WO2005119237A1

WO2005119237A1 - Biosensor for detecting nitrate ion and measuring method using the same

Info

Publication number: WO2005119237A1
Application number: PCT/KR2005/001529
Authority: WO
Inventors: Jong-Kwang Lee; Seok-Jae Lee; Moo-Hoon Kim; Yongho Yu; Hee Kim; Doo-Hyun Park; Ha-Su Song; Hyung-Soo Park; De Quan; Hak-Hyun Nam; Geun-Sig Cha
Original assignee: Samsung Engineering Co., Ltd.
Priority date: 2004-05-24
Filing date: 2005-05-24
Publication date: 2005-12-15
Also published as: JP2007510890A

Abstract

A biosensor for detecting nitrate ions and a measuring method using the same are provided. The biosensor includes a working electrode having a nitrate reductase (NaR) immobilized thereon, a counter electrode and a reference electrode. The method is used to measure the concentration of nitrate in a sample using the biosensor. There is further provided a nitrate ionization reactor which enables the bio sensor to measure the concentration of total nitrogen in a sample by converting nitrogen compounds other than nitrates into nitrates. The biosensor for detecting nitrate ion can conveniently measure an accurate concentration of nitrate ions using an electrochemical method. In addition, the biosensor has a simple structure and can be inexpensively manufactured, and thus is differentiated from existing measuring apparatuses and is advantageous in terms of price. The nitrate ionization reactor can conveniently convert nitrogen compounds other than nitrates into nitrates and can be used in combination with the biosensor to simply and conveniently measure total nitrogen in a sample.

Description

BIOSENSOR FOR DETECTING NITRATE ION AND MEASURING METHOD USING THE SAME

TECHNICAL FIELD The present invention relates to a biosensor for detecting nitrate ions and a measuring method using the same. More particularly, the present invention relates to a biosensor which is portable and can accurately measure the concentration of nitration ions in a sample and a method of measuring the concentration of nitrate ions using the same. The present invention also relates to a nitrate ionization reactor in which nitrogen compounds other than nitrates can be converted into nitrates to easily measure the concentration of total nitrogen in the sample using the biosensor.

BACKGROUND ART A biosensor is a sensor which converts chemical information in a sample into a manageable electric signal by systematically combining a biological material with an electronic measuring instrument. Since biosensors can selectively provide quantitative information on chemical species in real time without the use of a complicated chemical or biological treatment, they are being actively developed and are widely used in medical and environmental fields. Nitrate ions are a well-known water pollutant. Contamination by nitrate ions seriously threatens the environment and human health. Thus, analysis of nitrate ions is very important. Intake of a high concentration of nitrate ions over a long period of time increases the probability of inducing digestive system cancer. Many countries prescribe the maximum concentration of nitrate in drinking water in the range of

400-800 μ M and the EPA (Environmental Protection Agency) prescribes the maximum concentration at 700 μ M. Chemical measurement of the concentration of nitrate ions is primarily carried out in three manners. Direct measurement is achieved by colorimetry, the use of an ion-selective electrode, ion chromatography, etc. Indirect measurement is achieved by polarography, etc. In addition, after nitrate is reduced to nitrite, ammonia, nitrogen oxide, etc., the measurement of the concentration of nitrate ions can be achieved (Sah, R.N. Commun. Soil Sci. plant Anal. 1994, 25, 2841). These methods have poor specificity as a result of many ions interfering with the measurement. Thus, a pretreatment is required before the measurement, which is inconvenient and time consuming. In addition to the above-mentioned chemical measurement methods, the concentration of nitrate ions can also be measured using a biosensor method. The biosensor method uses the specificity and sensitivity of an enzyme in combination with the sensitivity of optical, electrochemical and thermal analysis methods, and thus is particularly effective in the medical and environmental fields. The biosensor method is advantageous in that an apparatus used can be simply fabricated, inexpensive and convenient and the pretreatment of a sample is not required, thereby significantly reducing analysis time relative to the chemical method. Although papers regarding the measurement of the concentration of nitrate ions with a biosensor using nitrate reductase (NaR), this method is not very actively studied. U.S. Patent No. 5,776,715 discloses a biosensor detecting nitrate ions using an optical method. If nitrate ions are reduced by the NaR included in the biosensor, NADPH is oxidized to produce NADP⁺. At this time, a photoluminescent directly or indirectly produces photons which are detected by a photodetector. However, the optical method requires a proper luminescent, is disturbed by external light, depends on the amount of a light-emitting reagent in the sample, which makes it difficult to manufacture a small apparatus, and must convert light signals into detectable electric signals in the photodetector. Current methodologies used for measuring nitrate ion concentrations using NaR were first reported in 1994 (S., Cosnier, Innocent, C. and Jouanneau, Y., Anal. Chem. 1994, 66, 3198). However, since NaR mediates a reduction reaction, it is significantly affected by oxygen. To reduce the affect of oxygen, the measurement is carried out using conventional electrodes under Ar in a laboratory. Among conventional biosensor methods of measuring nitrate ions, there are no sensors for measuring nitrate ion concentrations which can be directly used in locations other than a laboratory. The main problem with sensors for measuring nitrate ions on-site is the affect of oxygen. When the sensor is used on-site, its most important use is not measurement of only the concentration of nitrate ions but measurement of the quantity of nitrogen compounds, i.e., the total amount of nitrogen. However, conventional apparatuses for measuring the quantity of nitrogen compounds can only be used accurately in a laboratory since pretreatment for long term and complicated analysis processes are required. Thus, direct on-site monitoring is impossible. The present inventors intensively studied the problems of the conventional technology and discovered that when a reagent containing an oxygen scavenger is applied to a biosensor in which a working electrode having an immobibilzed nitrate reductase, and a counter electrode and a reference electrode around the working electrode are arranged using a screen-printing method, nitrate ions can be simply and conveniently electrochemically detected on-site. Further, a nitrate ionization reactor capable of easily converting nitrogen compounds other than nitrates into nitrates was developed and it was determined that the nitrate ionization reactor can be used in combination with the biosensor to solve the problems of the conventional technology.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a disposable electrode used in a biosensor according to an embodiment of the present invention; FIG. 2 is a series of cross-sectional views illustrating a conventional screen-printing process for preparing an electrode on a strip; FIG. 3 is a graph illustrating reduction current with respect to working potential; FIG. 4 is a graph illustrating change in current with respect to PVA content in a biosensor according to an embodiment of the present invention; FIG. 5 is a graph illustrating change in current with respect to immobilized NaR content in a biosensor according to an embodiment of the present invention; FIG. 6 is a graph illustrating current with respect to the pH of a sample in a biosensor according to an embodiment of the present invention; FIG. 7 is a graph illustrating the sensitivity of a biosensor according to an embodiment of the present invention with respect to the concentration of a buffer (3-(N-morpholino)propanesulfonic acid: MOPS); FIG. 8 is a graph illustrating current with respect to the concentration of an electron carrier (methyl viologen: MV) in a biosensor according to an embodiment of the present invention; FIG. 9 is a graph illustrating current with respect to the concentration of an oxygen scavenger; FIG. 10 is a graph illustrating current with respect to the concentration of an oxygen scavenger (sulfite) in a biosensor according to an embodiment of the present invention; FIG. 11 is a calibration curve with respect to the concentration of nitrate ions of a biosensor according to an embodiment of the present invention; FIG. 12 is a perspective view of a nitrate ionization reactor according to an embodiment of the present invention and a plan view of a lower electrode structure of the nitrate ionization reactor; FIG. 13 is a schematic diagram of an apparatus in which a detachable power supply unit and a timer capable of controlling reaction time are integrated for operating the nitrate ionization reactor of FIG. 12; FIG. 14 is a graph illustrating the degree of nitrate ionization of nitrogen compounds other than nitrates with respect to time; FIG. 15 is a graph illustrating the degree of nitrate ionization of nitrogen compounds other than nitrates with respect to voltage; FIG. 16 schematically illustrates the oxidation of ammonium ions to nitrate ions through an electrochemical catalytic oxidation reaction; and FIG. 17 schematically illustrates the conversion of an organic nitrogen compound into ammonia through an electrochemical catalytic oxidation reaction.

DETAILED DESCRIPTION OF THE INVENTION Technical Goal of the Invention The present invention provides a biosensor for detecting nitrate ions capable of monitoring the concentration of nitrate ions on-site. The present invention also provides a nitrate ionization reactor capable of simply and conveniently converting nitrogen compounds other than nitrates into nitrates. The present invention also provides a system capable of simply and conveniently measuring the concentration of total nitrogen to monitor the concentration of total nitrogen on-site. The present invention also provides a method of measuring the concentration of nitrate ions using the biosensor for detecting nitrate ions.

Disclosure of the Invention According to an aspect of the present invention, there is provided a biosensor for detecting nitrate ions, including: a working electrode, and a counter electrode and a reference electrode separated from the working electrode by a predetermined distance, in which the working electrode is a carbon electrode having a nitrate reductase immobilized thereon and electrochemically measures the reduction of nitrate ions contained in a sample caused by the nitrate reductase . According to another aspect of the present invention, there is provided a nitrate ionization reactor including: a reaction vessel; at least one titanium oxide (TiO₂) electrode within the reaction vessel; at least two Pt electrodes within the reaction vessel; an inlet for supplying reactants into the reaction vessel; and an outlet for discharging products from the reaction vessel. According to another aspect of the present invention, there is provided a system for measuring the concentration of the total nitrogen, including the biosensor for detecting nitrate ions and the nitrate ionization reactor. According to another aspect of the present invention, there is provided a method of measuring the concentration of nitrate ions, including: introducing a sample into a sample inlet of a biosensor; applying a voltage to the working electrode of the biosensor; and measuring the current flowing between the working electrode and the counter electrode.

Effect of the Invention The disposable biosensor for detecting nitrogen ions according to an embodiment of the present invention can conveniently measure an accurate concentration of nitrate ions in actual places using an electrochemical method. Further, the biosensor has a simple structure, can be inexpensively fabricated and is portable, and thus is differentiated from conventional measurement products and advantageous in terms of price. The nitrate ionization reactor according to an embodiment of the present invention conveniently converts nitrogen compounds other than nitrate into nitrate and is in combination with the biosensor for detecting nitrate ion to conveniently measure the concentration of the total nitrogen in a sample. BEST MODE FOR CARRYING OUT THE INVENTION A biosensor for detecting nitrate ion according to an embodiment of the present invention will now be described in more detail. Generally, a biosensor including an electrochemical sensor strip is roughly divided into two parts: a sensing electrode and a portable measurement system. In the present invention, the biosensor narrowly means the sensing electrode in a narrow sense, but broadly includes the portable sensing system equipped with the sensing electrode. The portable sensing system includes a strip-shaped sensing electrode, a potentiostat for measuring current flowing to the sensing electrode, etc. A biosensor for detecting nitrate ion according to an embodiment of the present invention includes a working electrode, and a counter electrode and a reference electrode separated from the working electrode by a predetermined distance. The working, counter and reference electrodes are arranged on a strip-shaped substrate using a screen-printing method. A sample inlet can be formed on the working electrode, and the working electrode can be a carbon electrode having a nitrate reductase immobilized by polymer entrapment. The working electrode electrochemically measures a degree of reduction of nitrate ions contained in a sample caused by the nitrate reductase. A nitrate reductase (Sigma, USA) can be used in the biosensor for detecting nitrate ions. The enzyme promotes the reduction of nitrate ions to produce nitrite ions, and has relatively high activity. Various enzymes can be used depending on a sample to be analysed. For example, nitrate reductase, nitrite reductase (oxidase), ammonium oxidase (reductase), etc. can be used. FIG. 1 is a schematic diagram of a disposable electrode used in the fabrication of a sensing electrode of a biosensor according to an embodiment of the present invention. The sensing electrode includes a sample inlet 1 , a counter electrode 2, a working electrode 3, a reference electrode 4, an insulating layer 5, a lead wire 6 and a lead terminal 7. A disposable screen-printed carbon paste electrode (SPCE) is formed on a strip-shaped insulating substrate using a screen-printing method such that the counter electrode 2 and the reference electrode 4 surround the working electrode 3. The counter, working and reference electrodes 2, 3 and 4 are connected to the lead terminal 7, which is located at the end of the substrate, through the lead wire 6 and the sample inlet 1 is formed in a circular form on the counter, working and reference electrodes 2, 3 and 4, thereby preparing the biosensor. When the strip-type sensor is incorporated into measuring equipment, the concentration of a certain material in a sample can be determined by converting electrical signals. The working electrode 3 has an enzyme immobilized thereon and senses current resulting from a reduction reaction. The working electrode 3 is a carbon electrode and may be composed of a carbon paste or a modified carbon paste. The carbon paste can be disposable due to the low price thereof, can be mass-produced and can relatively stably immobilize enzymes. In combination with the working electrode 3, the counter electrode 2 allows current to flow and may be composed of materials similar to those of the working electrode 3. The reference electrode 4 maintains a constant working voltage between the working electrode 3 and the counter electrode 2 without current flow, and is primarily composed of Ag/AgCI. To prepare the reference electrode 4, an Ag paste layer is oxidized with a saturated FeCI₃ solution, and then the resultant is washed with distilled water and dried in air. The carbon paste and the Ag paste can be obtained from Asahi (Japan). In the biosensor of the present embodiment, a polymer for immobilizing the NaR may be polyvinyl alcohol (PVA), polyacrylamide (PAA), polyethylene glycol (PEG), agarose, starch gels, nylon, Silastic gels, polypyrrole, etc. Preferably, 1-5% by weight of PVA may be mixed with the NaR since PVA is water soluble due to many -OH groups and is an environmental friendly material. In the biosensor of the present embodiment, the amount of the immobilized NaR may be 6.25-25 mU. When the amount of the NaR is less than 6.25 mU, the strength of signals produced by the biosensor is low, and thus limitations in measurement may arise. When the amount of the NaR is greater than 25 mU, signals do not increase in spite of the increase in the amount of NaR, which is not cost effective. 1 -10 mL of a mixture of 6.25-25 mU of NaR and PVA can be immobilized on the working electrode. The substrate may be composed of a flexible and strong material. Polymers such as polycarbonate, polystyrene, polyethylene may be used and polyethylene is preferable. The electrode strip is formed on the substrate according to a general screen-printing method. Referring to FIG. 2, a mask having a pierced electrode pattern is placed on a substrate and a paste (ink) for forming electrodes is sequentially sprayed on an electrode forming region. Then, the paste is scrubbed with a squeegee and the mask is removed to form a strip. The thickness of the mask may be 0.1-0.25 mm. The fabrication of an electrochemical biosensor strip in which quantitative analysis of a very small quantity of nitrate ions is possible using printed circuit board (PCB)-applied technology is disclosed in U.S. Patent No. 5,437,999. In the biosensor of the present embodiment, the pH of a sample may be 5.5-8.0. The maximum signal appears at a pH of 6.5, but the range of pH values is selected in consideration of the effect of an oxygen scavenger. The sample may include a buffer such as 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES) and phosphate. Preferably, 0.05-0.2 M MOPS is used since this is a physiological buffer having a proper pH range and is stable even at a significantly high working voltage (-700 - -900 mV vs. Ag/AgCI). When the concentration of a buffer is greater than 0.2 M, the immobilized enzyme becomes unstable, and when the concentration of a buffer is less than 0.05 M, sensing time is relatively long. The sample may further include an electron carrier such as phenothiazines, triphenylmethanes, sulfonphthaleines, etc. for mediating transition of electrons in the reduction reaction. For example, a 10-30 mM methyl viologen (MV) may be used as the electron carrier since it has better electrochemical reversibility than other acceptable electron carriers. When the concentration of MV is less than 10 mM, signal strength is relatively low and when the concentration of MV is greater than 30 mM, the immobilized enzyme becomes unstable. Unlike glucose oxidase for measuring blood glucose concentration, NaR is significantly affected by oxygen. To reduce the affect of oxygen on NaR, the sample may further include an oxygen scavenger such as sulfite (SO₃ ^2"), metabisulfite (S₂O₅ ^2"), bisulfite (HSO₃ ^~), etc. For example, a 0.5-4 mM sulfite can be used as the oxygen scavenger since it can remove oxygen more effectively than the other possible oxygen scavengers. When the concentration of the oxygen scavenger is too low, the amount of oxygen removed is insufficient and when the concentration of the oxygen scavenger is too high, the immobilized enzyme layer becomes unstable. To achieve another object of the present invention, there is provided a nitrate ionization reactor in which a sample is pretreated for using the biosensor according to the previous embodiment of the present invention. That is, to measure the concentration of the total nitrogen included in a sample, it is necessary to convert nitrogen compounds other than nitrates, for example, amino acids, amines, imines, amides, ammonium ions, nitrite ions, etc., into nitrates. The nitrate ionization reactor is provided to obtain this requirement by converting nitrogen compounds other than nitrates into nitrates. The nitrate ionization reactor includes: a reaction vessel; at least one titanium oxide (TiO₂) electrode within the reaction vessel; at least two Pt electrodes within the reaction vessel; an inlet for supplying reactants into the reaction vessel; and an outlet for discharging products from the reaction vessel. To convert nitrogen compounds other than nitrates into nitrates, an oxidant and a catalyst for promoting the conversion are required. To this end, a material capable of acting as a catalyst in the conversion is used to form as an electrode in the nitrate ionization reactor, and electrons flowing through the sample between electrodes act as an oxidant. The sample may include organic nitrogen compounds, inorganic nitrogen compounds or both. The material capable of acting as a catalyst is selected according to whether the sample includes an organic nitrogen compound or an inorganic nitrogen compound. A Pt electrode and a titanium oxide electrode can be used as a cathode electrode and an anode electrode, respectively, for pre-treatment of inorganic nitrogen compounds. In this case, the conversion of nitrogen compounds other than nitrates into nitrates is schematically illustrated in FIG. 16. The titanium oxide electrode acts a catalyst for oxidation and the oxidation reaction is further facilitated in an alkaline condition. Both of the cathode electrode and the anode electrode for pretreatment of organic nitrogen compounds can be composed of Pt. In this case, the conversion of nitrogen compounds other than nitrates into nitrates is schematically illustrated in FIG. 17. Pt, which forms the electrodes, acts as a catalyst for the oxidation reaction of organic nitrogen compounds. Due to the oxidation reaction, a carbon skeleton of organic nitrogen compounds undergoes decomposition and nitrogen is converted into nitrates through intermediates such as ammonium ions, etc. Thus, at least one titanium oxide electrode and at least two Pt electrodes are required to properly convert a sample including both organic nitrogen compounds and inorganic nitrogen compounds into nitrates. The nitrate ionization reactor may include: two Pt electrodes facing each other disposed horizontally; and a Pt electrode and a titanium oxide electrode facing each other disposed vertically. To increase a surface area for reaction, the electrodes may have protrusions. The protrusions of each of the electrodes preferably do not contact the protrusions of the other electrodes to prevent short circuits. The protrusions of facing electrodes may be alternately extended to have an overlapping pattern without physical contact. Such a configuration is illustrated in FIG. 12B. Power may be simultaneously supplied to all of the electrodes of the nitrate ionization reactor or, for example, may be alternately supplied to each pair of electrodes at predetermined intervals of time. The reaction vessel may be made of any chemical resistant material which does not react with reactants in the sample. The shape and structure of the reaction vessel can be those known in the art. The capacity of the reaction vessel is not particularly restricted and may be varied depending on the amount of sample. For example, the capacity of the reaction vessel may be 0.1 μl to 10 mL. The nitrate ionization reactor may further include a dilution vessel capable of diluting reactants which may be connected to the reaction vessel through the inlet. The dilution vessel is used to dilute products resulting from nitrate ionization and may be disposed on the reaction vessel. The dilution vessel is marked with a scale to quantify a liquid such as a reactant. The material, shape and structure of the dilution vessel may be those known in the art. For example, any chemical-resistant material which does not react with a sample may be used. Since the concentration of nitrate ions in raw water such as domestic wastewater, sewage and industrial wastewater empirically constitutes 40-80% of total nitrogen, a dilution factor can be easily determined by measuring the concentration of nitrate ions. Further, in most cases, the concentration of nitrate ions in water discharged after treatment is at least 90% of total nitrogen. However, a concentration ratio of organic nitrogen to nitrogen ions present in wastewater when it is transferred from a factory to a treatment plant can be recorded by a wastewater treatment supervisor. Thus, the dilution factor after nitrate ionization can be determined empirically or using prior-information, or can be obtained by actually applying various dilution concentrations. The outlet may be located in a lower portion of the reaction vessel so that products can be discharged through gravity. The nitrate ionization reactor of the present embodiment may be connected to an external power supply or include its own power supply unit. When the nitrate ionization reactor includes its own power supply unit, it may further include a timer capable of controlling the supply of power. To achieve another object of the present invention, there is provided a system capable of simply and conveniently measuring the concentration of total nitrogen in a sample. The system for measuring the concentration of total nitrogen in a sample can be achieved by coupling the nitrate ionization reactor and the biosensor for detecting nitrate ion as one body. The method of coupling the nitrate ionization reactor and the biosensor for detecting nitrate ions is not particularly restricted. For example, the outlet of the reaction vessel of the nitrate ionization reactor may be connected to the sample inlet of the biosensor for detecting nitrate ions. To achieve another object of the present invention, there is provided a method of measuring the concentration of nitrate ions, including: introducing a sample into a sample inlet of a biosensor; applying a voltage to a working electrode of the biosensor; and measuring the intensity of current flowing between the working electrode and a counter electrode. In the method, a working voltage of -700 to -900 mV (vs. Ag/AgCI) is applied to the working electrode and the intensity of current is measured by cyclic voltammetry (Kissinger et al., J. Chem. Ed. 60 (9), 702-706). The method of measuring the concentration of nitrate ions may further include, before introducing a sample into the sample inlet of the biosensor, introducing the sample into a nitrate ionization reactor and applying a voltage to the nitrate ionization reactor to convert nitrogen compounds other than nitrates into nitrates. In this case, the concentration of total nitrogen in the sample can be measured. When an acidic or alkaline material is added to the nitrate ionization reactor to obtain an acidic or alkaline solution, the conversion of nitrogen compounds into nitrates occurs more rapidly. However, since an acidic material may cause corrosion of electrodes, an alkaline material such as sodium hydroxide can be used. The concentration of sodium hydroxide may be 0.1 to 2.0 N. When the concentration of sodium hydroxide is less than 0.1 N, the conversion of nitrogen compounds into nitrates is not effectively facilitated. When the concentration of sodium hydroxide is greater than 2.0 N, the sample must be neutralized before being analyzed with the biosensor. The nitrate ionization may be carried out for 5 to 30 minutes. When carried out for less than 5 minutes, errors in subsequent measurement results can be caused due to the reaction occurring insufficiently. When carried out for more than 30 minutes, the nitrate ionization is already almost completed, thus cost-consuming. The voltage applied to the nitrate ionization reactor may be 5-30 V. When the voltage is less than 5 V, the reaction does not occur easily. When the voltage is greater than 30 V, the applied voltage does not increase the reaction rate additionally, and thus is cost-consuming, and damage and overheating of the electrodes may occur. The voltage can be proportional to the size of the reaction vessel. In the method, the pH of the sample may be 5.5-8.0 as described above. In the method, the sample may include a buffer such as 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES) or phosphate. 0.05-0.2 M MOPS may be used for the reasons described above. When the concentration of the buffer is too high, the immobilized enzyme becomes unstable, and when the concentration of buffer is too low, sensing time is relatively long. In the method, the sample may further include an electron carrier such as phenothiazines, triphenylmethanes, sulfonphthaleines, etc. for mediating the transition of electrons in the reduction reaction. For example, a 1.0-3.0 mM methyl viologen (MV) may be used as the electron carrier. When the concentration of MV is less than 10 mM, the intensity of signals is relatively low, and when the concentration of MV is greater than 30 mM, the immobilized enzyme becomes unstable. Unlike glucose oxidase for measuring blood glucose concentration, NaR is significantly affected by oxygen. To reduce the effect of oxygen on NaR, the sample may further include an oxygen scavenger such as sulfite (SO₃ ^2"), metabisulfite (S₂O₅ ^2"), bisulfite (HSO₃ ^"), etc. For example, a 0.5-4 mM sulfite can be used as the oxygen scavenger. When the concentration of the oxygen scavenger is too low, the effect of removing oxygen is insufficient, and when the concentration of the oxygen scavenger is too high, the immobilized enzyme layer becomes unstable. The present invention will now be described in greater detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 : Electrochemical characteristics of nitrate reductase An electrochemical experiment was carried out in a three-electrode system. A glassy carbon disk electrode (3 mm) was used as a working electrode, a spiral Pt wire was used as an aid electrode and Ag/AgCI (3 M KCI) was used to form a reference electrode. A cDAQ- 604 potentiostat (Elbio, Korea) was used in preparing a cyclic voltammogram (CV) and an l-V curve. A working potential of -0.4 to 1.0 mV (vs. Ag/AgCI) was applied to the working electrode to obtain the l-V curve. FIG. 3 is a graph illustrating reduction current with respect to working potential obtained through cyclic voltammetry using nitrate reductase and various concentrations of substrate (nitrate ion) in the biosensor according to an embodiment of the present invention. Referring to FIG. 3, when the nitrate reductase immobilized on the working electrode reacted with the nitrate and an electron carrier (methyl viologen: MV), the reduction current increased with the concentration of substrate, which indicates that the nitrate reductase reduces nitrates to nitrites and MV acts as an electron carrier. Nearly identical curve patterns were obtained when the concentrations of substrate were 50 μ M and 100 μ M. It can be seen from the experimental results that when MV continuously facilitates electrochemical reduction, the reduction current caused by nitrate ions can be obtained and the unknown concentration of a substrate can be measured.

Example 2: Fabrication and optimization of sensing electrode sensor strip A disposable screen-printed carbon paste electrode (SPCE) was fabricated on a polyethylene (PE) plate to a width of 12.8 mm and a length of 65 mm using a screen-printing method illustrated in FIG. 1. 3 μl of a solution containing an oxygen scavenger, an electron carrier, MOPS and a substrate was brought into contact with the end of the disposable electrode having an enzyme immobilized thereon by means of a pipet. After the solution was sucked into the electrode, a voltage of -700 to -900 mV (vs. Ag/AgCI) was applied to the electrode and a cDAQ-1604 potentiostat (Elbio, Korea) was used to collect data. In the fabrication of the disposable strip-type sensor, PVA, which has good stability and storage properties, was used to easily immobilize an enzyme on the electrode. 1 wt%, 2.5 wt%, 5 wt%, 10 wt%, 15 wt% and 20 wt% of PVA were used, and it was found that the optimal content of PVA was in the range of 1-5 wt%. The noise produced increased when the amount of PVA was increased. When less than 1 wt% of PVA was used, the signal intensity was too low. FIG. 4 is a graph illustrating change in current (Δl) with respect to PVA content of the biosensor. To determine the optimum amount of enzyme required to generate electrode signals, 6.25, 12.5, 25 and 50 m\ IZ.μl of the enzyme were immobilized. FIG. 5 is a graph illustrating change in current (Δl) with respect to the nitrate reductase content of the biosensor. Although the intensity of the signal increased as the amount of the immobilized enzyme increased, the rate of increase significantly decreased as the amount of enzyme increased. The optimum amount of the immobilized enzyme was found to be in the range of 6.25 to 25 mU/3 μl. To investigate the pH dependency of electrode, MES and MOPS were used as buffers. FIG. 6 is a graph illustrating current with respect to the pH of a sample.

When the pH was in the range of 5.5-6.0, MES buffer was used, and when the pH was in the range of 6.5-8.0, MOPS buffer was used. Data obtained using MES were normalized by comparing data obtained using MES at a pH of 6.5 with data obtained using MOPS at a pH of 6.5. It can be seen from the experimental results that the optimum pH is in the range of 6.5 to 7.0. To examine the effect of the concentration of a buffer, MOPS was used as a buffer in concentrations of 0.05, 0.1 , 0.2, 0.3, and 0.4 M at pH 7.0. FIG. 7 is a graph illustrating the sensitivity of the biosensor with respect to the concentration of MOPS.

Referring to FIG. 7, similar signals were obtained at various concentrations of MOPS.

The signals converged on a mean sensitivity of 7.13 with a standard deviation of 1.4%.

The optimum concentration of the buffer was determined to be in the range of 0.05 to 0.2 M since the immobilized enzyme was relatively unstable at higher concentrations of the buffer. To determine the optimum concentration of MV (electron carrier), 1 to 5 mM MV was used. FIG. 8 is a graph illustrating the apparent current with respect to the concentration of MV as an electron carrier. Referring to FIG. 8, the current linearly increased with the concentration of MV. In consideration of the amount of the immobilized enzyme and the amount of the substrate used, the optimum concentration of MV was 1.0 to 3.0 mM. To remove the effect of oxygen on NaR, sulfite, which was found to be an optimum oxygen scavenger, was used in concentrations of 0.5, 1 , 2, 4, 6, 8 and 10 mM. The oxygen removal reaction facilitated by SO₃ ^2" is as follows: 2SO₃ ^2" + O₂ → 2SO ^2". FIG. 10 is a graph illustrating the current with respect to the concentration of sulfite as an oxygen scavenger. Considering that oxygen in air was dissolved in a buffer while oxygen was removed, the optimum concentration of the oxygen scavenger ranges from 0.5 to 4 mM. To obtain a calibration curve of an electrode for measuring the concentration of nitrate ions, the substrate was used in concentrations of 50, 100, 150, 200, 250 and 300 mM and the experiment for measuring current was repeatedly carried out (30 times). FIG. 11 is a graph illustrating the current variation (Δl) generated in the electrode system (strip-type sensor) with respect to the concentration of nitrate ions. Referring to FIG. 11 , a linear equation of Y = 5.42X - 38.43 (r² = 0.996) was obtained.

Example 3: Optimization of nitrate ionization of non-nitrate ions FIG. 12 illustrates a nitrate ionization reactor for ammonium ions and organic nitrogen compounds. The nitrate ionization reactor can oxidize ammonium ions and organic nitrogen to produce nitrate ions by applying a DC voltage of 5-30 V depending on the concentration of a reactant for 5-30 minutes, in the presence of 0.1-2.0 N sodium hydroxide as an electrolyte, without an additional oxidant or catalyst. The capacity of the reactor was adjusted to 0.1 mL in consideration of the amount and concentration of nitrate ions that can be measured by the biosensor, and dilution and neutralization were required to obtain a sample appropriate for the sensing ability of the biosensor. The reactor included a 20 mL dilution vessel for diluting the sample with a buffer solution. FIG. 13 is a side view of a power supply unit for operating the nitrate ionization reactor. The power supply unit was manufactured such that an applied voltage and a reaction time could be freely controlled and a current was automatically controlled depending on the concentration of a reactant. The current was maximized at 5 A so that it could increase or decrease in proportion to the concentration of a reactant, and the maximum voltage was adjusted to 30 V. FIG. 14 is a graph illustrating the nitrate ionization efficiency (%) of the nitration ionization reactor for non-nitrate ions - ammonia and organic nitrogen compound (sodium glutamate)- at a voltage of 20 V with respect to time. Non-nitrate ion concentrations of the tested substances were 100 ppm. Referring to FIG 14, there was no substantial increase in the reaction efficiency after 10 minutes. Thus, a proper on-site treatment time was determined to be 10 minutes in consideration of time for preparation, reaction, accurate measurement, etc. When various samples are treated at a time, since multiple reactors can be used, the total treatment time will not be significantly increased. FIG. 15 is a graph illustrating the nitrate ionization efficiency (%) of electrochemical catalytic oxidation for non-nitrate ions - organic nitrogen compound (sodium glutamate)- with respect to voltage. The non-nitrate ion concentration of the tested substance was 100 ppm. Referring to FIG. 15, a voltage required for sufficient oxidation within 10 minutes was about 25 V, which is 2 times the battery voltage for vehicles. Thus, it seems that a voltage of 15 V is sufficient considering the concentration of the treated substance and the fact that the amount of organic nitrogen compound contained in natural water is small. Table 1 shows nitrate ionization efficiencies for ammonia nitrogen, organic nitrogen compound, river water, sewage available from Jungrang Sewage Treatment Plant, etc. determined using a nitrate ionization reactor according to an embodiment of the present invention and the concentration of the total nitrogen measured using the biosensor for measuring nitrates. As can be seen from Table 1 , the nitrate ionization efficiency of ammonia nitrogen was a very high value of greater than 97%. Also, the nitrate ionization efficiencies of natural river water and raw water introduced into the sewage treatment plant, sewage which was being treated, etc. were generally about 85-95%, which are relatively high values. It seems that this result is obtained because natural river water and sewage contain more ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, etc. than organic nitrogen. Further, the concentration of the total nitrogen measured using the biosensor was very similar to that measured using ion chromatography (IC), which indicates that the nitrate ionization of a sample used in conjunction with the biosensor can be used to measure the concentration of all the nitrogen in a sample.

Table 1

(a) - this indicates total nitrogen measured using HACH. (b) - measured using IC. (c) - measured using the biosensor of the present invention. The nitrate measured by the biosensor was obtained by converting non-nitrates into nitrates by means of the nitrate ionization reactor of the present invention and 100 times dilution. It can be seen from Table 1 that the accuracy of the nitrate ionization reactor and the biosensor for detecting nitrate ions has an error within 2-3%, and thus is very accurate. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A biosensor for detecting nitrate ions, comprising a working electrode, and a counter electrode and a reference electrode separated from the working electrode by a predetermined distance, in which the working electrode is a carbon electrode having a nitrate reductase immobilized thereon and electrochemically measures the reduction of nitrate ions contained in a sample caused by the nitrate reductase.

2. The biosensor of claim 1 , wherein the working electrode is a carbon paste electrode.

3. The biosensor of claim 1 , wherein 1 to 5% by weight of polyvinyl alcohol (PVA) is mixed with the nitrate reductase to immobilize the nitrate reductase.

4. The biosensor of claim 1 , wherein an amount of the immobilized nitrate reductase is 6.25 to 25 mU.

5. The biosensor of claim 1 , wherein the pH of the sample is 5.5 to 8.0.

6. The biosensor of claim 1 , wherein the sample comprises 0.05-0.2 M

3-(N-morpholino)propanesulfonic acid (MOPS) as a buffer.

7. The biosensor of claim 1 , wherein the sample comprises 1.0-3.0 mM methyl viologen (MV) as an electron carrier.

8. The biosensor of claim 1 , wherein the sample comprises 0.5-4 mM sulfite as an oxygen scavenger.

9. A nitrate ionization reactor comprising: a reaction vessel; at least one titanium oxide (TiO₂) electrode within the reaction vessel; at least two Pt electrodes within the reaction vessel; an inlet for supplying reactants into the reaction vessel; and an outlet for discharging products from the reaction vessel.

10. The nitrate ionization reactor of claim 9, wherein electrodes have protrusions formed thereon.

11. The nitrate ionization reactor of claim 10, wherein the protrusions of the electrodes are alternately extended to have an overlapping pattern without physical contact.

12. The nitrate ionization reactor of claim 9, wherein electrodes include two Pt electrodes facing each other and disposed horizontally; and a Pt electrode and a titanium oxide electrode facing each other and disposed vertically.

13. The nitrate ionization reactor of claim 9, further comprising a dilution vessel.

14. A system for measuring the concentration of the total nitrogen, comprising the biosensor for detecting nitrate ions of claim 1 and the nitrate ionization reactor of claim 9.

15. A method of measuring the concentration of nitrate ions, comprising: introducing a sample into a sample inlet of the biosensor of any one of claims

1-8; applying a voltage to the working electrode of the biosensor; and measuring the current flowing between the working electrode and the counter electrode.

16. The method of claim 15, further comprising, before introducing a sample into the sample inlet of the biosensor: introducing the sample into a nitrate ionization reactor; and applying a voltage to the nitrate ionization reactor to convert nitrogen compounds other than nitrates contained in the sample into nitrates.

17. The method of claim 16, further comprising introducing 0.1-2.0 N sodium hydroxide into the nitrate ionization reactor.

18. The method of claim 16, wherein the power is supplied alternately to each of two pairs of electrodes included in the nitrate ionization reactor.

19. The method of claim 16, wherein nitrate ionization is carried out for 5-30 minutes.

20. The method of claim 16, wherein the applied voltage is 5-30 V.

21. The method of claim 15, wherein the pH of the sample is 5.5-8.0 when the concentration of nitrate ion is measured.

22. The method of claim 15, wherein the sample comprises 0.05-0.2 M

3-(N-morpholino)propanesulfonic acid (MOPS) as a buffer.

23. The method of claim 15, wherein the sample comprises 1.0-3.0 mM methyl viologen (MV) as an electron carrier.

24. The method of claim 15, wherein the sample comprises 0.5-4 mM sulfite as an oxygen scavenger.