KR101990703B1 - Method for measuring the concentration of target substance in a testing sample using a electrochemical sensor for point of care self-diagnosis - Google Patents

Abstract

The present disclosure relates to a method for measuring the concentration of a target substance in a sample using an electrochemical sensor for site self-diagnosis, the sensor comprising an enzyme immobilized on the surface of the working electrode,
Controlling the temperature of the sample to a constant temperature (T) determined by the following method;
Immersing the working electrode in the sample for a predetermined time (t) or longer;
Applying a redox voltage to the immersed working electrode; And
Calculating the concentration of the target substance in the sample from the current value measured corresponding to the voltage,
Here, the predetermined temperature (T)
(i) preparing an electrochemical sensor including a working electrode to which a sample containing a target substance and an enzyme reactive with the target substance are immobilized;
(ii) after immersing the working electrode in the sample adjusted to _{10 ℃ ≤T 1, T 2,} ..., T n of different temperatures _{(T 1, T 2, ...} , T n) ≤100 ℃ Measuring a current value with respect to a voltage at each temperature through a cyclic voltammetry (CV) method;
(iii) determining a maximum current value (I _max ), which is a maximum value of the measured current values, and a corresponding voltage (V _CV ); And
(iv) determining a temperature (T) having a current value (I _cv ) that is at least 90% of the maximum current value (I _max ) at the same voltage as the determined voltage (V _CV ) The temperature (T) relates to a method for measuring the concentration of a target substance in a sample by using an electrochemical sensor for in-situ self-diagnosis, which is determined by a temperature determination method in which the temperature (T) - 20 |

Description

[0001] The present invention relates to a method for measuring the concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis,

The present invention relates to a method for measuring the concentration of a target substance in a sample using an electrochemical sensor for site self-diagnosis, and more particularly, to a method for measuring an enzyme reaction temperature and an incubation time optimized for a site self-diagnosis using a cyclic voltammetry (CV) And a method for measuring the concentration of the target substance in the sample according to the temperature and time.

On-site self-diagnosis means that patients who have been diagnosed before they have to go to the hospital are examined by the patient themselves on the spot (where the patient is present) without the help of specialized medical personnel. For the purposes of diagnosis and prognosis of disease, determination of health condition, judgment of treatment effect of disease, prevention, etc., it is possible to test using a small amount of sample collected from human body.

For such field self-diagnosis, many electrochemical sensors or biosensors have been researched which measure the electrical signals from the chemical reaction of the target substance in the sample and calculate the concentration of the target substance in the sample. In recent years, there have been many studies on electrochemical sensors that calculate the concentration of a target substance in a sample by measuring the movement of an electron generated by an oxidation / reduction electrode reaction of a target material on a working electrode, that is, an electric current. In particular, recently, there is a demand for an electrochemical sensor for on-site self-diagnosis at low cost, simplicity, and ease of miniaturization. Therefore, researches on an electrochemical sensor having a sensor having a small size and easy portability and excellent sensitivity It is progressing.

On the other hand, an immuno-related substance (for example, an antigen or an antibody) as an object to be measured by an electrochemical sensor has a very low electrical conductivity and a low electrochemical reactivity. In the case of a target substance having no redox function in addition to such immuno-related substances, it is difficult to perform the analysis through the oxidation / reduction electrode reaction as described above. In order to measure the concentration of the target substance, introduction of a marker capable of electrochemically activating the target substance is necessary. As such a marker, an enzyme capable of reacting with the target substance to produce a product having high electrochemical activity is generally used.

However, these enzymes each have different optimal activity conditions. Unlike catalysts, which generally increase activity with increasing kinetic energy as the temperature increases, there is an optimal temperature for the enzyme to be maximally activated. The activity of the enzyme is proportional to the sensitivity of the electrochemical sensor. When the sensitivity of the sensor is taken into consideration, it is most preferable to perform the measurement at a temperature at which the activity of the enzyme becomes maximum. However, for the purpose of site self-diagnosis, adjusting the sample temperature to the maximum active temperature is not preferable in view of time and cost.

In addition, for the sensing by the electrochemical sensor, the reaction products of the samples and enzymes having high electrochemical activity generated by the enzyme must reach a certain concentration, and the incubation time of the sample and the enzyme also affects the sensitivity of the sensing It is an important factor. For the purpose that the incubation time is also for the field self-diagnosis, it is not preferable in terms of time to have a long incubation time until the concentration of the enzyme reaction product on the working electrode reaches the saturated state.

There is no prior literature on the electrochemical methods that can determine the reaction temperature conditions of the enzyme and the incubation time conditions of the enzyme which can meet the purpose of the site self-diagnosis and have excellent sensing sensitivity.

For example, the prior art KR 10-1740098 B1 relates to a biofuel cell comprising an aldose saccharide dehydrogenase, wherein the activity of the aldehyde dehydrogenase is greatly changed according to the reaction temperature, Compare the cyclic voltammetry curves at 25 ° C and 75 ° C. However, this is merely a demonstration of higher activity at 75 ° C for aldose dehydrogenase, which has activity at higher temperatures, and does not teach desirable enzyme reaction temperature conditions for site self-diagnosis.

The prior patent US 6413411 B1 also relates to a current measurement analysis method using a new disposable electroanalytic cell for determining the quantitation of biologically important compounds from body fluids, which suggests a specific incubation time for glucose, This is only a sufficient time to saturate to the maximum, and does not teach the optimal incubation time conditions for site self-diagnosis.

Therefore, from the viewpoint of the present disclosure, the enzyme reaction temperature and the incubation time, which meet the purpose of the field self-diagnosis and have excellent sensitivity, are determined for each target substance and enzyme, and the concentration of the target substance in the sample And a method of measuring by using an electrochemical sensor.

A method for measuring the concentration of a target substance in a sample using an electrochemical sensor for site self-diagnosis to achieve the first aspect of the present disclosure, the sensor comprising an enzyme immobilized on the surface of the working electrode, Controlling at a constant temperature (T) determined by the following method; Immersing the working electrode in the sample for a predetermined time (t) or longer; Applying a redox voltage to the immersed working electrode; And calculating the concentration of the target substance in the sample from the current value measured corresponding to the voltage, wherein the predetermined temperature (T) is a temperature of the sample, wherein (i) the sample containing the target substance and Preparing an electrochemical sensor including an enzyme-immobilized working electrode; (ii) after immersing the working electrode in the sample adjusted to _{10 ℃ ≤T 1, T 2,} ..., T n of different temperatures _{(T 1, T 2, ...} , T n) ≤100 ℃ Measuring a current value with respect to a voltage at each temperature through a cyclic voltammetry (CV) method; (iii) determining a maximum current value (I _max ), which is a maximum value of the measured current values, and a corresponding voltage (V _CV ); And (iv) determining a temperature (T) having a current value (I _cv ) that is greater than or equal to 90% of the maximum current value (I _max ) at a voltage equal to the determined voltage (V _CV ) (T) is determined by the temperature determination method, which is the temperature at which the temperature (T) - 20 | has the minimum value. According to an embodiment of the present disclosure, the predetermined time (t) includes: (a) preparing an electrochemical sensor including a sample containing a target substance and a working electrode to which an enzyme reacting with the target substance is immobilized; (B) the electrode is heated for 15 seconds? T ₁ , t ₂ , ..., t _m (T ₁ , t ₂ ,..., T _m ) of ≤60 min., The current value for the voltage supply time with respect to each immersion time is measured by a time-of-current (Chronoamperometry, CA) Measuring; (C) calculating an integrated value of the current value from 10 seconds to 15 seconds after the voltage is applied using the data of the current value for the measured time for each immersion time; (D) determining a minimum time (t) in which the variation coefficient of the integral values when the immersion time is equal to or greater than the time (t) in the calculated integrated values is within 2% is determined by the immersion time determination method .

According to one embodiment of the present disclosure, the sample is a body fluid obtained from a human body.

According to one embodiment of the present disclosure, the target substance is one selected from the group consisting of an antigen, an antibody, a protein, an immunomolecule, DNA, and RNA.

According to one embodiment of the present disclosure, the enzyme is an enzyme capable of converting a target substance into an enzyme reaction product capable of oxidation / reduction reaction.

According to one embodiment of the present disclosure, the enzyme is selected from the group consisting of Horseradish peroxidase (HRP), Alkaline phosphatase (ALP), Glucose Oxidase, Luciferase, Beta-di-Galactosidase -galactosidase, malate dehydrogenase (MDH), and acetylcholinestrerase.

According to one embodiment of the present disclosure, the working electrode is indium tin oxide (ITO).

According to one embodiment of the disclosure, the enzyme is immobilized on the surface of the working electrode through a linking compound.

According to one embodiment of the present disclosure, n is an integer of 3? N? 20.

According to one embodiment of the present disclosure, m is an integer of 3? M? 15.

According to one embodiment of the present disclosure, the voltage supply time in step (b) is 40 to 60 seconds.

According to one embodiment of the present disclosure, the applied redox voltage is a constant voltage or a circulating voltage.

By measuring the concentration of the target substance in the sample of the present disclosure, it is possible to determine the enzyme reaction temperature at a temperature as close as possible to room temperature while keeping the sensitivity of the enzyme excellent. This makes it possible to eliminate or minimize the unnecessary temperature controller in the electrochemical sensor for on-site self-diagnosis, thereby reducing the cost.

In addition, through the method of the present disclosure, it becomes possible to determine the optimum incubation time to perform the electrochemical measurement, so that it is possible to measure more rapidly than the conventional field self-diagnosis sensor while having excellent sensing sensitivity.

Unlike the method of measuring the absorbance using the color change used in the quantitative measurement of the conventional sample, it is necessary to separately prepare the sample having the same number of times of inspection due to the color change of the enzyme reaction product, It is possible to determine the enzyme reaction temperature and incubation time which are suitable for the self-diagnosis of the enzyme in each case more quickly and easily.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a mechanism of an enzyme reaction and a redox reaction of a target substance according to one embodiment of the present disclosure; FIG.
Figure 2 is a schematic view of an enzyme immobilized on a working electrode surface according to one embodiment of the present disclosure;
3 is a graph of current values versus time measured by the cyclic voltammetry at each temperature according to one embodiment of the present disclosure;
4 is a graph of current values versus voltage supply times measured by the time zone current method for each immersion time according to one embodiment of the present disclosure;
FIG. 5 is a graph showing an integrated value of a current value for 10 to 15 seconds measured by the time-of-day current method according to one embodiment of the present disclosure; FIG.

The objectives, specific advantages, and novel features of the present disclosure will become more apparent from the following detailed description and preferred embodiments, which are to be read in connection with the accompanying drawings, wherein the disclosure is not necessarily limited thereto. In the following description of the present invention, detailed description of known related arts will be omitted when it is determined that the gist of the present disclosure may be unnecessarily obscured.

The present invention relates to a method for measuring the concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis, wherein the sensor includes an enzyme immobilized on the surface of the working electrode, ; Immersing the working electrode in the sample for a predetermined time (t) or longer; Applying a redox voltage to the immersed working electrode; And calculating the concentration of the target substance in the sample from the current value measured corresponding to the voltage.

The sample used in the measurement in this disclosure may be a body fluid obtained from an animal. More specifically, the sample may be a body fluid obtained from a human body. Herein, the body fluid refers to, for example, blood, urine, saliva, and the like, and is not limited to the above example, but can be used if the target substance to be analyzed exists in the body fluid.

In the present disclosure, a target substance to be measured using an electrochemical sensor may be an antigen, an antibody, a vitamin, a protein, an immunological molecule, DNA, RNA, and the like. According to one embodiment of the present disclosure, the target material may be a substance that can not undergo an oxidation reaction or a reduction reaction itself. For this purpose, the methods of the present disclosure involve the use of the markers described below. The target substance may be, for example, ascorbic acid-2-phosphate or 4-aminophenyl phosphate, but is not limited thereto.

The labeling agent used in the present disclosure is an enzyme, and the enzyme is an enzyme capable of converting a target substance into an enzyme reaction product capable of oxidation / reduction reaction. For example, the enzyme may be selected from the group consisting of alkaline phosphatase (ALP), horseradish peroxidase (HRP), glucose oxidase, luciferase, beta-di-galactosidase (? -D-galactosidase), malate dehydrogenase (MDH), acetylcholinesterase, and the like. Particularly preferably, the enzyme may be ALP or HRP. ALP and HRP have the advantages of highly sensitive reactivity, and in particular, HRP is inexpensive and has an advantage in terms of cost. There is also an advantage in that ALP has a long signal retention time of about 24 to 48 hours.

Illustratively, the mechanism of the enzyme reaction by the ALP and the redox reaction of the enzyme reaction product is shown in FIG. Referring to FIG. 1, ascorbic acid-2-phosphate (AAP), which is a target substance, is converted to ascorbic acid (AA) as an enzyme reaction product by separating phosphate functions by ALP. The ascorbic acid is oxidized to dehydroascorbate by oxidation voltage. The electrochemical sensor can quantitatively measure a target substance in a sample by an electrical signal generated during the oxidation reaction, that is, by electrons. In FIG. 1, ascorbic acid-2-phosphate itself, which is a target substance to be measured for concentration, is accompanied by an enzyme reaction process in which ALP is converted into ascorbic acid capable of oxidation-reduction reaction because oxidation- .

The working electrode used in the present disclosure is not particularly limited as long as the enzyme can be immobilized on the working electrode and current can flow through the enzyme reaction product. For example, the working electrode may be a mercury electrode, an amalgam electrode, a noble metal electrode such as a Pt, Au, Pd or Rh electrode, a pyrolytic graphite electrode, a glassy carbon electrode, A carbon electrode such as indium tin oxide (ITO) electrode, or the like. Preferably, the working electrode may be an ITO electrode. In particular, when the ITO electrode is applied to a biosensor, a noise signal can be reduced, and a substrate on which an ITO thin film is deposited on an organic substrate can be used without a separate adhesive layer. Since the ITO substrate can utilize the thin film substrate manufacturing process having a high and stable electric conductivity characteristic on a large substrate in the display industry or the like, the ITO substrate has an advantage that the electrode can be manufactured at a very low cost.

In the present disclosure, the enzyme is immobilized on the working electrode. According to one embodiment of the present disclosure, the enzyme may be immobilized on the working electrode through a linking compound. Here, the term " linking compound " refers to a compound that links a working electrode and an enzyme. Biotin-avidin or biotin-streptavidin, which is a biochemical reactive compound, may be used as the linking compound. Also, as the chemical covalent bond, a carbodiimide crosslinking agent or a succinimide crosslinking agent which binds to the primary amine of the protein can be used. Preferably, a biotin-avidin binding compound can be used. In the case of using a chemical covalent bond, there is a randomness of the orientation of the enzyme to be immobilized. However, in the case of using the biotin-avidin binding compound, the control of the surface orientation direction of the binding site of the immobilized enzyme is easy, By appropriately controlling the direction of the enzyme on the surface, it is possible to maximize the expression of the surface area of the binding site of the enzyme, thereby maximizing the detection ability of the target substance in the sample.

 Figure 2 is a schematic image of a platform of a working electrode according to one embodiment of the present disclosure showing the ALP immobilized through the binding of biotin-avidin on the ITO electrode.

In the present disclosure, the temperature of the sample can be controlled at a constant temperature (T) during the sample inspection using the electrochemical sensor. The constant temperature (T) can be determined by the following method:

As a method of determining the temperature (T)

(i) preparing an electrochemical sensor including a working electrode to which a sample containing a target substance and an enzyme reactive with the target substance are immobilized;

(ii) after immersing the working electrode in the sample adjusted to _{10 ℃ ≤T 1, T 2,} ..., T n of different temperatures _{(T 1, T 2, ...} , T n) ≤100 ℃ Measuring a current value with respect to a voltage at each temperature through a cyclic voltammetry (CV) method;

(iii) determining a maximum current value (I _max ), which is the maximum value of the current values, and a corresponding voltage (V _CV ); And

(iv) determining a temperature (T) having a current value (I _cv ) that is at least 90% of the maximum current value (I _max ) at the same voltage as the determined voltage (V _CV ) The temperature (T) is the temperature at which the temperature (T) - 20 | has the minimum value.

According to one embodiment of the present invention, the determination of the predetermined temperature (T) can be performed preferentially, apart from measuring the concentration of the target substance in the sample using the electrochemical sensor for on-site self-diagnosis. Since the determination of the constant temperature T requires a plurality of measurements at various temperatures, it is preferable that the determination is performed in a laboratory rather than a site requiring rapid diagnosis.

First, an electrochemical sensor having a working electrode is prepared by selecting a target material, preparing a sample containing the target material, and fixing an enzyme capable of reacting with the target material to generate a redox reaction product. For the target substance, sample, and enzyme, the embodiments of the present disclosure described above are applicable, but are not limited thereto.

Subsequently, the sample is adjusted to different temperatures (T ₁ , T ₂ , ..., T _n ), and then the working electrode is immersed in the sample. The immersion is performed for incubation of the sample and the enzyme. For the determination of the constant temperature (T) of the present disclosure, the immersion is preferably carried out for the same period of time.

According to one embodiment of the present disclosure, the different temperatures may be T _{n -1} < T _n . Where T _n is the temperature at which the next current measurement is performed after the current measurement at T _{n -1} . According to another embodiment of the _{disclosure, T n -o-1 <T} n <T n - may be a _o. Where o is an integer of 1 ≤ o ≤ n-2.

According to one embodiment of the present disclosure, T _n -T _{n -1} may be constant. At this time, it is preferable that the above-mentioned T _n -T _{n -1} is 10 ° C, more preferably 5 ° C or less from the viewpoint of accuracy of the constant temperature (T) crystal. According to another embodiment of the present disclosure, T _n -T _{n -1} may be different. Determining a constant temperature (T) is a bar in the measurement of high current between the _m may be estimated, in which case, optimization than _- previously measured while going through the above-Temperature measurement T _{-m n-1} and T _n Lt; _{RTI ID = 0.0 & gt;} Tn _- Tn _-1 &lt; / RTI &gt;

According to one embodiment of the present disclosure, the different temperatures T ₁ , T ₂ , ..., T _n may be 10 ° C ≤ T ₁ , T ₂ , ..., T _n ≤ 100 ° C, n is an integer of 3? n? 20. Preferably, n is 4? N? 15, more preferably 5? N? 10. If n is less than 3, a problem may occur that the reliability against the maximum current value I _max is lowered. On the other hand, if n exceeds 20, time and cost problems due to excessive repetitive measurement may occur.

The working electrode immersed in the sample at the different temperature can be measured for the voltage at each temperature through the circulating voltage method. The circulating voltage method is a method of measuring the current by circulating the voltage of the working electrode at a constant rate, thereby obtaining a cyclic voltage / current curve as shown in FIG. The redox reaction occurring on the electrode surface can be grasped by the cyclic voltammetry. In the cyclic voltammetry, the initial voltage and the reverse voltage can be appropriately set for each enzyme so that the redox signal of the electrode can be observed. As shown in FIG. 3, a voltage of 0 to 0.6 V can be set to grasp the redox reaction of AA produced by ALP, for example.

Conventionally, quantitative determination of a target substance is mainly performed by measuring the reflectance or transmittance of light using a photometer using a dye source that changes color when a target substance or an enzyme reaction product is oxidized. In this case, there is a problem that it is necessary to separately prepare the same samples as the number of quantification times. On the contrary, when the cyclic voltammetry used in the present disclosure is used, it is possible to carry out continuous quantification with the sample to be loaded in one container, so that the constant temperature (T ) Can be determined. On the other hand, it has been confirmed that the method for determining the enzyme reaction temperature suitable for the field self-diagnosis using this cyclic voltammetry has not been disclosed before the filing of this disclosure.

The maximum current value (I _max ) of the enzyme can be determined by measuring the current value with respect to the voltage at each temperature through the circulating voltage method. The maximum current value I _max means the highest current value measured for all measurement results at different temperatures. The generation of the most enzyme reaction product means the highest current value, and it is possible to specify the maximum activation temperature at which the enzyme can be most activated through the maximum current value I _max .

Further, the voltage (V _CV ) applied when the maximum current value (I _max ) is determined can also be specified together. It is possible to compare the current value when the voltage equal to the voltage (V _CV ) is applied at the specific temperature from the measured data to the maximum current value (I _max ). Accordingly, the current value I _CV at the voltage V _CV is 90% or more, preferably 92% or more, more preferably 95% or more, and most preferably 99% or more of the maximum current value I _max . (%) &Lt; / RTI &gt; At the same time, the temperature (T) is the temperature at which the temperature (T) -20 is the minimum value, which is required to be nearest to the room temperature in order to meet the purpose of the on-site self-diagnosis. If the current value I _CV is less than 90% of the maximum current value I _max , the temperature may be more approximate to room temperature, but the performance of the sensing sensitivity may be reduced. As a result, The accuracy may be lowered in the process of measuring the concentration of the target substance in the sample.

On the other hand, in the present disclosure, at the time of sample inspection using an electrochemical sensor, the working electrode can be incubated or immersed in the sample for a predetermined time (t). The predetermined time t may be determined by the following method:

As a method of determining the immersion time (t)

(A) preparing an electrochemical sensor including a sample containing a target substance and a working electrode to which an enzyme reacting with the target substance is immobilized;

(B) The electrode is heated for 5 seconds? T ₁ , t ₂ , ..., t _m (T ₁ , t ₂ ,..., T _m ) of ≤60 min., The current value for the voltage supply time with respect to each immersion time is measured by a time-of-current (Chronoamperometry, CA) Measuring;

(C) calculating an integrated value of the current value from 10 seconds to 15 seconds after the voltage is applied using the data of the current value for the measured time for each immersion time;

(D) determining a minimum time (t) within which the coefficient of variation of the integral values when the immersion time is equal to or greater than the time (t) among the calculated integrated values is within 2%.

According to one embodiment of the present invention, the determination of the predetermined time t may be performed preferentially, separately from the measurement of the concentration of the target substance in the sample using the electrochemical sensor for site self-diagnosis. Since the determination of the predetermined time t requires a plurality of measurements at various immersion times, it is preferable that the determination is performed in a laboratory rather than a site requiring rapid diagnosis.

First, an electrochemical sensor having a working electrode is prepared by selecting a target material, preparing a sample containing the target material, and fixing an enzyme capable of reacting with the target material to generate a redox reaction product. For the target substance, sample, and enzyme, the embodiments of the present disclosure described above are applicable, but are not limited thereto.

Then, the immersed in the sample during the working electrode a different time _{(t 1, t 2, ...} , t m). For determination of the constant immersion time (t) of the present disclosure, the temperature of the sample is preferably carried out at the same temperature. According to one embodiment of the present disclosure, the temperature may be a temperature determined by the determination method at a predetermined temperature (T) described above.

According to one embodiment of the present disclosure, the different times may be t _{m -1} < t _m . Where t _m is the time at which the working electrode for the next current measurement is immersed after the current measurement at t _{m -1} . According to another embodiment of the present _{disclosure, t m -p-1 <t} m <t m - may be a _p. Here, p is an integer satisfying 1? P? M-2.

According to one embodiment of the disclosure, t _m -t _{m -1} may be constant. At this time, the t _m -t _{m -1} to 2 minutes, preferably 1 minute, more preferably preferably from the point of view of accuracy of the crystal is not more than 30 seconds a predetermined time (t). According to another embodiment of the present disclosure, t _m -t _{m -1} may be different. In the course of the measurement according to the immersion time, there may be an optimal immersion time between the previously measured t _{m -p-1} and t _{m -p} . In this case, in order to determine a certain time (t) t _m -t _m-1 may be different.

According to one embodiment of the present disclosure, the different times t ₁ , t ₂ , ..., t _m may be 15 seconds t ₁ , t ₂ , ..., t _m 60 minutes, where m Is an integer of 3? M? 15. Preferably, m is 4? M? 12, more preferably 5? M? 10. If m is less than 3, the reliability of the coefficient of variation may be lowered. On the other hand, if m exceeds 15, temporal and cost problems due to excessive repetitive measurement may occur.

The current value for the voltage supply time can be measured through the time zone current method for each immersion time in the working electrode immersed for the different time. The time zone current method is a method in which a voltage of a sufficiently large value that can induce an electrochemical reaction is applied to a balanced electrode in steps so that a current flow is observed. . &Lt; / RTI &gt; As a result, the current curve of the time zone as shown in FIG. 4 can be obtained. In the time-domain current method, the applied constant voltage can be appropriately set for each enzyme so that the current signal by the oxidation-reduction reaction can be observed.

As described above, the quantification of the conventional target substance is mainly performed by measuring the degree of change of color by using a dye source that causes a change in color when a target substance or an enzyme reaction product is oxidized by using a photometer to measure light reflectance or transmittance Respectively. In this case, there is a problem that it is necessary to separately prepare the same samples as the number of quantification times. Alternatively, in the case of using the time-period current method used in the present disclosure, it is preferable to continuously immerse a sample in a container for a predetermined time after immersing the sample for a predetermined time and then applying a constant voltage, observing a change in current, It is possible to determine the constant temperature (T) of the enzyme reaction which is suitable for the self-diagnosis of the enzyme by the enzyme more quickly and easily. On the other hand, it has been confirmed that there has not been disclosed a method of determining the immersion time of the working electrode suitable for the field self-diagnosis by using the time zone current method.

It is possible to calculate the integral value of the current value for a specific time through the data by measuring the current value with respect to the different immersion time through the time zone current method. According to one embodiment of the present disclosure, the integrated value of the current value from 10 seconds to 15 seconds after the voltage is applied can be individually calculated for each immersion time using the data of the current value for the measured time have. In the case of 10 seconds after the application of the voltage, there is a problem that the integration value of the immersion time is not accurate due to the noise generated when the constant voltage is applied. On the other hand, when the integration value is calculated based on the time zone after 15 seconds, the amount of electrons emitted or absorbed in the reaction is reduced due to the progress of the oxidation reduction reaction, .

The integrated values of the immersion time with respect to time t and the variation coefficient of the integrated values when the immersion time is equal to or longer than the time t are within 2% (T) within 1.8%, more preferably within 1.5%. In order to meet the purpose of on-site self-diagnosis, it is advantageous that the immersion time is short with excellent sensing sensitivity, and it is important to determine the minimum time while satisfying the limit range of the coefficient of variation.

Herein, the coefficient of variation is defined as the standard deviation divided by the arithmetic mean. In this disclosure, the standard deviation of the integration values between 10 and 15 seconds is calculated from the results of the time- The coefficient of variation was calculated by dividing by the average.

When the coefficient of variation exceeds 2%, the enzyme reaction product is sufficiently deposited on the working electrode, that is, it has not reached the saturation state, which causes a problem of error in quantitative measurement.

The enzyme reaction temperature and working electrode immersion time determined by the above determination methods can be applied to a method for measuring the concentration of a target substance in the sample of the present disclosure.

Thereafter, the redox voltage is applied to the working electrode immersed for the predetermined time (t) or more. The applied voltage may be appropriately applied to each enzyme reaction product so as to induce a redox reaction.

The current value can be measured corresponding to the applied voltage. According to one embodiment of the present disclosure, a cyclic voltage method or a time zone current method may be used for measuring the current value. From the measured current value, the concentration of the enzyme reaction product can be calculated in consideration of the oxidation-reduction reaction formula of the enzyme reaction product, and the concentration of the target substance in the sample can be calculated from the concentration of the enzyme reaction product.

In the electrochemical sensor used in the present disclosure, an appropriate enzyme reaction temperature and an immersion time can be predetermined in correspondence with a target substance in a sample to be measured and an enzyme reacting therewith. If desired, the electrochemical sensor may include a temperature controller. Conventional sensors have a high temperature which has to be elevated through a temperature controller in consideration of maximizing the activity of the enzyme, and thus the time required for the enzyme is long. Thus, the electrochemical sensor of the present disclosure has an enzyme reaction Determining the temperature has the advantage of reducing the energy and time required for the temperature regulator. Particularly preferably, when the enzyme reaction temperature is room temperature, the electrochemical sensor may not include a temperature control device. On the other hand, with regard to the immersion time, it is possible to provide a manual or the like through immersion for a predetermined period of time or more determined by the method of the present disclosure, thereby making it possible for the user to enjoy the convenience in the field.

Hereinafter, preferred embodiments are described to facilitate understanding of the present disclosure, but the following embodiments are provided for easier understanding of the disclosure, and the present disclosure is not limited thereto.

Example

Manufacturing of platform

2, the surface of the ITO electrode (24 mm ² ) was subjected to sonication treatment in 10 mL of each of Trichlorethylene solution, ethanol solution and DI water for 15 minutes. Then, an ammonium hydroxide solution, hydrogen peroxide, DI water at a ratio of 1: 1: 5, and the electrode was immersed in a 75 ° C thermostat for 1 hour and 30 minutes. Then, the electrode was washed in DI water, and the OH group was finally activated on the electrode surface. Mu] g / ml avidin at room temperature for about 2 hours. Through hydrophobic interaction, avidin was physically adsorbed on the ITO electrode surface. Then, the electrode and 15 의 of 1% bovine serum albumin (BSA) were reacted at 4 캜 for about 24 hours. Then, the electrode and 15 μl of biotin-adhered ALP (10 μg / ml) were reacted at 4 ° C. for about 30 minutes.

Determination of Enzyme Reaction Temperature of Electrochemical Sensor for Field Self-Diagnosis

Ascorbic acid-2-phosphate (AAP) was used as the target substance, and ALP was used as the enzyme. After the temperature of the sample containing the AAP was adjusted to 15 캜, the previously prepared platform was immersed as a working electrode in the sample. After the immersion, the current value with respect to the voltage was measured using the circulating voltage method. The voltage was cycled between 0 and 0.6V.

1 mM solid AAP powder was dissolved in a buffer solution prepared by using 50 mM Tris-HCl (pH 9.6), 10 mM MgCl ₂ and water, and the sample was heated at 20 ° C, 25 ° C and 30 ° C After adjusting, the current value with respect to the voltage was measured in the same manner as above. The cyclic voltage-current curve thus obtained is shown in Fig.

When the working electrode containing ALP is immersed in the sample, the ALP and the AAP in the sample react to form ascorbic acid (AA), which is deposited on the ITO surface. Then, when a voltage is applied on the electrode, AA is oxidized into dehydrocysteic acid salt, and current is generated. The larger the current value measured, the better the oxidation reaction was, and the greater the amount of AA was generated in the sample. This indicates that the enzyme was activated at a certain temperature during the same immersion time.

As shown in FIG. 3, the current value with respect to the voltage at each temperature has a maximum current value (I _{max at 30 ° C.} = 6.10 μA) _{at 30 ° C. and} 0.34 V, respectively. It can be seen that a current value (I = 6.09 μA) of 90% or more of the maximum current value (I _{max at 30 ° C.} = 6.10 μA) was measured _{at 25 ° C.} have. In contrast, it was confirmed that the current values (I = 2.51 μA, I = 4.64 μA) at the time of application of 0.34 V at 15 ° C. and 20 ° _C. were less than 90% of the maximum current value (I _{max at 30 ° C.} = 6.10 μA).

From the above measurement results, it is determined that the reaction temperature of the enzyme suitable for on-site self-diagnosis is about 25 캜.

Determination of immersion time of electrochemical sensor for field self-diagnosis

A target substance, a sample, and an enzyme which are the same as the determination of the enzyme reaction temperature were used. The AA formed by the reaction of ALP and AAP is immersed on the surface of ITO until the surface of ITO reaches the saturation state. Considering this, the minimum time for the ITO surface to reach the saturation state can be regarded as the optimum immersion time required for the electrochemical sensor for field self-diagnosis. The change in the current value was measured through the time-of-day current method using the platform thus fabricated as the working electrode, with the time during which the working electrode was immersed in the sample as a variable. The voltage applied between the working electrode and the counter electrode was a constant voltage of 0.45 V at the Ag / AgCl reference electrode.

After immersing the working electrode in the sample for 5 seconds, the voltage was applied. The change of the current value was measured for about 50 seconds through the time zone current method. The voltage application was then stopped and immersed for the next immersion time. This was repeated at 30 seconds, 1 minute, 1 minute, 30 seconds, 2 minutes, 3 minutes, 5 minutes and 7 minutes. The time-period current curve measured according to the time-of-day current method is as shown in FIG.

Then, the integrated value of the current value in the interval of 10 to 15 seconds was calculated for each immersion time in the curve of FIG. The integration value for each immersion time is shown in Fig.

The calculated values of the respective coefficient of variation are shown in Table 1 below.

Stability estimation based on the integral value variation coefficient of the current value with each incubation time The average of the integration values by immersion time in the 10-15s interval
(N = 3) * 5s 30s 1m 1.5m 2m 3m 5m 7m Average Standard Deviation Coefficient of variation (CV) Immersion time 5s-7m 1.47E-05 1.72E-05 1.86E-05 1.93E-05 2.01821E-05 2.05796E-05 2.06213E-05 2.09043E-05 1.90E-05 2.15E-06 11.33% Immersion time
30s-7m 1.72E-05 1.86E-05 1.93E-05 2.01821E-05 2.05796E-05 2.06213E-05 2.09043E-05 1.96E-05 1.36E-06 6.91% Immersion time
1m-7m 1.86E-05 1.93E-05 2.01821E-05 2.05796E-05 2.06213E-05 2.09043E-05 2.00E-05 8.87E-07 4.43% Immersion time
1.5m-7m 1.93E-05 2.01821E-05 2.05796E-05 2.06213E-05 2.09043E-05 2.03E-05 6.11E-07 3.01% Immersion time
2m-7m 2.01821E-05 2.05796E-05 2.06213E-05 2.09043E-05 2.06E-05 2.97E-07 1.44%

* Using the average values of the currents using three electrode platforms manufactured in the same way.

Here, it is confirmed that the coefficient of variation of the integral values when the immersion time is 2 minutes or more is 2% or less.

On the other hand, the coefficient of variation of the integral values when the immersion time is 1 minute and 30 seconds or more exceeds 2%. Therefore, it is determined that the immersion time of the electrochemical sensor for field self-diagnosis when the target substance is ALP high enzyme is about 2 minutes.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it is to be understood that the present invention is not limited to the specific embodiments thereof, It will be understood that various modifications may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Claims (12)

A method for measuring the concentration of a target substance in a sample using an electrochemical sensor for field self-diagnosis, the sensor comprising an enzyme immobilized on the surface of the working electrode,
Controlling the temperature of the sample to a constant temperature (T) determined by the following method;
Immersing the working electrode in the sample for a predetermined time (t) or longer;
Applying a redox voltage to the immersed working electrode; And
Calculating the concentration of the target substance in the sample from the current value measured corresponding to the voltage,
Here, the predetermined temperature (T)
(i) preparing an electrochemical sensor including a working electrode to which a sample containing a target substance and an enzyme reactive with the target substance are immobilized;
(ii) after immersing the working electrode in the sample adjusted to _{10 ℃ ≤T 1, T 2,} ..., T n of different temperatures _{(T 1, T 2, ...} , T n) ≤100 ℃ Measuring a current value with respect to a voltage at each temperature through a cyclic voltammetry (CV) method;
(iii) determining a maximum current value (I _max ), which is a maximum value of the measured current values, and a corresponding voltage (V _CV ); And
(iv) determining a temperature (T) having a current value (I _cv ) that is at least 90% of the maximum current value (I _max ) at the same voltage as the determined voltage (V _CV ) A method for measuring the concentration of a target substance in a sample using an electrochemical sensor for in-situ self-diagnosis, wherein the temperature (T) is determined by a temperature determination method having a temperature at which the temperature (T) - 20 | The method according to claim 1,
The predetermined time (t)
(A) preparing an electrochemical sensor including a sample containing a target substance and a working electrode to which an enzyme reacting with the target substance is immobilized;
(B) the electrode is heated for 15 seconds? T ₁ , t ₂ , ..., t _m (T ₁ , t ₂ ,..., T _m ) of ≤60 min., The current value for the voltage supply time with respect to each immersion time is measured by a time-of-current (Chronoamperometry, CA) Measuring;
(C) calculating an integrated value of the current value from 10 seconds to 15 seconds after the voltage is applied using the data of the current value for the measured time for each immersion time;
(D) determining a minimum time (t) within which the coefficient of variation of the integral values when the immersion time is equal to or greater than the time (t) among the calculated integrated values is within 2% A method for measuring the concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis. The method according to claim 1,
And measuring the concentration of the target substance in the sample using the electrochemical sensor for on-site self-diagnosis, wherein the sample is a body fluid obtained from a human body. The method according to claim 1,
Wherein the target substance is one selected from the group consisting of an antigen, an antibody, a protein, an immunomolecule, DNA, and RNA, and measuring the concentration of the target substance in the sample using the electrochemical sensor for on-site self-diagnosis. The method according to claim 1,
Wherein the enzyme is an enzyme capable of converting a target substance into an enzyme reaction product capable of oxidation / reduction reaction. The method for measuring the concentration of a target substance in a sample using an electrochemical sensor. The method according to claim 1,
The enzyme may be selected from the group consisting of HRP (Horseradish peroxidase), ALK (alkaline phosphatase), glucose oxidase, luciferase,? -D-galactosidase, MDH malate dehydrogenase, acetylcholinesterase, and acetylcholinesterase. The method for measuring the concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis. The method according to claim 1,
Wherein the working electrode is ITO (Indium Tin Oxide). A method for measuring a concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis. The method according to claim 1,
Wherein the enzyme is immobilized on the surface of the working electrode through a linking compound. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein n is an integer satisfying 3 &lt; = n &lt; = 20, wherein the concentration of the target substance in the sample is measured using an electrochemical sensor for on-site self-diagnosis. The method of claim 2,
and m is an integer satisfying 3? m? 15. 2. A method for measuring a concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis. The method of claim 2,
Wherein the voltage supplying time in the step (b) is 40 to 60 seconds. A method for measuring a concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis. The method according to claim 1,
Wherein the applied redox voltage is a constant voltage or a circulating voltage. A method for measuring a concentration of a target substance in a sample using an electrochemical sensor for on-site self-diagnosis.

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