Enzymes are naturally occurring biological macromolecules that are made up of proteins. They are responsible for accelerating, or catalyzing, chemical reactions in many biological systems.
Enzymes convert substrates, which are molecules that the enzyme reacts with into different molecules, known as products.
Using enzymes to test for nitrate content can allow people to: maximize crop yields, protect water quality, protect livestock from nitrate toxicity, and also make sure that organizations stay in regulatory compliance with the mandated safe nitrate levels.
Enzymes only react with their specific substrate, which makes them excellent candidates for quantifying specific molecules in a sample, even in complex mixtures.
They do this by having a very specific "binding-site", where the substrate will fit, like a lock-and-key or two puzzle pieces would. When the substrate is released, it can react with other chemicals to quantify the amount of substrate in the sample.
Enzymes are present in nearly every species of animal, plant, and fungi. NECi started by extracting Nitrate Reductase from corn seedlings, then purifying it for commercial sale. This is known as a native enzyme, since it was extracted from its natural source.
Since the DNA sequence of nitrate reductase is known, a recombinant form was designed in order to harness desired properties and allow for more efficient production using a vector yeast, Pichia pastoris. Recombinant protein technology allows for modification of native DNA for optimized properties and the ability to produce the enzyme outside of its native source.
No! Enzymes are essential for almost every organism to perform routine functions. They speed up chemical reactions in all sorts of biological systems, and react under those gentle conditions. They're in our bodies and in the food that we eat, and are completely non-toxic to handle, ship, and dispose of, but please don't eat them!
Nitrate is a negatively charged ion (anion) composed of one nitrogen atom and three oxygen atoms. Its formula weight is 62 g/mol and is very soluble in water.
Nitrite is a negatively charged ion (anion) composed of one nitrogen atom and two oxygen atoms. Its formula weight is 46 g/mol and is very soluble in water.
Nitrate is a naturally occurring anion. It's essential for plant growth, amino acid production, and more. Although nitrate is found naturally, humans have played a huge role in altering nitrate concentrations in the environment.
Nitrate in the environment can occur from a number of sources, including agricultural fertilizer inputs, industrial processing waste, municipal waste, and more.
Concentrations in ambient (undisturbed) sources of water are generally at or below 2 ppm nitrate-N.
Nitrate measurement may be recorded in different units depending on region, and results may vary from laboratory to on-site analysis. Measurements can be reported in ppm (parts per million) of nitrate or nitrate-N. Most of the U.S. reports in nitrate-N, which only measures the amount of nitrogen in the nitrate molecule. California and Europe report in simply units of nitrate, meaning the entire molecule - so the reading will be higher than if it were in units of nitrate-N.
Most laboratories and the scientific community describe a quantity of nitrate in molarity (M, mM), or moles per liter (m/L) of solution. The conversion is as follows: 1 ppm nitrate-N = 4.4 ppm nitrate = 71 mM nitrate.
There are three main areas of concern when considering nitrate concentrations in potable/drinking water, food, and the ocean. The U.S. Environmental Protection Agency (EPA) sets the maximum contaminant level at 10 ppm nitrate-N for safe drinking water. This is the maximum level which is considered safe for human consumption. Livestock, animals, and other ruminants may handle a slightly higher level than this, but many sources will provide different information on what is considered safe depending on the species.
Because plants take up nitrate in order to grow and function, it's naturally present in many vegetables, especially leafy greens, stalk and root crops such as spinach, celery, beets and carrots. Nitrate is also used to prevent bacterial growth in cured meats. There is still controversy on how much nitrate is acceptable for human consumption in foods.
In the ocean, anything above 2 ppm nitrate-N begins to kill many coral species, and contributes to algal growth, which may cause toxic blooms that create dead zones in concentrated areas. Once nitrate is present in excess levels, it is very difficult to remove it from water since it's very soluble and there is currently no easy way to isolate and remove it from a such a vast area.
Nitrate consumption can affect certain groups of individuals differently depending on health, age, or chronic exposure to elevated nitrate levels. When humans (and most animals) take up nitrate in food or water, most of it is quickly eliminated from the body through urine. Nitrate is water soluble and does not accumulate in body fat. However, if there is a constant intake of nitrate, as might be in the case if your drinking water contains high nitrate levels, there will always be some nitrate present in the body, which is when health issues may occur. The US EPA sets the maximum limit for nitrate in drinking water at 10 ppm nitrate-N, which is regulated through the Safe Drinking Water Act. Although this is regulated in state laboratories and enforced in waste water treatment plants, the US EPA does not regulate private wells, and therefore it is the homeowners responsibility to test their wells for nitrate contamination. Nitrate must be ingested in order to have a negative impact on human health, it is not absorbed through the skin.
When nitrate is consumed, bodily bacteria converts it to nitrite, which is then absorbed and stored in cells until conversion to nitric oxide which relaxes blood vessels and increases blood flow. Nitrite can also bind to haemoglobin to form methaemoglobin, which negatively impacts the blood's ability to carry oxygen throughout the body. This is especially a concern for infants, and causes a condition known as methaemoglobinaemia or "blue-baby syndrome". The elderly population and individuals with gastrointestinal infections are also more susceptible to this condition.
A peer-reviewed study in Environmental Health Perspectives published April 2016 indicated that nitrate may also affect the thyroid. Nitrate is considered to be a sodium/iodide symporter (NIS) inhibitor which blocks iodide uptake to the thyroid, therefore inhibiting thyroid function. According to this study, thyroid dysfunction may adversely affect certain stages of child development.
Phosphate is a molecule in which one phosphorus atom is bound to four oxygen atoms.
It's the naturally occurring form of the element phosphorus, which is mined for agriculture and other industrial processes.
Phosphate is used in agriculture to help stimulate plant growth through the application of fertilizers. Phosphorus is a finite resource, and world phosphate rock reserves are believed to be in a steep decline. This is driving the price of phosphate based fertilizers up. It is important to monitor phosphate levels wherever it is being applied in order to prevent crop phosphate deficiency, reduce excess phosphate from running off into aquatic systems, and to conserve phosphate resources. Targeting optimal phosphate levels in the soil prevent crop phosphate deficiency, water pollution and wasted resources from excess runoff.
Phosphorus in excess contributes to harmful toxic algal blooms which can create dead zones in surface waters. Excess phosphate can enter environmental systems through industrial processing waste or from a variety of other contaminating processes. Our kits can detect concerning levels of phosphate in the environmental and industrial effluent.
The levels of phosphate measured in environmental systems depends on a number of factors including proximity to human influence,
Phosphate is a macro-nutrient that is vital to plant growth. It is a component of nucleic acids, so it plays an important role in plant reproduction.
For certain crops such as grains, it is essential to farmers that the plant has the ability to reproduce effectively.
Phosphorus (P) is an unstable element, meaning it has a tendency to react with other chemicals in nature. Although phosphorus is found in its elemental state in the form of phosphate rock, most of the phosphorus in nature exists in an oxidized form such as phosphate (PO4). When considering how phosphate interacts with plants and animals, there are two main types to be aware of: organic & inorganic phosphate. There are various forms of inorganic phosphate, and all of these types are also referred to as orthophosphate. Orthophosphate (inorganic phosphate) is the only form of phosphate that is available to plants. Organic phosphate is the form that exists in plant or animal tissue.
In order for organic phosphate to be converted to inorganic, so that plants can use it as a nutrient, it must first be decomposed by microorganisms. As if this weren't already enough to remember, the forms of phosphate that are able to move around in soil or water, are constantly changing between soluble and insoluble. This means that some phosphate is attached to particles such as soil, or sand, while some is dissolved in water where it can be taken up by plants, animals or microorganisms. So, when measuring phosphate, it is important to know if it is organic or inorganic, soluble or insoluble.
Some examples of traditional methods for measuring phosphate include ion chromatography, the ascorbic acid method, the stannous chloride method, and digestion methods. The traditional methods of measuring phosphate invlove expensive equipment, unstable/dangerous reagents, high temperatures, and are often inaccurate.
NECi's enzymatic phosphate determination method is accurate, fast, and does not involve any harmful reagents. This method is formatted for both on-site and laboratory use. View our phosphate analysis products to learn more!
Enzymes are catalysts that drive complex biological reactions. They happen to be excellent reagents for analytical chemistry because they are reliable, accurate, sensitive, selective, and non-toxic.
Enzyme reactions occur in gentle, biological conditions with no organic solvents, heavy metals, high heat or pressure involved.
They are sensitive, which means they have low detection limits in complex mixtures.
They will ONLY react with a specific substrate, using their highly specialized binding site that only fits THEIR substrate . Once their substrate is bound, catalysis changes the substrate into a new product. After this is complete, the enzyme releases the newly formed product and is ready to begin the process again!
Analyzing samples with enzymes will result in accurate and consistent data without the need for any hazardous materials or methods, unlike methods employing cadmium or zinc for nitrate analysis.
NECi Superior Enzymes has developed a variety of products to fit the needs of users with various backgrounds and experience. Each product has been formatted for its specific application so that anyone, from elementary students to laboratory scientists, can find a product that fits their application.
On-Site test kits are designed for taking quick measurements of nitrate or phosphate in soil, water, or plant samples where they are collected. They are simplified versions of the lab kits, providing accurate and reliable results without the need for laboratory equipment. However, we've designed an affordable portable, smartphone linked photometer for these kits to provide digital data in the field, increasing accuracy of the results.
Laboratory test kits are for users with knowledge of basic laboratory procedures and access to laboratory equipment (test tubes, spectrophotometer, pipettes, etc.)
Enzyme reagent packs are designed for experienced users that prefer to supply their own buffers and color reagents (if necessary), and are available with an optimized amount of co-factor included or as stand-alone enzyme.
All reagents have been designed and developed to be used with spectrophotometers, automated liquid handling equipment, discrete analyzers, etc.
Most importantly, all test kits were designed for ease of use and reliability, to get the best results with the safest and most environmentally friendly reagents in any commercial nitrate test kit available today!
NECi's nitrate test kits use an enzyme called nitrate reductase to convert the nitrate in a sample to nitrite. Once the Griess color reagents are added, nitrite becomes visible as a pink solution. This visible pink color is analyzed by comparison to a color chart, or by measuring absorbance at 540 nm in a spectrophotometer, or NECi's handheld dual wavelength photometer. The darker the shade of pink is, the more nitrate is in the sample.
NECi's enzyme based phosphate test kits work in a similar manner: the enzyme reacts with all of the phosphate in a sample to convert it to a new molecule, however there are no color reagents added, and therefore no color change occurs. Instead, absorbance is measured in the UV range at 340 nm using a spectrophotometer or NECi's handheld dual wavelength photometer, detecting the amount of the newly formed product, which correlates to the amount of phosphate in a sample.
We recommend that laboratory users begin by purchasing a complete test kit for convenience of pre-made buffers and detailed instructions. This limits error and helps with troubleshooting. Once users are familiar with the assay, economical reagent packs are available which includes only the enzyme, co-factor, and enzyme reconstitution buffer.
All on-site kits are simplified, pre-calculated versions of the laboratory methods and are the best choice for fast and accurate analysis anywhere on-site. These kits require no laboratory equipment or chemistry experience and can be used to analyze water, plant, soil, forage, and aqueous process effluent samples.
For high throughput automated laboratory analysis, we have developed reagent packs complete with instructions customized to the users equipment. These reagent packs include enzyme, co-factor, and enzyme reconstitution buffer, complete with instructions for instruments such as discrete analyzers, flow through analyzers, and more. Contact NECi today to develop a custom kit for your automated laboratory instrument.
NECi offers on-site phosphate kits formatted for Water and Soil Samples, with the testing range of between 0 and 20 ppm phosphate for standard-range kits. There is also a low-range water option good between 0.5 and 5 ppm.
For the test tube formatted phosphate lab kits customers can choose from a low-range option measuring from 0.5 to 5 ppm phosphate and a standard-range formatted kit good from 0-20 ppm phosphate.
If you need a different testing range, you can contact NECi and, if possible, a kit can be formatted to meet that need.
Laboratory test kits are supplied with an optimized amount of enzyme, co-factor, a standard, and all other necessary reagents. Some lab kits may come with additional materials, depending on the kit.
Lab kit users should have lab quality water and 3N HCl available.
NECi's nitrate test kits use an enzyme (NaR) and natural reducing agent (NADH) for highly accurate and easy to use nitrate testing. The enzyme used in our nitrate test kits and reagents is a purified form of nitrate reductase (NaR). Other test kits use the toxic metals cadmium or zinc. NECi’s NaR is more stable and unlike other forms of NaR it does not require expensive NADPH as its cofactor. Instead, NADH is used.
The NaR reaction is diagrammed like this: NADH + NITRATE → NITRITE + NAD+ + OH-
NADH (nicotinamide adenine dinucleotide) is a derivative of a B vitamin. The resulting nitrite reacts with Color Reagents (the Griess Reaction) to form a pink color easily detected by eye or by a photometer at 540nm ± 10nm. In most kits, you’ll end by comparing the color to that of the nitrate standards (supplied with each kit) to determine the nitrate content of your samples.
Enzymes can be trusted for clinical diagnostic tests because they're accurate, reliable, sensitive, selective, and generally environmentally benign and non-toxic.
Enzymes are ideal for nitrate detection in particular because most other commercial nitrate analysis methods use a heavy metal, such as cadmium or zinc, for reducing nitrate to nitrite. These metals, especially cadmium, are toxic and so present a health risk for both the person doing the analysis and for the environment. Cadmium disposal is also difficult.
NECi NTK systems use natural NaR and NADH for nitrate analysis which are both user and environmentally safe. Since NECi NTK systems are as accurate, if not more so, than other commercial systems, NaR/NADH based systems are a better choice.
The user can choose to analyze nitrate in the Standard Range of 0.5 to 10 ppm nitrate-N or the Low Range of 0.01 to 1.0 ppm nitrate-N.
Since NECi NTK systems all use a photometric method for evaluating the nitrate content of your samples, results are highly accurate.
Included in seawater kits for Low Range nitrate (less than 1 ppm nitrate-N, or 7 micromolar) are nitrate standards in seawater and the AtNaR form of nitrate reductase, which is not sensitive to Cl.
For lab and micro plate kits the standard curve used for quantifying your results must be made using standards that contain salt water. Inform us that you are testing salt water when you order and we will be happy to provide a saltwater solution for sample preparation!
If you bought the Standard Range kit, no changes are required.
The few necessary changes required for the instructions in the test tube format kits are included on the standard instruction sheets, in the blue font.
YNaR is a recombinant form of our NaR produced in yeast (short for Yeast Nitrate Reductase). AtNaR is also a recombinant form of nitrate reductase. While YNaR is a yeast form, AtNaR is derived from Arabidopsis, a small plant in the Brassica family.
YNaR is moderately inhibited by Cl-, but is still highly effective for EPA or standard range nitrate levels as well as seawater testing.
AtNaR is not inhibited by the complex organic acids that can be found in plants (humics, tannins), and functions efficiently at higher temperatures (peak activity at 35°C). AtNaR also works well for soil extracts in the presence of 2M KCl
Because YNaR is moderately inhibited by Cl- we recommend AtNaR for all plant extracts, high salt samples, and very low nitrate samples (below 1 ppm nitrate-N).
For onsite analysis NECI offers a range of complete, packaged kits that require NO additional materials. With the exception of the Dry Forage Kits, kits come with the requisite amounts of enzyme, a standard, and ALL other necessary reagents.
For Dry Forage Kits users need only supply a kitchen blender and distilled water.
Users can achieve fast and accurate results from water and other aqueous samples (such as extracts of food, plants, and soil). Non-aqueous solvents interfere with the enzyme catalyzed reaction and must NOT be used.
Saltwater samples CAN be analyzed with many NTK and PTK systems and this option should be specified when ordering so kits can be adjusted.
Physiological samples such as blood and plasma CAN analyzed because generally their salt and protein content presents no significant interference.
Turbid or "colored" samples can be analyzed in standard range systems but samples must be filtered and or centrifuged for low range results.
No, they cannot be used interchangeably because while both kits measure nitrate content in animal feed and forage (from a range of 0 ppm – 9000 ppm nitrate (0 ppm – 2000 ppm Nitrate-N)) they each require a different method of sample preparation.
The Green Forage Nitrate Test Kit is designed to analyze samples when sap or a wet extract is available by squeezing with a garlic press or pair of pliers.
If samples are too dry to squeeze sap from, the Dry Forage Nitrate Test Kit is recommended, where the sample will be blended using a grinder then added to water to make a sample extract.
Each kit contains specific materials that are required for the sample preparation step. After sample preparation, however, the chemistry and kits are formulated the same.
NO additional materials are required for the majority of onsite testing kits.
Users will need to supply a kitchen blender and distilled water for Dry Forage Kits.
Most components should be stored cold and dark: see below for outlines for each of the components’ cold-storage requirements.
With regards to storage equipment, it is important to note that NADH and Enzyme (AtNaR, YNaR) are sensitive to oxidizing agents and thus clear glass, PP (polypropylene), or PE (polyethylene) tubes and vials should be used.
The enzyme packs contain the enzyme and NADH, freeze-dried in cuvettes and containers, which are then vacuum-sealed in foil pouches.
There will be a 6-month expiration date on all kits, but packs consistently are stable for more than 6 months stored at any temperature below 22C; stability study samples have been effective even after multiple years. The enzyme can remain stable for years if stored in these packets at -20C.
Because NADH oxidizes to NAD+ over time it will eventually be unable to supply the electrons needed for the nitrate reduction step.
Use these precautions for NADH: store portioned into small aliquots - the volume you would use in a day's work. Store the aliquots in a fridge (at least one week) or frozen (one month). You may see longer activity retention - a month or more - but one or two weeks is safe.
The assay buffer, when stored cold, is stable for one month.
Both Color Reagent 1 and Color Reagent 2 are stable enough for use after being stored for up to one month cold, at or below 4C, and dark.
Color reagent 2 (NED reagent) will start to turn brownish after some weeks after the addition of water. This will cause a loss in sensitivity as the background in blanks becomes too high.
There will be a 6-month expiration date on all kits. Packs consistently are stable for more than 6 months stored at any temperature below 22C; stability study samples have been effective even after multiple years.
The enzyme can be stable for years if stored in these packets at -20C.
Once the kit process has been started you should aim to complete it in a timely fashion, being sure to FOLLOW STATED TIMES carefully. The most common errors are either stopping the reduction reaction too soon, or not proceeding to the next step in time.
DO NOT let the solutions you prepare sit for extended periods of time. Once started, the kit process should be completed that day.
If you had reason to expect nitrate in this sample, the most likely cause is you left out one of the reagents. Each tube must get: buffer, NADH, and NaR (enzyme) and then the two color reagents.
Exclusion of buffer or color reagents will result in that sample having a significantly smaller volume, while if NADH or NaR are left out the volume will look the same.
SOLUTION: Repeat the analysis of the sample or standard being sure to get every component into the tube.
If this problem of light coloration is occurring, you might have stopped the reaction too soon by preemptively adding the Color Reagent.
SOLUTION: Allow enough time for the reactions to occur and carefully follow the time-frames outlined in the instructions.
For Simple NTKs: Be sure to wait AT LEAST 10 MINUTES AFTER adding samples to the reaction/standard cuvettes BEFORE adding the Color Reagent. If necessary, adding more time DURING the reaction step, BEFORE the color reagent is added will not negatively affect results.
Are you sure there IS nitrate in the samples? If you confirmed the presence of nitrate with another system, there may be an interfering factor(s).
SOLUTION: Try to make a standard by diluting it with the sample and then testing for nitrate in comparison to a standard prepared with nitrate-free deionized water. If you get no color development in the standard prepared with your sample but did get color in the normal standard, then you have interference with the enzymatic reaction catalyzed by NaR.
The sample has too much nitrate and must be diluted and reanalyzed.
SOLUTION: Dilute the sample with nitrate-free deionized water 1:4 (five-fold dilution) and 1:9 (ten-fold dilution) and analyze the nitrate again. If the sample is still off the standard curve (ie. higher in absorbance than the highest standard), then you will have to dilute it more, even up to 50- or 100-fold.
Most probably, there is nitrate or nitrite in your deionized water.
SOLUTION: obtain new deionized water.
This could most likely be attributed to pipetting error(s), either in preparing the standard or depositing it to the tubes. Accurate and careful pipetting is essential for getting good results with ALL of NECi Superior Enzyme Products.
SOLUTION: Repeat the standard curve using the same diluted standards. If still getting a non-linear standard curve, then prepare new diluted standards and repeat the analysis.
Simply leave the NADH and NaR out when you prepare the sample and replace them with deionized water. Then do the color development.
Following the stated times is VERY IMPORTANT. Be especially sure to give the enzyme reaction time to fully complete: not allowing the reaction time to finish is the most common user mistake.
It is best to keep components cold and sealed until use.
DO NOT open or reconstitute ANY component until ready to use your kit.
As supplied in freeze-dried form, PNP is stable for months when stored at -20 C or colder.
Once reconstituted, PNP should be used that day.
As supplied in freeze-dried form, MESG is stable for months when stored at -20 C or colder.
Once reconstituted, MESG should be used that day.
The buffer can be stable for months when stored at 4 C.
Do not open until ready to use.
The phosphate test kits reaction remains clear, with out a visible color change.
In order to analyze samples, it's necessary to use our handheld photometer or any spectrophotometer which accepts standard square cuvettes capable of reading at 360 nm.
At this time, the only sample types that have demonstrated notable interference would be samples containing high levels of Arsenic.
You must blank the NECi Photometer each time you turn on the device, or switch between readings of nitrate or phosphate.
Ensure that the photometer is properly paired with your android device before you begin using the NECi Photometer app.
Navigate to your Android device’s Bluetooth settings, and make sure that your photometer is paired. If it is paired and you are still seeing connection failures, unpair your photometer and re-pair it within the Bluetooth settings.
First, make sure that your photometer is paired to your Android device. If it is, exit the “Selected Photometer” list, and select it again, your device should now show up.
The NECi Photometer and NECi Easy Test Kits have a combined margin of error of less than or equal to 10%.
Ensure that the battery is sufficiently charged. A full charge is indicated by the top LED on the front of the photometer. When plugged into a charger, the LED will appear green when fully charged, and red when not fully charged.
If the battery is sufficiently charged and the problems are still occurring, contact NECi for customer service.
We will do our best to resolve your issue as quickly as possible. We are usually available from 10am-5pm EST M-F.
Enzyme Based Analytical Chemistry
Test kits for laboratories, agriculture, homeowners, and more
334 Hecla Street, Lake Linden, MI 49945
Phone: (906) 296-1115
Fax: (906) 296-8003
Tech Support: email@example.com