KR20190127349A - A method for electrochemically measuring the concentration of biomarkers in the urine - Google Patents

Abstract

The present disclosure relates to a method for measuring the concentration of antigen in urine using an electrochemical sensor, wherein the sensor comprises an antibody immobilized on the surface of a working electrode. The method comprises the steps of: measuring the pH of the urine; dipping the working electrode in the urine; treating the immersed working electrode with an electrical redox enzyme, and then immersing in a solution containing an electrical redox substrate, followed by application of a redox voltage to measure an electrical signal generated by the redox reaction; and correcting the measured electrical signal with an electrical signal at a predetermined pH using the trend of antigen and antibody binding according to pH.

Description

A method for electrochemically measuring the concentration of biomarkers in the urine

The present disclosure relates to a method of electrochemically measuring the concentration of biomarkers of bladder cancer or prostate cancer present in urine, and more particularly, in consideration of the binding degree of antigen antibodies according to pH. It relates to a method of electrochemically measuring the concentration.

The study of urine disease diagnosis began to attract attention in 1958 when urine test papers were developed to test sugars and proteins. Research is ongoing. When urine is used to diagnose a variety of diseases, including prostate or bladder cancer, it is possible to obtain samples by non-invasive methods, unlike invasive methods such as blood collection, thereby reducing the physical and physical burden on the patient. Has an advantage.

In addition, in contrast to the conventional urine test paper to determine the presence of a disease, depending on the visual judgment such as discoloration, in the case of measuring the concentration of the biomarker of the disease present in the urine using an electrochemical sensor, the presence or absence of the disease as a numerical value It is possible to determine the reliability can be further increased.

However, the pH of urine may vary depending on the patient and the condition of the patient, the pH may have a value between up to 4.6 to 7.5. This difference in pH affects the degree of binding of the biomarker in the electrochemical sensor and the antibody to which the biomarker binds through an antigen antibody reaction. The error caused by the difference in the binding degree is finally the error of the concentration value of the biomarker in the urine, it may have an important effect on the diagnosis of the presence or absence of the disease. Despite the problem of accuracy due to the pH as described above, a method for measuring the concentration of the biomarker in consideration of the binding of the antigen antibody according to the pH of the urine is not developed yet, there is a difficulty in a more accurate diagnosis It is true.

Prior art US 2017/0199181 A1 discloses an electrochemical method for modifying a urine sample for detection of an immune sensor. However, the prior art is only to adjust the pH of the urine to the optimal pH of the ALP enzyme to optimize the activity of the ALP enzyme used for electrochemical redox reaction after antigen antibody binding, the degree of binding of the antigen antibody according to pH It is not considered.

Prior art KR 10-1758355 discloses an ion-sensitive urine sensor capable of detecting analytes in urine in a non-invasive way in urine. However, the prior art only experiments with the solution of the actual human urine and pH 10 in the embodiment, it is not aware of any change in the electrochemical signal of the biomarker in the urine with pH.

Therefore, there is still a need for the development of an electrochemical method for measuring the concentration of biomarkers in urine in consideration of the change in antigen-binding degree of pH.

Accordingly, an aspect of the present disclosure is to provide a method for measuring the concentration of antigen in the urine using an electrochemical sensor while considering the trend of antigen antibody binding according to pH in order to diagnose bladder cancer or prostate cancer.

A method for measuring the concentration of antigen in urine using an electrochemical sensor to achieve the aspects of the present disclosure includes the antibody comprising an antibody immobilized on the surface of the working electrode, measuring the pH of the urine; Dipping a working electrode in the urine; Treating the immersed working electrode with an electrical redox enzyme, and then immersing in a solution containing an electrical redox reaction substrate and applying a redox voltage to measure an electrical signal according to a redox reaction; And correcting the measured electrical signal with an electrical signal at a predetermined pH using a trend of antigen antibody binding according to pH.

According to one embodiment of the present disclosure, the antigen is a biomarker for diagnosing prostate cancer or bladder cancer.

According to one embodiment of the present disclosure, the biomarker is Metrix Metallo Peptidase-9 (MMP-9), Apolipoprotein A-1 (ApoA1), prostate-specific antigen (PSA), Prostate specific membrane antigen (PSMA), Annexin A3 (ANX A3), nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA), and urinary bladder carcinoma antigen (UBC).

According to one embodiment of the present disclosure, the antibody is immobilized via a linking compound on the surface of the working electrode.

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

According to one embodiment of the present disclosure, the electrical redox enzyme is HRP (Horseradish peroxidase), ALP (Alkaline phosphatase), glucose oxidase (Glucose Oxidase), luciferase, beta-di-galactosidase (β-D-galactosidase), malate dehydrogenase (MDH), and one type of enzyme selected from the group of acetylcholinestrerase.

According to an embodiment of the present disclosure, the electrical signal is measured by a cyclic voltage (CV) method, and the electrical signal is a maximum current value measured by the cyclic voltage method.

According to one embodiment of the present disclosure, the trend of antigen antibody binding according to the pH is: n buffers having different pH, including a biomarker, and 4 ≦ pH ₁ , pH ₂ , ..., pH _n ≦ 10. Preparing a solution; Measuring an electrical signal according to the binding of the biomarker and the antibody in the respective buffer solution using an electrochemical sensor immobilized on the surface of the working electrode; And it is determined by the method of determining the trend of antigen antibody binding according to pH comprising calculating a trend line of the degree of binding according to pH between the biomarker and the antibody from the measured electrical signal.

According to an embodiment of the present disclosure, n is an integer of 5 or more.

According to one embodiment of the present disclosure, the buffer is citric acid, phosphate buffered saline, or carbonate buffered saline buffer.

According to one embodiment of the present disclosure, the coefficient of determination (R ² ) of the trend line is at least 0.95.

According to one embodiment of the present disclosure, the trend of antigen antibody binding according to the pH is predetermined before measuring the electrical signal according to the redox reaction of the antigen in the urine.

Through the method of measuring the concentration of the antigen in the urine of the present disclosure, it is possible to minimize the effect of the patient-specific pH in measuring the concentration of the biomarker in the urine. Thus, the accuracy of biomarker detection in urine can be improved. Finally, it can greatly improve the early diagnosis / prognosis and treatment of the disease.

In addition, compared to a technique for adjusting the pH of the sample itself, by correcting the electric signal value after measurement using the trend of antigen antibody binding according to pH, it is possible to reduce the use of the buffer required for pH control, It can have advantages in terms of time, cost and effort. In addition, in the case of adjusting the pH of the sample itself, there is a possibility that it may affect the antigen present in the sample to reduce the activity of the antigen antibody reaction in the process of adjusting the pH, but the method of the present disclosure can avoid such a risk. have.

1 is a schematic diagram of an antibody immobilized on a working electrode surface, a biomarker binding to the antibody, and an electrical redox enzyme treated on the biomarker according to one embodiment of the present disclosure;
2 illustrates an electrical redox enzyme and its redox mechanism according to one embodiment of the present disclosure;
3 is a graph showing trend lines of antigen antibody binding according to pH calculated according to one embodiment of the present disclosure; And
Figure 4 is a graph plotting the current value according to the concentration of MMP-9 in pH 7.4 conditions.

The objectives, specific advantages, and novel features of the present disclosure will become more apparent from the following detailed description and preferred embodiments in conjunction with the accompanying drawings, but the present disclosure is not necessarily limited thereto. In addition, in describing the present disclosure, when it is determined that the detailed description of the related known technology may unnecessarily obscure the subject matter of the present disclosure, the detailed description thereof will be omitted.

The present disclosure provides a method for measuring the concentration of antigen in the urine using an electrochemical sensor, the sensor comprises an antibody immobilized on the surface of the working electrode, measuring the pH of the urine; Dipping a working electrode in the urine; Treating the immersed working electrode with an electrical redox enzyme, and then immersing in a solution containing an electrical redox reaction substrate and applying a redox voltage to measure an electrical signal according to a redox reaction; And correcting the measured electrical signal with an electrical signal at a predetermined pH using a trend of antigen antibody binding according to pH.

The antigen may be a biomarker that is an indicator for diagnosing disease. According to an embodiment of the present disclosure, the antigen may be a biomarker for diagnosing prostate cancer or bladder cancer. Specifically, the biomarker is Metrix Metallo Peptidase-9 (MMP-9), Apolipoprotein A-1 (ApoA1), prostate-specific antigen (PSA), Prostate specific membrane antigen (PSMA), Annexin A3 (ANX A3), nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA), and urinary bladder carcinoma antigen (UBC) may be one selected from the group consisting of. In the present disclosure, preferably, the biomarker may be MMP-9.

MMP-9 is mainly produced in tumor cells itself and is known as an important enzyme that degrades extracellular matrix in the process of cancer cell metastasis. Today, MMP-1, 2, 9, etc. are mainly studied in relation to bladder cancer, and in particular, MMP-2, 9 and Tissue inhibitor of metalloproteinases 2 (TIMP-2) are involved in the recurrence of high grade, advanced stage bladder cancer and bladder cancer. This is being reported. The cut-off value of the MMP-9 is 8.7 ng / ml (European urology, 2007, (52), 1388-1397). If there is more than the MMP-9 in the urine, it is diagnosed as bladder cancer.

In the present disclosure, the antibody is an antibody capable of binding to the biomarker, and means an antibody capable of capturing the biomarker on the working electrode. The type of capture antibody is not particularly limited as long as it is possible to capture the biomarker. The antibody can be immobilized directly or indirectly on the surface of the working electrode. In the present disclosure, direct fixation or coupling means that two objects are fixed or combined without the presence of a separate medium, material, or object between the two objects. Also, indirect fixation or coupling means that a separate medium, substance, or object exists between two objects to fix or couple them. According to one embodiment of the present disclosure, the antibody may be indirectly immobilized via a linking compound on the surface of the working electrode. Herein, the linking compound means a compound connecting the working electrode and the antibody, and a biochemical reaction binder biotin-avidin or biotin-streptavidin may be used as the linking compound. In addition, as chemical covalent bonds, carbodiimide crosslinkers or succinimide crosslinkers that bind to the primary amine of the protein may be used.

Referring to FIG. 1, a capture antibody immobilized indirectly on the surface of the working electrode is shown, specifically the antibody is immobilized on the surface of the working electrode using biotin-avidin as the linking compound. In the present disclosure, a biotin-avidin binding compound may be preferably used as the linking compound. In the method of immobilizing an enzyme using chemical covalent bonds, there is a randomness of the aromaticity of the enzyme to be immobilized, but when using a biotin-avidin binding compound, it is easy to control the surface area expression direction of the binding site of the enzyme to be immobilized. Appropriate directional control of the enzyme on the surface can maximize the surface area expression of the binding site of the enzyme, thereby maximizing the detection ability of the target material in the sample.

In order to correct an error occurring according to pH in the present disclosure, first, it is necessary to know the pH of the urine of the patient. For measuring the pH of the subject urine for which the antigen concentration is to be measured, a known pH measuring method may be applied without particular limitation. For example, an acid-base indicator, pH test paper, or an electrochemical method may be used to measure the pH of the urine. The measured pH of urine is used to calculate the electrical signal of the biomarker at the specific pH desired along with the trend line of antigen antibody binding according to the pH described below.

Apart from the pH measurement of the urine, the working electrode of the electrochemical sensor is immersed in the urine obtained from the patient. 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 the 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 Pt, Au, Pd, Rh electrode, pyrolytic graphite electrode, glassy carbon electrode, carbon paste electrode, carbon fiber electrode It may be a carbon electrode, an indium tin oxide (ITO) electrode, and the like.

Referring again to FIG. 1, an ITO electrode is used as the working electrode. The working electrode used in the present disclosure may preferably be an ITO electrode. In the case of an ITO electrode, when applied to a biosensor, it is possible to reduce a noise signal and to use a substrate on which an ITO thin film is deposited on an organic substrate without a separate adhesive layer. The ITO substrate has the advantage of being able to be manufactured as an electrode at a very low cost because the ITO substrate can use the thin film substrate manufacturing process having a high and stable electrical conductivity on a large substrate in the display industry.

By immersing the working electrode in urine, an antigen that functions as a biomarker of a particular disease present in the urine and an antibody immobilized on the surface of the working electrode are bound through an antigen antibody reaction. As described below, the antigen antibody response may vary in activity of urine according to the pH of the urine. Thus, even if the concentration of the antigen present in the urine varies depending on the patient or the condition of the patient, the antigen antibody response may be different. It matters.

After immersing the working electrode in urine, the immersed working electrode is treated with an electrical redox enzyme. Thereafter, the treated working electrode is immersed in a solution containing an electrical redox substrate and a redox voltage is applied to measure an electrical signal according to the redox reaction. In the present disclosure, the electrical redox enzyme refers to a substance that activates the redox substrate to enable the redox reaction. In addition, the electrical redox substrate in the present disclosure means a material that is activated, and when a voltage is applied through the working electrode, the redox reaction proceeds by the applied voltage to generate or absorb an electron to generate a current. do.

Referring again to FIG. 2, where an embodiment according to the present disclosure is illustrated, for example, in FIG. 2, alkaline phosphatase (ALP) is used as an electrical redox enzyme, and ascorbic acid-2- as an electrical redox substrate. phosphate) is used. The AAP is separated from the phosphate functional group by ALP and converted into AA (ascorbic acid) as an enzyme reaction product. The AA is oxidized to dehydroascorbate by an oxidation voltage applied through the working electrode. An electrical signal generated during the oxidation reaction, for example, a current value is measured, from which the concentration of a desired target substance in the sample can be calculated.

The electrical redox reaction enzyme may be processed to the working electrode immersed in the urine and bound to the antigen as shown in FIG. 1. Herein, the treatment of the enzyme is not particularly limited as long as it is possible to uniformly bind the enzyme to the antigen bound to the antibody immobilized on the surface of the working electrode. For example, the enzyme may be a known method such as spraying, dipping and soaking. It can be combined with the antigen through.

Since enzymes have substrate specificity, the antigens and enzymes in the present disclosure can be directly or indirectly bound. When indirectly bound, a secondary antibody capable of binding to an antigen different from the primary antibody immobilized on the surface of the working electrode and an electrical redox attached with an electrical redox enzyme that binds the secondary antibody Reactive enzyme-linked antibodies can be used.

For example, referring to FIG. 1, an MMP-9 Antibody different from an MMP-9 Biotinylated Antibody is used as a secondary antibody, and an ALP-attached Anti-mouse IgG as an electrical redox enzyme-linking antibody capable of binding to the secondary antibody. Used.

The redox reaction of the electrical redox substrate is performed only when the electrical redox enzyme binds to the antigen bound to the antibody immobilized on the working electrode. Therefore, the electrical signal measured through the electrical redox reaction is urine. It depends on the concentration of the antigen in the body, and thus it is possible to invert the concentration of the antigen in the urine from the electrical signal.

By way of example, the electrical redox enzymes of the present disclosure may include ALP, HRP (Horseradish peroxidase), glucose oxidase (Glucose Oxidase), luciferase, beta-di-galactosidase (β-D-galactosidase), Malate dehydrogenase (MDH), acetylcholinesterase, and the like. Especially preferably, the enzyme may be ALP or HRP. ALP and HRP have the advantage of having very sensitive reactivity, in particular HRP is inexpensive and cost-effective. In addition, ALP is advantageous in that it has a long signal holding time of about 24 to 48 hours.

In addition, as the electric redox substrate of the present disclosure, for example, a substrate corresponding to the electric redox enzyme, such as AAP activated by ALP, is used.

In the measurement of the electrical signal, a known electrochemical measuring method can be used. According to one embodiment of the present disclosure, a cyclic voltage (Cyclic Voltammetry, CV) method can be used. The cyclic voltage method is a method of measuring a current by circulating a voltage applied to the working electrode at a constant speed, through which a cyclic voltage current curve can be obtained. Here, the electrical signal measured by the cyclic voltage method means the maximum value of the current value shown in the cyclic voltage current curve.

The electrical signal measured by the above method can be corrected by the electrical signal at a predetermined pH using the trend of antigen antibody binding with pH. This is because, as described above, the pH of the patient's urine is different, the pH affects the binding of the antigen and the antibody, and the biomarker at the predetermined pH, which is a criterion for the presence of disease through the correction, It is possible to calculate the concentration. According to one embodiment of the present disclosure, the measured electrical signal may be corrected to the electrical signal when the pH is 7.4 through the correction.

In the present disclosure, the trend of antigen antibody binding according to pH can be obtained through the following steps.

The method for determining the trend of antigen antibody binding according to the pH of the present disclosure comprises preparing a buffer solution having a different pH of 4 <pH ₁ , pH ₂ , ..., pH _{n <} 10 step; Measuring an electrical signal according to the binding of the biomarker and the antibody in the respective buffer solution using an electrochemical sensor immobilized on the surface of the working electrode; And calculating a trend line of the degree of binding according to pH between the biomarker and the antibody from the measured electrical signal.

The buffer solution contains the same biomarker at the same concentration. Here, the biomarker is the same substance as the antigen to be measured in the method of measuring the concentration of the antigen in the urine of the present disclosure.

The buffer solution may be n buffer solutions with different pHs, preferably 5 ≦ pH ₁ , pH ₂ ,..., PH _n ≦ 9. Wherein n may be an integer greater than or equal to 5, for example 5, 6, 10, 12, 20, and 50.

The buffer solution is not particularly limited as long as control to the desired pH is possible without affecting the antigen antibody reaction of the biomarker with the corresponding antibody. According to one embodiment of the present disclosure, the buffer solution may include citric acid buffer, phosphate buffered saline buffer, or carbonate buffered saline buffer. Adjustment of the buffer solution to pH 4 to 8 may use citric acid buffer or phosphate buffered saline buffer. In addition, the adjustment of the buffer solution to a pH above 8 to 10 may be used carbonate buffered saline buffer.

For each of the buffer solutions having different pHs, including the biomarker, an electrical signal due to the binding of the biomarker and the antibody is measured using an electrochemical sensor in which the antibody is immobilized on the working electrode surface. The measurement of the electrical signal may be performed in the same manner as the method for measuring the concentration of the antigen in the urine of the present disclosure described above. That is, the working electrode is immersed in a buffer solution to perform an antigen antibody binding reaction between the biomarker and the antibody, and after the immersed working electrode is treated with an electrical redox enzyme, the solution is contained in a solution containing an electrical redox substrate. By dipping and applying a redox voltage, an electrical signal according to a redox reaction may be measured. This can be done repeatedly in each buffer solution at different pHs to determine the electrical signal due to binding of biomarkers and antibodies to different pHs.

The measurement of the electrical signal can also be performed using cyclic voltammetry. When using the cyclic voltammetry, the electrical signal means a current value generated at a certain same voltage with respect to the working electrode immersed in each buffer solution, and the specific voltage indicates that the reaction of the enzyme and the substrate is maximized. It is preferable that it is a voltage which can be used. By measuring the current value at the voltage at which the reaction of the enzyme and substrate can be activated to the maximum, the other variables are minimized to increase the reliability of the trend of the electrical signal due to antigen antibody binding in buffer solutions with different pH. Can be.

For example, when ALP is used as the electrical redox enzyme, it is preferable to determine the trend using the respective current values measured at a voltage of 0.34V, as disclosed in the prior patent application KR 10-2017-0154148. .

Using the electrical signals measured from the buffer solutions having different pHs, it is possible to calculate trend lines with the x-axis as the pH value and the y-axis as the electrical signal. According to an embodiment of the present disclosure, the crystal coefficient R ² of the trend line may be 0.95 or more, preferably 0.97 or more, and more preferably 0.99 or more. As the value of the crystal coefficient approaches 1, the reliability of the trend of the degree of binding according to pH between the biomarker and the antibody increases.

On the other hand, the determination of the antigen antibody binding trend according to the pH may be performed in advance before the implementation of the method for measuring the concentration of the antigen in the urine according to the present disclosure. Once the trend of antigen antibody binding according to pH is determined through the above trend determination method for a specific antigen, a separate experiment for further trend determination is not required for the specific antigen. After performing pH titration on the sample, it is more advantageous than measuring the electrical signal.

Hereinafter, preferred examples are provided to aid in understanding the present disclosure, but the following examples are provided only to facilitate understanding of the present disclosure, and the present disclosure is not limited thereto.

Example

Production Example  1. Preparation of Electrochemical ELISA Platform

After ITO electrode surface (24mm ² ) was sonicated for 10 minutes in 10 mL of Trichloroethylene solution, ethanol solution and DI water for 15 minutes, the solution was mixed with Ammonium hydroxide solution, Hydrogen peroxide, and DI water in 1: 1: 5. After soaking and reacting for 1 hour and 30 minutes in a 75 ℃ thermostat, the electrode was washed with DI water to finally activate the OH group on the electrode surface. 15 μl of avidin at 100 μg / ml was reacted at room temperature for about 2 hours. Through hydrophobic interactions, avidin was physically adsorbed on the ITO electrode surface. Thereafter, 15 μl of 1% BSA (bovine serum albumin) was reacted at 4 ° C. for about 24 hours. The electrode was reacted with 15 μl of Human / Primate MMP-9 Biotinlated Antibody (BAF911, manufactured by R & D Systems, 10 μg / ml) at 4 ° C. for about 30 minutes.

Experiment 1. Antigen Antibody Binding According to pH Trendline  decision

A buffer solution of pH 4, 5, 6, 7, 8, 9 containing 100ng / mL of Recombinant human MMP-9 (911-MP-010, manufactured by R & D Systems) as a biomarker was prepared. Citric acid buffer or phosphate buffered saline buffer was used for the preparation of the buffer solution of pH 4 to 8, and carbonate buffered saline buffer was used for the preparation of the buffer solution of pH 9.

The working electrode prepared in Preparation Example 1 was immersed in the buffer solution of pH 4 prepared at room temperature of 25 ° C. for 2 minutes.

Thereafter, 10 µg / ml of Human / Primate MMP-9 Antibody (MAB936-100, manufactured by R & D systems) and Anti-Mouse IgG (whole molecule) -Alkaline Phosphatase antibody (A3562, manufactured by Sigma Aldrich) Sequentially contacted with the working electrode surface for 30 minutes at 25 ℃.

The treated working electrode was immersed for 2 minutes in a mixed solution in which 1 mM solid AAP powder was dissolved in a solution containing 50 mM Tris-HCl (pH 9.6), 10 mM MgCl ₂ , and water. Was used to measure the current value against the voltage. The voltage was cycled between 0 and 0.6V and the current values at 0.34V are shown in Table 1 below.

The same procedure was repeated for buffer solutions with different pH values of 5, 6, 7, 8, and 9, and the current values at 0.34 V of the current values measured in the experiments in the respective buffer solutions are shown in the following table. 1 is shown. In addition, the trend line was calculated on a graph in which the x-axis is the pH and the y-axis is the current value using the data of pH 5 to pH 9 in Table 1 below. This is illustrated in FIG. 3, and referring to FIG. 3, it can be seen that the trend line has a relational expression of a linear function and the coefficient of determination (R ² ) is also 0.95 or more.

The equation of the trend line of FIG. 3 is as follows.

Equation 1.

Where pH z is the pH value of the solution containing the biomarker, y is the current value according to the trend when pH z.

The current value at pH 7.4, which is a general in vivo pH, is about 0.23 mA from Equation 1, and the deviation of the current value at each pH is shown in Table 1 below.

Current value of electrical redox reaction at different pH and deviation from current value at pH 7.4 pH z pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 Current value
(@ 0.34V) 0.17 0.29 0.27 0.25 0.22 0.19 % deviation from current value at pH 7.4 -27.23 +22.55 15.74 7.23 -7.66 -10.85

From the results of Equation 1 and Table 1, the maximum deviation of the electrical signal in the electrochemical analysis of the biomarker according to pH is 22.55%, so that the error of the final diagnosis within 22.55% through the correction of the electrical signal using the trend line It can be seen that it can be improved.

according to pH Current value  Correction and diagnosis of bladder cancer

The current value according to the electrochemical measurement of the biomarker in the urine of the patient can be corrected using Equation 1 and Equation 2 below.

Equation 2.

Here, y _{pH calibrated} is the current value corrected by Equation 2, y (measurement) is the current value measured from the urine of the patient, y (trend) is the current value according to the trend calculated by Equation 1 it means. In addition, with respect to '±' in Equation 2, when z is larger than 7.4, it is calculated as '+', and when z is smaller than 7.4, it is calculated as '-'.

Thus, if the pH and current value of the patient's urine is measured, it is possible to calculate the corrected current value using the equations (1) and (2).

On the other hand, Figure 4 is a graph plotting the current value according to the concentration of MMP-9 at pH 7.4 conditions. Formula of the graph shown in Figure 4 is as follows.

Equation 3.

Where x is the concentration of MMP-9 and y _{(pH 7.4)} is the current value according to the concentration of a particular MMP-9 under conditions of pH 7.4.

Substituting the corrected current value calculated using Equations 1 and 2 into Equation 3 above allows inversion to calculate the concentration of MMP-9 in the urine of the patient.

In addition, as described above, in the diagnosis of bladder cancer, the cut-off value of MMP-9 is 8.7 ng / ml. Therefore, it is better to diagnose the bladder cancer of the patient by comparing the calculated value of MMP-9 in the urine of the patient. It is possible.

Although the preferred embodiments of the present disclosure have been shown and described above, the present disclosure is not limited to the specific embodiments described above, and the present disclosure is not limited to the specific embodiments described above. Various modifications may be made by those skilled in the art, and these modifications should not be individually understood from the technical spirit or prospect of the present disclosure.

Claims (12)

As a method of measuring the concentration of antigen in the urine using an electrochemical sensor,
The sensor comprises an antibody immobilized on the surface of the working electrode,
Measuring the pH of the urine;
Dipping a working electrode in the urine;
Treating the immersed working electrode with an electrical redox enzyme, and then immersing in a solution containing an electrical redox reaction substrate and applying a redox voltage to measure an electrical signal according to a redox reaction; And
And correcting the measured electrical signal with an electrical signal at a predetermined pH using a trend of antigen antibody binding according to pH. The method according to claim 1,
Said antigen is a biomarker for diagnosing prostate cancer or bladder cancer. The method according to claim 2,
The biomarker is Metrix Metallo Peptidase-9 (MMP-9), Apolipoprotein A-1 (ApoA1), prostate-specific antigen (PSA), Prostate specific membrane antigen (PSMA), Annexin A3 (ANX A3), nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA), and urinary bladder carcinoma antigen (UBC) is a method for measuring the concentration of antigen in the urine, characterized in that one. The method according to claim 1,
And said antibody is immobilized via a linking compound on the surface of the working electrode. The method according to claim 1,
The working electrode is ITO (Indium Tin Oxide) method for measuring the concentration of the antigen in the urine. The method according to claim 1,
The electrical redox enzymes include HRP (Horseradish peroxidase), ALP (Alkaline phosphatase), glucose oxidase (Glucose Oxidase), luciferase, beta-di-galactosidase (β-D-galactosidase), malic acid A method for measuring the concentration of antigen in urine, characterized in that it is one enzyme selected from the group of dehydrogenase (MDH: malate dehydrogenase) and acetylcholinestrerase. The method according to claim 1,
The measurement of the electrical signal using a cyclic voltage (CV) method, the electrical signal is a method for measuring the concentration of antigen in the urine, characterized in that the maximum current value measured by the cyclic voltage method. The method according to claim 1,
The trend of antigen antibody binding according to the pH is:
Preparing n buffer solutions having different pH, wherein the biomarkers include 4 ≦ pH ₁ , pH ₂ ,..., PH _n ≦ 10;
Measuring an electrical signal according to the binding of the biomarker and the antibody in the respective buffer solution using an electrochemical sensor immobilized on the surface of the working electrode; And
Measuring the concentration of antigen in the urine, characterized in that determined by the trend determination method of antigen antibody binding according to pH comprising the step of calculating the trend of the binding degree according to pH between the biomarker and the antibody from the measured electrical signal How to. The method according to claim 8,
N is an integer greater than or equal to 5, the method of measuring the concentration of the antigen in the urine. The method according to claim 8,
The buffer is a method for measuring the concentration of antigen in the urine, characterized in that the citric acid, phosphate buffered saline, or carbonate buffered saline buffer The method according to claim 8,
The determination coefficient (R ² ) of the trend line is a method for measuring the concentration of antigen in the urine, characterized in that more than 0.95. The method according to claim 1,
The trend of antigen antibody binding according to the pH is a method for measuring the concentration of the antigen in the urine, characterized in that predetermined before the measurement of the electrical signal according to the redox reaction of the antigen in the urine.

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