The development of microfluidic platforms for environmental analysis

There is currently a gap in the use of centrifugal microfluidics in the field environmental sensing. The purpose of this thesis was to develop new and innovative centrifugal microfluidic platforms, which could enhance current environmental monitoring strategy limitations; portability and in-situ capability, cost-effectiveness, generical design for multi-analyte detectability, and the minimal required end-user interaction. Included in the main body of the thesis will be a review article, providing the theoretical perspectives which have been demonstrated for microfluidic applications in other domains and recommendations for adaptation towards environmental sensing using centrifugal microfluidics, and three novel papers on the staged development of a multi- toxin detection platform aimed to be incorporated within the fully deployable MariaBox (Marine environmental in-situ assessment and monitoring toolBox, co-funded by the European Commission: contract no.614088) system. The aspects covered across these three original articles includes the development of a centrifugal microfluidic platform with complementary fluorescence detection system as an initial test bed for toxin bio- assay integration on-disc, progression of current centrifugally-automatable pneumatic microvalve mechanisms for increased actuation predictability, and the further combination of both of these detection and microvalve mechanisms for a complete on- disc, multi-toxin detection platform which has been designed specifically to be compatible with the deployable MariaBox platform

Optical enhancement strategies on centrifugal microfluidic water sensors for detection of phosphate

Phosphorous is one of the principle elements contributing to eutrophication[1] in coastal, marine and fresh water. This study aims to develop new technologies that can enabled near real-time, rapid, reliable and robust analysis of water nutrient levels, such as phosphate, in water systems. Herein, describes an enhancement study of a previously demonstrated lab-on-a-disc (LOAD) centrifugal microfluidic device for the detection of phosphate in freshwater. The LOAD device utilizes a microfluidic sample processing to enable high precision metering and reagent mixing, followed by colorimetric analysis (at 880 nm) of the resultant complex. A customisable and complementary, in-house analysis system was also developed to enhance user interaction and enable rapid analysis. This analysis system delivers both disc centrifugation and automated colourimetric detection of the LOAD device, with recording of data transmitted via PC interface. The aim of this study is to maintain the same level of sensitivity of the current[2] system with a reduced pathlength. The limit of detection (LOD) and limit of quantification (LOQ) for this new revised system are as follows: The blackened chip obtained the best sensitivity with an LOD and LOQ of 6 and 19 μg L-1 respectively

Marine inspired textured materials for reduction of biofouling on surfaces

Biofouling on deployed in-situ sensors without regular removal or cleaning can disrupt sensor data collected. The current replacement antifouling (AF) materials under development are largely unsuited to sensor technologies as they have been developed with large scale applications in mind, such as those required by the shipping industry. Therefore, a strategy for the development of novel, sustainable, antifouling materials for sensor applications is required. Bio-inspiration refers to adapting strategies already developed in the natural world to problems encountered in modern science and technology. Engineered surfaces capable of controlling cellular behaviour under natural conditions are challenging to design due to the diversity of attaching cell types in environments such as marine waters, where many variations in cell shape, size and adhesion strategy exist. Nevertheless, understanding interactions between a cell and a potential substrate for adhesion, including topographically driven settlement cues, offers a route to designing surfaces capable of controlling cell settlement. Biomimetic design of artificial surfaces, based upon microscale features from natural surfaces, can be utilized as model surfaces to understand cell-surface interactions. In this study it was hypothesized that an AF effect could be induced through the replication of a synthetic surface. Scophthalmus rhombus (Brill) is a small flatfish occurring in marine waters of the Mediterranean as well as in Norway and Iceland. It inhabits sandy and muddy coastal waters from 5 to 80 metres. Its skin changes colour depending on the environment but is generally brownish with light and dark freckles and a creamy underside. S. rhombus is oval in shape and its flesh is white[1], [2]. In this study, the micro topography of the brill scale is characterized for the first time which may serve as a trend for the design of a marine inspired biomimetic surface texture. Natural dermal scales of S. rhombus are artificially replicated using 3-D printing and mould casting technologies. The replication methods are then tested for initial colonization of fouling species using 3 h immersion testing using diatom species, CCAP 1052/1B, Phaeodactylum tricornutum. The aim of this study was to discover the potential of using textured surfaces inspired by nature in particular marine organisms to combat fouling. This work identifies simple textures that can reduce fouling in its early stages which can contribute to antifouling coatings on sensors for monitoring in the marine environment

Design features for enhancing optical detection on lab-on-a-disc platforms

Centrifugal microfluidics has undergone a massive growth surge over the past 15 years, evident by the number of comprehensive reviews currently available, with special regard towards Lab-On-A-Disc (LOAD) diagnostic solutions.1–3 The potential of a LOAD system is dependent on its ability to mimic the specific laboratory protocols with which are required to conduct sample-to-answer analysis. This would include sample handling and manipulation (such as mixing and separation), sample modification (including heating and redox reactions), as well as reaction detection (such as optical, electrochemical, or as required by user). Optical detection strategies on LOAD platforms has been largely successful in both the fields of biological and chemical sensing.4 Herein, will demonstrate the optical optimisations which were carried out on a biological fluorescent-based5 and a chemical absorbance-based6 LOAD detection platforms. This will include the identification and optimisation of LED-photodiode selection, the effects of detection orientation and pathway-length fluorophore selection. Also covered will be a comparison between the microfluidic architecture for incorporating either detection methods as well as their reported limits of detection

A novel optical sensing lab-on-a-disc platform for chromium speciation

The determination chromium speciation in the field is a significant analytical challenge. While chromium exists in oxidation states from 0 to VI, it is predominantly found in the (III) and (VI) states [1]. Industry effluent (e.g. textile/electroplating) is a common source of chromium pollution in the environment. Due to corrosion inhibitors used in pipes, and contamination leaching from sanitary landfills, drinking water supplies can become contaminated also [2]. The bioavailability and toxicity of chromium is largely dependent the oxidation state of the element [2]. Consumption of Cr (III) is an essential component in human diet, as it is responsible for maintaining glucose, lipid and protein metabolism [3]. In contrast, Cr (VI) is strongly oxidizing, exhibiting high toxicity, with carcinogenic and mutagenic properties [4]. It is recommended by the World Health Organisation (WHO) that the maximum allowable concentration of chromium (VI) in drinking water is 0.05 mg L−1 [5]. Handheld colourimeters for on-site measurements are a convenient option for frequent water monitoring; however the limit of detection (LOD) of these devices is typically higher than the recommended limit. Microfluidic ‘lab-on-a-disc’ technologies were used in the development of an optical sensor for chromium speciation in water. The principal behind these devices is to minimize laboratory processes onto a microfluidic system that can be brought to the sampling site for rapid sample-to-answer analyses. The objective for this device was to design and fabricate a fully integrated optical sensor for on-site measurement of both trivalent and hexavalent chromium in freshwater. A strong focus was placed on maximizing sensitivity in order to achieve a low LOD

Development of an autonomous algal toxin analytical platform for aquatic monitoring

Cyclic peptide cyanobacterial toxins, in particular Microcystis aeruginosa, pose a serious health risk to humans and animals alike [1], [2]. Occurring mostly in fresh and brackish water, they have been identified to cause cancer promotion and liver damage [3]. Herein, we describe a portable, microfluidic-based system for in-situ detection of algal toxins in fresh water. The technology development presented here is a fully integrated and portable sample-to-answer centrifugal microfluidics-based system for the detection of toxic cyanobacteria – Microcystin-LR in fresh water. Our unique system employs highly-specific recombinant chicken anti-microcystin antibodies, prepared in-house, with a 3D-printed ‘LASER-photo¬diode’ fluorescent detection technique, also developed in-house. The system has high analytical specificity and sensitivity for detection of toxins below the regulatory limit with intra/inter day coefficient of variation of less than 20%. Dissolvable-film based valving technique was used for flow actuation and integration of multiple assays on the centrifugal cartridge. This new approach forms the basis of a cost efficient, USB-controlled water quality monitoring system. Technically, this integrated system consists of two components; a microfluidic disc (figure 1.A), the disc-holder fabricated and assembled from a 3D-printed casing, with electronic components housed in device. The 5-layered microfluidic disc consists of five reservoirs (figure 1.B), each with a separate venti-lation, aligned radially with inter-connected microchannels. A competitive immunoassay format is utilised to detect free toxin (figure 1.C). Sensitivity, reproducibility and ease-of-use are key features of this monitoring device. The ‘top-down’ optical detection system has been modified for improved detection sensitivity, as well as the elimination of external noise

A centrifugal lab-on-a-disc device for the in situ determination of dissolved reactive phosphate in water.

Phosphorus (P) is an important nutrient to monitor in natural waters as it is a growth limiting nutrient. When levels of phosphorus are elevated, excessive growth of algae occurs. This can lead to hypoxic or anoxic waters, potential release of harmful toxins and it impacts negatively on the ecosystem. Phosphorus measurement in water involves the collection of grab samples, transport and storage of samples, and analysis using expensive analytical instrumentation. This is costly, time and labour intensive and is carried out infrequently as a result. In this work, P is measured as soluble reactive phosphate (SRP), to give an indication of P levels in fresh water. A device for in situ analysis of SRP was fabricated. This device consists of a centrifugal microfluidic lab-on-a-disc platform, with a housing unit. The disc operates by mixing the sample with ascorbic acid method reagents using centrifugal force. The coloured product is presented to an optical detection system consisting of a laser and photodiode. The limit of detection has been optimised by modification of the optical path length. The housing unit is a 3D printed portable box with a built in motor for disc rotation. This box also houses the optical detection system which consists of a laser and photodiode. The box facilitates the alignment of the detection system with the optical pathway of the disc for absorbance measurements in an environment free from ambient light. This device allows for rapid analysis times, compactness, ease of use, low cost analyses and low reagent consumption. Keywords: Phosphate, microfluidics, lab-on-a-disc, optical senso

Application of the geometric morphometrics approach in the discrimination of morphological traits between brown trout lineages in the Danube Basin of Croatia

Brown trout is a salmonid fish with a natural range extending throughout western Eurasia and North Africa. Due to its commercial value, it has also been introduced worldwide. In continental Croatia, introduced trout of the Atlantic lineage hybridizes with native trout of the Danubian lineage, threatening the native genetic diversity. The geometric morphometrics approach was used in this study to analyse changes in shape between native trout, introduced trout and their hybrids, classified a priori by molecular phylogenetic analyses. A total of 19 landmarks and semi-landmarks were used to capture the shape of 92 trout individuals belonging to two lineages and their hybrids. Canonical variate analysis and discriminant function analysis were used to analyse and describe shape variation. A significant difference was found between the shape of the Atlantic lineage trout and both Danubian lineage trout and hybrids, with the most prominent differences in body depth, head length and eye size. No statistically significant shape differences were observed between Danubian lineage trout and the hybrids. The observed significant differences in shape could be the result of genetic diversity or trout phenotypic plasticity. Further studies are needed to clarify the origin of this variation in shape

Atalanta: The autonomous analytical algal toxin platform

Cyclic peptide cyanobacterial toxins, in particular Microcystis aeruginosa, pose a serious health risk to humans and animals alike [1], [2]. Occurring mostly in fresh and brackish water, they have been identified to cause cancer promotion and liver damage [3]. Herein, we describe a portable, microfluidic-based system for in-situ detection of algal toxins in fresh water. The Atalanta system is a novel, portable and sample-to-answer platform for the detection of toxic cyanobacteria – Microcystin-LR in fresh water. Atalanta utilises the partnership of highly-specific recombinant chicken anti-microcystin antibodies, prepared in-house, with a 3D-printed ‘LASER-photo¬diode’ fluorescent detection method, also developed in-house. A competitive immunoassay format is utilised to detect free toxin. Furthermore, dissolvable-film (DF) based flow-actuation facilitates full assay inte¬gration. This new approach will form the basis of a cost efficient, USB-controlled water quality monitoring system. The Atalanta detection system consists of two components; the microfluidic Atalanta disc and the disc-holder. The Atalanta disc-holder was fabricated and assembled from a 3D-printed casing, with electronic components housed in device. The 5-layered microfluidic disc consists of five reservoirs, each with a separate venti¬lation, aligned radially with inter-connected microchannels. Each reservoir represents a functional assay step. First, microcystin conjugate is coated to the functionalised surface of the reservoir 3 prior to assembling the disc. A freshwater sample in reservoir 1 is pre-incubated with recombinant antibodies labelled with fluorophore (Alexa 647) in reservoir 2. This is then spun into reservoir 3 for detection through a competitive immunoassay using Microcystin-LR. Low fluorescence signal indicates high Microcystin-LR concentration in the sample