are ideally suited for PDT. In a process that can be considered as cancer therapy using a ‘Trojan horse’, when the nanoparticle conjugates are incubated with HeLa cells (a cervical cancer cell line), they are taken up thus delivering the phthalocyanine photosensitizer directly into the cell interior. Irradiation of the nanoparticle conjugates within the HeLa cells induced substantial cell mortality through the photodynamic production of singlet oxygen. The PDT efficiency of the nanoparticle conjugates, determined using colorimetric assay, was twice that obtained using the free phthalocyanine derivative. Following PDT with the nanoparticle conjugates, morphological changes to the HeLa cellular structure were indicative of cell mortality via apoptosis. Further evidence of apoptosis was provided through the bioluminescent assay detection of caspase 3/7. Our results suggest that gold nanoparticle conjugates are an excellent vehicle for the delivery of surface bound hydrophobic photosensitizers for efficacious photodynamic therapy of cultured tumour cells.

The thioctic acid amides of 2′-aminoethyl α-D-mannopyranoside and 2′-aminoethyl α-1,3-D-mannopyranosyl (α-1,6-D-mannopyranosyl)-α-D-mannopyranoside presented on both planar and nanoparticle gold surfaces give higher specific and lower non-specific protein binding than the related 2′-thioethyl glycosides.

The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins.

Photoswitching of ‘on’ and ‘off’ coordination sites in a self assembled monolayer containing the 4-(arylazo)pyridine chromophore has been achieved using the evanescent field and illustrated through coordination and photon induced release of zinc tetraphenylporphyrin.

Recently there has been considerable interest in using surface plasmon resonance (SPR) for the measurement of conformational changes of immobilized biomolecules that are induced by an exogenous analyte. While a number of studies have shown the analytical utility of such measurements, there has been no report which characterizes the specific secondary structure that actuates the change in SPR signal. The use of SPR to indicate the type of secondary structure present in two immobilized polypeptides, poly-L-lysine (PL) and poly-L-glutamic acid (PGA), and a globular protein, concanavalin A (Con A) is described in this report. The PL, PGA and Con A were modified with N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP) to introduce disulfide groups to facilitate the attachment onto gold-coated surfaces via self-assembly. Ethanol and 2,2,2-trifluoroethanol (TFE) were used to induce changes in the secondary structure of the immobilized polypeptides and the protein respectively. Using both circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, it has been demonstrated that it is possible to correlate the signal changes observed in SPR to the secondary conformation of the biomolecule. Both CD and FTIR showed that a decrease in SPR signal corresponded to a high content of β, turn or unordered structures while an increase corresponded to a high α-helical content. The sensitivity of the SPR technique is comparable to that obtained in solution with CD and FTIR spectroscopies. These results are the first demonstration that SPR can be used to characterize secondary structures. There is potential, therefore, for SPR to be used as a technique to study secondary conformational changes of immobilized polypeptides and proteins.

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l−1 (2.18 μM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol–gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075–1.250 μM was achieved, with a limit of detection of 0.075 μM. In order to increase the speed of response, a sol–gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol–gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 °C.

Aged fingermarks are difficult to develop using traditional fingermark methods. Here we use anti-cotinine magnetic particle conjugates combined with fluorescently tagged secondary antibody fragments to develop fingermarks, deposited on glass by a smoker volunteer, that have been aged under four different conditions: at room temperature in the light; room temperature in the dark; at an elevated temperature in the dark (55 °C); and stored within a freezer (−20 °C) in the dark. The anti-cotinine magnetic particle conjugates bound to the ridges of the latent fingermarks of the smoker to produce high resolution brightfield and fluorescence images that were suitable for identification purposes. The antibody-particle conjugates were used to develop fingermarks aged for up to 4 weeks stored under the 4 environmental conditions. The best quality images were obtained when the fingermarks were aged at an elevated temperature of 55 °C. This successful development of aged fingermarks using anti-cotinine magnetic particle conjugates suggests that this method is an excellent alternative to powders or cyanoacrylate (superglue) fuming that are typically used for the development of aged fingermarks.

With the commercial availability of recombinant human erythropoietin (rHuEPO), there is significant scope for athletes, especially those competing in endurance sports, to illicitly enhance their performance by increasing their aerobic capacity through enhanced erythrocyte production and hence oxygen transport. While such abuse has been confirmed in a number of human sports, there is also the possibility that rHuEPO can be abused in animal based sports such as thoroughbred horseracing. The direct detection of rHuEPO abuse, using either urine or blood samples, is challenging as the recombinant glycoprotein is similar to that produced endogenously and typically can only be measured above background levels within 4 days of administration. However, it is known that an immune response occurs when horses are doped with rHuEPO. The production of a specific antibody in response to doping with rHuEPO provides a target analyte that is not only different to endogenous species but one which resides in the body for considerably longer than the glycoprotein itself, significantly extending the measurement window. Here we have developed a glycoprotein microarray which exploits the antibody–antigen interaction to provide a means of detecting rHuEPO abuse in animals through the measurement of erythropoietin (EPO) antibodies (anti-HuEPO antibodies). Three commercially available isoforms of rHuEPO (Eprex®, Aranesp® and NeoRecormon®) were arrayed onto the planar surface of a nitrocellulose-coated microarray slide to act as the capture molecule in the assay. The assay was achieved by incubation of the microarray with solutions containing the anti-HuEPO antibody, followed by incubation with a fluorescently tagged secondary antibody. This ‘sandwich’ based assay enabled the fluorescent based detection of anti-HuEPO antibodies using an array-scanner. The EPO glycoprotein microarray was shown to be specific for anti-HuEPO antibodies. To detect anti-HuEPO antibodies in spiked serum samples an optimal dilution of the serum with buffer of 1:4 was established. Using Eprex®-10,000 IU as the capture molecule, the lowest concentration of anti-HuEPO antibody which was detected using the microarray was 148 pM, suggesting that the developed microarray platform could be used as a screen of EPO abuse.

A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent α2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25:75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.

We describe a gold nanoparticle based assay that can rapidly determine the crosslinking of DNA duplexes by ligands. Such compounds have potential in targeting highly compacted DNA such as that found in the nucleosome.