Periodic Reporting for period 1 - GrapheneBiosensor (Electrochemical Graphene Sensors as Early Alert Tools for Algal Toxin Detection in Water) Reporting period: 2017-09-26 to 2019-09-25 Summary of the context and overall objectives of the project Episodes of harmful algal blooms (HAB) of cyanobacteria, known as blue-green algae, occur frequently in bodies of water worldwide as a consequence of eutrophication resulting from anthropogenic activities such as agricultural run-off, urban waste, and manufacturing of detergents and global warming. Cyanobacterial HAB often produce undesirable color, odor, and tastes but most importantly, it also produces harmful toxins (i.e. cyanotoxins), which is a significant hazard for human health and the ecosystem in drinking water, recreational water, and aquaculture. The coastlines of many European countries (including UK) are frequented by large scale of HAB events. Following a significant HAB event, there is an urgent need to establish when a water source is safe to use or to evaluate the level of treatment required to make a source safe. Among cyanotoxins, microcystin-LR (MC-LR) is the most frequently occurring variant throughout the world. It had been confirmed that microcystins were responsible for some poisonings of animals and humans where water sources contained toxic cyanobacteria blooms. Acute or prolonged exposure to microcystins would cause liver damage, followed by a massive intrahepatic haemorrhage and probably leading to death. In 1998, the provisional guideline concentration limit of 1 μg/L MC-LR in drinking water was assigned by the World Health Organization (WHO). The development of reliable methods for monitoring MC-LR in water resources is of great interest to determine the occurrence and to prevent exposure to the toxin. Several methods have been developed to detect MC-LR, such as high-performance liquid chromatography/mass spectrometry (HPLC/MS), which require long processing times, sophisticated instruments, complex procedures, or high processing cost and are in general used in the laboratory, not in situ. In the last decade, electrochemical sensors have become a mature discipline with some outstanding commercial success. They are suitable devices for in situ monitoring, due to their possible miniaturisation, portability and automation. A sensitive, specific, simple, and rapid method for monitoring MC-LR could help to prevent exposure to the toxin. The overall aim of this project is to design, fabricate and validate electrochemical sensors based on flexographic-printed vertically aligned graphene (VAG) electrodes for the rapid, sensitive and reliable detection of MC-LR in water. This was realized by achieving the following main project objectives:1. VAG electrode fabrication under controlled laboratory conditions2. VAG biosensor platforms optimisation for sensitive and selective sensing design3. Optimisation of graphene based biosensing platforms for MC-LR detection in laboratory4. Performance validation of developed VAG biosensor in drinking water sources Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this work, graphene interdigitated electrodes were fabricated using roll-to-roll flexographic printing of ball-milled graphene nanoplatelet (GNP)/ethyl cellulose (EC) ink. Photonic annealing on flexographic printed graphene-ethyl cellulose composite was used to partially remove the polymeric binder (i.e. EC) and produce vertically-aligned graphene (VAG) structures, which maximized the specific surface area of graphene for ultra-sensitive detection of algal toxin (i.e. MC-LR) in drinking water. A spray coating technique was employed to functionalize the biosensor in order to preserve the VAG structures. Prior to immobilization of MC-LR antibodies on the biosensor, a uniform coverage of APTES, which acted as an insulating self-assembly monolayer (SAM) for nonfaradaic electrochemical impedance spectroscopy (EIS) biosensors, was spray-coated onto the VAG. The effectiveness of the spray-coated APTES was studied using energy-dispersive x-ray spectroscopy (EDX) and cyclic voltammetry (CV) techniques. The change in phase from the non-faradic EIS measurement was obtained at a frequency of 167 mHz. At this frequency, an optimal response from the binding of antibody-antigen was observed at the VAG biosensors, which were subsequently tested at different MC-LR concentrations between 0.001 and 10 µg/L in PBS. The results showed a linear correlation established between the change in phase and logarithm of MC-LR concentration. The biosensor exhibited excellent sensitivity of 0.46 degree per decade and limit of detection (LoD) of 1.2 ng/L with good reproducibility, selectivity and stability. Finally, the VAG biosensor was validated using local tap water samples (Swansea, UK) and experienced minimal matrix effects from other factors, such as metal ions, in the water. Such low-cost, label-free, easy to use and ultra-sensitive VAG biosensor is ideal for large-scale early screening of contaminations in drinking water. The key results have been presented in three conferences and workshops:• Invited presentation at UK-India Newton-Bhabha Fund RSC Researcher Links Workshop, IIT Bombay, Mumbai, India, 18th to 21st November 2019. • Invited presentation at Xinghai Youth Scholar Forum, Dalian University of Technology, Dalian, China, 15th-17th May 2019.• Oral Presentation at NANO.IL.2018 Jerusalem, Israel, 9th-11th October 2018.Key research outcomes have also been published and submitted in two refereed journal papers:• L, Wang, W. Zhang*, S. Samavat, D. Deganello, K. S. Teng, Vertically-aligned graphene biosensor prepared by photonic annealing for ultra-sensitive algal toxins detection in drinking water, Biosensor and Bioelectronics, submitted, 2020.• W. Zhang*, M. B. Dixon, C. Saint, K. S. Teng, H. Furumai, Electrochemical biosensing of algal toxins in water: The current state-of-art, ACS Sensors, 2018, 3, 1233-1245. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This work and its produced results are at the forefront of nanomaterials, chemo-/bio-sensor development, and water quality assessment to develop innovative analytical technology through an interdisciplinary approach. This research outcome will impact on the safety of water and provide better protocols for water pollutant control. This will address a real need to develop on-line detection technologies for application in the water industry. Impact of project outcomes has been delivered in three major directions: 1) industry, 2) academic and 3) commercial route: 1) Dwr Cymru Welsh Water are now looking at potential adaptation of developed sensor technology with follow-up joint grant applications (e.g. EPSRC, ERC and KESS East) and expand the technology to a wider range of water contaminants on-site monitoring. This will help local water bodies in Wales shape or improve their policy making on water quality monitoring in relation to algal bloom events and toxin contamination occurrences. 2) Through attending prestigious international conferences and workshops, project outcomes have been disseminated to a wider range of academic audiences and results have been published in high-impact international journals in the area of environmental and material science. On-line research communities such as Linkedin has been used to widen the online presence of the outputs and maximize the impact of this publicly-funded research on science and industry end users. 3) developed graphene-based biosensor technology will be further exploited through IP identification and filing. The Fellow will continue to work with the commercialisation manager at Swansea University to disseminate the technology to industry through their regular Knowledge Transfer Network and Technology Showcase events. MC-LR biosensor detection
