The influx of nutrients, such as phosphate and nitrate, to environmental waters can result in ecosystem degradation caused by excessive plant and algae growth. This in turn results in a depletion of the available oxygen in waterways that can result in fish-kills and damage to other waterborne fauna. The combination of these, and many other effects, reduce or destroy a waterways ability to provide a source of food for human or animal consumption, livestock drinking water, recreational activities and agricultural irrigation. Identification and prevention of this nutrient loading problem can be addressed with measurement technology that can be deployed in the environment. To be useful, the technology must be reliable and inexpensive to use. This project will investigate the feasibility of using a unique analytical system for the measurement of nutrients in environmental waters. The system will consist of a device that is small, light-weight, easy to deploy, and cost efficient to own and maintain. The proposed Phase I feasibility work involves the assembly of a first-generation prototype and will be limited in scope to the analysis of phosphate in environmental waters. Our goal is to eventually expand the scope of nutrients analyzed to also include ammonium, nitrate, and silica. OBJECTIVES: This Phase I study will be limited to demonstrating the feasibility of using a micro total analysis system for the colorimetric analysis of orthophosphate in environmental waters. The system will be an integration of a sampling device, microfluidic network, chemical reagents, and an absorbance detector. The analyzer will be deployable and will operate autonomously but it will not be submersible in this embodiment. The following 6 objectives will be specifically addressed during the project: 1) Design and test an absorbance-based optical detection system that provides adequate sensitivity, is compatible with microliter volumes of analytical solution, and can be interfaced to a microfluidic-based flow injection system. This detection system will have analytical figures of merit that are comparable to existing deployable nutrient analyzers and that are adequate for the determination of nutrient levels found in environmental waters; 2) Adapt existing chemistries for phosphate analysis for use on a microfluidic platform. The colorimetric method will be similar to that described previously in published methods or research literature. The uniqueness of the work will be in the optimization of reagent and sample volumes for use on the microfluidic scale; 3) Design and test a microfluidic-based flow injection system that is compatible with a microliter-volume optical detector and that can be used for colorimetric phosphate detection. The microfluidic chip along with associated fixturing and control structures will be designed to minimize reagent use and to be compatible with the sample introduction device and detector; 4) Design and test a sample introduction device that is pneumatically operated and is compatible with a microfluidic network. A pneumatically operated device will use the same gas source as that used for actuating the microfluidic control components and will only require power consumption for simple valve mechanisms; 5) Integrate the technologies to comprise an analyzer system that it is easy to use and deploy. The results of objectives 1-4 will be integrated into a compact and user-friendly analyzer. Components will occupy a cylinder-like enclosure measuring 36cm in length x 12cm in diameter and having a mass of less than 4kg; 6) Deploy and test the analyzer in the field in a body of water. This is a crucial part of the feasibility study where the system performance will be evaluated in the application for which it is intended. The results of this objective are expected to indicate how well all components interact, ways of optimizing reagent and power usage, and how much of an influence biofouling has on the sample introduction device and microfluidic network. The results will lend insight for future work in a Phase II project where modifications and optimizations will be made to the component technologies and where the system will be expanded to include the additional nutrients of nitrate/nitrite, silica, and ammonium. APPROACH: Demonstration of feasibility of the micro total analysis system for orthophosphate determinations will be accomplished by integrating work done in the technology areas of optical absorbance detectors, chemical reagents, microfluidic networks, and sampling devices. Optical detection will be carried out using a micro-volume waveguide system based on simple optical absorbance or evanescent wave attenuation (attenuated total internal reflection). Micro-optics and fiber optics will be used to couple light into and out of the waveguides. Design considerations will be such that the volume of the detector is compatible with a microfluidic platform. An LED light source will be used to measure the absorbance of the reacted analyte. Light detection will be done using a light-to-frequency converter with enhanced long wavelength response. The optical absorber in this application is a molybdenum blue dye formed by the reaction of orthophosphate with a molybdate salt. The colorimetric phosphate analysis will be done in a way similar to that in published methods and research literature. Sampled water to be analyzed is combined with an acidic solution consisting of ammonium molybdate and an antimony salt to form molybdophosphoric acid. This compound is then reduced by mixing with an ascorbic acid solution to form molybdenum blue dye. The amount of dye formed is proportional to the amount of phosphate originally present in the sample. The absorbance of the dye is measured at 880nm and is then related to the amount of phosphate in the sample. Reagent stability, chemical compatibility with analyzer components, and packaging will be also be studied. All chemical reactions related to colorimetric determinations will be performed in a liquid handling network composed of microfluidic structures. We will use a proprietary method of fabricating the microfluidic structures in a durable and reusable polymer material. The structures will measure approximately 60mm x 60mm and will be approximately 0.3 mm thick. The fluid channels will range in size from 100-200 micrometers in diameter. Polymer valves will be integrated with the microfluidic for liquid control. Additionally, there will be structures used for mixing reagents and an interface for the optical detector and sample introduction structures. Fluid control for the microfluidic and sample introduction will be done pneumatically using proprietary methods. The final stages of the project will involve integrating optical detection, chemistries, the microfluidic network, and the sample introduction device into a complete system. Initial testing will be done in the laboratory. Final testing will be done using a deployed unit at a wetland site associated with Ohio State University. During this time the analytical characteristics of the deployed unit will be compared to laboratory methods. Effects associated with biofouling will be investigated in relation to instrument performance.
