Construction and Characterization of NitrateReductase-Based Amperometric Electrode andNitrate Assay of Fertilizers and Drinking Water

Scott A. Glazier*

Biomolecular Materials Group, Biotechnology Division, National Institute of Standards and Technology,Gaithersburg, Maryland 20899

Ellen R. Campbell and Wilbur H. Campbell

The Nitrate Elimination Company, Inc., 334 Hecla Street, Lake Linden, Michigan 49945

The construction and characterization of a nitrate reduc-tase-based amperometric electrode for determination ofnitrate ion is described. The electrode consisted of nitratereductase held by dialysis membrane onto a Nafion-coatedglassy carbon electrode. Methyl viologen was allowed toabsorb into the Nafion layer, which acted as a reservoirfor the electron mediator. The utility of the electrode toassay fertilizer and water sample for nitrate was demon-strated. The assays conducted with this electrode com-pared well with colorimetric and potentiometric assays ofthe same samples.

Nitrate ion is an important analyte in such diverse materialsas fertilizers, foods, livestock feeds, wastewater, and drinkingwater. The methods for determination of nitrate can be dividedinto three main categories: direct, through a reduced nitrogenspecies, and indirect.1 Table 1 summarizes a variety of techniqueswhich fall into these categories.

Nitrate is a widespread contaminant of groundwater andsurface waters worldwide2,3 and is a potential human health threat,especially to infants, causing the condition known as methemo-globinemia, also called “blue baby syndrome”. Chronic consump-tion of high levels of nitrate may also cause other health problems,including some cancers and teratogenic effects. The data areinconclusive but are cause for concern.4,5 Nitrate concentrationis monitored in municipal water supplies worldwide and infoodstuffs to prevent exposure of populations to harmful or toxiclevels. The EPA maximum contaminant level is 10 ppm nitrate-nitrogen (7 × 10-4 mol/L nitrate) for potable water.

We are interested in the development of an amperometricbiosensor for nitrate determination via the selective reduction ofnitrate to nitrite by nitrate reductase enzyme.

Transduction of this reaction involves the monitoring ofreduction current of an electron mediator, methyl viologen, whichshuttles electrons between the enzyme and an electrode.

There have been a few reports of nitrate biosensors in theliterature. A patent on enzyme electrodes demonstrated theresponse of a electrode coated with a viologen-containing polymerto nitrate in the presence of nitrate reductase free in solution.6

Nitrate reduction was achieved with a nitrate reductase-basedelectrode which utilized a polythiophene bipyridinium film as theelectron mediator. The enzymatic reaction could be observed inthis system only through prolonged electrolysis, which resultedin an accumulation of nitrite in solution. Catalytic reductioncurrents could not be distinguished from background, presumablydue to low enzymatic activity at the electrode surface.7 A similarstudy by the same group reported the ability of a bipyridiniumthiol bound to a gold electrode to act as an electron mediator for

(1) Sah, R. N. Commun. Soil Sci. Plant Anal. 1994, 25, 2841.(2) Hallberg, G. R. In Nitrogen Management and Groundwater Protection; Follet,

R. F., Ed.; Elsevier: Amsterdam, 1993; pp 35-74.(3) Puckett, L. J. Environ. Sci. Technol. 1995, 29, 408A.(4) Kross, B. C.; Hallberg, G. R.; Bruner, D. R.; Cherryholmes, K.; Johnson, J.

K. Am. J. Public Health 1993, 83, 270.(5) Bruning-Fann, C. S.; Kaneene, J. B. Vet. Human Toxicol. 1993, 35, 521.

(6) Gregg, B. A.; Heller, A. Patent WO 92/12254, 1992.(7) Willner, I.; Katz, E.; Lapidot, N.; Bauerle, P. Biochem. Bioenerget. 1992,

29, 29.

Table 1. Methods for Nitrate Determination

directnitration of phenols and colorimetryoxidation of organics and colorimetryion-selective electrode detectiondirect UV-absorbance spectrophotometrygas chromatography after derivatizationelectrophoreticnitration of salicylic acidion chromatography

reduced nitrogen speciesreduction to nitrite and

spectrophotometryelectrochemistry

reduction to ammonia andcolorimetrypotentiometryconductimetry

reduction to nitric oxide andchemiluminescence

indirectatomic absorption spectrophotometypolarography

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nitrate reductase.8 A large number of dyes and other redoxspecies were examined for their ability to act as mediators fornitrate reductase in amperometric electrodes from two differentbacterial sources.9 An excellent paper described an amperometricnitrate electrode in which nitrate reductase from Escherichia coliwas immobilized onto the electrode during electropolymerizationof a bipyridinium pyrrole monomer.10 The electrode exhibitedgood response times to changes in nitrate concentration andprovided low detection limits. Through the use of a novel light-activated quinone mediator, the reduction of nitrate by nitratereductase could be switched on and off at a thiol-covered goldelectrode.11 Finally, an optical biosensor for nitrate was con-structed using sol-gel-immobilized nitrate reductase.12 Thesensor responded to nitrate in the micromole-per-liter range.Unfortunately, no assays of nitrate in samples were conductedwith any of these biosensors.

We present here a first-generation amperometric electrode fornitrate based on a soluble nitrate reductase from corn seedlings.In addition to characterizing the electrode, it was used todetermine nitrate in fertilizer and drinking water samples. To ourknowledge, this is the first demonstration of a nitrate reductase-based electrode being used in the assay of nitrate in real matrixes.

EXPERIMENTAL SECTIONNote: Reference to commercial supplies and apparatus is for

completeness and does not constitute endorsement by the NIST.Chemicals and Apparatus. Nafion solution (mass fraction

of 5%), sodium nitrate, and sodium nitrite were purchased fromAldrich Chemical Co. (Milwaukee, WI). 3-(N-Morphlino)propane-sulfonic acid (MOPS), sodium chlorate and methyl viologen camefrom Sigma Chemical Co. (St. Louis, MO). Caution: Methylviologen is toxic, and direct contact should be avoided. The useof appropriate protective equipment is strongly advised. All otherchemicals were reagent grade. Deionized water was used toprepare all solutions.

NADH:nitrate reductase (EC 1.6.6.1; 10 units/mL) was purifiedfrom corn seedlings using monoclonal antibody-based immunoaf-finity chromatography, as described previously.13 The fertilizerswere water soluble and obtained locally.

An Orion nitrate ion-selective electrode (Orion, Beverly, MA)was used to assay fertilizers. A CV-1B potentiostat (BioanalyticalSystems, West Lafayette, IN) performed all other electrochemicalmeasurements.

Nitrate Reductase-Based Electrodes. The enzyme elec-trodes were constructed from glassy carbon electrodes (3-mmdiameter, Bioanalytical Systems). The electrodes were firstpolished with 1-µm diamond polish on nylon mesh (BioanalyticalSystems), followed by 15-min sonications in water and thenethanol. Next, they were coated with 1.0 × 10-2 mL of Nafion (5g/L in ethanol) and allowed to dry. Finally, 1.0 × 10-2 mL ofnitrate reductase (1 × 10-1 units) was secured behind the dialysis

membrane (Spectra/Por 2; 12 000-14 000 MWCO; Spectrum,Houston, TX).

Measurement Conditions with Nitrate Reductase-BasedElectrodes. All electrode work was done in a background buffercomposed of 5 × 10-2 mol/L MOPS, 1 × 10-3 mol/L EDTA, 5 ×10-1 mol/L potassium chloride, pH 7.4. Samples and standardswere contained in septum-stoppered bottles and delivered to theelectrochemical cell in gastight syringes. The electrochemicalcell was composed of the enzyme electrode, a platinum wireauxiliary electrode, and a Ag/AgCl reference electrode (Bioana-lytical Systems). Solutions in the electrochemical cell werethoroughly purged with nitrogen before a working potential wasapplied and stirred by nitrogen bubbling during all measurements.

Measurement of Responses to Nitrate and Interferences.Before these electrodes could be used, they were always allowedto soak in 1 × 10-3 mol/L methyl viologen (in working buffer)for at least 5 min. This allowed the cation-exchange sites in theNafion to load with viologen and act as mediator reservoirs. Then,the electrodes were placed into 10-mL volumes of buffer in theelectrochemical cell. After a working potential of -0.8 V vs Ag/AgCl was applied, the background current decayed, and ionstandards were added. The current changes from backgroundwere noted. The electrodes were allowed to soak in the viologensolution before each subsequent calibration. The electrode, whoseresponse to nitrate was monitored during a 7-day period, wasstored in working buffer at 4 °C when not in use.

Standard Addition Assay of Fertilizer Samples by NitrateReductase-Based Electrodes. Stock solutions of the fertilizersamples were prepared in buffer. The electrode was set up inthe electrochemical cell in the same manner as was mentionedfor nitrate calibration. The background current was allowed todecay, and then a small volume of fertilizer sample was added,followed by additions of nitrate standard. The samples wereeffectively diluted by a factor of 20 000 during the analyses. Thetotal changes in nitrate concentration in the cell were intentionallykept small, so that the electrode response was linear. Duringstandard additions, the total cell volume changed less than 0.03%.Once the slope of electrode response had been calculated betweenthe sample and standard additions, it was a simple matter toextrapolate back to background to obtain the nitrate concentrationin the samples. Then, a mass fraction of nitrate-nitrogen wascalculated for the original granular sample, as is customary forfertilizers.

Analyte Addition Assay of Drinking Water Samples byNitrate Reductase-Based Electrodes. In these assays, theenzyme electrode was set up for nitrate calibration as before.Buffered drinking water samples were prepared using the samesalt composition as the working buffer. Then, 1.42 × 10-4 mol/Lspikes of nitrate (in the form of sodium nitrate) were added tothe buffered samples to serve as internal standards. Severaladditions of nitrate standard followed by an addition of buffereddrinking water were made to the cell. The drinking water sampleswere effectively diluted by a factor of 111 during the analyses.Again, total changes in nitrate concentration in the cell wereintentionally kept small, so that the electrode response was linear.From two of the nitrate additions, the slope of the electroderesponse was calculated, and the nitrate concentration in thesample was obtained by extrapolation.

(8) Katz, E.; Itzhak, N.; Willner, I. J. Electroanal. Chem. 1992, 336, 357.(9) Strehlitz, B.; Grundig, B.; Vorlop, K. D.; Bartholmes, P.; Kotte, H.;

Stottmeister, U. Fresenius J. Anal. Chem. 1994, 349, 676.(10) Cosnier, S.; Innocent, C.; Jouanneau, Y. Anal. Chem. 1994, 66, 3198.(11) Doron, A.; Portnoy, M.; Lion-Dagan, M.; Katz, E.; Willner, I. J. Am. Chem.

Soc. 1996, 118, 8937.(12) Aylott, J. W.; Richardson, D. J.; Russell, D. A. Analyst 1997, 122, 77.(13) Hyde, G. E.; Wilberding, J. A.; Meyer, A. L.; Campbell, E. R.; Campbell, W.

R. Plant Mol. Biol. 1989, 13, 233.

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Colorimetric Assay of Nitrate in Fertilizer and DrinkingWater Samples. This enzyme-based assay is a variation of EPAstandard method 40 CFR 141 for determination of nitrate indrinking water and wastewater.14 The fertilizer samples wereassayed by construction of a calibration curve of absorbanceversus nitrate concentration. For the drinking water samples, astandard addition version of this assay was used. All the drinkingwater samples were buffered with the MOPS buffer. Here, thesamples were spiked with several levels of nitrate and reacted asbefore to give diazo product. With these data and those of theblank (no sample, buffer only), the concentrations of nitrate inthe samples could be obtained by extrapolation.

Assay of Fertilizer Samples by Nitrate Ion-Selective Elec-trode. The electrode was calibrated as per the manufacturer’sinstructions. Nitrate concentrations in the samples were deter-mined by comparison with a calibration curve, and, from these,the weight percentage of nitrate-nitrogen was calculated.

RESULTS AND DISCUSSIONMediator. In a recent report,15 several electron mediators

were tested for their ability to shuttle electrons from an electrodeto the corn seedling nitrate reductase and support its activity.Among those tested were azure A, safranin T, neutral red,bromophenol blue, Cibacron blue, and methyl viologen. Throughpreliminary experiments, it was determined that methyl viologenwas an excellent choice for the nitrate reductase-based electrodeand -0.8 V vs Ag/AgCl a reasonable working potential.

The electrostatic attraction between the 2+ charge on methylviologen (1+ when reduced) and the ion-exchanger (Nafion),combined with hydrophobic interactions between the viologen andion-exchanger carbon skeleton, ensure that methyl viologen willbe immobilized at some percentage of saturation in the presenceof inorganic cations. This method of using Nafion as a mediatorreservoir has also served well for glucose16 and urate17 enzymeelectrodes.

Calibration and Lifetime of the Nitrate Reductase-BasedElectrode. Figure 1 shows the response of this electrode tonitrate on the first, second, and seventh days of electrode life.When the working potential was applied, a steady baseline currentwas reached within 3-4 min. The response time to nitrateadditions was less than 1 min. The upper limit of detection ofnitrate was approximately 1.2 × 10-2 mol/L, while the lower limit,as determined during the course of drinking water assays to bediscussed later, was consistently less than 3.0 × 10-6 mol/L (n )8; S/N ) 5.1). The linear range of electrode response, asobserved in the drinking water assays, extended from 3.0 togreater than 17.9 µmol/L of nitrate. A recent review1 on nitratedetermination listed limits of nitrate detection for a large numberof methods; the range was 0.003-13 µmol/L. Hence, the presentsystem compares well with others.

Note in Figure 1 that the magnitude of current responsedecreased as the electrode aged. As with all enzyme biosensors,

decay of enzyme activity with time is likely the cause. This effectwas even seen among different calibration curves obtained on thesame day. Experience with the enzyme preparation indicates thattotal inactivation occurs in 8 h at 1 unit/mL of enzyme free insolution at room temperature. At 10 units/mL (the nominalconcentration present under the dialysis membrane of theelectrode), inactivation is complete within 2 days at room tem-perature. The width of the error bars is an indication that themagnitudes of response are not that constant through the courseof a day. The reason for larger error bars on the second day, asopposed to the first, is not known at this time.

However, if the calibration curves are normalized to the highestcurrent, it is evident that the electrode response maintains thesame functional form during its lifetime. Figure 2 illustrates thispoint clearly. Here, all of the calibrations shown in Figure 1 werenormalized and then averaged. The uncertainty bars here arequite small in width. This effect of response “drift” during a givenday can likely be minimized by redesign of the electrode, withemphasis placed on enhanced enzyme stability, i.e., throughcovalent immobilization. Yet, the electrode can be used to performpractical nitrate assays by employing techniques which compen-sate for drifts in response from one sample to the next. This point

(14) Campbell, E. R.; Corrigan, J. S.; Campbell, W. H. In Symposium on FieldAnalytical Methods for Hazardous Wastes and Toxic Chemicals; Air WasteManagement Association Meeting, Las Vegas, NV, January 29-31, 1997;AWMA:

(15) Mellor, R. B.; Ronnenberg, J.; Campbell, W. H.; Diekmann, S. Nature 1992,355, 717

(16) Brown, R. S.; Luong, J. H. T. Anal. Chim. Acta 1995, 310, 419.(17) Jin, L.; Ye, J.; Tong, W.; Fang, Y. Mikrochim. Acta 1993, 112, 71.

Figure 1. Calibration of glassy carbon electrode: (9) day 1 (n )4), (0) day 2 (n ) 4), and (O) day 7 (n ) 2). Mean ( SE. Workingelectrode potential, -0.8 V vs Ag/AgCl.

Figure 2. Responses of glassy carbon electrode normalized to theresponse at the highest nitrate concentration. Mean of all normalizedcalibrations from day 1, day 2, and day 7 (n ) 10 total). Mean ( SE.

Analytical Chemistry, Vol. 70, No. 8, April 15, 1998 1513

will be illustrated later by the standard addition analysis offertilizers and the analyte addition analysis of drinking water.

Interferences. At a working potential of -0.8 V vs Ag/AgCl,the reduction of oxygen at the electrode surface would cause largebackground currents. The magnitude of this interference waseffectively decreased by purging samples and standard thoroughlywith nitrogen in septum-stopped vials and transferring them tothe electrochemical cell via gastight syringes. In addition, thebuffer in the cell was thoroughly purged with nitrogen beforepotential was applied and stirred by nitrogen bubbling during themeasurements.

Trace metals ions would have also caused an interference.Metals ions are notorious for their ability to denature enzymesand were controlled by the addition of EDTA. Chelation alsoprevented the metal ions from being concentrated on the cation-exchange sites of Nafion and successively reduced due to thenegative working potential.

In addition to oxygen and metal ions, interferences from othersolution species are possible. The responses of a newly madeelectrode to chlorate and nitrite are shown in Figure 3. The nitriteresponse is much smaller than that to nitrate. Hence, the enzymepreparation is apparently low in nitrite reductase activity, whichis quite often associated with nitrate reductase in living systems.The electrode response to chlorate was expected on the basis ofprevious knowledge of the enzyme’s selectivity.18 The halideanalogues of nitrate, chlorate, bromate, and iodate are all sub-strates for nitrate reductase. Higher oxidation state analogues,like perchlorate, are not reduced. The fact that chlorate is asubstrate could be significant in the drinking water analysis sinceit could be present as a result of chlorination.

In terms of inhibitors, chloride can cause some inhibition ofnitrate reductase. However, the electrode had a lower detectionlimit for nitrate, of less than 3.0 × 10-6 mol/L. Hence, theelectrode responded to nitrate when present at less than 6 × 10-6

times the chloride concentration. Similarly, in a report on anoptical approach to nitrate biosensing using a similar nitratereductase, no interference was found by phosphate, carbonate,hydrogen carbonate, or chloride at concentrations up to 0.5 mol/L.12

In contrast, the commercial nitrate ion-selective electrode usedhere has a selectivity pattern based on the well-known Hofmeisterseries. Hence, potential interferences for the ion-selective elec-trode, depending on their relative concentrations to nitrate, couldinclude perchlorate, iodide, chlorate, cyanide, bromide, nitrite,hydrosulfide, carbonate, chloride, bicarbonate, mono-, di-, andtribasic phosphates, acetate, fluoride, and sulfate.19 Interferenceby organic anions (e.g., benzoate) are likely as well.

Fertilizer Analysis. Table 2 outlines the results of themeasurement of nitrate in fertilizer samples. The nitrate reduc-tase-based electrode gave a mean percentage of nitrate-nitrogenwhich was between that found by the colorimetric and ion-selectiveelectrode techniques for sample A. However, the error of themean was larger than those for the other two techniques.Refinement of the nitrate reductase-based electrode and themeasurement method will likely increase the precision of theresults. For sample B, the enzyme electrode gave somewhat lowerassay than the ion-selective electrode but had better precision.The values given by both methods were consistent when theconfidence intervals were taken into account.

Drinking Water. Because of the importance of nitratemonitoring in groundwater and drinking water, a major goal ofthis work was to demonstrate that our first-generation electrodewas capable of measuring nitrate in such samples. Table 3illustrates the results of colorimetric and electrode assays fornitrate in two drinking water samples. Given the precision of bothtypes of assays, the nitrate values are quite comparable.

CONCLUSIONSA nitrate reductase-based electrode has been demonstrated

which employs nitrate reductase from corn seedlings and a Nafionreservoir to store the electron mediator, methyl viologen. Theelectrode exhibited a low detection limit for nitrate and rapidresponse to changing nitrate concentration. Most importantly, itdemonstrated the ability to assay real-world samples. Theelectrode does not require expensive/complex associated instru-mentation, unlike techniques such as ion chromatography, po-larography, fluorometry, direct spectrophotometry, or chemilu-

(18) Solomonson, J. P.; Vennesland, B. Plant Physiol. 1972, 50, 421. (19) The Fisher Catalog; Fisher Scientific Co.: Pittsburgh, PA, 1994; p 1359.

Figure 3. Responses of newly made glassy carbon electrode tohigh concentrations of (9) chlorate and (O) nitrite.

Table 2. Comparison of Assays for Nitrate in Fertilizersas Percent Nitrate-Nitrogena

electrodes

sample colorimetric enzyme ion-selective

A 6.7 ( 0.3 (n ) 3) 6.5 ( 1.3 (n ) 8) 6.0 ( 0.2 (n ) 3)B 15.7 ( 0.8 (n ) 4) 17.3 ( 1.0 (n ) 4)

a Mean ( SE.

Table 3. Comparison of Determinations ofConcentration of Nitrate in Drinking Watera

sample colorimetric (mol/L) enzyme electrode (mol/L)

A (1.1 ( 0.1) × 10-4 (n ) 3) (1.0 ( 0.1) × 10-4 (n ) 3)B (3.1 ( 0.3) × 10-4 (n ) 3) (3.3 ( 0.4) × 10-4 (n ) 4)

a Mean ( SE.

1514 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998

minescence, and could be adapted to field analysis. In addition,it has the inherent ability to operate in turbid samples, unlikecolorimetric methods. One significant improvement in electrodedesign could be the inclusion of components to reduce themagnitude of oxygen interference without the need for samplepurging.

ACKNOWLEDGMENTThis work was done under NIST CRADA CN-1105.

Received for review October 17, 1997. Accepted February15, 1998.

AC971146S

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