Monitoring a toxic bloom of Alexandrium minutum using novel microarray and multiplex surface plasmon resonance biosensor technology

Blooms of Alexandrium occur annually during the summer months in the North Channel of Cork Harbour on the south coast of Ireland. This study monitored an extensive bloom of the toxin producing Alexandrium minutum during the summer of 2011 with the use of the MIDTAL (Microarrays for the Detection of Toxic Algae) microarray and a prototype multiplex surface plasmon resonance (multi SPR) biosensor. Microarray signal intensities and toxin results from three testing platforms of the prototype multi SPR biosensor, commercial (CER) enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) were compared against light microscopy counts. The main aim was to demonstrate the use of these methodologies to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying the harmful phytoplankton community and their toxins in natural water samples. Both the microarray signals and multi SPR biosensor results followed a significant trend with light microscopy results and both techniques indicated detection limits of <4000 cells of A. minutum in natural seawater samples.

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. 'an artificial mouse'. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC20) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.

Development of a planar waveguide microarray for the monitoring and early detection of five harmful algal toxins in water and cultures

A novel multiplex microarray has been developed for the detection of five groups of harmful algal and cyanobacterial toxins found in marine, brackish, and freshwater environments including domoic acid (DA), okadaic acid (OA, and analogues), saxitoxin (STX, and analogues), cylindrospermopsin (CYN) and microcystins (MC, and analogues). The sensitivity and specificity were determined and feasibility to be used as a screening tool investigated. Results for algal/cyanobacterial cultures (n = 12) and seawater samples (n = 33) were compared to conventional analytical methods, such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Detection limits for the 15 min assay were 0.37, 0.44, 0.05, 0.08, and 0.40 ng/mL for DA, OA, STX, CYN, and MC, respectively. The correlation of data obtained from the microarray compared to conventional analysis for the 12 cultures was r(2) = 0.83. Analysis of seawater samples showed that 82, 82, 70, 82, and 12% of samples were positive (>IC20) compared to 67, 55, 36, 0, and 0% for DA, OA, STX, CYN, and MC, respectively, for conventional analytical methods. The discrepancies in results can be attributed to the enhanced sensitivity and cross-reactivity profiles of the antibodies in the MBio microarray. The feasibility of the microarray as a rapid, easy to use, and highly sensitive screening tool has been illustrated for the five-plex detection of biotoxins. The research demonstrates an early warning screening assay to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying harmful toxins in water samples.

The performance of a cathodic protection system in reinforced concrete structure: Monitoring and Service Life Modelling

The use of cathodic protection in reinforced concrete is becoming increasingly common with such systems being installed on a number of structures throughout the United Kingdom and Ireland. However the prescribed design lives (or service life) of each cathodic protection system vary widely. The aim of this project was to assess the effectiveness of a sacrificial anode cathodic protection system and to predict its design life through a series of laboratory based experiments. The experimental plan involved casting a number of slabs which represented a common road bridge structure. The corrosion of the steel within the experimental slabs was then accelerated prior to installation of a cathodic protection system. During the experiment corrosion potential of the steel reinforcement was monitored using half-cell measurement. Additionally the current flow between the cathodic protection system and the steel reinforcement was recorded to assess the degree of protection. A combination of theoretical calculations and experimental results were then collated to determine the design life of this cathodic protection system. It can be concluded that this sacrificial anode based cathodic protection system was effective in halting the corrosion of steel reinforcement in the concrete slabs studied. Both the corrosion current and half-cell potentials indicated a change in passivity for the steel reinforcement once sacrificial anodes were introduced. The corrosion current was observed to be sensitive to the changes to the exposure environment. Based on the experimental variables studied the design life of this sacrificial anode can be taken as 26 to 30 years.

Evaluation of the diagnostic potential of a novel field immunomagnetic separation-immunochromatographic lateral flow assay to detect Mycobacterium bovis cells in European badger (Meles meles) faeces

The European badger (Meles meles) is a natural reservoir for Mycobacterium bovis, the causative agent of Bovine Tuberculosis, and has consequently been implicated in transmission of the disease to cattle. This study describes application of a novel M. bovis-specific immunochromatographic (lateral flow) assay in combination with immunomagnetic separation (IMS-LFD), to test badger faeces samples. In total, 441 faeces samples from badgers of unknown disease status collected from latrines at 110 badger setts throughout Northern Ireland (NI) and 100 faeces samples from badgers of known infection status from Great Britain (GB) were tested. Faeces (approx. 1g) was homogenised in 9 ml phosphate buffered saline, filtered (70 µm), and then 6-8 ml subjected to the IMS-LFD test. Residual clarified faecal homogenates were subjected to automated IMS followed by MGIT™ liquid culture (AIMS-MGIT™ culture) and qPCR (AIMS-qPCR). Evidence for the presence of M. bovis was obtained for 78 (18%), 61 (14%) and 140 (32%) of 441 NI badger faeces samples, and 10 (10%), 41 (41%) and 56 (56%) of 100 GB badger faeces samples, by IMS-LFD, AIMS-MGIT culture and AIMS-qPCR tests, respectively. The IMS-LFD test was less sensitive than AIMS-qPCR for detection of M. bovis and was, therefore, detecting badgers shedding high numbers of M. bovis in their faeces only. However, these ‘super shedders’ may be primarily responsible for the spread of Bovine Tuberculosis so are, therefore, an important target. This non-invasive test could form the basis of a field surveillance tool to indicate infected badger groups which are actively spreading M. bovis.

Monitoring of saliva pH using miniaturized sensing tools as an indicator of oral health

The monitoring of oral disease is important, not alone for oral health, but for the detection and prevention of
systemic disease. The link between oral health and systemic disease is the focus of many studies, with
indications emerging of a causal link [1]. For disease diagnostics, blood has typically been the fluid of choice
for analysis, the retrieval of which is invasive and therefore unsuitable for wearable technology. Analysis of
saliva, however, is less invasive than that of blood, requires little or no pre-treatment and is abundantly
available. A strong correlation has been found between the analytes of blood and saliva [2] with saliva
containing biomarkers for diseases such as diabetes, oral cancer and cardiovascular disease. The development of
an implantable multi-parametric wireless sensor, to monitor both salivary analytes and changes in gingival
temperature, is the aim of this research project.
The aim of our current study is to detect changes in salivary pH, using a gold electrode with a pHsensitive
iridium oxide layer, and an Ion Sensitive Field Effect Transistor probe. Characterisation studies were
carried out in artificial saliva (AS). A salivary pH of between 4.5pH-7.5pH [3], and gingival temperature
between 35°C-38°C [4], were identified as the target range of interest for the human oral environment. Sensor
measurements were recorded in solutions of varying pH and temperature. An ISFET probe was then implanted
into a prototype denture and characterised in AS. This study demonstrates the suitability of ISFET and gold
electrode pH sensors for incorporation into implantable oral sensors.
[1] G. Taylor and W. Borgnakke, “Periodontal disease: associations with diabetes, glycemic control and
complications,” Oral Dis., vol. 14, no. 3, pp. 191–203, Apr. 2008.
[2] E. Tékus, M. Kaj, E. Szabó, N. L. Szénási, I. Kerepesi, M. Figler, R. Gábriel, and M. Wilhelm,
“Comparison of blood and saliva lactate level after maximum intensity exercise,” Acta Biol. Hung., vol. 63
Suppl 1, pp. 89–98, 2012.
[3] S. Naveen, M. L. Asha, G. Shubha, A. Bajoria, and A. Jose, “Salivary Flow Rate, pH and Buffering
Capacity in Pregnant and Non Pregnant Women - A Comparative Study,” JMED Res., pp. 1–8, Feb. 2014.
[4] A. F. Holthuis and F. S. Chebib, “Observations on temperature and temperature patterns of the gingiva. I.
The effect of arch, region and health,” J. Periodontol., vol. 54, no. 10, pp. 624–628, Oct. 1983

Hydrophilic Interaction Ultra-High Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry for Determination of Nucleosides and Nucleobases in Animal Horns

Traditional Chinese Medicines (TCMs) derived from animal horns are one of the most important types of Chinese medicine. In the present study, a fast and sensitive analytical method was established for qualitative and quantitative determination of 14 nucleosides and nucleobases in animal horns using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadruple tandem mass spectrometry (HILIC-UPLC-QQQ-MS/MS) in selective reaction monitoring (SRM) mode. The method was optimized and validated, and showed good linearity, precision, repeatability, and accuracy. The method was successfully used to determine contents of the 14 nucleosides and nucleobases in 25 animal horn samples. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed and the 25 samples were thereby divided into two groups, which agreed with taxonomy. The method may enable quick and effective search of substitutes for precious horns.

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3-6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH. © 1990 Kluwer Academic Publishers.

Experimental analysis of air flow profiles in a double skin facade in a maritime climate

Glazed Double Skin Facades (DSF) offer the potential to improve the performance of all-glass building skins, common to commercial office buildings in which full facade glazing has almost become the standard. Single skin glazing results in increased heating and cooling costs over opaque walls, due to lower thermal resistance of glass, and the increased impact of solar gain through it. However, the performance benefit of DSF technology continues to be questioned and its operation poorly understood, particularly the nature of airflow through the cavity. This paper deals specifically with the experimental analysis of the air flow characteristics in an automated double skin façade. The benefit of the DSF as a thermal buffer, and to limit overheating is evaluated through analysis of an extensive set of parameters including air and surface temperatures at each level in the DSF, airflow readings in the cavity and at the inlet and outlet, solar and wind data, and analytically derived pressure differentials. The temperature and air-flow are monitored in the cavity of a DSF using wireless sensors and hot wire anemometers respectively. Automated louvre operation and building set-points are monitored via the BMS. Thermal stratification and air flow variation during changing weather conditions are shown to effect the performance of the DSF considerably and hence the energy performance of the building. The relative pressure effects due to buoyancy and wind are analysed and quantified. This research aims to developed and validate models of DSFs in the maritime climate, using multi-season data from experimental monitoring. This extensive experimental study provides data for training and validation of models.

Rapid-bTB: a novel lateral flow test able to differentiate Mycobacterium bovis from Mycobacterium tuberculosis in sputum

Background: A novel lateral flow, immunochromatographic assay (LFD) specific for Mycobacterium bovis, the cause of bovine tuberculosis and zoonotic TB, was recently developed at Queen’s University Belfast. The LFD detects whole M. bovis cells, in contrast to other commercially available LFD tests (BD MGITTM TBc ID, SD Bioline TB Ag MPT 64, Capilia TB-Neo kit) which detect MPT64 antigen secreted during growth. The new LFD test has been evaluated in the veterinary context, and its specificity for M. bovis in the broadest sense (i.e. subsp. bovis, subsp. caprae and BCG) and sensitivity to detect M. bovis in positive MGIT™ liquid cultures was demonstrated comprehensively.
Methods: Preliminary work was carried out by researchers at Queen’s University Belfast to optimise sputum sample preparation, estimate the limit of detection (LOD) of the LFD with M. bovis-spiked sputum samples, and check LFD specificity by testing a broad range of non-tuberculous Mycobacterium spp. (NTM) and other bacterial genera commonly encountered in sputum samples (Haemophilus, Klebsiella, Pseudomonas, Staphylococcus). In the Cameroon laboratory direct detection of M. bovis in human sputa was attempted, and 50 positive sputum MGIT™ cultures and 33 cultures of various Mycobacterium spp. originally isolated from human sputa were tested.
Results: Sputum sample preparation consisted of digestion with 1% NALC for 30 min, centrifugation at 3000g for 20 min, PBS wash, centrifugation again, and pellet resuspended in KPL blocking buffer before 100 µl was applied to the LFD. The LOD of the LFD applied to M. bovis-spiked sputum was estimated to be 104 CFU/ml. A small number of confirmed Ziehl-Neelsen ‘3+’ M. bovis positive sputum samples were tested directly but no positive LFD results were obtained. All of the sputum MGIT™ cultures and mycobacterial cultures (including M. tuberculosis, M. africanum, M. bovis, M. intracellulare, M. scrofulaceum, M. fortuitum, M. peregrinum, M. interjectum) tested LFD negative when read after 15 min except for the M. bovis cultures, thereby confirming specificity of LFD for M. bovis in the clinical microbiology context.
Conclusions: Results indicate that the ‘Rapid-bTB’ LFD is a very specific test, able to differentiate M. bovis from M. tuberculosis, M. africanum, and a range of NTM isolated from human sputa in MGITTM liquid cultures. However, the LFD lacks sufficient sensitivity to be applied earlier in the diagnostic process to directly test human sputa.

Assessment Of various sensors for structural health monitoring for bridge weigh-in-motion (B-WIM).

In recent years, Structural Health Monitoring (SHM) systems have been developed to monitor bridge deterioration, assess real load levels and hence extend bridge life and safety. A road bridge is only safe if the stresses caused by the passing vehicles are less than the capacity of the bridge to resist them. Conventional SHM systems can be used to improve knowledge of the bridges capacity to resist stresses but generally give no information on the causes of any increase in stresses (based on measuring strain). The concept of in Bridge Weigh-in-Motion (B-WIM) is to establish axle loads, without interruption to traffic flow, by using strain sensors at a bridge soffit and subsequently converting the data to real time axle loads or stresses. Recent studies have shown it would be most beneficial to develop a portable system which can be easily attached to existing and new bridge structures for a specified monitoring period. The sensors could then be left in place while the data acquisition can be moved for various other sites. Therefore it is necessary to find accurate sensors capable of capturing peak strains under dynamic load and suitable methods for attaching these strain sensors to existing and new bridge structures. Additionally, it is important to ensure accurate strain transfer between concrete and steel, the adhesives layer and the strain sensor. This paper describes research investigating the suitably of using various sensors for the monitoring of concrete structures under dynamic vehicle load. Electrical resistance strain (ERS) gauges, vibrating wire (VW) gauges and fibre optic sensors (FOS) are commonly used for SHM. A comparative study will be carried out to select a suitable sensor for a bridge Weigh in Motion System. This study will look at fixing methods, durability, scanning rate and accuracy range. Finite element modeling is used to predict the strains which are then validated in laboratory trials.