Development of an affinity sensor for the detection of aflatoxin M1 in milk

Much research has been done on aflatoxins since their discovery in the 1960’s where it was concluded that aflatoxins have carcinogenic, mutagenic, teratogenic and immunosuppressive properties. Aflatoxin M1 exists in milk and since milk is a major component of the diet of infants, the maximum permissible limit set by the EU is 50 parts per trillion (ng L -1 ). Current methods of analysis for aflatoxin M1 is primarily based around techniques such as HPLC and TLC which require extensively trained operators and equipped laboratories. Using antibodies as receptors in an enzyme linked immunosorbent assay (ELISA), the analysis costs can be reduced and simplified, however, an equipped laboratory is still required. Hence there is a need for a low cost, rapid, portable instrument which is easy to use at the point of source for the detection of aflatoxin M1. This thesis describes the development of affinity sensors to meet these requirements. Firstly the design and optimisation of an ELISA method was carried out, utilising a commercially sourced monoclonal antibody. Once the antibodies suitability for sensing aflatoxin M1 was determined the antibody was successfully employed as the receptor for a screen printed HRP/TMB based immunosensor. Upon the analysis of milk it was observed that milk caused extensive interference and through a series of chemical extractions the interference was attributed to whey proteins in the milk with suspicion towards a- lactalbumin. A simple pre-treatment technique of adding calcium chloride was performed and the interference from the whey proteins was removed. The resulting immunosensor achieved a sensitivity of 39 ng L -1 (Figure 3.26), however, poor reproducibility was observed due to the screen printed electrode production (%CV = 21% variance for screen printed electrode production). Gold cell on a chip microelectrode arrays were used to replace the screen printed electrodes and the successful covalent attachment of the antibody to the microelectrode array through PDITC cross linking compound was monitored using atomic force microscopy and scanning electron microscopy. It was shown that the majority of the antibodies during immobilisation orientate in a ‘side on’ orientation and therefore a cheap capture polyclonal antibody was first immobilised before the addition of the sensing anti-aflatoxin M1 monoclonal antibody. Using the microelectrode array an improvement of the sensitivity as well as a reduction of the milk interference was shown. Sensitivity was improved to 8 ng L -1 in milk (Figure 4.23). Further work was performed to substitute the fragile antibody used in the sensing layer for a robust synthetic peptide receptor. Initially a virtual library of synthetic peptides was created using de novo design techniques in silico. Further computational techniques were performed to determine the best peptide from the library. This peptide had a sequence of PVGPRP. From literature a peptide (LLAR) was reported with affinity for aflatoxin B1. This sequence along with the de novo design peptide was synthesised and tested using a host of techniques and immobilisation chemistries such as optical waveguide lightmode spectroscopy (OWLS), BIAcore and enzymatic techniques using EDC/NHS, glutaraldehyde and BS 3 cross linking methods. The affinity of both peptides to aflatoxin M1 was demonstrated however further work is required to quantify the affinity and to incorporate the peptides into the microelectrode array.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of an affinity sensor for ochratoxin A

Ochratoxin A is a contaminant in wine and known to be immunosuppressive and possibly carcinogenic. Therefore, the development of a rapid and sensitive method for field analysis is required for risk assessment and management. The work presented in this thesis reports the construction of a sensor platform capable of fulfilling these requirements. As a sensor platform, screen-printed thick film electrodes and microelectrodes on a silicone support were investigated for sensor development. As biological recognition elements, an antibody specifically binding ochratoxin A and a peptide receptor that was designed using computational modelling were examined. A disposable immunosensor for ochratoxin A was developed based on screen-printing technology. An indirect competitive immunoassay format was used on bare screen printed gold electrode (SPGE). The performance of this sensor was compared to carboxmethylated dextran (CMD) modified SPGE. Detection was performed by chronoamperometry monitoring the reaction of tetramethylbenzidine and hydrogen peroxide catalysed by horseradish peroxidase. The SPGE-based immunosensor achieved a detection limit of 100 ng L-1 and the CMD-modified SPGE immunosensor 10 ng L-1. The latter has been used for ochratoxin A determination in wine samples and was validated against standard HPLC and a commercial immunoassay test kit. Wine sample analysis involved the sample pre-treatment using immunoaffinity chromatography, electrochemical wine component characterisation and interference control. The immunosensor format was transferred to a gold microelectrode array based on a silicone support for the purpose of signal sensitivity enhancement and miniaturisation in the prospect of field analysis. Preliminary data showed the characterisation of the microelectrode array immunosensor construction and characterisation. Further optimisation is needed to establish a calibration curve with the required sensitivity. The second part of the work comprised the design of a peptide receptor for ochratoxin A using computational methods by screening de novo designed peptide libraries. An octapeptide (CSIVEDGL) and a 13-peptide (GPAGIDGPAGIRC) were selected for synthesis and affinity characterised for ochratoxin A recognition using a surface plasmon resonance biosensor (BiacoreTM). The peptide receptors showed good sensitivity for ochratoxin A of 10 μg L-1. Preliminary affinity characterisation resulted in KA = 63 mM-1 for the 13-mer peptide and KA = 84 mM-1 for the octapeptide, which appears to be binding with higher strength to ochratoxin A. The affinity values correspond to the binding score (binding energy) calculated by computational modelling. This work shows the potential of designing peptide receptors for small molecules (e.g. ochratoxin A) and suggests their application in affinity sensors for detecting ochratoxin A contamination.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biosensor development for the analysis of food quality

This thesis describes the development and evaluation of a number of biosensors for food applications. The first part of this thesis deals with the development of Surface Plasmon Resonance (SPR) biosensor systems, coupled with Polymerase Chain Reaction (PCR) for the detection of GMO related amplified nucleic acids in foodstuffs. The first SPR Biosensor described, used streptavidin-biotin linkage chemistry to attach a P35S nucleic acid probe on dextran-coated SPR transducer chips. Methodologies were developed for both the PCR stage and post-PCR sample preparation for the sensitive, rapid and cost-effective detection of GMO-specific amplified DNA sequences. The final embodiment of the method was an asymmetric PCR amplification system with a simple sample processing step (0.3 M NaOH for 30 min in 20 % v/v formamide). The developed PCR-SPR system was successfully applied to the screening of samples of GMO origin. The second SPR biosensor reported herein, is based on a SPR chip immobilised single-stranded thiolated DNA. The thiolated probe exhibited a hybridisation capacity of 95 RU (Resonance Units) for 100 nM of complementary DNA target and a detection limit of 5 nM. The potential of the current probe system for the detection of symmetrically amplified DNA sequences of short length was subsequently confirmed. The second part of this thesis involved preliminary studies into the development of simple, disposable screen-printed electrodes for the electrochemical determination of glucose and L-amino acids in horticultural products. The dynamic range of the developed biosensors was up to 10 mM for glucose and up to 1 mM for L-leucine determination. The developed glucose biosensor exhibited encouraging analytical performance in fresh fruit samples. However, the L-amino acid oxidase electrodes consistently underestimated the amino acid content of the fruit samples. The latter observation was found to be primarily due to inhibitory components in the matrix.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Large-Scale CO Maps of the Lupus Molecular Cloud Complex

Fully sampled degree-scale maps of the 13CO 2-1 and CO 4-3 transitions toward three members of the Lupus Molecular Cloud Complex - Lupus I, III, and IV - trace the column density and temperature of the molecular gas. Comparison with IR extinction maps from the c2d project requires most of the gas to have a temperature of 8-10 K. Estimates of the cloud mass from 13CO emission are roughly consistent with most previous estimates, while the line widths are higher, around 2 km/s. CO 4-3 emission is found throughout Lupus I, indicating widespread dense gas, and toward Lupus III and IV. Enhanced line widths at the NW end and along the edge of the B228 ridge in Lupus I, and a coherent velocity gradient across the ridge, are consistent with interaction between the molecular cloud and an expanding HI shell from the Upper-Scorpius subgroup of the Sco-Cen OB Association. Lupus III is dominated by the effects of two HAe/Be stars, and shows no sign of external influence. Slightly warmer gas around the core of Lupus IV and a low line width suggest heating by the Upper-Centaurus-Lupus subgroup of Sco-Cen, without the effects of an HI shell.Comment: 54 pages, 27 figures, 5 tables. To appear in ApJS. Preprint also available (with full-size figures) from http://www.astro.ex.ac.uk/people/nfht/publications.html Datacubes available from http://www.astro.ex.ac.uk/people/nfht/resources.htm

Geysers in the Lagoon: new Herbig-Haro objects in M8

Aims: We search for direct evidence of ongoing star formation by accretion in the Lagoon Nebula (M8), using optical wide-field narrow-band imaging obtained at La Silla Observatory. Methods: We examine [SII] and Halpha images for line-emission features that could be interpreted as signatures of outflow activity of the exciting sources. Results: We discover five new Herbig-Haro objects, study in detail their morphology and attempt to identify their potential driving sources among the population of T Tauri stars and embedded sources in the surroundings. Conclusions: The results reported here conclusively demonstrate the existence of very young stars going through the accreting phase in the M8 region.Comment: 9 pages, 6 postscript figures (one in color). Accepted, Astronomy and Astrophysic

Pre-main sequence stars in the Lagoon Nebula (M8)

We report the discovery of new pre-main sequence (PMS) stars in the Lagoon Nebula (M8) at a distance of 1.25 kpc, based on intermediate resolution spectra obtained with the Boller & Chivens spectrograph at the 6.5-m Magellan I telescope (Las Campanas Observatory, Chile). According to the spectral types, the presence of emission lines and the lithium 6708A absorption line, we are able to identify 27 classical T Tauri stars, 7 weak-lined T Tauri stars and 3 PMS emission objects with spectral type G, which we include in a separated stellar class denominated "PMS Fe/Ge class". Using near-infrared photometry either from 2MASS or from our own previous work we derive effective temperatures and luminosities for these stars and locate them in the Hertzsprung-Russell diagram, in order to estimate their masses and ages. We find that almost all of our sample stars are younger than 3 10^6 years and span over a range of masses between 0.8 and 2.5 Msun. A cross-correlation between our spectroscopic data and the X-ray sources detected with the Chandra ACIS instrument is also presented.Comment: 18 pages, 15 figures, MNRAS, in pres

Electrochemical method for the determination of arsenic 'in the field' using screen-printed gold electrodes

This project describes development and problem solving efforts to realise a viable portable sensor for arsenic, applicable to drinking water. The work is the first dedicated effort towards this goal, after the preliminary investigations previously conducted at Cranfield University (Cooper, 2004 and Noh, 2005). Using polymeric gold ink BQ331 (DuPont Microcircuit Materials, Bristol, UK) as working electrode on screen printed strips, the electrochemical procedure was studied. Due to the wealth of research on electrochemical and non electrochemical methods for arsenic determination, this project attempts to capitalise on the unique advantages of the screen-printed gold surface. In particular, the issues surrounding the performance of the sensor were evaluated by electrochemical and spectroscopic means (including infrared, nuclear magnetic resonance and X-ray photoelectron spectroscopy). A number of custom screen printed electrodes were prepared in house comparing sensor performance on compositional factors. An interference coming from silver interaction with chloride in the reference electrode was identified. As such, the design of the sensor needs to change to include either an immobilising layer, such as Nafion, over the silver, or to omit screen-printed silver altogether. The Nafion was presumed to work by excluding (or at least much reducing) the passage of negatively charged chloride ions to the silver surface preventing formation of soluble silver chloride complexes. The design of the sensor was considered in light of performance and sensitivity. The screen-printed electrodes were cut to facilitate a microband design lending favourable diffusive to capacitive current characteristics. With this design, As(III) detection was demonstrated comfortably at 5 ppb (in a copper tolerant 4 M HCl electrolyte) without electrode need for additional preparation procedures. This is below the World Health Organisation (WHO) guideline and United States Environmental Protection Agency (USEPA) regulation level of 10 ppb in drinking water. The electrode materials are already mass manufacturable at an estimated cost less than £ 0.5 per electrode. Themicroband design could, in principle, be applied to mercury and other metal ions. The procedure for As(V) either with chemical or electrochemical reduction and determination still needs to be assessed. However, the presented electrode system offers a viable alternative to the colorimetric test kits presently employed around the world for arsenic in drinking water. Also, the Nicholson Method (Nicholson, 1965a), used for characterising electron transfer kinetics at electrode surfaces, was extended for application to rough surfaces using a fractal parameter introduced by Nyikos and Pajkossy (1988). This work includes mathematical derivation and numerical evaluation and gives a number of predictions for electrochemical behaviour. These predictions could not be tested experimentally, as yet, since the physical conditions must be carefully controlled.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Raman spectroscopy of biological tissue for application in optical diagnosis of malignancy

The use of Raman spectroscopy in the detection and classification of malignancy within the human larynx and lymph nodes of the head and neck has been evaluated. Currently histopathology is considered the diagnostic gold standard. The potential for Raman spectroscopy to be used as an in vivo diagnostic tool in the detection of dysplasia and malignancy has been demonstrated. A consensus opinion from three expert histopathologists has been obtained and spectral diagnostic models developed by correlation with these results. The ability of Raman spectroscopy to differentiate between disease entities and normal tissue within the larynx has been shown. Raman spectroscopy was able to identify non-neoplastic vocal cord mucosa (sensitivity 85 %, specificity 95%) from laryngeal mucosa showing neoplastic change (sensitivity 95 %, specificity 85%) with an increase in sensitivity to 89% for the non-neoplastic tissue and a reduction to73% in tissues showing neoplastic changes after cross-validation. For the first time benign changes in the structure of vocal cords such as those exhibiting hyperkeratosis and hyperplasia, where also identified with sensitivity of 97.9% for tissue exhibiting hyperplasia/hyperkeratosis and 100% for normal squamous cell epithelium. Research into the ability of Raman spectroscopy to interrogate lymphoid tissue in order to differentiate reactive nodes (sensitivity 90 %, specificity 88%) from those containing cancer (sensitivity 88 %, specificity 90%) was successful and fully independently validated. This work was further developed and the efficacy of Raman spectroscopy in differentiating between squamous cell carcinoma (sensitivity 76%, specificity 95%), adenocarcinoma (sensitivity 93 %, specificity 99%), Hodgkin‘s lymphoma (sensitivity 80%, specificity 90%) and reactive lymph nodes (sensitivity 81%, specificity 88%) was shown. This model was also independently cross-validated by node producing further improvements to give a spectral performance of sensitivity/specificity for SCC of 75/97%, adenocarcinoma 100/99%, Hodgkin‘s lymphoma 83/92% and reactive lymph nodes 85/86%.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Attenuation of quorum sensing using computationally designed polymers

It is generally accepted that the majority of Gram-negative and Gram-positive bacteria communicate via production and sensing of small signal molecules, autoinducers. The ability of bacteria to sense their population density is termed quorum sensing (QS). Quorum sensing controls certain phenotypic traits, particularly virulence factors and biofilm formation. In this project a new solution for the attenuation of quorum sensing which involves selective sequestering of the signal molecules using rationally designed synthetic polymers was explored.EThOS - Electronic Theses Online ServiceGBUnited Kingdo