AUTONOMOUS MICROFLUIDIC SENSORS FOR NUTRIENT DETECTION: APPLIED TO NITRITE, NITRATE, PHOSPHATE, MANGANESE AND IRON Vincent J. Sieben1*, Alexander D. Beaton2, Cedric F.A. Floquet2, Samer Abi Kaed Bey2, Iain R.G. Ogilvie2, Edward M. Waugh2, Jennifer K.C. Ang1, Matthew C. Mowlem2 and Hywel Morgan1 1
Nanoscale systems integration group, University Of Southampton, UK and 2 National Oceanography Centre, University Of Southampton, UK
ABSTRACT We present the design, fabrication, analysis and characterisation of autonomous high accuracy and sensitivity microfluidic nutrient sensors. A stand-alone sensor platform with integrated sub-systems is demonstrated, which is portable and capable of in-situ reagent-based nutrient analysis. The system is based on a low cost optical detection method, together with an automated microfluidic delivery system that is able to detect nitrite (e.g.) with a Limit of Detection (LOD) of 14nM. The platform was operated in-situ at Southampton Dockhead for 37 hours, performing 284 measurements. KEYWORDS: nutrient detection, microfluidic system integration, spectrophotometry, environment INTRODUCTION The application of microfluidics to in-situ colourimetric analysis provides several advantages over the traditional large-scale “macro” systems [1]. These include a significant reduction in reagent consumption, smaller physical size, low power consumption, with significantly longer lifetime, and increased system integration [2]. Such advantages enable both high-resolution temporal and spatial data sets that would be logistically impossible using traditional sampling techniques [3]. Often, previously demonstrated microchip-based chemical analysers have required extensive support infrastructure thus making them unsuitable for remote and on-site deployment [4]. Here we demonstrate an advanced level of integration to achieve stand-alone sensors that integrate all the sub-systems required to realise portable and on-site nutrient analysers based on microfluidics. The sensors will be deployed on buoys or profiling floats (eg. ARGO floats [5]) to monitor biogeochemical processes in ocean waters. The development of long-life (>12months), fully autonomous multi-analyte nutrient sensors opens a vast number of deployment scenarios (rivers, estuaries, water quality, etc.). The implementation of microfluidics as a versatile monitoring tool in the extreme conditions of the world’s oceans is extremely challenging. Typical water temperatures can range from 36°C in tropical waters to -2°C in the polar seas, while hydrostatic pressure increases linearly with depth, reaching approximately 1100 times atmospheric pressure at its deepest point (Challenger Deep in the Mariana Trench). Such diverse conditions have the potential to alter reaction kinetics, fluid flow rates, mixing dynamics, sub-component alignment and the behaviour of optical components. ARGO floats typically collect temperature and salinity profiles in the top 2000m of the world’s ice free oceans, meaning they experience regular (bi-weekly) pressure cycling over 200 atmospheres and a temperature range that approaches 30°C. A significant amount of research has therefore been devoted toward engineering and testing ruggedized microfluidic chemical analysers that are stable in such harsh environmental conditions. The sensors shown in Figures 1 have been developed for analysis of Nitrite, Nitrate, Phosphate, Iron and Manganese. They operate using a common technology platform of measuring the optical absorption of a coloured reagent, where the adsorption is proportional to the concentration of the analyte. For example, nitrite is measured using an Azo dye, based on the Griess reaction [4], whilst nitrate is measured by reduction to nitrite using a miniature integrated copper cadmium column. All fluidics are handled by ruggedized valves bonded to micro-machined PMMA microfluidic chips. They have been tested with extensive temperature, pressure cycling, as well as at dock-side.
Figure 1: Left: schematic of the colourimetric sensor platform. The device measures 12cm by 6cm. All channels were 150μm wide and 300μm deep, except the optical absorption cells which were 300μm wide and 300μm deep. Right: photograph of the autonomous nitrite and nitrate microfluidic sensor.
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14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands
EXPERIMENTAL The microfluidic platform shown in Figure 1 is based on a machined block of PMMA. The system is 12cm long, 6cm wide, 12cm tall and operates by mixing a water sample with a known volume of Griess reagent, creating a coloured Azo dye. The optical absorbance of the complex is proportional to the concentration of nitrite. The chip incorporates a fluidic manifold that permits sample selection (either ultra pure water blank, a 5µm nitrite standard carried on board, or a seawater sample), followed by a 250mm long serpentine mixer and three separate absorbance cells (two of 2.5cm path length, and one of 0.5cm path length; for further details see reference [4]). The microchip was machined in 5.0mm thick tinted-PMMA (Plexiglas GS 7F60, Röhm, Darmstadt, Germany) by micro-milling (LPKF Protomat S100 micromill). A solvent vapour bonding procedure was used to polish the channel surfaces and to bond two halves [6]. Fluid connectors, optical alignment grooves, valve mounts, and syringe mount were all milled into the lid or chip. Fluid handling was performed using seven micro-inert valves (LFNA1250325H, Lee Products Ltd., UK) and a custom built syringe pump driving two 500µL Hamilton 1750CX syringes (Nevada, USA). These were mounted to the bonded microchip. There was no interconnect tubing between the chip, valves and pump, thereby minimising dead-volume. Hall effect sensors were used for syringe pump feed-back an optical components were fixed with Norland optical adhesive 68 (NJ, USA). The microfluidic chip with pump and valves was housed in a water-tight acrylic tube, 160mm outer-diameter and 0.5m height. This tube was large enough to accommodate four 500mL volume ethylene-vinyl-acetate reagent storage bags (Nutrinox, Devon, UK) that held: Griess reagent, on-board standard, MilliQ blank, and system waste. Although the tube housing was large, most of this volume was not used. The sea-water sample was filtered using a 0.22µm Durapore membrane filter (GVWP01300, Millipore, Ireland). The sensor was controlled using custom electronics and a National Instruments Digital Acquisition Device PCI 6289 card installed in a ruggedised PC (AAEON BOXER 6920, Acal Tech. Ltd., UK) running Labview 2009. A labview state machine performed sampling as follows: withdraw sample; inject sample; wait and measure (i.e. a stopflow setup). A wait time of 150 seconds to ensure that the reaction had completed and sufficient time was given for the colour to develop [4]. This approach provided a calibrated and accurate method of measuring nitrite concentrations. The volume of sample and Griess reagent used was 250µL per sampling point (flow rate of 200µL/min). All reagents were prepared as described in our previous work [4]. RESULTS AND DISCUSSION The micro-sensor system required 1.5W of power, orders of magnitude lower than the high power consumption (typically 10-150W) of commercially available systems (such as the SubChemPak Analyzer, SubChem Systems, USA) [7]. Figures 2 shows the calibration and the linear performance of a nitrite sensor, demonstrating excellent performance over a concentration range of 30nM – 50µM, with a limit of detection of 14nM. Figure 3 is field data for the nitrite sensor from a in situ deployment, demonstrating both the stability and sensitivity of the system.
Figure 2: Left: the photodiode output voltage for a sequence of nitrite samples (varying concentrations; 30nM to 50µM), flowing through the microfluidic device. MilliQ blank sample was injected for 5min, followed by nitrite sample for 5min. Right: comparison of measured absorption by the nitrite microchip sensor and a Hitachi U-2800 spectrophotometer with varying concentrations. The linear fit illustrates the accurate performance of the micro-sensor.
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Figure 3: Sensor field data. The on-board standard, sea-water sample, and water height (tide) were measured. The stability of the system is demonstrated by the small deviation in the measurements of the on-board 5µm standard, which had a standard deviation of 13.4nM over 37 hours. The microchip sensor detected changes in nitrite concentration due to tidal influence. CONCLUSION We have demonstrated a microfluidics stand-alone, automated colourmetric sensor. The system has a power consumption that is orders of magnitude lower than conventional systems, and a reagent consumption measured in µL per sample. The nitrite sensor was operated in-situ for 37 hours and made 284 discrete measurements in the Southampton dock. In the future, data-logging electronics will be integrated and the sensor platform will be deployed on buoys and profiling floats (eg. ARGO floats) to monitor biogeochemical processes in ocean waters. ACKNOWLEDGEMENTS The authors acknowledge funding from ESPRC, NERC and also the University of Southampton. V. Sieben is supported in part by the Natural Sciences and Engineering Research Council of Canada. The authors would also like to thank Professor Peter Statham, Lee Fowler and Rob Brown for support on this project. REFERENCES [1] G. M. Greenway, et al., Characterisation of a micro-total analytical system for the determination of nitrite with spectrophotometric detection, Analytica Chimica Acta, 387, 1-10 (1999) [2] J. Cleary, et al., An autonomous microfluidic sensor for phosphate: On-site analysis of treated wastewater, Ieee Sensors Journal, 8, 508-515 (2008) [3] T. S. Moore, et al., Marine Chemical Technology and Sensors for Marine Waters: Potentials and Limits, Annual Review of Marine Science, 1, 91-115 (2009) [4] V. J. Sieben, et al., Microfluidic colourimetric chemical analysis system: Application to nitrite detection, Analytical Methods, 2, 484-491 (2010) [5] J. Gould, et al., Argo Profiling Floats Bring New Era of In Situ Ocean Observations, EOS, Transactions American Geophysical Union, 85, 185 (2004) [6] I. R. G. Ogilvie, et al., Reduction of surface roughness for optical quality microfluidic devices in PMMA and COC, Journal of Micromechanics and Microengineering, 20, 065016 (2010) [7] A. K. Hanson, A new in situ chemical analyzer for mapping coastal nutrient distributions in real time, Oceans 2000 Mts/Ieee - Where Marine Science and Technology Meet, Vols 1-3, Conference Proceedings, 1975-1982 (2000) CONTACT *Dr. V.J. Sieben, T: +44 (0)23 8059 6003, E:
[email protected]
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