Demonstration of an optical biosensor for the detection of faecal indicator bacteria in freshwater and coastal bathing areas.

ColiSense, an early warning system developed for Escherichia coli detection, is assessed using environmental samples. The system relies on the detection of ß-glucuronidase (GUS), a biomarker enzyme for E. coli. In contrast with other rapid GUS-based methods, ColiSense is the only method that uses 6-chloro-4-methyl-umbelliferyl-ß-D-glucuronide (6-CMUG) as a fluorogenic substrate. The system measures a direct kinetic response of extracted GUS, and the detection was carried out in the absence of particles or bacteria. It is necessary to evaluate the system with environmental samples to establish the relationship between faecal indicator bacteria E. coli and the response measured by the ColiSense. This paper presents the results of tests carried out with the ColiSense system for 2 trials, one conducted with freshwater samples collected from rivers in the Dublin area and a second conducted with seawater samples from coastal areas collected over the bathing season. A positive linear correlation was found between E. coli (MPN 100 mL-1) and ColiSense response (R2 = 0.85, N = 125, p < 0.01) for the seawater sample. A ColiSense response threshold was identified as 0-1.8 pmol min-1 100 mL-1, equivalent to 0-500 E. coli 100 mL-1. Using this threshold, 96.8% of the samples were correctly classified as being above or below 500 E. coli 100 mL-1 by the ColiSense system. Results presented demonstrate that the ColiSense system can be used as an early warning tool with potential for active management of bathing areas by providing results in 75 min from sample collection.

A Low-Cost Smart Sensor Network for Catchment Monitoring.

Understanding hydrological processes in large, open areas, such as catchments, and further modelling these processes are still open research questions. The system proposed in this work provides an automatic end-to-end pipeline from data collection to information extraction that can potentially assist hydrologists to better understand the hydrological processes using a data-driven approach. In this work, the performance of a low-cost off-the-shelf self contained sensor unit, which was originally designed and used to monitor liquid levels, such as AdBlue, fuel, lubricants etc., in a sealed tank environment, is first examined. This process validates that the sensor does provide accurate water level information for open water level monitoring tasks. Utilising the dataset collected from eight sensor units, an end-to-end pipeline of automating the data collection, data processing and information extraction processes is proposed. Within the pipeline, a data-driven anomaly detection method that automatically extracts rapid changes in measurement trends at a catchment scale. The lag-time of the test site (Dodder catchment Dublin, Ireland) is also analyzed. Subsequently, the water level response in the catchment due to storm events during the 27 month deployment period is illustrated. To support reproducible and collaborative research, the collected dataset and the source code of this work will be publicly available for research purposes.

Novel Microfluidic Analytical Sensing Platform for the Simultaneous Detection of Three Algal Toxins in Water.

Globally, the need for "on-site" algal-toxin monitoring has become increasingly urgent due to the amplified demand for fresh-water and for safe, "toxin-free" shellfish and fish stocks. Herein, we describe the first reported, Lab-On-A-Disc (LOAD) based-platform developed to detect microcystin levels in situ, with initial detectability of saxitoxin and domoic acid also reported. Using recombinant antibody technology, the LOAD platform combines immunofluorescence with centrifugally driven microfluidic liquid handling to achieve a next-generation disposable device capable of multianalyte sampling. A low-complexity "LED-photodiode" based optical sensing system was tailor-made for the platform, which allows the fluorescence signal of the toxin-specific reaction to be quantified. This system can rapidly and accurately detect the presence of microcystin-LR, domoic acid, and saxitoxin in 30 min, with a minimum of less than 5 min end-user interaction for maximum reproducibility. This method provides a robust "point of need" diagnostic alternative to the current laborious and costly methods used for qualitative toxin monitoring.

ChromiSense: A colourimetric lab-on-a-disc sensor for chromium speciation in water.

The development of a centrifugal device for quantitative analysis of both chromium (III) and (VI) species in water is reported. ChromiSense is a colourimetric sensor system that has been applied to the measurement of chromium in spiked river water samples. For analysis, the sample is loaded into a reservoir on the disposable microfluidic disc, along with reagents. A centrifugal force is created by spinning the disc to pump liquids through microchannels, causing them to mix and react to form a coloured product. The coloured product is then presented to a low-cost optical detection system, where absorbance measurements can be recorded. The optical detection system consists of a light emitting diode (LED) and photodiode (PD) couple. Chromium (III) was measured using 2,6-pyridine dicarboxylic acid as a ligand, forming a complex that was measured at 535nm and at 335nm. While measuring at 535nm allowed for the use of a low cost LED, the sensitivity was improved 2.5 times by measuring at 335nm. However, 335nm also yielded a diminished linear range with little improvement in limit of deteciton (LOD), and required a lengthier manufacturing process due to the need for a UV-transparent material. Chromium (VI) was detected using 1,5-diphenyl carbazide (DPC). This standard analysis method was simplified for automation on-disc, and optimised to achieve a low LOD. The LOD for trivalent and hexavalent chromium using this device were 21mgL-1 and 4µgL-1, respectively. The linear range for quantitative analysis was found to be 69-1000mgL-1 for Cr(III) and 14-1000µgL-1 for Cr (VI). While this range is high for Cr(III), incorporation of an off-disc pre-concentration method would make this technology suitable for environmental sample analysis. The device is simple to use, low in cost, and could provide rapid on-site measurements, with results comparable to those obtained using a benchtop spectrophotometer.

Protocol for the recovery and detection of Escherichia coli in environmental water samples.

To achieve active management of bathing areas and to reduce risk associated with the presence of fecal pollution, tests capable of rapid on-site assessment of microbiological water quality are required. A protocol for the recovery and detection of fecal pollution indicator bacteria, E. coli, using ß-glucuronidase (GUS) activity was developed. The developed protocol involves two main steps: sample preparation and GUS activity measurement. In the sample preparation step, syringe filters were used with a dual purpose, for the recovery and pre-concentration of E. coli from the water matrix and as µL reactors for bacteria lysis and GUS extraction. Subsequently, GUS activity was measured using a continuous fluorometric method developed previously. The optimum GUS recovery conditions for the sample preparation step were found to be 100 µL PELB (supplemented with 1 mg mL-1 lysozyme and 20 mM DTT) at 37 °C for 30 min. The protocol was evaluated on environmental samples (fresh and seawater) against an establish GUS assay method (Coliplage®). GUS activities corresponding to samples containing as low as 26 MPN E. coli 100 mL-1 were detected for the seawater sample and as low as 110 MPN E. coli 100 mL-1 for the freshwater samples. By comparison with the Coliplage® method, this protocol offered an improvement in the measured GUS activities of 3.1 fold for freshwater samples and 4.1 fold for seawater samples. Furthermore, the protocol developed here, has a time-to-result of 75 min, and successfully addresses the requirement for tests capable of rapid assessment of microbiological water quality.

3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography.

The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm.

ColiSense, today's sample today: A rapid on-site detection of ß-D-Glucuronidase activity in surface water as a surrogate for E. coli.

A sensitive field-portable fluorimeter with incubating capability and triplicate sample chambers was designed and built. The system was optimised for the on-site analysis of E. coli in recreational waters using fluorescent based enzyme assays. The target analyte was ß-D-Glucuronidase (GUS) which hydrolyses a synthetic substrate 6-Chloro-4-Methyl-Umbelliferyl-ß-D-Glucuronide (6-CMUG) to release the fluorescent molecule 6-Chloro-4-Methyl-Umbelliferyl (6-CMU). The system was calibrated with 6-CMU standards. A LOD of 5 nM and a resolution of less than 1 nM was determined while enzyme kinetic tests showed detection of activities below 1 pmol min(-1) mL(-1) of sample. A field portable sample preparation, enzyme extraction protocol and continuous assay were applied with the system to analyse freshwater and marine samples. Results from a one day field trial are shown which demonstrated the ability of the system to deliver results on-site within a 75 min period.

Continuous fluorometric method for measuring ß-glucuronidase activity: comparative analysis of three fluorogenic substrates.

E. coli ß-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-ß-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-ß-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-ß-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

A low-cost autonomous optical sensor for water quality monitoring.

A low-cost optical sensor for monitoring the aquatic environment is presented, with the construction and design described in detail. The autonomous optical sensor is devised to be environmentally robust, easily deployable and simple to operate. It consists of a multi-wavelength light source with two photodiode detectors capable of measuring the transmission and side-scattering of the light in the detector head. This enables the sensor to give qualitative data on the changes in the optical opacity of the water. Laboratory tests to confirm colour and turbidity-related responses are described and the results given. The autonomous sensor underwent field deployments in an estuarine environment, and the results presented here show the sensors capacity to detect changes in opacity and colour relating to potential pollution events. The application of this low-cost optical sensor is in the area of environmental pollution alerts to support a water monitoring programme, where multiple such sensors could be deployed as part of a network.