Niamh Gilmartin

Mastitis detection: current trends and future perspectives

Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided.

Nanobiotechnologies for the detection and reduction of pathogens.

Advances in the manipulation of nanomaterials has permitted the development of nanobiotechnology with enhanced sensitivities and improved response times. Low levels of infection of the major pathogens require the need for sensitive detection platforms and the properties of nanomaterials make them suitable for the development of assays with enhanced sensitivity, improved response time and increased portability. Nanobiotechnologies focusing on the key requirements of signal amplification and pre-concentration for the development of sensitive assays for food-borne pathogen detection in food matrices will be described and evaluated. The potential that exists for the use of nanomaterials as antimicrobial agents will also be examined.

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. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from naïve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.

Microfiber coupler based label-free immunosensor

Optical microfibers and related structures which incorporate large evanescent field and minimal size offer new opportunities for biosensing applications. In this paper we report the development of an immunosensor based on a tapered microfiber coupler embedded in a low refractive index polymer. Biomolecules adsorbed on the microfiber coupler surface modify the surrounding refractive index. By immobilizing antigens on the surface of the sensing area, the microfiber coupler was able to operate as a label-free immunosensor to detect specific antibodies. We experimentally demonstrated for the first time the sensing ability of this sensor using a fibrinogen antigen-antibody pair. By monitoring the spectral shift in the wavelength domain, the sensor was shown to be capable of detecting the specific binding between fibrinogen and anti-fibrinogen. The detected signal was found to be proportional to the anti-fibrinogen present.

Differential internalin A levels in biofilms of Listeria monocytogenes grown on different surfaces and nutrient conditions

Listeria monoctyogenes is a foodborne pathogen containing the surface protein, internalin A (InlA). The expression of this protein permits the invasion of L. monocytogenes into intestinal epithelial cells expressing the receptor E-cadherin, thus crossing the intestinal barrier and resulting in listerosis. The main aim of this work was to investigate InlA levels in different L. monocytogenes strains in both planktonic and sessile states using an anti-InlA antibody. Biofilms were grown in high and low nutrient environments on glass, stainless steel and polytetrafluoroethylene (PTFE). This study demonstrated that InlA levels varied greatly between strains and serotypes of L. monocytogenes. However, the serotypes 1/2a, 1/2b and 4b, associated with the largest number of outbreaks of listerosis consistently showed the highest InlA levels, regardless of nutrient content or planktonic or sessile state. Differences in InlA levels were also observed in biofilms grown on different surfaces such as glass, stainless steel and PTFE, with a significant reduction in InlA levels observed in biofilms on PTFE. Interestingly, although a large number of the total cells observed in biofilms formed in tap-water were non-cultivable, the virulence factor, InlA, was expressed at levels between 78 and 85%, thus indicating that these cells may still be virulent. A greater understanding of the factors that affect the levels of InlA on the surface of L. monocytogenes, is essential in the appreciation of the role of InlA in the persistence of biofilms containing L. monocytogenes and their potential to cause food borne disease.

Antibodies, enzymes, and nucleic acid sensors for high throughput screening of microbes and toxins in food

It is well established that the contamination of produce destined for human consumption by pathogenic microbes and/or toxins poses a considerable risk to public health and has serious economic consequences. Hence, it is imperative that such analytes are detected through the implementation of rapid, reproducible, and sensitive detection strategies. In this chapter, we provide a critical assessment of three methodologies that incorporate antibody-, enzyme-, or nucleic acid-based probes as biorecognition elements and discuss how these can be applied for high throughput screening of pathogens and toxins in complex food sample matrices.

CHAPTER 4:Nanoparticle Technologies in Detection Science

Over the past two decades, the synthesis and characterization of nanoparticles for sensing applications (environmental and biomedical) has garnered considerable attention in the scientific community and in industry. Advances in nanoparticle technologies in detection science has permitted the development of sensors with enhanced sensitivities and improved response times. This chapter aims to review research in this area, with a particular focus on biosensors. A description of the most commonly used nanoparticles in detection science and a synopsis of the bioanalytical detection techniques using these types of nanoparticles is provided. Essential to enhanced sensitivities in detection science and biosensor development is the successful attachment of nanoparticles to relevant biomolecules and a summary of functionalization approaches to achieve this are discussed. While nanoparticle technologies offer many advantages, one of the key benefits of nanoparticle-based device performance is signal amplification. Detection techniques in both optical and electrical systems highlighting this benefit are described. Finally, the acceptance of nanoparticles in biomedical applications depends strongly on nanoparticle biocompatibility and toxicity. Current studies outlining the toxic effects of nanoparticles and the potential of harnessing these toxic effects as antimicrobial agents are therefore discussed at the end of the chapter.

Controllable Chemical Modification of Polyaniline Nanofibres

A method for simply and controllably modifying the surface of polyaniline nanofibres is described. The technique can be used to attach substituents bearing both acid and amine functional groups, making the materials suitable for further modification. Acid/amine functionalisation is achieved by a simple reflux reaction and therefore is a quick and easily scalable process. The modified nanofibres maintain their ability to switch between different states displaying distinctly different properties, thus making them suitable for adaptive sensing applications. As an example, we demonstrate how biomolecules can be attached to these functionalised nanofibres, to produce conducting polymer-based biosensors.