KR20210078001A - Label-Free Electrochemical Sensing Method for On-Site Self Diagnosis - Google Patents

Abstract

The present disclosure relates to a method for measuring impedance depending on the concentration of an antigen in a body fluid using a label-free electrochemical sensor. The electrochemical sensor comprises a working electrode, wherein the working electrode comprises an antibody immobilized on a surface of the working electrode. The method includes a step of immersing a working electrode in a body fluid; and a step of measuring the impedance change of the immersed working electrode using electrochemical impedance spectroscopy (EIS). The present invention provides a method for measuring impedance depending on the concentration of an antigen in a body fluid using a label-free electrochemical sensor and a method for on-site self-diagnosis of a disease using the method.

Description

Label-Free Electrochemical Sensing Method for On-Site Self Diagnosis

The present disclosure relates to a label-free electrochemical sensing method preferred for on-site self-diagnosis, and more particularly, to a label-free electrochemical sensing method preferred for on-site self-diagnosis using Electrochemical Impedance Spectroscopy (EIS). .

The on-site self-diagnosis refers to a rapid self-examination of a disease in the field (where the patient is) without the help of professional medical personnel, which could only be diagnosed by going to a hospital. For the purpose of diagnosis and prognosis of disease, determination of health status, determination of disease treatment effect, prevention, etc., it is possible to test using a small amount of body fluid collected from the human body.

For this on-site self-diagnosis, an electrochemical sensor or biosensor that calculates the concentration of a target substance in a body fluid by measuring an electrical signal generated from a chemical reaction of a target substance in a body fluid is being studied. Recently, many studies have been conducted on electrochemical sensors that calculate the concentration of a target material in a sample by measuring the movement of electrons generated by the oxidation/reduction electrode reaction of the target material on the working electrode, that is, the current. In particular, as low cost, convenience, and ease of miniaturization are recently required for on-site self-diagnosis electrochemical sensors, research on electrochemical sensors having excellent sensing sensitivity while making it easy to carry by miniaturizing the size of the sensor is actively conducted. is in progress

On the other hand, as a measurement target of the electrochemical sensor, an immune-related material (eg, an antigen or an antibody) has very low electrical conductivity and low electrochemical reactivity. In the case of a target material having no redox function in addition to such an immune-related material, it is difficult to analyze it through the oxidation/reduction electrode reaction as described above. Accordingly, in order to measure the concentration of the target substance, it is essential to introduce a labeling factor capable of electrochemically activating the target substance.

In the case of a biosensor using the cyclic voltammetry (CV) method and the chromoamperometry (CA) method, which are conventional electrochemical signal measurement methodologies, a second antibody to which the labeling factor is bound is required as a receptor in order to measure the concentration of the target substance. Do. The presence of the receptor causes an increase in cost as a separate antibody other than the first antibody fixed to the electrode is required as a receptor in the manufacture of the biosensor, and the time and measurement steps required for measurement in use increase, and thus There is a disadvantage in that it includes problems such as an increase in the possibility of error occurrence. Accordingly, there is a demand for a so-called unlabeled electrochemical sensor that does not require the bioreceptor as described above.

US 10-1990703 B1 US 10-2019-0127349 A

Accordingly, a first aspect of the present disclosure is to provide a novel electrochemical sensing method optimized for on-site self-diagnosis that does not require such a bioreceptor.

In addition, a first aspect of the present disclosure is to provide a new method for self-diagnosing the presence or absence of a corresponding disease in the field using such an electrochemical sensing method.

In order to achieve the first aspect of the present disclosure, there is provided a method for measuring impedance according to the concentration of an antigen in a body fluid using an unlabeled electrochemical sensor, wherein the electrochemical sensor includes a working electrode, wherein the working electrode is a An antibody immobilized on a surface, the method comprising: immersing a working electrode in the bodily fluid; and measuring an impedance change of the immersed working electrode using Electrochemical Impedance Spectroscopy (EIS).

According to one embodiment of the present disclosure, the bodily fluid is urine.

According to one embodiment of the present disclosure, the antigen is a biomarker for diagnosis of 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 on the surface of the working electrode via a linking compound.

According to one embodiment of the present disclosure, the working electrode is ITO (Indium Tin Oxide) glass.

According to one embodiment of the present disclosure, the measuring the impedance change of the immersed working electrode using the EIS includes immersing the working electrode immersed in the body fluid in a solution including a redox couple. to do; applying an AC voltage to the working electrode, wherein the AC voltage is applied while gradually decreasing a frequency value within a predetermined frequency range, and the AC voltage has a sinusoidal waveform; and analyzing the impedance for each of the frequency values.

According to one embodiment of the present disclosure, the redox pair is ferricyanide/ferrocyanide.

According to one embodiment of the present disclosure, the range of the frequency is 1 to 100000 Hz.

In order to achieve the second aspect of the present disclosure, a method for on-site self-diagnosis of a disease using a non-labeled electrochemical sensor includes a method for measuring an impedance according to the first aspect of the present disclosure, wherein the on-site self-diagnosis method comprises: Comparing the impedance change measured by the impedance measuring method according to the first aspect of the disclosure with the previously measured disease reference impedance data, determining whether there is a disease or not.

Through the measurement method of the present disclosure, it is possible to avoid the use of a bioreceptor labeled with an electrochemical signal factor, thereby reducing the cost and simplifying the measurement procedure according to the use of the bioreceptor, thereby further enhancing user convenience. The effect exists. These advantages are more consistent with the purpose of the present disclosure for point-of-care self-diagnosis.

1 is a schematic diagram of manufacturing a working electrode for use in one embodiment of the present disclosure;
2 is a graph plotting the results of Example 1 of the present disclosure;
3 is a graph plotting the results of Example 2 of the present disclosure.

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

Method for measuring impedance according to the concentration of antigen in a sample using an unlabeled electrochemical sensor

A first aspect of the present disclosure is a method for measuring impedance according to the concentration of an antigen in a body fluid using an unlabeled electrochemical sensor, wherein the electrochemical sensor includes a working electrode, wherein the working electrode is on a surface of the working electrode. An antibody that is immobilized, the method comprising: immersing a working electrode in the bodily fluid; and measuring an impedance change of the immersed working electrode using Electrochemical Impedance Spectroscopy (EIS).

The bodily fluid used for measurement in the present disclosure may be a bodily fluid obtained from an animal. More specifically, the bodily fluid may be a bodily fluid obtained from a human body. Here, the bodily fluid means, for example, blood, urine, saliva, etc., and is not limited to the above example, and any bodily fluid in which the target substance to be analyzed exists may be used.

The antigen of the present disclosure may be a biomarker that is an index for diagnosing the presence or absence of a disease. According to one embodiment of the present disclosure, the antigen may be a biomarker for diagnosis of prostate cancer or bladder cancer. As a specific example, 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), It may be one selected from the group consisting of nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA), and urinary bladder carcinoma antigen (UBC). In the present disclosure, preferably, the biomarker may be ApoA1.

ApoA1 is a biomarker of bladder cancer, and when the concentration of ApoA1 in body fluid is 0.645 nM or more, bladder cancer is diagnosed.

In the present disclosure, an antibody refers to a specific antibody capable of generating an antigen-antibody reaction with the antigen, and refers to an antibody that is fixed on a working electrode and capable of capturing a biomarker. The type of the capture antibody is not particularly limited as long as it is possible to capture the corresponding biomarker. The antibody may be immobilized directly or indirectly on the surface of the working electrode. In the present disclosure, direct fixing or coupling means that two objects are fixed or coupled without the presence of a separate medium, material, or object between the two objects. In addition, indirect fixing or coupling means that a separate medium, material, 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 on the surface of the working electrode through a linking compound. Here, the linking compound refers to a compound that connects the working electrode and the antibody, and as the linking compound, biotin-avidin or biotin-streptavidin, which is a biochemical reaction compound, may be used. In addition, as a chemical covalent compound, a carbodiimide crosslinking agent or a succinimide crosslinking agent that binds to the primary amine of a protein may be used.

Referring to FIG. 1 showing a schematic diagram of the manufacturing of the working electrode used in one embodiment of the present disclosure, it can be confirmed that the capture antibody is immobilized on the surface of the working electrode through biotin-avidin as a linking compound. In the present disclosure, a biotin-avidin binding compound may be preferably used as the linking compound. In the case of a method of immobilizing an antibody using a chemical covalent bond, there is randomness in the directionality of the antibody to be immobilized, but when a biotin-avidin binding compound is used, it is easy to control the directionality of the expression of the surface area of the binding site of the antibody to be immobilized, By controlling the proper directionality of the antibody on the surface, the surface area of the binding site of the antibody can be maximized, thereby maximizing the detection ability of the target substance in the body fluid.

In the method of the present disclosure, the working electrode to which the antibody is immobilized is immersed in a bodily fluid obtained from an animal, human, specifically a patient, more specifically, urine obtained from a patient. The working electrode used in the present disclosure is not particularly limited in its type as long as the antibody can be immobilized on the working electrode and current can flow to the antigen. 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 paste electrode, a carbon fiber electrode It may be a carbon electrode, such as an indium tin oxide (ITO) electrode, and the like.

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

By immersing the working electrode in a pre-collected body fluid, an antigen present in the body fluid and functioning as a biomarker of a specific disease and an antibody immobilized on the surface of the working electrode are bound through an antigen-antibody reaction.

In the measurement method of the present disclosure, a change in impedance due to an antigen bound to the working electrode is measured by using electrochemical impedance spectroscopy (EIS) of a working electrode immersed in a body fluid. According to the CA method or CV method used as a conventional measurement method, in order to measure the antigen concentration, a second antibody to which a separate labeling factor is attached (that is, an antibody that binds to an antigen at a different location from the fixed antibody of the working electrode) ) was required as a receptor. However, in the measurement method of the present disclosure, this disadvantage can be solved by using EIS, and thus it has an advantageous advantage that it is possible to use a non-labeled electrochemical sensor.

Measuring the impedance change of the working electrode using the EIS of the present disclosure may include: immersing the working electrode immersed in the body fluid in a solution containing a redox couple; applying an AC voltage to the working electrode, wherein the AC voltage is applied while gradually decreasing a frequency value within a predetermined frequency range, and the AC voltage has a sinusoidal waveform; and analyzing the impedance for each of the frequency values.

EIS is an analysis method that applies a sine wave (sine wave) to the sample in turn from high frequency to low frequency, measures the change in amplitude and phase according to the response sine wave coming out through the sample, and then analyzes the impedance. In order to apply the EIS to the method of the present disclosure, the working electrode on which the antigen-antibody binding reaction has been performed is immersed in a solution containing a redox pair. The solution containing the redox couple is an electrolyte solution, which supplies power to the solution in a later step so that the sine wave can be transmitted to the working electrode. The redox pair is not particularly limited as long as an oxidation-reduction reaction can be performed when a voltage is applied to the solution. The redox pair used in the present disclosure may preferably be an iron redox pair, more preferably a ferricyanide/ferrocyanide.

Next, in a state in which the working electrode is immersed in a solution containing the redox pair, applying an alternating voltage to the working electrode is performed. The AC voltage has a wavelength of a sine wave. The AC voltage may be applied while gradually decreasing a frequency value from a high frequency to a low frequency. 2 and 3, and as will be described later, looking at the Nyquist plot in which the impedance change of the present disclosure is plotted, the impedance value at a specific frequency is indicated as one point in the graph. Accordingly, it is possible to observe variations in the impedance of the surface of the working electrode in the case of having a specific concentration of antigen while changing the frequency from high to low.

According to one embodiment of the present disclosure, the range of the frequency may be about 1 to about 100000 Hz. The change in the impedance of the working electrode surface in the corresponding frequency range exhibits a trend, for example, as shown in FIG. 3 . Measurement in a range exceeding the frequency has a problem in that reliability of the result of the measured impedance value may be deteriorated due to the strong influence of noise.

Also, according to one embodiment of the present disclosure, the AC voltage may be applied in a voltage range of about 1 mV to about 10 mV. When the AC voltage is less than 1 mV, there is a possibility that the impedance value cannot be measured because the applied voltage value is too small. On the other hand, when the AC voltage is applied in excess of 10 mV, the value of the impedance due to the antigen-antibody binding product bound to the surface of the working electrode is very small compared to the applied voltage, so it is related to the concentration of the antigen in the body fluid. Without it, impedance values are similarly analyzed, which may cause a problem in that meaningful analysis of the impedance for each antigen concentration becomes difficult.

The impedance value at each frequency measured as described above may be analyzed through the Nyquist plot as described above.

Furthermore, since the Nyquist plot for each antigen concentration has a unique plot as exemplarily shown in FIG. 3 , it is possible to self-diagnose whether or not a patient has a disease as described later through comparison between them.

A method for self-diagnosis of disease sites using unlabeled electrochemical sensors

As shown in exemplary FIG. 3 , for a specific antigen concentration, impedance analysis through EIS within the frequency range is expected to have a unique Nyquist plot. Therefore, if the Nyquist plot at the concentration of a specific biomarker, which is a criterion for judging the presence or absence of a specific disease, is known in advance, by comparing the Nyquist plot result based on the concentration of the biomarker in the patient's body fluid and It is possible to judge

The present disclosure provides a method for on-site self-diagnosis of a disease using a non-labeled cell chemical sensor as a second aspect of the present disclosure based on the above facts. The method may include the impedance measurement method of the first aspect of the present disclosure described above, including determining whether a disease exists by comparing the impedance change measured through the measurement method with pre-measured specific disease reference impedance data do. Here, the specific disease reference impedance data means impedance data previously measured at a known minimum concentration or maximum concentration of a biomarker that is determined to have a disease.

According to one embodiment of the present disclosure, the comparison between the measured impedance change value and the reference impedance data may be performed through the comparison of the impedance value. In the Nyquist plot, the impedance value (Z) can be calculated by the following equation (1).

More specifically, for example, it is known that the standard diagnostic concentration of ApoA1 for bladder cancer in urine is 0.645 nM. Accordingly, by comparing the results of the Nyquist plot of ApoA1 at 0.645 nM and the Nyquist plot of the sensor immersed in the urine of a patient, the Z value within the frequency range of 1 to 100,000 Hz is the impedance when the concentration of ApoA1 is 0.645 nM If the value is _{greater than or equal to the Z criterion} , it can be determined that the patient has bladder cancer.

Hereinafter, preferred examples are presented to help the understanding of the present disclosure, but the following examples are provided for easier understanding of the present disclosure, and the present disclosure is not limited thereto.

Example

Preparation Example 1. Preparation of unlabeled electrochemical working electrode platform

Sonication treatment of the ITO electrode surface (24mm ² ) in 10 mL of trichlorethylene solution, ethanol solution, and DI water for 15 minutes, immersing the electrode in 30% KOH solution for 10 minutes, washing the electrode in DI water, and finally the electrode OH groups were activated on the surface. After that, the working electrode was treated with 3-Aminopropyl)triethoxysilane (APTES) and Glutaraldehyde (GA) and reacted with 15 μl of 100 μg/ml of avidin at room temperature for about 2 hours. Avidin was physically adsorbed on the ITO electrode surface through hydrophobic interaction. Then, the electrode and 15 μl of 1% bovine serum albumin (BSA) were reacted at 4° C. for about 24 hours. Then, the electrode and 15 μl of ApoA1 Biotinlated Antibody (10 μg/ml) were reacted at 4° C. for about 30 minutes.

The impedance change according to each surface treatment was observed during the manufacturing process of the platform, and each Nyquist plot was integrated into one graph and shown in FIG. 2 . As can be seen from FIG. 2 , it can be seen that the impedance slightly decreases due to the etching effect of the ITO glass surface during the KOH treatment, but the impedance gradually increases as the surface treatment proceeds thereafter. In addition, by finally measuring the impedance value after being fixed to the antibody, the default impedance value of the working electrode before antigen-antibody binding can be confirmed.

Example 1. Measurement of impedance values for each concentration of ApoA1 bladder cancer biomarker

An electrochemical sensor including a working electrode prepared in Preparation Example 1 was used, and samples having ApoA1 concentrations of 0, 0.01, 0.1, and 1 nM, respectively, were prepared. After immersing the working electrode in each sample and leaving it for a sufficient time for the antigen-antibody binding reaction to proceed, the electrode is immersed in Ferri-Ferro solution, and an alternating voltage of 1 to 10 mV is applied to the working electrode, EIS analysis was performed while gradually decreasing the frequency from 100000 Hz to 1 Hz. The Nyquist plot according to the frequency change for each sample was integrated into one graph, which is shown in FIG. 3 of the present disclosure.

As described above, the reference concentration for diagnosing bladder cancer with respect to ApoA1, a biomarker of bladder cancer, is 0.645 nM or more. Meanwhile, referring to FIG. 3 , it can be seen that, according to the method of the present disclosure, an analysis of impedance within the range of at least 0.01 nM and 1 nM is effectively possible. Also, it can be seen from FIG. 3 that the impedance value at the same frequency gradually increases as the concentration of ApoA1 in the body fluid increases.

From the above results, it can be seen that the presence or absence of a disease in a patient can be easily determined by comparing the impedance value of the biomarker concentration in the patient's body fluid with the impedance value of the disease reference biomarker concentration.

Although the preferred embodiments of the present disclosure have been illustrated and described above, the present disclosure is not limited to the specific embodiments described above, and it is common in the technical field to which the disclosure belongs without departing from the gist of the present disclosure as claimed in the claims. Various modifications may be made by those having the knowledge of, of course, and these modifications should not be individually understood from the technical spirit or perspective of the present disclosure.

Claims (10)

A method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, the method comprising:
wherein the electrochemical sensor comprises a working electrode, wherein the working electrode comprises an antibody immobilized on a surface of the working electrode;
The method is
immersing a working electrode in the bodily fluid; and
Method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, comprising the step of measuring an impedance change of the immersed working electrode using Electrochemical Impedance Spectroscopy (EIS) . The method according to claim 1,
The method for measuring impedance according to the antigen concentration in the body fluid using an unlabeled electrochemical sensor, characterized in that the body fluid is urine. The method according to claim 1,
The method for measuring the impedance according to the concentration of the antigen in a body fluid using an unlabeled electrochemical sensor, characterized in that the antigen is a biomarker for diagnosis of prostate cancer or bladder cancer. 4. The method according to claim 3,
The biomarkers are 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 method for measuring impedance according to the concentration of antigen in body fluid using a non-labeled electrochemical sensor, characterized in that one selected from the group consisting of urinary bladder carcinoma antigen (UBC) . The method according to claim 1,
The method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, characterized in that the antibody is immobilized on the surface of the working electrode through a connection compound. The method according to claim 1,
The method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, characterized in that the working electrode is made of ITO (Indium Tin Oxide) glass. The method according to claim 1,
Measuring the impedance change of the immersed working electrode using the EIS comprises:
immersing the working electrode immersed in the body fluid in a solution containing a redox couple;
applying an AC voltage to the working electrode, wherein the AC voltage is applied while gradually decreasing a frequency value within a predetermined frequency range, and the AC voltage has a sinusoidal waveform;
A method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, comprising the step of analyzing the impedance for each of the frequency values. 8. The method of claim 7,
The redox pair is a method of measuring impedance according to the antigen concentration in a body fluid using an unlabeled electrochemical sensor, characterized in that ferricyanide/ferrocyanide. 8. The method of claim 7,
The method for measuring impedance according to the concentration of antigen in a body fluid using an unlabeled electrochemical sensor, characterized in that the frequency range is 1 to 100000 Hz. A method for on-site self-diagnosis of a disease using an unlabeled electrochemical sensor, comprising:
The on-site self-diagnosis method comprises a method of measuring the impedance according to any one of claims 1 to 9,
The on-site self-diagnosis method comprises:
Using an unlabeled electrochemical sensor, comprising the step of determining the presence or absence of a disease by comparing the impedance change measured by the method for measuring impedance according to any one of claims 1 to 9 with the previously measured disease reference impedance data A method of on-site self-diagnosis of the disease.

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