Ducrée, Jens

Fitzgerald, Jenny

Heery, Brendan

Maguire, Ivan

Murphy, Caroline

Nwankire, Charles

O'Kennedy, Richard

Regan, Fiona

Name not available

Important notes:  Do NOT write outside the grey boxes. Any text or images outside the boxes will be deleted.  Do NOT alter the structure of this form. Simply enter your information into the boxes. The form will be automatically processed – if you alter its structure your submission will not be processed correctly.  Do not include keywords – you can add them when you submit the abstract online.   Title: In-Situ detection of microcystin in a pre-lysed freshwater sample using an integrated centrifugal microfluidics platform  Authors & affiliations: I. Maguire*, J. Fitzgerald*, B. Heery*, C. Murphy*, C. Nwankire**, R. O’Kennedy*, J. Ducreé* and F. Regan*. * School of Chemistry, Dublin City University, Glasnevin, Dublin 9, Ireland **MESTECH, Dublin City University, Glasnevin, Dublin 9, Ireland.  Abstract:  (Your abstract must use Normal style and must fit in this box.  Your abstract should be no longer than 300 words. The box will ‘expand’ over 2 pages as you add text/diagrams into it.) Preparation of Your Abstract 1. The title should be as brief as possible but long enough to indicate clearly the nature of the study. Capitalise the first letter of the first word ONLY (place names excluded). No full stop at the end. 2. Abstracts should state briefly and clearly the purpose, methods, results and conclusions of the work. Introduction: Clearly state the purpose of the abstract Methods: Describe your selection of observations or experimental subjects clearly Results: Present your results in a logical sequence in text, tables and illustrations Discussion: Emphasize new and important aspects of the study and conclusions that are drawn from them Introduction: Cyanobacterial toxins from Microcystis aeruginosa predominantly manifest in brackish and fresh water sources and have been shown to promote hepatic disease and cancer [1 - 3]. Autonomous and continuous monitoring of these hepatotoxic cyanobacterial toxins is particularly challenging, and thus critical to environmentally monitor them. Here, we present a portable and cost-effective centrifugal microfluidics-based integrated system for in-situ detection of toxins from microcystis aeruginosa. Our unique system includes an in-house developed, and optimised, competitive assay for microcystin toxins, with a top-down LED-photodiode detection system also developed and 3D printed in-house. Preliminary results demonstrate that the system detects cyanobacteria toxins in pre-lysed freshwater at World Health Organisation recommended detection limits of 1ng.ml-1 [2]. Methods: The centrifugal microfluidic-based system, A.K.A. ToxiSense, consists of two components; a multi-layered microfluidic disc for fluidic control and multiplexed assay integration; and a detection system. Also included is a software controlled motor for disc spinning as well as a top-down fluorescent detection technique for Alexa 430.   Figure 1: The microfluidic detection system Important notes:  Do NOT write outside the grey boxes. Any text or images outside the boxes will be deleted.  Do NOT alter the structure of this form. Simply enter your information into the boxes. The form will be automatically processed – if you alter its structure your submission will not be processed correctly.  Do not include keywords – you can add them when you submit the abstract online.    The microfluidic disc is manufactured from poly-(methyl-methacrylate) (PMMA) sheets and pressure-sensitive-adhesive (PSA) (ARseal™90880), sourced from Radionics™ and Adhesives Research™ respectively. The Disc consists of five reservoirs, each with separate ventilation systems, in radial alignment connected by microchannels. Each reservoir is a single step in the assay protocol (shown below), with reservoir four and five acting as the control and waste reservoirs, respectively.   Figure 2: The bio-activated microfluidic disc  Results and Discussion:                                            Table 1: Microcystin competitive assay      Using the ToxiSense platform, the results in Table 1 illustrate that analyte (microcystin) concentration is inversely proportional to fluorescence response. This simple, easy to use system offers a novel approach to in-situ observation of microcystin. The entire system including the assay, microfluidic platform and detection system will continue to be optimised in order to achieve an autonomous and continuous environmental monitoring system.   References [1] C. MacKintosh, et al., “Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants,” FEBS Lett., vol. 264, no. 2, pp. 187–192, 1990. [2] World Health Organization, Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management. 1999. [3] J. M. Rinta-Kanto, et al., “Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake,” Harmful Algae, vol. 8, no. 5, pp. 665–673, 2009.  Microcystin concentration (ng/mL) Normalised Relative fluorescence (a.u.) 10000.0 0.8640 1111.1 0.8953 41.2 0.94825 13.7 1.0024 0 1 Important notes:  Do NOT write outside the grey boxes. Any text or images outside the boxes will be deleted.  Do NOT alter the structure of this form. Simply enter your information into the boxes. The form will be automatically processed – if you alter its structure your submission will not be processed correctly.  Do not include keywords – you can add them when you submit the abstract online.    

In-Situ detection of microcystin in a pre-lysed freshwater sample using an integrated centrifugal microfluidics platform

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In-Situ detection of microcystin in a pre-lysed freshwater sample using an integrated centrifugal microfluidics platformI. Maguire1, J. Fitzgerald1, B. Heery1, C. Murphy2, C. Nwankire3, R. O’Kennedy2, J. Ducrée4and F. Regan11School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland2School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland3Marine Environmental detection Technology Hub, Dublin City University, Glasnevin, Dublin 9, Ireland4School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland1.Introduction: General Toxicology• What is a Toxin?• According to Merriam-Webster: “A poisonous substance that is a specific product of the metabolic activities of a living organism. It is usually very unstable and toxic when introduced into the tissues. It also typically capable of inducing antibody formation”.Biologist Chemist PhysicistBAD**Should probably developsome sensors then?Of course you should!....1.Introduction: MicrocystinMICROCYSTIN:Produced from: Microcystis Aeruginosa,freshwater cyanobacteriaClassification: Potent hepatotoxin (hepato = Liver)Predominant Congener: Microcystin-LR (also most toxic variant)Regulator limit in drinking water: 1 ng mL-1It results from harmful algal blooms which can cause ecological and economical disastersMicrocystin-LR Chemical structure1.Introduction: Microcystis Aeruginosa BloomsLake Erie in October 2011: the lakes worst cyanobacteria blooms in decades. Caused by eutrophicationLake Erie in July 2015: 2-ToxiSense system concept2-ToxiSense system concept: Lab-On-A-Chip Vs. Lab-On-A-Disc platforms• Lab-On-A-Chip• Requires accurate pumping mechanisms, often at very high cost• Chip is stationary for studies• Easier to simulate and control fluid flow• Lab-On-A-Disc• Requires a motor, often relatively inexpensive• Disc is in motion for studies• More difficult to simulate and control fluid flowCost was a decision factor in this project: Lab-On-A-Disc platform was selected 2-ToxiSense system concept: Introduction to ToxiSense system• The ToxiSense System consists of two components.The ToxiSense Microfluidic Disc The ToxiSense detection system2-ToxiSense system concept: Microfluidic DiscThe ToxiSense Microfluidic Disc:• On-board microfluidics (Lab-On-A-Disc platform)• Manufactured from poly(methyl methacrylate) (PMMA)  ( Radionics™) and pressure sensitive adhesive (Adhesives Research Inc. ™)• Easily modifiable• Microcystin-LR detection: 5-step assay.• Low sample size• High sensitivity• Cheap to manufacture2-ToxiSense system experiments: Off-Site Fluidic studiesThe fluidic movement can be studied to confirm fluid is obeying the assay procedure correctlyObservation CCD cameraStrobe LightMotor for centrifugation of discViewing MonitorMotor Controller displayCamera Phase CaptureSpin stand with computer observationSingle assay proof of concept using pneumatic pressure valvingRadial Flow directionPre-lysed Sample inlet and air ventAir ventilation controls fluid flowMicrochannelsChamber 3. Test ReservoirChamber 5. Waste ReservoirChamber 4. Control ReservoirChamber 1. Sample loadingChamber 2. Antibody mixingChamber 1. Sample loadingChamber 2. Antibody mixChamber 3. Test ReservoirBefore (left) and after (right)4Control R servoir Unbound anti-body Bound anti-body pair 2-ToxiSense system concept: detection SystemThe ToxiSense detection system:• 3D-Printed fluorescent detection system• Casing manufactured from acrylonitrile butadiene styrene (ABS)• Detection system developed in-house.• Easily modifiable• Microcystin-LR detection: Alexa fluor 430• Powered by mains • Communications either via USB to PC or with Wireless dongle (in-house model)2-ToxiSense system concept: detection method2-ToxiSense system concept: Video demonstration3-ToxiSense system studies3-ToxiSense system studies: Initial microcystin detection resultsNext stage is to test full inverse assayy = 329.14x + 394.6R² = 0.9747y = 288.49x + 310.7R² = 0.957301002003004005006000 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4Response unitsConcentration (mg.ml-1)Fluorescent response units vs concentration of fluorescent abs in sedimentationTest DetectorControl DetectorLinear (Test Detector)Linear (Control Detector)3-ToxiSense system studies: Fluorescent Microscopy images of test reservoirs0 μg.mL-1 2 μg.mL-1 10 μg.mL-1Microcystin Concentration bound to reservoir surface4-Conclusions4-Conclusions: Summary of the ToxiSense system• The ToxiSense detection system is:• A highly sensitive and portable toxin detection system.• A flexible and easily modifiable system.• An easy-to-use and cost effective solution to in-situ toxin detection• The first step in developing a fully autonomous and in-situ toxin detection systemFurther information can be found on the poster: P1.134 – “Enhancing the capabilities of a biosensor for microcystin-LR”AcknowledgementsDr. Jenny FitzgeraldBrendan HeeryDr. Caroline MurphyDr. Charles NwankireProf. Jens DucréeProf. Richard O’ KennedyProf. Fiona ReganMESTECH

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In-Situ detection of microcystin in a pre-lysed freshwater sample using an integrated centrifugal microfluidics platform

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