Source: NECi: The Nitrate Elimination Company, Inc submitted to
NITRATE AND PHOSPHATE BIOSENSOR FOR HANDHELD MONITORING OF WATER, SOIL AND CROPS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1015977
Grant No.
2018-33610-28272
Cumulative Award Amt.
$100,000.00
Proposal No.
2018-00638
Multistate No.
(N/A)
Project Start Date
Aug 1, 2018
Project End Date
Mar 31, 2019
Grant Year
2018
Program Code
[8.4]- Air, Water and Soils
Project Director
Campbell, W. H.
Recipient Organization
NECi: The Nitrate Elimination Company, Inc
334 Hecla Street
Lake Linden,MI 49945
Performing Department
(N/A)
Non Technical Summary
NECi proposes to develop a low cost, handheld device similar in form and function to the glucose meters used by diabetics, but for measurement of the plant nutrients nitrate and phosphate. The inexpensive meter will use disposable sensor strips. Sensing is based on immobilized enzyme films "printed" onto the electrode material of the sensor strips. Recombinant enzymes are the sensing agents: nitrate reductase for nitrate, purine nucleoside phosphorylase for phosphate. The enzymes confer analytical specificity and selectivity: an enzyme can find "a needle in a haystack", or 10 ppm nitrate in a drop of liquid. As the enzyme binds and reacts with its target analyte, the device detects the tiny electrical signal produced, and transmits this data to smartphones for transfer to any spreadsheet or ag software package. Little or no sample preparation is required.This product line has been NECi's goal since its founding in 1993: Point of Care technology for testing agricultural materials. Growers can monitor nutrient status of crops, thus maximizing yield and quality of fruit, seed, or biofuel feedstock. Soil or runoff testing shows nutrient content of soils, saving money on excess fertilizer addition while protecting water resources. Laboratory analysis will always have a place in farm management. These new tools add timely, on-site information. NECi believes that simple tools that yield accurate data will enable better practices in nutrient management.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020210100085%
6010110208015%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management; 102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil; 0210 - Water resources;

Field Of Science
2080 - Mathematics and computer sciences; 1000 - Biochemistry and biophysics;
Goals / Objectives
The goal of this project is to move beyond test kits into one-step electronic devices for nitrate and phosphate testing, with data sent and recorded on smartphones for use in any spreadsheet or ag software package. This product line has been NECi's goal since our founding in 1993. The point of this technology is to replace test kits for nitrate and phosphate for non-chemists. We propose to develop a low cost, handheld device similar in form and function to the glucose meters used by diabetics. The inexpensive meter will use disposable sensor strips. Sensing is based on immobilized enzyme films "printed" onto the electrode material of the sensor strips. The enzymes are the sensing agents: nitrate reductase for nitrate, PNP for phosphate. The enzymes confer analytical specificity and selectivity - translated, this means an enzyme can find "a needle in a haystack", or 10 ppm nitrate in a drop of liquid. As the enzyme binds and reacts with its target analyte, the device detects the tiny electrical signal produced. Little or no sample preparation is required, no reagents, no test tubes and no pipets or droppers. A small drop of liquid is all users need.This is Point of Use technology for testing agricultural materials. The technology barrier that prevented earlier implementation was oxygen interference of the electrode signal. This barrier was overcome under a prior USDA SBIR project: NECi scientists developed a two-enzyme system for removal of oxygen from liquids open to the air. A patent on this oxygen removal technology was granted in November 2015 (Campbell, Campbell, and Plumeré, 2015). The oxygen removal system eliminates the need to purge samples with argon; it is enabling technology for biosensors in general. NECi has all required enzymes cloned and optimized for expression. The oxygen system patent protects the core IP. This proposal involves testing formulations of the enzyme films, the next step to development of manufacturing methods, and validation of the sensor technology in ag samples (water, soil, and plant extracts).
Project Methods
This is a multidisciplinary project. Methods include protein expression, enzyme biochemisitry, electrochemistry, electronics design and signal detection, and advances in the newer small scale manufacturing systems (additive manufacturing, or 3D printing). NECi's experience in nutrient analysis in a wide array of sample types, from seawater to corn stalks, ensure that device results will be accurate. NECi will contact online training sessions via webinars. Partnering with STEM projects at all education levels from middle school and up are planned. Requests from nonprofits for pilot testing are under evaluation.The ideal situation for a farmer or farm manager is to be able to self-analyze a field at low cost and to immediately obtain information on nutrient requirements to optimize crop productivity. The design requirements of the desired analytical system are as follows:The analysis must be performable by a non-trained operator. It must be error proof and rely on simple instructions for operation;The analysis must be cheap: low cost sensor and meter, automatic data transmission and automatic fertilizer input recommendation; andThe analysis must be fast: no transport of sample (in-field), fast (or no) sample preparation, no need for calibration, fast response time and data analysis.Electrochemical biosensors have great potential in meeting these criteria owing to their low cost construction and extreme selectivity.

Progress 08/01/18 to 03/31/19

Outputs
Target Audience:This is a Phase I proof of concept project to develop new technololgy that enables onsite and accurate nitrient testing in plants, soils and water. NECi personnel made presentations on the project at four conferences over the course of the project. Dr WH Campbell presented at a Biosensor conference. ER Campbell made presentations at NEMC 2018, an EPA/Lab association conference, at SCIEX 2018, and at PittCon 2019. Audiences centered on analytical chemistry, citizen science, and regulated analytical labs. Market target includes agriculture. NECi's strategy is to solidify credibility from the scientific community prior to market entry for new products. Changes/Problems:This project went well. Financial changes were minor. We chose not to use payroll resources to compensate Dr Campbell, NECi co-owner. The Travel nudget was exceeded as more abstracts to conferences were accepted. NECi's poster presentation at the Pittsburgh Conference (PittCon 2019) was chosen for the new Flash Poster session. Conference organizers chose 20 posters (out of hundreds) for 5 minute presentations on the conference Sunday. ER Campbell made this presentation. What opportunities for training and professional development has the project provided?NECi hired three undegraduate student interns from Michigan Technological University to participate on this project. All three were female STWM students on biochemistry/chemistry related fields. They were trained in enzyme assays including stability studies. Two students worked on the oxygen removal system, which is intergral to the sensor technology. How have the results been disseminated to communities of interest?This grant was cited on three accepted journal publications and presented at four scientific conferences over the term of the project. Presentations included: Campbell, W. H. (2019) Rediscovering Phosphate-Phosphorus with Molecular Sensors", Special Issue Celebrating 350th Anniversary of Discovery of Phosphorus of the Journal of Environmental Quality, submitted and accepted after revision. Campbell, W. H., & E. R. Campbell, Kopiec, G., N. Plumeré (2018) Nitrate and Phosphate Enzyme Sensors for Electrochemical Detection in Environmental Water and Ag Crops, poster presented at International Congress on Biosensors, Miami, FL. Campbell, E. R. & W. H. Campbell (2019) Bioelectrochemistry for On-Site Detection of Nitrate and Phosphate", poster and talk will be presented at Pittcon 2019, Philadelphia PA. Kopiec, G., K. Starzec; J. Kochana; T. Kinnunen-Skidmore; W. Schuhmann; W. H. Campbell; A. Ruff; & N. Plumeré (2018) Bioelectrocatalytic and Electrochemical Cascade for Phosphate Sensing with up to 6 Electrons per Analyte Molecule, Biosensors and Bioelectronics 117: 501-7. Letter of support included in the Phase II proposal show high interest from the agricultural community in the products envisioned. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In Objective 1, feasibility of building a phosphate sense was shown by demonstration of the detecting and quantifying phosphate by electrochemical analysis using Standard Phosphate of known concentration and phosphate content of a forage sample which was equivalent to that found using NECi Phosphate Test Kit (Kopiec et al., 2018). We also designed the electrode system for the NECi Phosphate Sensor. Direct detection of phosphate by electrochemistry is not possible since it is electrochemically silent. Therefore, we used NECi recombinant PNP and natural nucleoside Inosine as substrate. PNP catalyzes reaction of Inosine with Inorganic Phosphate to yield Hypoxanthine and Ribose-1-Phosphate (R-1P), in equal amounts with the R-1-P in proportion to the phosphate in the sample. Objective 2 was accomplished by design and manufacture of NECi Prototype Nitrate Meter with app running in the smart phone. Essentially, the current prototype Meter is capable of running the Phosphate Sensor once the software app is updated to include both analyte analysis. Smartphone is connected to prototype NECi Nitrate Meter wired via USB port of phone. Smartphone runs NECi Nitrate Biosensor app, which provides controlled direct current for coulometric analysis of nitrate and collects data from the analysis. Raw data is automatically baseline corrected and area under curve is integrated to calculate charge Q. Based on Standard calibration for Nitrate content of the sample, result of the nitrate analysis is displayed on smartphone and stored.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: NEMC 2018, New Products Demonstration session presentation by ER Campbell
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: SCIEX/FACSS conference 2018, presentation on handheld instruments suited for Citizen and Open Source Science
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Campbell, W. H., & E. R. Campbell, Kopiec, G., N. Plumer� (2018) Nitrate and Phosphate Enzyme Sensors for Electrochemical Detection in Environmental Water and Ag Crops, poster presented at International Congress on Biosensors, Miami, FL.
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Campbell, W. H. (2019) Rediscovering Phosphate-Phosphorus with Molecular Sensors, Special Issue Celebrating 350th Anniversary of Discovery of Phosphorus of the Journal of Environmental Quality, submitted and accepted after revision.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Campbell, E. R. & W. H. Campbell (2019) Bioelectrochemistry for On-Site Detection of Nitrate and Phosphate, poster and talk presented at Pittcon 2019, Philadelphia PA.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kopiec, G., K. Starzec; J. Kochana; T. Kinnunen-Skidmore; W. Schuhmann; W. H. Campbell; A. Ruff; & N. Plumer� (2018) Bioelectrocatalytic and Electrochemical Cascade for Phosphate Sensing with up to 6 Electrons per Analyte Molecule, Biosensors and Bioelectronics 117: 501-7.