Source: THE NITRATE ELIMINATION COMPANY, INC. submitted to
NITRATE BIOSENSOR FOR AGRICULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0211055
Grant No.
2007-33610-18480
Project No.
MICK-2007-02431
Proposal No.
2007-02431
Multistate No.
(N/A)
Program Code
8.4
Project Start Date
Sep 1, 2007
Project End Date
Aug 31, 2010
Grant Year
2007
Project Director
Campbell, W. H.
Recipient Organization
THE NITRATE ELIMINATION COMPANY, INC.
334 HECLA STREET
LAKE LINDEN,MI 49945
Performing Department
(N/A)
Non Technical Summary
Improved control of nitrate utilization will both help farmers and protect water quality. The goal is to research and develop the prototype Ag Nitrate Biosensor System (ANS) with disposable Screen-Printed Carbon Electrodes with NECi Superior Stock Nitrate Reductase and dye immobilized on the electrode surface by vacuum drying (SPCE/NaR/dye) for quantitative nitrate detection using a Field Portable and Handheld Meter.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110200050%
1020210200050%
Goals / Objectives
A nitrate biosensor based on the enzyme nitrate reductase will be formulated for agricultural applications. Screen printed carbon electrode (SPCE) for nitrate biosensor with NECi Superior Stock Nitrate Reductases (YNaR1 & AtNaR2) and using dyes as electron carrier will be developed. Formulation of SPCE-NaR will be done by varying enzyme drying method, selecting most effective and safe electrode-carrying dye, and establishing an effective sample reagent pack with buffer system containing sulfite for eliminating oxygen interference. The nitrate biosensor will be calibrated using nitrate standards and nitrate content of real world samples extracted from crop leaves and soils will be determined. The results of testing the combined system components will be used to develop a prototype nitrate biosensor for application in agriculture. This device will assist farmers and agricultural consultants with nutrient management by providing rapid field analysis.
Project Methods
An optimized formulation for the disposable Nitrate Sensor consisting of Screen-Printed Carbon Electrode with NECi Superior Stock Nitrate Reductase and dye immobilized on the electrode surface via vacuum drying (SPCE/NaR/dye) will be developed by comparing different NECi NaR forms and different dyes. Optimized reagent pack for sample analysis which will include buffer components, pH, and oxygen scavenger, will be formulated. New oxygen scavengers will be tested. Nitrate Meter (ie. hand-held, portable potentiostat) component of the NECi Ag Nitrate Biosensor System Prototype (ANS) will be developed based on a modification of a published design. The optimized Nitrate Sensor with the Nitrate Meter will be calibrated using nitrate standards, and real world samples tested for nitrate content. Emphasis here will be placed on performance characteristics of the nitrate electrode component with the potentiostat component. Formulation of Nitrate Sensor and the Nitrate Meter will be refined based on initial test results to yield final prototype ANS. Computer software component for reporting nitrate content of samples by ANS will be developed. Field testing of the final prototype ANS with Ag samples from crops and soil will be done. The key evaluation will be based on ease of use of the system in the field. Manufacturable design of the prototype Ag Nitrate Biosensor System will be finalized.

Progress 09/01/07 to 08/31/10

Outputs
OUTPUTS: CRIS form AD-421 Outputs The goal of this project was to develop a handheld meter for nitrate detection suitable for use by agricultural consultants, farmers and others in the field. Working prototypes were developed and described in the Final Report. Prototypes were evaluated using lab standards and a variety of ag samples such as water and plant materials. Livestock feed samples were obtained locally (corn, grass), and from Dr L J Snyder, at Purdue. Wheat and barley grass samples were obtained from Northern Star Integrated Services and Dr C MacKown, USDA-ARS in El Reno, OK. A number of novel enzyme immobilization chemistries were developed and investigated as new techniques for attaching nitrate reductase (NaR) to electrode surfaces were required. This aspect of the project derived from Dr N Plumere's thesis work; Dr Plumere was the post-doctoral associate on this grant. An unanticipated outcome of the project was development of an enzymatic system for removal of oxygen from the biosensor reagent mix. Oxygen interference is a major technology barrier for reductase type sensors. The reagent system developed under this grant may be a valuable discovery. NECi intends to investigate the market potential of the new oxygen removal system in 2011. This system itself may have sufficient value to warrant patent protection. In late 2009, YSI Inc (ysi.com) began negotiations with NECi on licensing its patent for simplified nitrate reductase. A License Agreement between YSI and NECi was fully executed in May 2010, and an up-front fee was paid to NECi. The Agreement gives YSI exclusive rights to use NECi's S-NaR in nitrate biosensors for environmental markets. They anticipate having S-NaR-based products on the market in 2011. YSI will purchase this protein from NECi and also pay a royalty on each sensor sold. YSI has a very good reputation in their field, and great marketing capabilities. YSI's approach to nitrate biosensor development differs from that taken in this project; NECi is free to develop the technology developed under this grant. So this is a win-win for NECi and the USDA. Dissemination Ellen Campbell of NECi remains active in explaining the benefits of nitrate testing to the agricultural community. She gave invited presentations on nitrate testing and NECi's nitrate test kits at the Michigan Agri-Business Association's Winter Meetings in Jan 2008 and 2009. She was invited to present on enzyme-based nitrate analysis at the American Chemical Society's Annual Green Chemistry Conferences in June 2009 and 2010. NECi has not submitted papers or abstracts on biosensor development in 2010 due to time constraints as well as the licensing commitment by YSI. We anticipate discussing IP protection options with our intellectual property attorneys later this year or early in 2011. Dr N Plumere, post-doc on this project, returned to Germany with a new position on the faculty of the University of Bochum. He presented a talk on the oxygen removal system at conference called Electrochemistry 2010, in Sept 2010 in Bochum (http://www.gdch.de/vas/tagungen/tg/5407__e.htm). PARTICIPANTS: Dr. Nicolas Plumere, who served as post-doctoral associate on this project, is now on the faculty of the University of Bochum, Germany (Ruhr-Universitat Bochum, Zentrum fur Elektrochemie). We will continue to collaborate with Dr Plumere on enzyme/electrochemistry research. TARGET AUDIENCES: In April 2009, ER Campbell presented NECi's ideas on nitrate testing and products to a focus group of Extension personnel, grain elevator operators, and crop consultants at an event organized by Au Sable LLC, a company the consults and distributes to the ag industry. ER Campbell presented a poster on the potential for phosphate test kits or hand held sensors at a conference on Phosphate in Windsor, Ontario, Canada, in July 2009. PROJECT MODIFICATIONS: In the Budget, the only significant revision was reallocation of funds from payment to Lab Assistants (high school or college students working as trainees) to salaries for other employees on the project. We had anticipated expenditures of $9100 for this category, but spent $2920 on workers at this level of experience. A bit less than anticipated was paid to one of the technicians; she moved away from the area toward the end of the second year of the project. Other categories were within $1000 - 2000 as budgeted. NECi began negotiations with a suitable marketing partner for licensing our technology for their own proprietary nitrate biosensor in late 2009. A License Agreement, which included a Supplier's Agreement whereby the company will buy enzyme from NECi, was finalized in April 2010. This company is well on the way to having nitrate detection devices on the market which incorporate NECi enzymes and technical know-how; they anticipate market introduction in late 2011 or early 2012. They are developing monitoring equipment, not the small, hand-held devices envisioned in the proposal. They have the marketing, production and distribution capabilities to make the most of NECi's technology. ER and WH Campbell devoted major effort (outside this project) to solidification of this relationship, which is the culmination of years of contact. Revenues from sales of this company's nitrate sensor will be used to pursue NECi's alternate approach to biosensor signal detection technology. In addition, this company may expand into the handheld market in the future. The company is strong in environmental, industrial process, and agricultural monitoring, a very good match with NECi's interests and markets. We took the opportunity afforded by this relationship to explore in greater depth enzyme immobilization chemistries as well as the potentially valuable oxygen removal system, in order to best utilize Dr Plumere's expertise. NECi and Dr Plumere will continue to collaborate on R&D of direct electron transfer between enzyme and electrode surfaces for biosensor technology. Design of the device and signal processing of enzyme electrode signals was finalized as described in the goals. Improvements in sensor fabrication need to be made in conjunction with a suitable commercial partner in the screen-printed electrode field. NECi continues to work with suitable partners in the US and EU. Finding the right partner and developing a good design for the disposable electrode is the key to commercial success for the handheld nitrate meter.

Impacts
NECi is an environmental biotechnology company dedicated to the application of enzymes to the solution of environmental problems. NECi has focused on nitrate detection since its founding in 1993. Nitrate is a critical plant nutrient, but excess nitrate is a water quality problem in rural and urban areas worldwide. On another front, many of the lab techniques used for detection of analytes of environmental concern are themselves toxic. NECi believes that development and introduction of enzyme-based analytical chemistry will benefit users at all levels, from laboratories to citizen water quality monitoring groups. Enzymes perform their functions under gentle conditions in aqueous solution. Enzymatic reactions are fast, specific, and generally safe. The benefits of reducing or eliminating hazardous or toxic chemicals from test kits and laboratories are self-evident. Through market research efforts including focus groups, it is clear that the agricultural community needs to see fast and easy devices before adopting nutrient detection technology. NECi will continue to develop its marketing capabilities and product lines in nitrate and phosphate test kits while biosensor technology is perfected. The increasing cost of nutrient inputs makes nitrate testing of higher interest to producers, but ease of use - which means requiring as little time as possible - is a barrier for the extremely busy people in this market. Users want to see a small device, something that fits in a pocket, that requires little time to learn to use and provides a digital readout plus advice on what to do with the information (e.g. the field is adequately fertilized, the runoff from a manure pond is in regulatory compliance, the feed is safe for my livestock, how many pounds of N should be added for maximum corn yield). NECi's goal is to enable nitrate testing that is easy and accurate enough to provide information of real monetary value to agricultural producers. In this way, the nitrate biosensor can also play a role in protection of water quality.

Publications

  • No publications reported this period


Progress 09/01/07 to 08/31/08

Outputs
OUTPUTS: Two key aspects of the NECi Ag Nitrate Biosensor have been investigated to obtain working components for the system. First, the disposable Screen-Printed Carbon Electrode (SPCE) has been designed in cooperation with an American vendor, Conductive Technologies. The SPCE will be manufactured with a carbon working electrode, a carbon counter electrode and a silver-silver chloride reference electrode on a plastic strip. The SPCE will have a removable cover which will allow NECi to drop coat the working electrode surface with a mixture of Nitrate Reductase (AtNaR2) and an electrode carrying dye. Then when the cover is placed over the 3 electrodes, it will form a 10 microliter well which can be filled with the reaction solution by capillary action. This solves a major problem with the prototype SPCE used in the Phase I project. The second aspect was the design and construction of the prototype Nitrate Meter, which was carried out via a subcontract with a local electrical engineer. Working from the design and schematic presented in the Phase II proposal, he prepared an improved design with more up-to-date components and ordered 5 printed circuit boards, which are 7 x 10 cm. He then populated one board with the chips and resistors specified in the design, which resulted in a working potentiostat with the appropriate range of current detection for the Nitrate Meter. This prototype Nitrate Meter was tested with SPCE used in the Phase I project and shown to report current responses to nitrate as shown previously with the BAS potentiostat used in the Phase I study. At this time, the output from the Nitrate Meter, via its USB port, is fed directly to a laptop computer running Microsoft Excel and the current observed on the SPCE is collected every 0.1 sec (actually an average of 10 reading of the current is reported to Excel). Further testing of the first prototype is underway and the other 4 boards are being populated for use in the project. The next stage is to design a case and LCD screen for the Nitrate Meter to make it more user friendly. Improvements in production of Superior Stock Nitrate Reductase (ATNaR2) by fermentation of the NECi Pichia pastoris cell line were made. Three fermenter runs were done in the summer, and improvements in protocols with the goal of increased final active enzyme yield were made. The AtNaR2 was purified using NECi standard protocols, and stored for future use in this project. Sufficient AtNaR2 for tens of thousands of individual nitrate electrodes was accumulated in this work. Finally, additional work to test applications of the final project were begun. A seasonal study of nitrate levels in wheat grass related to protein content in the harvested grain and another study following nitrate levels in sweet corn are near completion in early October 2008. ER Campbell gave talks or presentations at the Michigan Agribusiness Association Winter Meeting, Lansing, MI. NECi presented posters at the 2007 Agronomy, Crop & Soil Science Society conference and at the Pittsburgh Conference in March 2008. PARTICIPANTS: Instead of hiring a computer science intern for development of the electronics portion of the biosensor device, NECi hired an electrical engineer as consultant. Dan Kemppainen is employed full time at a local small business and does this kind of work to supplement his income. Dan's work also replaces that of Thomas A Rodeheffer, who passed away this summer from pancreatic cancer. Melissa Richards, MSc Biotechnology from UW Madison, began working for NECi part time in August 2008. She assists with protein production and purification, and on systematic studies for new agricultural applications of enzyme-based nitrate analysis. The arrival of Dr Nicolas Plumere, post-doctoral student on this project, is delayed until 4th Quarter 2008 due to scheduling conflicts for his thesis defense. In the meantime, he has prepared the derivatized methyl viologen compounds to be used in electrode development, and has two papers accepted for publication on this work. NECi is maintaining collaborative relationships with Dr Charles Patton, USGS, and Dr Charles MacKown, USDA ARS, and others for continuing external verification and validation of our methods. TARGET AUDIENCES: ER Campbell was invited by the USDA SBIR program to speak about business development at a conference in Portland, ME in October 2007. Poster presentation by ER Campbell and NECi at the ASSA/CSA/SSA conference in New Orleans in November 2007. The poster was on use of NECi's nitrate test kits for agriculture. ER Campbell gave an invited talk on NECi's line of nitrate test kits for agriculture at the Winter Meeting of the Michigan Agri-Business Association, January 15 , 3008, Lansing MI. NECi presented a poster on enzyme-based nitrate analysis for Discrete Analyzer equipment at the 2008 Pittsburgh Conference, also in New Orleans, in March. ER Campbell gave a short talk on commercialization of basic research at a local Sustainable Living organization in Houghton, MI. ER Campbell has been invited to speak about Advanced Nitrogen Utilization at the 2009 MABA Winter Conference in January 2009. PROJECT MODIFICATIONS: As noted in the Participants section above, Dr Plumere's arrival has been delayed from December 2007 until November 2008. This change has caused NECi to concentrate on the electronics of the biosensor device design in Year 1 of the project instead of during Year 2. This has worked out well, since consultant Mr Kemppainen is able to perform two functions on the project, replacing Mr Rodeheffer as well as a computer science intern. Opportunities for investigations into interesting applications of nitrate testing for agriculture arose in 2008, providing us with sample sets of red winter wheat and sweet corn. Publications in trade journals especially are planned based on these samples. An article for submission to Crop Science is also planned. Because the salaries of Dr Plumere or Mr Rodeheffer were not expended in Year 1, we have approx 25 per cent of Year 1 funds remaining. Expenditures will accelerate in Year 2.

Impacts
Improved enzyme production protocols were developed for the AtNaR2 nitrate reductase form. Custom SPCE electrodes designed for use in enzyme-based biosensors were developed in collaboration with the SPCE manufacturer. The electronics for the prototype handheld meter for detection of the signal produced by the SPCE-nitrate electrodes was developed and tested.

Publications

  • No publications reported this period