Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milk

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e BiomédicasBovine mastitis is an economic burden for farmers mostly because of decreased milk yield, premature culling and cost of veterinary treatments. The identification of mastitis pathogens is of major importance in order for adequate control measures to be taken, to reduce the risk of appearance of chronic infections, and to target antimicrobial therapy. The aim of this study was to develop and validate a sensitive method for magnetic detection of Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis in raw milk samples. Mastitic milk samples were collected aseptically from 81 cows with subclinical mastitis, from 12 Portuguese dairy farms. Ninety one quarter milk samples were selected based on bacteriological results. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analyzed using a lab-on-a-chip magnetoresistive cytometer, with microfluidics sample handling. This immunological recognition was able to detect bacterial presence above 100 cfu/ ml, depending on antibody and targeted bacteria. Comparison with PCR results showed sensitivities of 73% and 41%, specificity values of 25% and 57%, and PPV values of 35% and 54% for magnetic identification of streptococci species with an anti-S. agalactiae antibody and an anti-GB Streptococcus antibody, respectively. Regarding staphylococci species, the sensitivity values found were of 57.1% and 79.3%, specificities of 75% and 50%, and PPV values of 40% and 95.8% for magnetic identification with an anti-S. aureus antibody and an anti-Staphylococcus spp. antibody, respectively. Both bacterial genus studies translated a fair expectation for a “cow-side” use application, making this integrated platform of potential use after further improvements for fast bacteriological infection screening. Some constraints are described as well as the method´s limitations in bacterial quantification.RESUMO - Plataforma portátil “lab-on-chip” para diagnosticar mastite bovina em leite crú - A mastite bovina representa um custo económico relevante para os produtores de leite principalmente devido ao decréscimo da produção leiteira, abate prematuro e custos associados ao tratamento veterinário. Consequentemente, a identificação atempada dos agentes etiológicos é crítica para a implementação de medidas de controlo adequadas, redução do risco de infecções crónicas e aplicação de uma terapia microbiana específica. O objectivo deste estudo foi desenvolver e validar um método de detecção magnética capaz de identificar Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus e Staphylococcus epidermidis em amostras de leite crú. As amostras de leite mastítico utilizadas foram recolhidas de 81 animais com mastite subclínica, de 12 explorações leiteiras nacionais. As amostras de leite de 91 quartos de úbere foram selecionadas tendo em conta os resultados bacteriológicos. Todas as amostras foram analisadas por PCR e pelo citómetro magnetoresistivo “lab-on-chip”, tendo sido necessário neste caso, adicionar uma solução com partículas magnéticas funcionalizadas com anticorpos específicos. Este reconhecimento imunológico detectou presença bacteriana acima das 100 ufc/ml, dependendo do anticorpo e da bactéria-alvo. Comparando com os resultados da análise por PCR, este método de detecção magnética apresentou sensibilidades de 73% e 41%, valores de especificidade de 25% e 57%, e valores VPP de 35% e 54% para identificar espécies de Streptococcus com os anticorpos anti-S. agalactiae e anti-GB Streptococcus, respectivamente. No que diz respeito às espécies de Staphylococcus, os valores de sensibilidade encontrados foram de 57.1% e 79.3%, de 75% e 50% para a especificidade, e de 40% e 95.8% para VPP com os anticorpos anti-S. aureus e anti-Staphylococcus spp., respectivamente. Os dois estudos apontam para uma potencial utilização do tipo “cow-side”, tornando a plataforma integrada potencialmente utilizável para uma rápida monitorização de infecção bacteriológica, após melhorias futuras. O método desenvolvido apresenta algumas restrições e limitações relativamente à quantificação bacteriana

Biosensors

A biosensor is defined as a detecting device that combines a transducer with a biologically sensitive and selective component. When a specific target molecule interacts with the biological component, a signal is produced, at transducer level, proportional to the concentration of the substance. Therefore biosensors can measure compounds present in the environment, chemical processes, food and human body at low cost if compared with traditional analytical techniques. This book covers a wide range of aspects and issues related to biosensor technology, bringing together researchers from 11 different countries. The book consists of 16 chapters written by 53 authors. The first four chapters describe several aspects of nanotechnology applied to biosensors. The subsequent section, including three chapters, is devoted to biosensor applications in the fields of drug discovery, diagnostics and bacteria detection. The principles behind optical biosensors and some of their application are discussed in chapters from 8 to 11. The last five chapters treat of microelectronics, interfacing circuits, signal transmission, biotelemetry and algorithms applied to biosensing

Applications of bioluminescence in biotechnology and beyond.

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin-luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin-luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist

The development of biosensing systems incorporating eukaryotic cells for rapid toxicity assessment

This thesis describes the development of biosensing systems incorporating eukaryotic cells. The ultimate objective of this work was to design devices capable of rapidly assessing the toxicity of effluents and environmental pollutants. Although much work remains to be done in order to achieve this goal, the work reported here demonstrates, in principle, the approaches adopted. The first approach exploited the reducing nature of healthy biological cells. So called 'redox mediated whole cell biosensors' have been described before. In this work, an algal toxicity test of short duration was developed and sensors incorporating cultured fish cells were described for the first time. The sensitivity of biosensors incorporating the green alga Selenastrum capricornutum, to diuron and pentachlorophenol, was found to compare favourably with that from other standard ecotoxicological tests. However, although the sensitivity of biosensors incorporating immobilised BF-2 fish cells was found to compare well with that of other fish cell-based toxicity tests, it appeared that whole organism tests were much more sensitive to the compounlds tested. The second approach involved the genetic manipulation of fish cells in order to incorporate luminescent reporter genes. Although this work is less well advanced, it demonstrates that the luc reporter gene can be successfully inserted into BF -2 fish cells and that these transformed cells can produce a luminescent response when incubated with luciferin substrate. Preliminary investigations have indicated that the sensitivity of luc-transformed BF-2 cells to 4-chlorophenol is comparable to that of some standard whole organism ecotoxicological tests and although much work is still required to validate this approach, it could eventually provide a simple, sensitive and rapid route to toxicity assessment

Large-Scale Kinetic Analyses of Protein-Protein Interactions: Advancing the Understanding of Post Translational Modifications in Biological Regulation

abstract: Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Definition of a near real time microbiological monitor for space vehicles

Efforts to identify the ideal candidate to serve as the biological monitor on the space station Freedom are discussed. The literature review, the evaluation scheme, descriptions of candidate monitors, experimental studies, test beds, and culture techniques are discussed. Particular attention is given to descriptions of five candidate monitors or monitoring techniques: laser light scattering, primary fluorescence, secondary fluorescence, the volatile product detector, and the surface acoustic wave detector

The use of analytical techniques for the rapid detection of microbial spoilage and adulteration in milk

Milk is an important nutritious component of our diet consumed by most humans on a daily basis. Microbiological spoilage affects its safe use and consumption, its organoleptic properties and is a major part of its quality control process. European Union legislation and the Hazard Analysis and the Critical Control Point (HACCP) system in the dairy industry are therefore in place to maintain both the safety and the quality of milk production in the dairy industry. A main limitation of currently used methods of milk spoilage detection in the dairy industry is the time-consuming and sometimes laborious turnover of results. Attenuated total reflectance (ATR) and high throughput (HT) Fourier transform infrared (FTIR) spectroscopy metabolic fingerprinting techniques were investigated for their speed and accuracy in the enumeration of viable bacteria in fresh pasteurized cows' milk. Data analysis was performed using principal component-discriminant function analysis (PC-DFA) and partial least squares (PLS) multivariate statistical techniques. Accurate viable microbial loads were rapidly obtained after minimal sample preparation, especially when FTIR was combined with PLS, making it a promising technique for routine use by the dairy industry. FTIR and Raman spectroscopies in combination with multivariate techniques were also explored as rapid detection and enumeration techniques of S. aureus, a common milk pathogen, and Lactococcus lactis subsp cremoris, a common lactic acid bacterium (LAB) and potential antagonist of S. aureus, in ultra-heat treatment milk. In addition, the potential growth interaction between the two organisms was investigated. FTIR spectroscopy in combination with PLS and kernel PLS (KPLS) appeared to have the greatest potential with good discrimination and enumeration attributes for the two bacterial species even when in co-culture without previous separation. Furthermore, it was shown that the metabolic effect of L. cremoris predominates when in co-culture with S. aureus in milk but with minimal converse growth interaction between the two microorganisms and therefore potential implications in the manufacture of dairy products using LAB. The widespread and high consumption of milk make it a target for potential financial gain through adulteration with cheaper products reducing quality, breaking labeling and patent laws and potentially leading to dire health consequences. The time consuming and laborious nature of currently used analytical techniques in milk authentication enabled the study of FTIR spectroscopy and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) as rapid analytical techniques in quantification of milk adulteration, using binary and tertiary fresh whole cows', goats' and sheep's milk mixture samples. Chemometric data analysis was performed using PLS and KPLS multivariate analyses. Overall, results indicated that both techniques have excellent enumeration and detection attributes for use in milk adulteration with good prospects for potential use in the dairy industry.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Miniaturised analytical systems with chemiluminescence detection for environmental applications

This thesis details the use of microfluidic devices and chemiluminescence detection in order to develop a portable method of analysis for measuring chemical species in the environment.Chapter 1 outlines microfluidic technology, including fabrication techniques, fluid manipulation and mixing within the devices. Their advantages for analytical environmental purposes are demonstrated along with a review of their uses in environmental applications. Chemiluminescence detection provides a sensitive method of analysis for measuring chemical species in the environment and chemiluminescence theory and reagents are addressed.Chapter 2 details the development of a portable battery operated chemiluminescence detection system, which can be used in conjunction with microfluidic devices. The fabrication of the micro fluidic devices used in this work is documented. Different micro fluidic channel manifolds were investigated for chemiluminescence reactions and a serpentine design (200 μm width, 65 μm depth) with a channel length of 206 mm was selected as the most suitable design. Methods of fabrication for incorporating immobilised reagents on solid supports within a microfluidic device were also designed.Chapter 3 documents the investigation of the luminol-cobalt(II) chemiluminescence reaction within a microfluidic device using the portable chemiluminescence detection system to produce a miniaturised analytical system for the determination of hydrogen peroxide in rainwater. Enhancement of the chemiluminescence signal by 132 %was achieved by means of using the mirror reaction to apply a reflective surface directly VI to the top of the micro fluidic device. Immobilisation techniques for immobilising luminol using adsorption and covalent attachment onto a solid support were investigated as a means of producing a reagentless system, however poor sensitivity was observed and this was not progressed for the analytical system.Using the luminol-cobalt(II) chemiluminescence reaction within a microfluidic device a method of measuring hydrogen peroxide in the low micromolar concentrations was achieved, producing a limit of detection of 4.7 nmol L⁻¹ with a small sample volume (10 μL min⁻¹). A small reagent consumption size (1.2 mL per hour) and a low waste production size (2.4 mL per hour) were also achieved. This system was then used for the determination of hydrogen peroxide in rainwater samples during rainfall events showing the hydrogen peroxide concentration varied from 0.1 to 3.2 μmol L⁻¹. The method was also applied to the analysis of hydrogen peroxide in snow demonstrating the hydrogen peroxide concentration varied from 0.2 to 0.5 μmol L⁻¹ in samples taken at ground level.Chapter 4 details the development of a heterogeneous (two site) sandwich immunoassay within a microfluidic device to produce a miniaturised analytical system for the determination of E. coli bacteria in seawater. There is a need for rapid sensitive methods of analysis to measure E. coli in seawater as an indicator of faecal contamination. A review of traditional methods and current research on the area is presented. Immunological techniques based on using antibodies to specifically bind to their respective antigens were found to be the most amenable method of analysis for E. coli and an outline of how they work is shown. HRP was selected as the sensitive enzyme label for the antibody in the sandwich immunoassay. The chemiluminescence detection of HRP using the luminol-hydrogen peroxide VB chemiluminescence reaction was investigated within a microfluidic device, the detection was optimised and p-iodophenol was selected as an enhancer for the reaction. The investigation into the immobilisation of E. coli specific antibodies using covalent attachment onto controlled pore glass is presented, an optimal loading of 1.5 μg g⁻¹ was achieved. The development of an ELISA method for the purpose of screening the antibody for their specificity towards different isolates of E. coli and their non-specificity towards other bacteria is detailed. Finally, a microfluidic immunoassay was developed. Regeneration of the immobilised antibodies was achieved using 0.5 mol L⁻¹ sodium hydroxide, allowing the immobilised antibodies to be reused. The microfluidic immunoassay provided a rapid method for the determination of E. coli with an analysis time of 13 min for each sample. The assay also used a low reagent consumption and waste production. This would enable the rapid testing of a number of small samples to provide high temporal and spatial resolution data. Sensitivity provided a problem with the immunoassay and ways to overcome this were addressed