JP2007510890A - Biosensor for detecting nitrate ion and measuring method using the same - Google Patents

Abstract

The present invention provides a biosensor for detecting nitrate ions and a measurement method using the biosensor.
The disposable biosensor for detecting nitrate ions produced according to the present invention can conveniently measure the exact concentration of nitrate ions in the field using electrochemical methods. In addition, since the structure and manufacturing cost are low and portable, it is different from existing measurement products and has high price competitiveness.
The nitrate ionization reactor of the present invention is a device that conveniently converts nitrogen compounds that are not nitrates into nitrates. By combining the nitrate ionization reactor and a biosensor for detecting nitrate ions, the total nitrogen in the sample is converted. It can be measured conveniently.

Description

  The present invention relates to a biosensor for detecting nitrate ions and a measurement method using the biosensor, and more specifically, a biosensor capable of accurately measuring the concentration of nitrate ions in a sample while being carried conveniently and using the same. Relates to a method for measuring the nitrate ion concentration. The present invention also relates to a nitrate ionization reactor that enables measurement of the concentration of total nitrogen by switching a nitrogen compound that is not nitrate to nitrate.

  A biosensor refers to a sensor in which a biological substance is organically coupled to an electronic measurement device and switches to an electrical signal that can easily process chemical information in an arbitrary sample. The biosensor has been widely developed due to the advantage that quantitative information of a desired chemical species can be selectively obtained in real time without the need for complicated chemical and biological processing. Widely used in medical, environmental fields.

  Nitrate ions are one of the known water pollutants. Extensive contamination with nitrate ions is a serious threat to the environment and human health. Therefore, the analysis of nitrate ions is very important. Ingesting high concentrations of nitrate for long periods of time is very likely to induce cancer of the digestive system. In many countries, the maximum concentration of nitrate in drinking water is specified as 400 to 800 μM, and the US Environmental Protection Agency (EPA) specifies up to 700 μM. There are mainly three methods for chemical measurement of nitrate ions, but direct measurement methods include colorimetric analysis, ion selective electrodes, ion chromatography, and indirect measurement methods include polarography. There is a method of measuring nitrate after it is reduced to nitrite, ammonia, or nitrogen oxide (Non-patent Document 1). Such a method is inferior in specificity, and many ions interfere with it. Therefore, it is inconvenient because a pretreatment process is required before measurement, and a long time is required.

  In addition to the chemical measurement method described above, nitrate ions can also be measured by a biosensor method. The biosensor method is particularly effective in the medical and environmental fields because it combines the specificity and sensitivity of the enzyme with sensitivities such as optical, electrochemical and thermal analysis methods. The biosensor method has advantages in that the device is easy and economical to manufacture compared to the chemical method, is convenient to use, and does not require a sample pretreatment process, so that the analysis time can be greatly shortened. Although a paper was published in which nitrate ions were measured by a biosensor using nitrate reductase (hereinafter abbreviated as NaR), it has not been actively studied.

Patent Document 1 discloses a biosensor that detects nitrate ions by an optical method. When nitrate ions are reduced by NaR contained in the biosensor, the light emitting agent generates photons directly or indirectly while NADPH is oxidized to NADP ⁺ , and this is detected by a photodetector. However, since the optical method requires an appropriate luminescent agent, is disturbed by light in the surrounding environment, and depends on the amount of the luminescent reagent, it is difficult to make the optical signal small. There is a problem that it is necessary to switch to a detectable electrical signal.

  The galvanostatic measurement of nitrate ion using NaR was first published in 1994 (Non-Patent Document 2), but NaR mediates the reduction reaction, so it was greatly influenced by oxygen. Measured in the laboratory using a common electrode on argon to reduce oxygen interference. Of the biosensor methods for nitrate ion measurement that have been published so far, no sensor for current method measurement that can be used immediately in the field other than the laboratory has been reported. The biggest difficulty with sensors for in situ measurement of nitrate ions is how to reduce oxygen interference.

  Also, the most important application in actual use in the field is to measure the amount of total nitrogen compounds, that is, the amount of total nitrogen, rather than measuring the concentration of nitrate alone. However, the conventional apparatus for measuring the total amount of nitrogen compounds is a laboratory-scale apparatus that requires a long time of pretreatment and a complicated analysis process, so that it cannot be immediately monitored in the field. there were.

  As a result, the present inventors have intensively studied to overcome the problems of the prior art, and as a result, a fixed working electrode in which nitrate reductase is immobilized, a relative electrode and a reference electrode around the working electrode are screened. When applying a sample containing an oxygen scavenger to a three-electrode biosensor arranged by a printing technique, it is confirmed that nitrate ions can be easily and electrochemically detected in the field, and the present invention is completed. It came. In addition, a nitrate ionization reactor that easily switches a nitrogen compound that is not a nitrate to a nitrate was developed, and it was confirmed that the above-described problems of the prior art can be solved by appropriately combining this with the biosensor.

US Pat. No. 5,776,715 Sah, R.A. N. Commun. Soil Sci. plant Anal. 1994, 25, 2841 S. Cosnier, Innocent, C.I. and Jouanneau, Y. et al. , Anal. Chem. 1994, 66, 3198

  The problem to be solved by the present invention is to provide a biosensor for detecting nitrate ions capable of in situ monitoring the concentration of nitrate ions.

  Another problem to be solved by the present invention is to provide a nitrate ionization reactor capable of easily switching a nitrogen compound that is not a nitrate to a nitrate.

  Still another problem to be solved by the present invention is to provide a total nitrogen concentration measurement system that can be easily measured so that the total nitrogen concentration can be monitored in the field.

  Still another problem to be solved by the present invention is to provide a measurement method for measuring nitrate ion concentration using the biosensor for detecting nitrate ions.

  In order to solve the above problems, the present invention provides a biosensor in which a working electrode, a relative electrode spaced apart from the working electrode and a reference electrode are arranged, and the working electrode is a carbon electrode fixed with nitrate reductase The biosensor for detecting nitrate ions is characterized by electrochemically measuring the degree to which nitrate ions contained in a sample are reduced by the nitrate reductase.

In order to solve the other problems, the present invention provides a reaction vessel, at least one titanium oxide (TiO ₂ ) electrode immersed in the reaction vessel, and at least two platinum (Pt) immersed in the reaction vessel. ) A nitrate ionization reactor having an electrode, an inlet through which a reactant can be introduced into the reaction tank, and an outlet through which the reactant can be discharged from the reaction tank.

  The present invention provides a total nitrogen concentration measuring system including the biosensor for detecting nitrate ions and the nitrate ionization reactor, in order to solve the still another problem.

  In order to solve the further another problem, the present invention provides a step of introducing a sample into a sample injection port of the biosensor, a step of applying a voltage to the working electrode of the biosensor, and a working electrode in the applying step. And a method for measuring a nitrate ion concentration, comprising: measuring an intensity of a current flowing between the first electrode and a relative electrode.

Biosensors for nitrate ion detection In general, biosensors including electrochemical sensor strips are roughly classified into two parts. One is the sensing electrode part and the other is a portable measuring device. In the present invention, a biosensor means a portion of the sensing electrode in a narrow sense, but broadly means a portable measuring device equipped with the sensing electrode. The portable measuring device may include a constant potential device for measuring a current flowing through the sensing electrode with the strip-shaped sensing electrode portion inserted therein.

  To achieve the object of the present invention, the present invention provides a working electrode on a strip-shaped substrate, a relative electrode spaced apart from the working electrode by a screen printing technique, and arranged on the working electrode. In the biosensor provided with a sample inlet, the working electrode is a carbon electrode in which nitrate reductase is immobilized by a polymer capture method, and nitrate ions contained in the sample are reduced by the nitrate reductase. A biosensor for detecting nitrate ions is provided.

  Nitrate reductase (Sigma, USA) can be used for the biosensor for nitrate ion measurement of the electrochemical method used in the present invention. This enzyme promotes the reaction of reducing nitrate ions to nitrite ions, but has relatively good activity. The enzyme used varies depending on the sample to be analyzed. For example, nitrate reductase, nitrite reductive (oxidation) enzyme, ammonium oxidization (reduction) enzyme, and the like can be used.

  FIG. 1 is a schematic diagram of a disposable electrode used for manufacturing a sensing electrode of a biosensor as one embodiment of the present invention. Reference numeral 1 is a sample injection port, 2 is a relative electrode, 3 is a working electrode, 4 is a reference electrode, 5 is an insulating layer, 6 is a lead wire, and 7 is a lead terminal. Disposable screen-printed carbon paste electrode (SPCE) electrodes are formed on a strip-shaped insulating substrate by a screen printing technique, and arranged so that the relative electrode 2 and the reference electrode 4 surround the working electrode 3. The electrodes 2, 3 and 4 were connected to a lead terminal 7 at the end of the substrate through a lead wire 6, and a circular sample inlet 1 was formed on the electrodes 2, 3 and 4 to manufacture a biosensor. Such a strip type sensor can be inserted into a measurement equipment to convert an electrical measurement signal and measure a substance concentration in a sample.

  In the biosensor of the present invention, the working electrode serves to sense an electric current due to a reduction reaction with an enzyme immobilized, and the material is preferably a carbon paste electrode or a deformed carbon paste electrode as a carbon electrode. This is because carbon paste is inexpensive, can be used as a disposable, can be mass-produced, and an enzyme immobilized thereon is relatively stable.

  In the biosensor of the present invention, the relative electrode is combined with the working electrode to flow an electric current, and an electrode similar to the working electrode can be used. Further, the reference electrode has a function of maintaining a constant operating voltage between the working electrode and the relative electrode without flowing current, and the material is mainly Ag / AgCl.

Asahi (Japan) was used as the carbon paste and silver paste. In order to make a reference electrode, the silver paste layer was oxidized with an iron chloride (FeCl ₃ ) saturated solution, washed with distilled water, and then dried in air.

  In the biosensor of the present invention, the polymer for immobilizing the nitrate reductase is PVA (polyvinyl alcohol), PAA (polyacrylamide), PEG (polyethylene glycol), agarose, starch gel, nylon, silastic gel, polypyrrole. Desirably, a mixture of 1 to 5% by weight of PVA is desirable. This is because PVA has many —OH groups and is therefore water-soluble and environmentally friendly.

  In the biosensor of the present invention, the amount of immobilized nitrate reductase (NaR) is preferably 6.25 to 25 mU. If the amount of NaR is small, the sensor signal is small and the measurement limit increases so much. If the amount of NaR is large, the signal does not increase any more, which is uneconomical. Preferably, 6.25 to 25 mU of nitrate reductase is uniformly mixed with PVA, and then 1 to 10 mL can be fixed to the working electrode.

  In the biosensor of the present invention, the substrate material preferably has flexibility and strength. Desirably, polymer compounds such as polycarbonate, polystyrene, and polyethylene can be used, and polyethylene is particularly desirable.

  The electrode strip on the substrate is formed by placing a mask, which is formed in an electrode shape by a normal screen printing method as shown in FIG. If the mask is removed after rubbing with a squeegee, a strip is formed. A suitable thickness of the mask is about 0.1 to 0.25 mm. Reference can be made to US Pat. No. 5,437,999 for a technique for producing an electrochemical biosensor strip that can be quantitatively analyzed in a very small amount by an application technology of a printed circuit board (PCB).

  In the biosensor of the present invention, the pH of the sample is preferably 5.5 to 8.0. In the embodiment of the present invention, the maximum signal appeared at pH 6.5, but 5.5 to 8.0 was selected. The reason is that both the effects of the oxygen scavenger are considered.

  In the biosensor of the present invention, the sample may use a buffer such as MOPS (3- (N-morpholino) propanesulphonic acid), MES (2- (N-morpholino) ethansulfonic acid), phosphate, etc. The sample is characterized by using a MOPS buffer having a concentration of 0.05 to 0.2M. The reason for choosing MOPS is that the pH range is a suitable physiological buffer and is stable even at very high operating voltages (-700 to -900 mV vs. Ag / AgCl). If the buffer concentration is too high, the immobilized enzyme is unstable, and if the concentration is too low, the sensitivity time is relatively long.

  In the biosensor of the present invention, an electronic transition mediator such as phenothiazines, triphenylmethanes, sulfonephthaleins and the like can be added to the sample in order to mediate electronic transition of nitrate ion reduction reaction. In addition, MV (methyl viologen) having a concentration of 1.0 to 3.0 mM is further added to the sample as an electron transition medium. MV was chosen because it has the best electrochemical reversibility when compared to the electron transition mediator described above. If the MV concentration is too low, the signal is relatively low, and if the MV concentration is too high, the immobilized enzyme becomes unstable.

  In the biosensor of the present invention, unlike the conventional glucose oxidase for blood glucose measurement, the nitrate reductase as a reductase is greatly affected by oxygen. An oxygen scavenger such as sulfite, metabisulfite, or bisulfite can be further added to the sample, but it is desirable to use 0.5 to 4 mM sulfite as the oxygen scavenger in the sample. . Of the oxygen scavengers described above, the effect of sulfite is the best. If the concentration of the oxygen scavenger is too low, the effect of removing oxygen is insufficient, and if the concentration is too high, it is not necessary at a higher concentration, and the immobilized enzyme layer becomes unstable.

Nitrate Ionization Reactor To achieve another object of the present invention, there is provided a nitrate ionization reactor for pretreating a sample in order to utilize the biosensor according to the present invention.

  That is, in order to measure the total nitrogen contained in a sample using the biosensor of the present invention, nitrogen compounds that are not nitrates, such as amino acids, amines, imines, amides, ammonium salt ions, nitrite ions, etc. The nitrate ionization reactor is for satisfying such a requirement, and a nitrogen compound that is not a nitrate is switched to a nitrate.

The nitrate ionization reactor includes a reaction tank, at least one or more TiO ₂ electrodes immersed in the reaction tank, at least two or more Pt electrodes immersed in the reaction tank, and an input capable of charging a reactant into the reaction tank. And a discharge port through which the reactant can be discharged from the reaction vessel.

  In order to switch a nitrogen compound that is not a nitrate to a nitrate, an oxidizing agent and a catalyst that catalyzes the switching reaction are required. If a substance capable of the catalytic action is used as the electrode of the nitrate ionization reactor, the flow of electrons passing through the sample through the electrode acts as an oxidant, so that such a requirement can be satisfied. However, an organic nitrogen compound is also included in the sample, and an inorganic nitrogen compound can also be included. The substance capable of performing the catalytic action may vary depending on whether the sample is an organic nitrogen compound or an inorganic nitrogen compound.

In order to pretreat the inorganic nitrogen compound, a Pt material is desirable as the cathode electrode, and a TiO ₂ electrode is desirable as the anode electrode. At this time, the conceptual diagram of the reaction which switches the nitrogen compound which is not nitrate to nitrate is shown in FIG. Here, TiO ₂ plays a catalytic role for oxidation, and the oxidation reaction is further activated in an alkaline atmosphere.

  In order to pretreat the organic nitrogen compound, it is desirable that both the cathode electrode and the anode electrode are made of Pt material. At this time, the conceptual diagram of the reaction which switches the nitrogen compound which is not nitrate to nitrate is shown in FIG. The Pt forming the electrode catalyzes a reaction in which the organic nitrogen compound is oxidized. The organic nitrogen compound is decomposed in the carbon skeleton by the oxidation reaction, and the nitrogen is switched to nitrate through an intermediate such as an ammonium ion.

Therefore, in order to appropriately switch a sample containing both organic nitrogen compound and inorganic nitrogen compound to nitrate, it is required to include at least one TiO ₂ electrode and at least two or more Pt electrodes.

The electrode of the nitrate ionization reactor preferably includes two Pt electrodes facing in the lateral direction, and a Pt electrode and a TiO ₂ electrode facing in the vertical direction. The electrode preferably has a protrusion in order to increase the reaction surface area. The protrusions of the electrodes are preferably not in contact with the protrusions of other electrodes in order to prevent a short circuit in the external circuit, and the protrusions are alternately spaced apart to increase the reaction area without mutual contact. It may be in a form overlapping. An exemplary elevation view of such a configuration is shown in FIG.

  Power may be supplied to all the electrodes of the nitrate ionization reactor at the same time. For example, power may be supplied alternately in pairs at predetermined time intervals.

  Any material can be used for the reaction vessel as long as it has chemical resistance that does not cause a chemical reaction with the reactant, and is not particularly limited. Further, the form and structure of the reaction vessel are not particularly limited, and the form and structure known in the art can be used. The volume in which the reaction vessel can accommodate the reaction product is not particularly limited and can be changed depending on the amount of sample required, but is preferably 0.1 μl to 10 mL.

  The nitrate ionization reactor may further include a dilution tank capable of diluting a reactant, and the dilution tank may be connected through the reaction tank and a charging port. The dilution tank is used to dilute the concentration of the reactant after the nitrate ionization reaction, and is preferably installed in the upper part of the reaction tank. It is desirable to display a scale for quantifying a liquid such as a reactant in the dilution tank. The material, form, and structure of the dilution tank are not particularly limited, and may be a material, form, and structure known in the art. However, a chemically resistant material that does not chemically react with the sample is desirable.

  Empirically, the concentration of nitrate ions in raw sewage, sewage, and industrial wastewater accounts for 40 to 80% of total nitrogen, so the dilution factor can be easily measured by measuring the nitrate ion concentration with a nitrate ion biosensor. Can be determined. Further, the concentration of nitrate ions in the number of discharges processed through the treatment process is almost 90% or more of the total nitrogen. However, the concentration ratio between organic nitrogen and nitrate ions present in the wastewater during the process of moving from the factory to the treatment plant can be computerized through the factory wastewater treatment and management. Therefore, the dilution rate after the nitrate ionization reaction must be determined empirically, or determined using prior information, or a method in which all dilutions are applied to various concentrations.

  The discharge port is preferably installed at a lower portion of the reaction tank so that the reactant can be discharged using gravity.

  The nitrate ionization reactor of the present invention may be used by being connected to an external power supply device, or may be provided with a voluntary power supply device. In the case of further including a voluntary power supply device, it may further include a timer device that can supply power or stop the operation depending on the operation time.

Total Nitrogen Concentration Measurement System In order to achieve another object of the present invention, a total nitrogen concentration measurement system capable of easily measuring the total nitrogen concentration in a sample is provided.

  That is, the object can be achieved by combining the nitrate ionization reactor of the present invention and the biosensor for detecting nitrate ions of the present invention in an integrated manner. The connection relationship between the nitrate ionization reactor and the biosensor for detecting nitrate ions is not particularly limited. For example, an outlet of the reaction vessel of the nitrate ionization reactor is connected to a sample injection of the biosensor for detecting nitrate ions. It may be connected to the entrance and is not particularly limited.

Method for Measuring Nitrate Ion Concentration To achieve another object of the present invention, the present invention comprises a step of introducing a sample into the sample inlet of the biosensor according to the present invention, and a voltage applied to the working electrode of the biosensor. There is provided a method for measuring a nitrate ion concentration comprising: an applying step; and a step of measuring an intensity of a current flowing between a working electrode and a relative electrode in the applying step.

  In the measuring method of the present invention, an operating voltage of −700 to −900 mV (vs. Ag / AgCl) is applied to the working electrode in the second step, and in the third step, the current intensity is measured by a circulating voltage measuring method (Kissinger et al. , J. Chem. Ed. 60 (9), 702-706).

  Further, in the nitrate ion concentration measuring method of the present invention, in the first step, before the sample is introduced into the sample inlet of the biosensor, the sample is introduced into a nitrate ionization reactor, and the nitrate ionization reactor is introduced into the nitrate ionization reactor. Applying a power source to switching a non-nitrate nitrogen compound in the sample to nitrate. In this case, the concentration of all nitrogen compounds present in the sample of the main body can be measured.

  As described above, when a sample is put into a nitrate ionization reactor and a non-nitrate nitrogen compound is converted to nitrate, an acidic or alkaline substance is added to make the sample into an acidic or alkaline solution. Conversion takes place more quickly. However, since an acidic substance can cause corrosion of the electrode, an alkaline substance is desirable, and sodium hydroxide is particularly desirable. The preferred sodium hydroxide concentration is between 0.1 and 2.0N. If the concentration of sodium hydroxide is lower than 0.1N, the effect of promoting the conversion to nitrate is insignificant, and if the concentration of sodium hydroxide is higher than 2.0N, it is neutralized when applied to a biosensor. There is a disadvantage that an additional process must be added.

  The time for performing the nitrate ionization reaction is preferably 5 to 30 minutes. If the reaction time is shorter than 5 minutes, the reaction is not sufficiently performed, and an error may occur in subsequent measurement results. If the reaction time is longer than 30 minutes, the reaction is already sufficient, which is uneconomical. .

  The voltage of the power source applied for the nitrate ionization reaction is preferably 5 to 30V. When the voltage is lower than 5V, the reaction may not be performed satisfactorily because the voltage is low. When the voltage is higher than 30V, the effect due to the voltage is saturated and uneconomical. Undesirable because of the risk of damage and overheating. The voltage can be appropriately selected in proportion to the size of the reaction vessel.

  In the method for measuring nitrate ion concentration according to the present invention, the pH of the sample is preferably 5.5 to 8.0. The reason is as described above.

  In the method for measuring nitrate ion concentration of the present invention, the sample may use a buffer such as MOPS, MES, and phosphate, but preferably the sample uses a MOPS buffer having a concentration of 0.05 to 0.2M. It is characterized by that. The reason for selecting MOPS is as described above. If the buffer concentration is too high, the immobilized enzyme is unstable, and if the concentration is too low, the sensitivity time is relatively long.

  In the method for measuring nitrate ion concentration of the present invention, an electronic transition medium such as phenothiazines, triphenylmethanes, sulfonephthaleins and the like can be further added to the sample in order to mediate electronic transition of nitrate ion reduction reaction. Preferably, however, MV having a concentration of 1.0 to 3.0 mM is further added to the sample as an electron transition medium. If the MV concentration is too low, the signal is relatively low, and if the MV concentration is too high, the immobilized enzyme becomes unstable.

  In the method for measuring nitrate ion concentration according to the present invention, the nitrate reductase, unlike the conventional glucose oxidase for blood glucose measurement as a reductase, is greatly influenced by oxygen, and therefore, it interferes with oxygen against the reductase. In order to reduce the sample, an oxygen scavenger such as sulfite, metabisulfite and bisulfite can be added to the sample. Preferably, 0.5 to 4 mM sulfite is used as the oxygen scavenger in the sample. It is desirable to do. If the concentration of the oxygen scavenger is too low, the effect of removing oxygen is insufficient, and if the concentration is too high, it is not necessary at a higher concentration, and the immobilized enzyme layer becomes unstable.

  Hereinafter, the structure and effects of the present invention will be described in more detail with reference to specific examples and comparative examples. However, these examples are merely for the purpose of clearly understanding the present invention, and are within the scope of the present invention. Not trying to limit.

Electrochemical characteristics of nitrate reductase Electrochemical experiments were conducted with a three-electrode system. The working electrode was a glassy carbon disk electrode (3 mm), the auxiliary electrode was a helical Pt wire, and the reference electrode was Ag / AgCl (3M KCl). cDAQ-1604 potentiostat (Elbio, Korea) was used for cyclic voltage measurement (CV) and current-voltage recording. In experiments where current-voltage was recorded, an operating potential of -0.4 to 1.0 mV (vs. Ag / AgCl) was applied.

  FIG. 3 is a graph showing the results of circulating voltage measurement for each substrate (nitrate ion) concentration using nitrate reductase of the biosensor of the present invention, and showing the current intensity due to voltage change. In FIG. 3, it can be seen that the reduction current increases as the concentration of the substrate increases when nitrate reductase and an electron mediator are reacted with the working electrode. This means that nitrate reductase reduces nitrate to nitrite, and at the same time it can be seen that MV plays a mediator role. The same tendency was expressed when the concentration of the substrate was 50, 100 μM. According to the experimental results, if MV is continuously reduced electrochemically, a reduction current of nitrate ions can be obtained through an electrochemical method, and the concentration of an unknown substrate can be measured.

Production and Optimization of Sensing Electrode Sensor Strip Disposable SPCE electrode is screen-printed to fit the size of portable measuring instrument to 12.8mm wide and 65mm long, as shown in Fig. 1, polyethylene substrate (PE board) ) Made above. Pipette 3 μL of the solution containing oxygen scavenger, electron transition mediator, MOPS, and substrate onto the end of the disposable electrode with the enzyme immobilized. After the solution was aspirated, a voltage of -700 to -900 mV (vs. Ag / AgCl) was applied and data was collected using a cDAQ-1604 potentiostat (Elbio, Korea).

In order to fabricate a disposable strip type sensor of the enzyme in the present invention, first, PVA excellent in stability and storage stability was used in order to facilitate the fixation of the enzyme at the electrode site.
Experiments were performed separately for concentrations of 1% (wt), 2.5%, 5%, 10%, 15%, and 20%. The optimal PVA concentration was expressed as 1 to 5 wt%. The higher the concentration, the larger the noise, and the concentration lower than 1 wt% was not good because the signal was relatively low. FIG. 4 is a graph showing the amount of current change (ΔI) for each PVA concentration of the biosensor of the present invention.

  In order to determine the optimal amount of enzyme required for the generation of the electrode signal, experiments were performed with enzyme concentrations of 6.25, 12.5, 25, 50 mU / 3 μL. FIG. 5 is a graph showing the amount of current change (ΔI) depending on the concentration of nitrate reductase of the biosensor according to the present invention. The signal increases with an increase in the amount of immobilized enzyme, but the range of increase by concentration is greatly reduced as the enzyme concentration is higher. Desirably, the optimal amount of immobilized enzyme is set to 6.25 to 25 mU / 3 μL.

  In order to investigate the pH dependence of the nitrate measurement electrode, experiments were performed using MES buffer and MOPS buffer. FIG. 6 is a graph showing the amount of current depending on the pH of the sample of the biosensor of the present invention. Experiments were performed with MES buffer between pH 5.5 and 6.0, and MOPS buffer was used between 6.5 and 8.0. The data obtained from the MES buffer was normalized by comparing the data obtained from the pH 6.5, MES buffer with the data obtained from the pH 6.5, MOPS buffer. According to the experimental results, it is desirable to set the optimum pH to 6.5 to 7.0.

  In order to examine the effect of buffer concentration, experiments were conducted at pH 7.0 with MOPS buffer concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4M. FIG. 7 is a graph showing the sensitivity according to the concentration of the MOPS buffer of the biosensor of the present invention. Experimental results showed similar signals at all buffer concentrations. The signal was centered at 1.4% standard deviation based on an average sensitivity of 7.13. Since the stability of the immobilized enzyme is relatively poor at high buffer concentrations, the optimal buffer concentration was set to 0.05 to 0.2M.

  To determine the optimal MV (Electron Transition Mediator) concentration, the MV concentration was tested from 1 mM to 5 mM. FIG. 8 is a graph showing the apparent current according to the concentration of MV as an electronic transition mediator of the biosensor of the present invention. From the experimental results, the signal increased linearly as the concentration of MV increased. When comparing the amount of immobilized enzyme and the amount of substrate, the optimum MV concentration was preferably 1.0 to 3.0 mM.

In order to remove the enzyme's reaction to oxygen, experiments were conducted while varying the concentration of sulfurous acid known as the optimum oxygen scavenger at 0.5, 1, 2, 4, 6, 8, 10 mM. The oxygen removal reaction of SO ₃ ²⁻ is as follows: 2SO ₃ ²⁻ + O ₂ → 2SO ₄ ²⁻

  FIG. 10 is a graph showing the amount of current depending on the concentration of sulfite as an oxygen scavenger of the biosensor of the present invention. Considering simultaneously the oxygen that is dissolved in the buffer during the oxygen removal, the optimum oxygen scavenger concentration is preferably 0.5 to 4 mM.

  To obtain a calibration curve for the nitrate measuring electrode, duplicate experiments were performed with the substrate concentration varied between 50, 100, 150, 200, 250 and 300 mM (n = 30). FIG. 11 is a graph showing a change in the amount of current (ΔI) generated from the enzyme electrode system (strip type sensor) according to the nitrate concentration of the biosensor in the present invention. Looking at the experimental results, the linear equation was Y = 5.42X−38.43 (r2 = 0.996).

Optimization of Nitrate Ionization Reaction of Non-Nitrate Ionic Nitrogen FIG. 12 is a nitrate ionization reactor of ammonium salt ions and organic nitrogen compounds in the presence of 0.1 to 2.0 N sodium hydroxide as electrolyte. Depending on the concentration of the reactants, ammonium salt ions and organic nitrogen can be oxidized to nitrate ions in a reaction for 5 to 30 minutes using only a DC power source of 5 to 30 V and without an accompanying oxidant or catalyst. In consideration of the volume and concentration applicable to the biosensor, the reactor volume should be adjusted to 0.1 ml, and appropriate dilution and neutralization is necessary, preferably in the range of the biosensor's sensing ability. A 20 ml dilution tank was used as possible.

  FIG. 13 is a side view of a power supply device manufactured for the operation of the nitrate ionization reactor of the present invention. The power supply device is manufactured so that the supplied voltage and reaction time can be freely adjusted. It was adjusted automatically according to the concentration of the substance. The amount of current was adjusted to a maximum of 5 A and the maximum usable voltage to 30 V in order to allow a margin of increase / decrease in proportion to the concentration of the reactant.

  FIG. 14 is a graph showing the result of nitrate ionization of non-nitrate ion-ammonia and organic nitrogen (sodium glutamate) using the nitrate ionization reactor of the present invention, and shows the time required for the reaction at a voltage of 20V. This is a test result for determination, and the concentration of the substance used in the test was adjusted to 100 ppm. As a result, as shown in FIG. 14, no dramatic increase in reaction efficiency is shown after the reaction time of 10 minutes. Therefore, when testing in the field, it was determined that 10 minutes was appropriate considering the time for preparation, reaction, measurement, etc. When the number of samples is large, the number of reactors can be increased for processing, so it is determined that there is no significant increase in the overall processing time.

  FIG. 15 is a graph showing the result of nitrate ionization of non-nitrate ion-organic nitrogen (sodium glutamate) by an electrochemical catalytic oxidation method, and the concentration of the substance used in the test was adjusted to 100 ppm. As shown in FIG. 15, the voltage required for a sufficient oxidation reaction for 10 minutes is about 25 V, which corresponds to twice the vehicle battery voltage. Therefore, in view of the concentration of the treated substance and the small amount of organic nitrogen present in natural water, 15V is considered sufficient.

  Table 1 shows the efficiency of nitrate ionization of ammonia nitrogen, organic nitrogen, the number of rivers, sewage from a treatment plant, and the like measured by the biosensor for nitrate measurement of the present invention using the nitrate ionization reactor of the present invention. It summarizes the concentration of nitrogen. As can be seen from Table 1, the efficiency of nitrate ionization of ammoniacal nitrogen was as high as 97% or more. However, the efficiency of nitrate ionization of natural rivers, water flowing into sewage treatment plants, sewage being treated, etc. was generally about 85 to 95%, and the treatment efficiency was relatively high. This is presumably because natural rivers and sewage contain relatively higher amounts of ammonia, nitrite, nitrate nitrogen, etc. than the content of organic nitrogen. In addition, the total nitrogen concentration measured by the biosensor is very similar to the value measured by ion chromatography (IC), but this is a measurement method that links the nitrate ionization reaction of the sample and the biosensor. Represents that it can be reasonably applied for the measurement of total nitrogen.

  In Table 1, (a) is the amount of total nitrogen measured using hack (HACH), (b) is the amount of nitrate measured using ion chromatography (IC), (c ) Is the amount of nitrate measured using the biosensor of the present invention after being converted to nitrate using the nitrate ionization reactor of the present invention and diluted 100 times.

  As can be seen from Table 1, the accuracy of the nitrate ionization reactor of the present invention and the biosensor for detecting nitrate ions shows an error within 2 to 3%, which indicates that the accuracy is excellent. .

  Although preferred embodiments of the present invention have been described in detail as described above, those skilled in the art will recognize that the present invention can be variously modified without departing from the spirit and scope of the present invention as defined in the claims. Modifications could be made. Accordingly, changes to future embodiments of the invention will not depart from the technology of the invention.

  The disposable biosensor for detecting nitrate ions produced according to the present invention can conveniently measure the exact concentration of nitrate ions in the field using electrochemical methods. In addition, since the structure and manufacturing cost are low and portable, it is different from existing measurement products and has high price competitiveness.

  The nitrate ionization reactor of the present invention is a device that conveniently converts nitrogen compounds that are not nitrates into nitrates, and if combined with the nitrate ionization reactor and the biosensor for detecting nitrate ions, the total nitrogen in the sample Can be measured conveniently.

FIG. 4 is a one-time electrode model diagram used for manufacturing a sensing electrode of a biosensor as an example of the present invention. FIG. 5 is a flow chart showing a normal screen printing method for manufacturing the strip-shaped electrode of the present invention. It is a graph which shows the catalytic action of the nitrate reductase of the biosensor of this invention by the circulating voltage current method. It is a graph which shows the optimal PVA density | concentration of the biosensor of this invention. It is a graph which shows the quantity of optimal immobilization of nitrate reductase of the biosensor of the present invention. It is a graph which shows the optimal pH of the biosensor of this invention. It is a graph which shows the density | concentration of the optimal buffer (MOPS) of the biosensor of this invention. 2 is a graph showing the optimum concentration of electron transition mediator (MV) of the biosensor of the present invention. It is a graph which shows the result of having measured the change of the electric current by the density | concentration of an oxygen scavenger using CV. It is a graph which shows the density | concentration of the optimal oxygen scavenger (sulfite) of the biosensor of this invention. It is a graph which shows the calibration curve by nitrate concentration of a biosensor in the present invention. It is the perspective view of one example of the nitrate ionization reactor of this invention, and the top view of a lower electrode structure. It is a schematic diagram showing a detachable power supply device for operating the nitrate ionization reactor of the present invention and a timer integrated device capable of adjusting the reaction time. It is a graph which shows the grade of nitrate ionization of the nitrogen compound which is not nitrate by reaction time. It is a graph shown by the voltage which added the grade of nitrate ionization of the nitrogen compound which is not nitrate. It is a conceptual diagram which shows notionally the process in which ammonium salt ion is oxidized to nitrate ion by an electrochemical catalytic catalytic oxidation reaction. It is a conceptual diagram which shows notionally the process in which an organic nitrogen compound is converted into ammonia by an electrochemical catalytic catalytic oxidation reaction.

Claims (24)

  In a biosensor in which a working electrode, a relative electrode spaced apart from the working electrode and a reference electrode are arranged, the working electrode is a carbon electrode fixed with nitrate reductase, and the nitrate ion contained in the sample is A biosensor for detecting nitrate ions, wherein the degree of reduction reaction by nitrate reductase is electrochemically measured.   The biosensor for detecting nitrate ions according to claim 1, wherein the working electrode is a carbon paste electrode.   The biosensor for detecting nitrate ions according to claim 1, wherein 1 to 5% by weight of PVA is mixed in order to immobilize the nitrate reductase.   The biosensor for detecting nitrate ions according to claim 1, wherein the fixed amount of the nitrate reductase is 6.25 to 25 mU.   The biosensor for detecting nitrate ions according to claim 1, wherein the pH of the sample is 5.5 to 8.0.   The biosensor for detecting nitrate ions according to claim 1, wherein the sample uses a MOPS buffer having a concentration of 0.05 to 0.2M.   The biosensor for detecting nitrate ions according to claim 1, wherein MV having a concentration of 1.0 to 3.0 mM is further added to the sample as an electron transition medium.   The biosensor for detecting nitrate ions according to claim 1, wherein sulfite having a concentration of 0.5 to 4 mM is used as an oxygen scavenger in the sample. A reaction vessel, at least one or more TiO ₂ electrodes immersed in the reaction vessel, at least two or more Pt electrodes immersed in the reaction vessel, an input port through which a reactant can be introduced into the reaction vessel, and the reaction vessel A nitrate ionization reactor characterized by having a discharge port capable of discharging a reactant.   The nitrate ionization reactor according to claim 9, wherein the electrode has a protrusion.   11. The nitrate ionization reactor according to claim 10, wherein the protrusions are not in contact with each other and are alternately overlapped while being separated from each other. 10. The nitrate ionization reactor according to claim 9, wherein the electrode includes two Pt electrodes opposed in the horizontal direction, and a Pt electrode and a TiO ₂ electrode opposed in the vertical direction.   The nitrate ionization reactor according to claim 9, further comprising a dilution tank.   A total nitrogen concentration measurement system comprising: the biosensor for detecting nitrate ions according to claim 1; and the nitrate ionization reactor according to claim 9.   9. The step of putting a sample into the sample inlet of the biosensor according to claim 1, the step of applying a voltage to the working electrode of the biosensor, and the working electrode in the applying step Measuring the intensity of the current flowing between the electrode and the relative electrode, and a method for measuring a nitrate ion concentration.   A step of introducing the sample into a nitrate ionization reactor before being introduced into the sample inlet of the biosensor; The nitrate ion concentration measuring method according to claim 15, further comprising:   The method for measuring nitrate ion concentration according to claim 16, wherein sodium hydroxide is further added to the nitrate ionization reactor so that the sodium hydroxide concentration is 0.1 to 2.0N.   17. The nitrate ion concentration measuring method according to claim 16, wherein the power is supplied alternately in turn for each pair of electrodes.   The method for measuring nitrate ion concentration according to claim 16, wherein the reaction is performed for 5 to 30 minutes.   The method of measuring nitrate ion concentration according to claim 16, wherein the voltage of the power supply to be supplied is 5 to 30V.   The method for measuring a nitrate ion concentration according to claim 15, wherein the pH of the sample is 5.5 to 8.0 when the nitrate ion concentration is measured.   The method of claim 15, wherein the sample further includes 0.05 to 0.2M MOPS as a buffer.   The method of claim 15, wherein the sample further includes MV having a concentration of 1.0 to 3.0 mM as an electron transition medium.   The method for measuring nitrate ion concentration according to claim 15, wherein the sample further contains sulfite having a concentration of 0.5 to 4 mM as an oxygen scavenger.

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