Method for point of care testing and testing kit

Abstract

The present invention relates to a point-of-care diagnostic method and a diagnostic kit comprising the steps of: preparing a gold electrode bound to an aptamer that specifically binds to a specific protein and 6-mercapto-1-hexanol; contacting a sample with the gold electrode; and applying a voltage to the gold electrode and measuring the current flowing through the gold electrode. The invention has the advantage of enabling rapid on-site diagnosis and allowing for a quick response to diseases or illnesses.

Description

Technology Field

[0001] The present invention relates to a field diagnostic method and a diagnostic kit, and more specifically, to a field diagnostic method and a diagnostic kit capable of rapidly detecting a target protein and rapidly diagnosing it at the site. Background Technology

[0003] Point-of-care testing is a method that allows for rapid confirmation of diagnostic results on-site. By shortening the diagnostic time, it enables prompt treatment and therapy, and is being utilized in various fields such as blood glucose measurement, pregnancy determination, myocardial infarction diagnosis, and COVID-19 infection diagnosis.

[0004] Conventional point-of-care diagnostic tests are based on antigen-antibody binding reactions and have the disadvantage of a low detection limit because they use antibodies targeting the virus's spike protein as an antigen. Consequently, there is a possibility that a result may appear negative even if the patient is actually positive for the virus; such false negatives can lead to problems such as the spread of infection or the worsening of the disease due to a lack of prompt intervention and treatment.

[0005] Another principle of point-of-care diagnostic testing is based on the specific binding reaction between aptamers and proteins, which allows for diagnosis by evaluating the presence or absence of proteins that cause the disease.

[0006] However, these existing point-of-care diagnostic tests take approximately 8 to 15 minutes to produce results, making them insufficient for diagnosing diseases requiring rapid diagnosis, such as heart attacks. Therefore, there is a need to develop technology for new diagnostic methods that enable rapid on-site diagnosis. Prior art literature

[0008] Registered Patent No. 10-2180007 (Registered Nov. 11, 2020) The problem to be solved

[0009] The present invention aims to provide a field diagnostic method and a diagnostic kit capable of rapidly detecting target proteins and diagnosing them quickly at the site. means of solving the problem

[0011] One embodiment of the present invention for achieving the above-described purpose relates to a field diagnostic method comprising: a step of preparing a gold electrode bound to a double-stranded aptamer that specifically binds to a specific protein and 6-mercapto-1-hexanol; a step of contacting a sample with the gold electrode; and a step of applying voltage to the gold electrode and measuring the current flowing through the gold electrode.

[0012] When a specific protein in the above sample specifically binds to a double-stranded aptamer, a structural change into a single-stranded aptamer occurs, and charge flow may increase.

[0013] The analytical sensitivity of the above-mentioned field diagnostic method for the above-mentioned specific protein may be 1 pg / mL.

[0014] The change in current resulting from the specific binding of a double-stranded aptamer to a specific protein can occur within one minute.

[0015] Another embodiment of the present invention comprises: a sample injection unit; a detection unit communicating with the sample injection unit to detect a specific protein in the sample; and a current measuring device connected to the detection unit to measure a current, wherein the detection unit may include a gold electrode with a double-stranded aptamer and 6-mercapto-1-hexanol bonded to its surface. Effects of the invention

[0017] The on-site diagnostic method of the present invention has the advantage of enabling rapid diagnosis at the site, thereby allowing for a rapid response to diseases or illnesses. Brief explanation of the drawing

[0019] Figure 1 is a diagram briefly illustrating the reaction results between a double-stranded aptamer and a protein. Figures 2(A) to 2(D) are graphs of experimental results measured by cyclic voltammetry. Specific details for implementing the invention

[0020] Before describing the preferred embodiments of the present invention in detail below, it should be noted that the terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0021] Throughout this specification, when a part is described as “comprising” a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0022] In each step, identification codes are used for convenience of explanation and do not describe the order of the steps; the steps may be performed differently from the specified order unless a specific order is clearly indicated in the context. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the reverse order.

[0023] The present invention will be described in detail with reference to the embodiments and drawings. These embodiments are presented merely as examples to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments.

[0024] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains, and in the event of a conflict, the description in this specification, including the definitions, shall prevail.

[0025] To clearly explain the proposed invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been assigned similar reference numerals. Additionally, the term "part" as described in the specification refers to a single unit or block that performs a specific function.

[0026] Hereinafter, embodiments of the present invention are examined. However, the scope of the present invention is not limited to the following preferred embodiments, and a person skilled in the art to which the present invention pertains may implement various modified forms from the contents described in the specification.

[0027] The present invention relates to a field diagnostic method and a field diagnostic kit, and more specifically, to a field diagnostic method and a field diagnostic kit capable of rapid field response by enabling diagnosis or rapid measurement of specific biological signals at the site.

[0028] In particular, according to the present invention, detection is possible even when the concentration of the target is low, as it has an analytical sensitivity of 1 pg / mL. Unlike conventional point-of-care testing methods that require a detection time of 5 minutes or more, detection is completed within 1 minute, enabling rapid diagnosis that was previously unavailable. Accordingly, patients or guardians can quickly diagnose diseases and take prompt action even in the absence of medical personnel, thereby improving the efficiency of response to urgent diseases and conditions.

[0029] One embodiment of the present invention relates to a field diagnostic method, wherein the field diagnostic method according to the present embodiment comprises the steps of: preparing a gold electrode to which an aptamer that specifically binds to a specific protein and 6-mercapto-1-hexanol are bound; contacting a sample with the gold electrode; and applying a voltage to the gold electrode and measuring the current flowing through the gold electrode.

[0030] First, a step is performed to prepare a gold electrode bound to an aptamer that specifically binds to a specific protein and 6-mercapto-1-hexanol (MCH). Here, the aptamer may be a double-stranded aptamer.

[0031] The above gold electrode is prepared to detect a specific protein in a sample, and as long as both double-stranded aptamer and 6-mercapto-1-hexanol are bound to the surface of the gold electrode, the shape of the gold electrode or the shape of the kit equipped with such a gold electrode is not particularly limited.

[0032] The above aptamers are characterized by specifically binding to specific proteins, and aptamer-protein combinations include (Troponin Aptamer)-(Troponin), (Pegaptanib)-(VEGF-165 (vascular endothelial growth factor)), (AS1411)-(Nucleolin), (REG1)-(Coagulation factor IXa), (NOX-A12)-(Cell-derived factor 1), (ARC1779)-(von Willebrand Factor (vWF)), (ARC1905)-(Complement C5 protein), (NU172)-(Thrombin), (HER2 Aptamer)-(HER2), (EGFR Aptamer)-(Epidermal growth factor receptor (EGFR)), (PD-L1 Aptamer)-(PD-L1), (MUC1 Aptamer)-(MUC1), (PSA Aptamer)-(Prostate-specific antigen (PSA)), (TNF-α Aptamer)-(Tumor necrosis factor-alpha (TNF-α)), (IL-6 Aptamer)-(Interleukin-6 (IL-6)), (NF-κB Aptamer)-(NF-κB), (Complement C5 Aptamer)-(Complement C5 protein), (TGF-β1 Aptamer)-(Transforming growth factor-beta (TGF-β1)), (Thrombin Aptamer)-(Thrombin), (FXa Aptamer)-(Factor(HIV gp120 Aptamer)-(HIV gp120), (Influenza HA Aptamer)-(Hemagglutinin (HA) protein), (Hepatitis C Virus (HCV) NS5B Aptamer)-(NS5B RNA polymerase), (SARS-CoV-2 Spike Protein Aptamer)-(SARS-CoV-2 spike protein), (Beta-Amyloid (Aβ) Aptamer)-(β-amyloid peptide), (Tau Protein Aptamer)-(Tau protein), (α-Synuclein Aptamer)-(α-synuclein), (BACE1 Aptamer)-(β-secretase (BACE1)), (Insulin Aptamer)-(Insulin), (Glucagon Aptamer)-(Glucagon), (Oxytocin) Aptamer)-(Oxytocin), (CGRP Aptamer)-(Calcitonin gene-related peptide It may be at least one of (CGRP)), (Angiotensin II Aptamer)-(Angiotensin II), (Substance P Aptamer)-(Substance P), (Endorphin Aptamer)-(Endorphin), (Dynorphin Aptamer)-(Dynorphin), (Defensin Aptamer)-(Defensin), and (Cathelicidin Aptamer)-(Cathelicidin). The combinations listed herein are described in the form of (aptamer)-(protein).

[0033] Aptamers provided on the surface of a gold electrode can be provided in a double-stranded form. In this case, when the aptamer binds to a protein upon contact with a sample, one of the double strands detaches from the electrode surface, and the structure changes to a single-stranded form. Consequently, more of the electrode surface is exposed, and charge flow is enhanced; due to the resulting change in current, the presence or amount of a protein that specifically binds to the aptamer in the sample can be rapidly detected. This reaction process is briefly illustrated in Fig. 1.

[0034] Conventional point-of-care diagnostic methods utilizing antigen-antibody reactions or aptamer-protein binding reactions have the disadvantage of slow measurement speeds because they measure signals generated from the binding of antigens and antibodies or signals generated from the binding of aptamers and proteins. However, since the present invention measures changes in electrode charge flow that occur as double-stranded aptamers transform into single-stranded aptamers, significantly faster measurement is possible compared to existing methods.

[0035] For example, using conventional point-of-care diagnostic methods, it took 8 to 15 minutes to diagnose myocardial infarction using the aptamer-troponin binding reaction, but using the point-of-care diagnostic method of the present invention, it takes less than 1 minute, making very rapid diagnosis possible. In particular, an increase in blood troponin concentration is a signal that serves as a marker for myocardial infarction, and using the present invention allows for the diagnosis of an increase in blood troponin concentration within 1 minute. In the case of acute myocardial infarction, the prognosis varies significantly depending on treatment within the first 5 minutes; therefore, while it was difficult to respond early with conventional methods, it is expected that using the present invention will enable a more appropriate early response, thereby contributing to an increase in survival rates.

[0036] The above 6-mercapto-1-hexanol may be provided to prevent non-specific binding of proteins. Accordingly, specific binding of the aptamer to the protein is well achieved, and the charge change as described above can be effectively achieved.

[0037] Next, a step of contacting a sample with the gold electrode is performed. This step may be performed by any method as long as the sample is brought into contact with the gold electrode. For example, the method is not limited, such as immersing the gold electrode in the sample or dropping the sample onto the gold electrode.

[0038] As for the sample, any sample containing the protein to be detected, such as blood, saliva, tears, or sweat, may be used without special restrictions.

[0039] The step of applying voltage to the gold electrode and measuring the current flowing through the gold electrode is a step of measuring the change in current due to the specific binding of aptamer-protein, and the diagnostic method through this can be divided into two ways.

[0040] The first method involves contacting a sample with a gold electrode, applying voltage, and measuring the current; if the current is above or below a preset value, it determines the presence of a disease or disorder.

[0041] The second method involves applying voltage to a gold electrode to continuously measure the current, bringing a sample into contact with the electrode, and determining the presence of a disease or disorder based on changes in the current.

[0042] Both of these methods have the advantage of enabling rapid diagnosis because the change in current resulting from the specific binding of double-stranded aptamers and specific proteins occurs within one minute.

[0043] Meanwhile, another embodiment of the present invention relates to a field diagnostic kit, and field diagnosis can be performed in the manner described above according to one embodiment by using the field diagnostic kit according to this embodiment. Accordingly, redundant descriptions are omitted.

[0044] The above-described field diagnostic kit comprises: a sample injection unit; a detection unit communicating with the sample injection unit to detect a specific protein in the sample; and a current measuring device connected to the detection unit to measure the current, wherein the detection unit comprises a gold electrode with a double-stranded aptamer and 6-mercapto-1-hexanol bonded to its surface.

[0045] The sample injection section described above is the part into which a sample is injected, and the presence or concentration of a specific protein can be measured through a detection section. The detection section is designed to communicate with the sample injection section, and it is also possible to additionally provide a filter between the sample injection section and the detection section to filter out impurities within the sample.

[0046] The above detection unit includes a gold electrode, and the gold electrode may have a double-stranded aptamer and 6-mercapto-1-hexanol formed on its surface. Accordingly, the detection or concentration of a protein that specifically binds to the aptamer in the manner described above can be measured, and the presence of a disease or disorder can be diagnosed.

[0047] The above-described current measuring device is a device that is electrically connected to a detection unit, applies voltage to the detection unit, and measures the current. This device may be equipped with a display device that shows the measured current and changes in current, or may be equipped with a communication device that transmits the measured current and changes in current to a terminal, so that the measured results can be checked through the terminal.

[0048] Hereinafter, the specific operation and effects of the present invention will be explained through an embodiment of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the rights of the present invention is not limited according to the embodiment.

[0050] [Experimental Example]

[0051] Step 1

[0052] First, the gold electrode was cleaned with distilled water and ethanol, connected to a cyclic voltammetry device, and the change in current was measured under an electrolyte (5 mM K3Fe(CN)6 + 0.1 M KNO3). (Fig. 2(A))

[0053] Step 2

[0054] Next, an aptamer solution was prepared by diluting 1 μmol of double-stranded aptamer to a concentration of 1 μM (mol / L) in a 5 mM TCEP solution (in 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.4), and the gold electrode was immersed in 300 μL of the aptamer solution for 20 hours to bind the double-stranded aptamer to the surface of the gold electrode. Subsequently, the change in current was measured in the same manner as above and is shown in Fig. 2(B).

[0055] Step 3

[0056] Next, an MCH solution was prepared by diluting 6-mercapto-1-hexanol (MCH) to a concentration of 5 mM in 5 mM TCEP, and a gold electrode bound to a double-stranded aptamer was immersed in the solution for 1 hour, after which it was washed with distilled water. The change in current was measured in the same manner as above and is shown in Fig. 2(C).

[0057] Step 4

[0058] Next, 50 μg of troponin I was dissolved in 10 mM PBS to prepare a 1 pg / mL solution (troponin solution), and the current change of an electrode combined with a double-stranded aptamer and MCH was measured in the same manner as above, but the current change was measured by adding the troponin solution to the electrolyte, and the results are shown in Fig. 2(D). In this step, the measurement was performed by carrying out a total of 8 cycles.

[0059] In Figures 2(A) to 2(D), which are the results of this experiment, the area within the graph represents the amount of electric charge, and a larger area indicates a larger current.

[0060] As a result of the experiment, referring to Figures 2(A) and 2(B), it was observed that the current decreased when a double-stranded aptamer was bound to the electrode, and looking at Figure 2(C), it was observed that the current decreased further when an MCH was bound. On the other hand, looking at Figure 2(D), it was confirmed that the current increased compared to Figure 2(C), which is a change that occurred as the double-stranded aptamer reacted with Troponin I to change its structure into a single-stranded aptamer. Through these experimental results, it was confirmed that the presence or concentration of protein in a sample can be evaluated by measuring the change in current using the field diagnostic method of the present invention.

[0061] In addition, when measuring current changes in step 4, an average of 32 seconds was taken per cycle, and an incomplete graph was observed in the first cycle, while a stable graph appeared from the second cycle onwards. Through these results, it was confirmed that the field diagnostic method of the present invention enables rapid diagnostic evaluation within one minute.

[0062] The present invention is not limited to the specific embodiments and descriptions described above, and various modifications can be made by anyone with ordinary knowledge in the technical field to which the invention pertains without departing from the essence of the invention as claimed in the claims, and such modifications fall within the scope of protection of the present invention.

Claims

Claim 1 A point-of-care diagnostic method comprising: a step of preparing a gold electrode bound to a double-stranded aptamer that specifically binds to a specific protein and 6-mercapto-1-hexanol; a step of contacting a sample with the gold electrode; and a step of applying voltage to the gold electrode and measuring the current flowing through the gold electrode. Claim 2 A field diagnostic method according to claim 1, characterized in that a specific protein in the sample specifically binds to a double-stranded aptamer, causing a structural change into a single-stranded aptamer and increasing charge flow. Claim 3 A field diagnostic method according to claim 1, characterized in that the analytical sensitivity of the field diagnostic method for the specific protein is 1 pg / mL. Claim 4 A field diagnostic method according to claim 1, characterized in that the change in current resulting from the specific binding of a double-stranded aptamer and a specific protein occurs within 1 minute. Claim 5 A point-of-care diagnostic kit comprising: a sample injection unit; a detection unit communicating with the sample injection unit to detect a specific protein in a sample; and a current measuring device connected to the detection unit to measure an electric current, wherein the detection unit comprises a gold electrode with a double-stranded aptamer and 6-mercapto-1-hexanol bonded to its surface.

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