Analysis method, analysis device, and analysis system

Abstract

To provide an improved analysis method or the like capable of analysis using a specific bonding function of DNA.SOLUTION: The analysis method is an analysis method for analyzing a target substance in a sample using the sample that may contain the target substance, the analysis method including: Acquiring a first signal of the sample after the reaction, reacting the sample with a second test substance containing a DNA assembly consisting of DNA of one or more types of sequences and acquiring a second signal of the sample after the reaction, and analyzing the target substance in the sample based on a difference between the first signal and the second signal, wherein a dissociation constant between the DNA assembly and the target substance is larger than a dissociation constant between the aptamer and the target substance.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present disclosure relates to an analytical method, an analytical device, and an analytical system for analyzing a target substance. [Background technology]

[0002] DNA (deoxyribonucleic acid) not only encodes genetic information of living organisms, but also specifically binds to specific substances. For example, Patent Document 1 discloses a biosensor that can analyze target DNA in real time using capture molecules composed of DNA on a gold substrate, using fluorescence enhancement as an indicator. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2007 / 094817 Summary of the Invention [Problem to be solved by the invention]

[0004] As disclosed in Patent Document 1, the specific binding function of DNA can be used to analyze target molecules (hereinafter also referred to as target substances). However, there is room for improvement in such analyses using the specific binding function of DNA. In view of the above, the present disclosure provides an improved analytical method and the like that enables analysis using the specific binding function of DNA. [Means for solving the problem]

[0005] In order to solve the above problem, an analytical method of one embodiment of the present disclosure is an analytical method for analyzing a target substance in a sample that may contain the target substance, the method comprising: reacting the sample with a first test substance including an aptamer that binds to the target substance, and obtaining a first signal of the sample after the reaction; reacting the sample with a second test substance including a DNA assembly consisting of DNA of one or more types of sequences, and obtaining a second signal of the sample after the reaction; and analyzing the target substance in the sample based on the difference between the first signal and the second signal, wherein the dissociation constant between the DNA assembly and the target substance is greater than the dissociation constant between the aptamer and the target substance.

[0006] Furthermore, an analytical device according to one embodiment of the present disclosure is an analytical device for analyzing a target substance in a sample that may contain the target substance, the analytical device comprising a processor and a memory, wherein the processor uses the memory to perform an analysis of the target substance in the sample based on the difference between a first signal of the sample after reaction when the sample is reacted with a first test substance including an aptamer that binds to the target substance, and a second signal of the sample after reaction when the sample is reacted with a second test substance including a DNA assembly consisting of DNA of one or more types of sequences, and the dissociation constant between the DNA assembly and the target substance is greater than the dissociation constant between the aptamer and the target substance.

[0007] An analysis system according to an aspect of the present disclosure includes the above-described analysis device, a first acquisition unit that acquires the first signal, and a second acquisition unit that acquires the second signal. [Effects of the Invention]

[0008] According to the present disclosure, an improved analytical method and the like are provided that enables analysis using the specific binding function of DNA. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 is a diagram showing an example of the configuration of an analysis system according to the present embodiment. [Figure 2] FIG. 2 is a diagram showing the concept of analysis according to this embodiment. [Figure 3] FIG. 3 is a diagram showing the concept of analysis according to this embodiment. [Figure 4] FIG. 4 is a diagram illustrating the binding dissociation constants of the DNA aptamer and DNA assembly of this embodiment. [Figure 5] FIG. 5 is a diagram illustrating the binding dissociation constants of the DNA aptamer and DNA assembly of this embodiment. [Figure 6] FIG. 6 is a flowchart showing the analysis method of this embodiment. [Figure 7] FIG. 7 is a diagram for explaining Example 1 of the present embodiment. [Figure 8] FIG. 8 is a diagram for explaining Example 1 of the present embodiment. [Figure 9] FIG. 9 is a diagram for explaining Example 1 of the present embodiment. [Figure 10] FIG. 10 is a diagram for explaining Example 2 of the present embodiment. [Figure 11] FIG. 11 is a diagram for explaining Example 2 of the present embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] (Findings that formed the basis of this disclosure) The specific binding function of DNA can be used to analyze the presence or concentration of specific molecules. In other words, the specific binding function of DNA allows for qualitative or quantitative analysis of specific molecules. Furthermore, because DNA sequence design and amplification methods have been established, it is easy to determine the base sequence of DNA for specific components and to amplify the determined base sequence to the required amount of DNA (for testing or as a product).

[0011] Specifically, DNA is a chain-like polymeric product in which four types of deoxyribonucleotides, each containing one of the bases adenine, guanine, thymine, or cytosine, are linked in a specific sequence. Currently, DNA of any sequence can be produced by solid-phase synthesis using phosphoramidites, although there are limitations on its length. Furthermore, amplification of template DNA can be performed almost automatically by reacting template DNA (DNA containing the target sequence and primer sequences attached to both ends) with dNTPs (a mixture of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP)), primers, polymerase, etc., using the polymerase chain reaction (PCR).

[0012] On the other hand, DNA is known to be negatively charged due to the presence of phosphodiester bonds, which means that in addition to specific binding, it can also form nonspecific bonds with positively charged molecules.

[0013] There is a demand for the specific binding function of DNA aptamers to analyze specific molecules in biological samples (e.g., blood samples, urine samples, etc.). Blood and urine contain various metabolic products, and a healthy state is characterized by a healthy metabolic state. Conversely, the specific binding function of DNA aptamers can be used to analyze whether a metabolic state is abnormal, thereby determining whether a person is healthy or not. In biological samples, it is assumed that a wide variety of molecules (non-target substances) are present in addition to specific molecules of interest (hereinafter referred to as target substances), and these non-target substances may be present in significantly higher concentrations than the target substances. As mentioned above, DNA binds non-specifically to positively charged molecules. Therefore, if a sample contains a high concentration of positively charged non-target substances, these non-target substances may also bind non-specifically to DNA aptamers.

[0014] When a target substance is analyzed using a DNA aptamer, the degree to which the DNA aptamer binds to the target substance is detected as some kind of signal, so if the DNA aptamer also binds nonspecifically to a non-target substance, the amount of nonspecific binding will be added to the signal. As a result, the amount of added nonspecific binding is unknown, making it difficult to quantify the degree to which the DNA aptamer binds to the target substance.

[0015] In view of these circumstances, the present disclosure calculates the signal that should be obtained from the binding of the original target substance to the DNA aptamer by experimentally obtaining the signal corresponding to the nonspecific binding of the DNA aptamer to the non-target substance and subtracting it from the signal obtained as a test result for an actual sample containing a mixture of the target substance and the non-target substance. This allows for analysis using the specific binding function of the DNA aptamer to be performed with improved accuracy.

[0016] In this specification, acquiring a signal may mean generating a signal using a sensor or the like, or may mean acquiring data indicating a signal stored in memory via a network or the like.

[0017] (Summary of the Disclosure) The outline of the present disclosure for realizing the above is as follows.

[0018] An analytical method according to a first aspect of the present disclosure is an analytical method for analyzing a target substance in a sample that may contain the target substance, comprising: reacting the sample with a first test substance including an aptamer that binds to the target substance and obtaining a first signal from the sample after the reaction; reacting the sample with a second test substance including a DNA assembly consisting of DNA of one or more types of sequences and obtaining a second signal from the sample after the reaction; and analyzing the target substance in the sample based on the difference between the first signal and the second signal, wherein the dissociation constant between the DNA assembly and the target substance is greater than the dissociation constant between the aptamer and the target substance.

[0019] According to this analytical method, the effect of nonspecific binding between the DNA aptamer and the non-target substance on the first signal is reproduced by the second signal resulting from nonspecific binding between the DNA assembly and the non-target substance, and by subtracting this, the portion of the first signal relating to the specific binding between the DNA aptamer and the target substance can be calculated. Thus, an analytical method that is improved in terms of accuracy and that enables analysis using the specific binding function of DNA can be realized.

[0020] Furthermore, an analytical method according to a second aspect of the present disclosure is the analytical method according to the first aspect, wherein the dissociation constant between the DNA assembly and the target substance is 100 times or more the dissociation constant between the aptamer and the target substance.

[0021] According to such an analytical method, the binding of DNA assemblies to target substances is made less likely, and an analytical method that is improved in terms of accuracy can be realized.

[0022] Furthermore, an analytical method according to a third aspect of the present disclosure is the analytical method according to the second aspect, wherein the dissociation constant between the DNA assembly and the target substance is 1000 times or more the dissociation constant between the aptamer and the target substance.

[0023] According to such an analytical method, the binding of DNA assemblies to target substances is made less likely, and an analytical method that is improved in terms of accuracy can be realized.

[0024] Furthermore, an analytical method according to a fourth aspect of the present disclosure is the analytical method according to any one of the first to third aspects, in which the aptamer is DNA, and the number of bases in the DNA of the aptamer is equal to the number of bases in the DNA constituting the DNA assembly.

[0025] According to such an analytical method, the conditions for the reaction between the first test substance and the sample and the reaction between the second test substance and the sample are the same, so that the first signal and the second signal can be easily compared.

[0026] Furthermore, an analytical method according to a fifth aspect of the present disclosure is an analytical method described in any one of the first to fourth aspects, in which the concentration of the aptamer contained in the first test substance is equal to the concentration of DNA constituting the DNA assembly contained in the second test substance.

[0027] According to such an analytical method, the conditions for the reaction between the first test substance and the sample and the reaction between the second test substance and the sample are the same, so that the first signal and the second signal can be easily compared.

[0028] Furthermore, an analytical method according to a sixth aspect of the present disclosure is the analytical method according to any one of the first to fifth aspects, wherein the first test substance and the second test substance each contain gold nanoparticles, and the first signal and the second signal include at least one of absorbance at a specific wavelength within a wavelength range of 500 to 550 nm and absorbance at a specific wavelength within a wavelength range of 600 to 800 nm.

[0029] According to such an analysis method, an analysis method that is improved in terms of accuracy can be realized by using gold nanoparticles.

[0030] Furthermore, an analytical method according to a seventh aspect of the present disclosure is the analytical method according to any one of the first to fifth aspects, wherein the aptamer is immobilized on a first substrate, the DNA constituting the DNA assembly is immobilized on a second substrate, the first signal is the intensity of fluorescence emitted by detecting the sample after reaction on the first substrate using a fluorescent label, and the second signal is the intensity of fluorescence emitted by detecting the sample after reaction on the second substrate using a fluorescent label.

[0031] According to such an analysis method, by detecting the reactions on the first and second substrates based on the intensity of the fluorescent light emitted, an analysis method that is improved in terms of accuracy can be realized. Furthermore, an analytical method according to an eighth aspect of the present disclosure is the analytical method according to any one of the first to fifth aspects, wherein the aptamer is immobilized on a first substrate, the DNA constituting the DNA assembly is immobilized on a second substrate, the first signal is the intensity of an electrochemical signal obtained by detecting the sample after a reaction on the first substrate using an electrochemical label, and the second signal is the intensity of an electrochemical signal obtained by detecting the sample after a reaction on the second substrate using an electrochemical label.

[0032] According to such an analysis method, reactions on the first substrate and the second substrate are detected by electrochemical signals, thereby realizing an analysis method that is improved in terms of accuracy.

[0033] Furthermore, an analytical device according to a ninth aspect of the present disclosure is an analytical device for analyzing a target substance in a sample, using the sample that may contain the target substance, and includes a processor and a memory, wherein the processor uses the memory to perform analysis of the target substance in the sample based on the difference between a first signal of the sample after reaction when the sample is reacted with a first test substance including an aptamer that binds to the target substance, and a second signal of the sample after reaction when the sample is reacted with a second test substance including a DNA assembly consisting of DNA of one or more types of sequences, and the dissociation constant between the DNA assembly and the target substance is greater than the dissociation constant between the aptamer and the target substance.

[0034] Such an analysis device provides the same effects as the above-described analysis method.

[0035] An analysis system according to a tenth aspect of the present disclosure includes the analysis device according to the ninth aspect, a first acquisition unit that acquires a first signal, and a second acquisition unit that acquires a second signal.

[0036] Such an analysis system provides the same effects as the above-described analysis method.

[0037] These comprehensive or specific aspects may be realized as a system, a method, an apparatus, an integrated circuit, a computer program, or a recording medium such as a computer-readable CD-ROM, or may be realized as any combination of a system, a method, an apparatus, an integrated circuit, a computer program, and a recording medium.

[0038] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0039] The embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement and connection configurations, steps, and step sequences shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, among the components in the following embodiments, components that are not described in the independent claims that represent the highest concepts are described as optional components. Furthermore, the drawings are not necessarily strict illustrations. In the drawings, substantially identical components are designated by the same reference numerals, and redundant descriptions may be omitted or simplified.

[0040] Furthermore, in this disclosure, terms indicating the relationship between elements, such as parallel and perpendicular, terms indicating the shape of elements, such as rectangle, and numerical values ​​do not only represent the strict meaning, but also include a substantially equivalent range, for example, a difference of about a few percent.

[0041] Hereinafter, DNA having a specific base sequence that binds to a specific molecule will be referred to as a DNA aptamer, or simply as an aptamer.

[0042] (Embodiment) Hereinafter, the embodiment will be specifically described with reference to FIGS. 1 to 11. FIG.

[0043] [Analysis System] First, an analysis system according to an embodiment will be described with reference to Figures 1 to 5. Figure 1 is a diagram showing an example of the configuration of an analysis system according to an embodiment.

[0044] The analysis system 200 according to this embodiment includes the analysis device 100, a first acquisition unit 151, and a second acquisition unit 152. The analysis system 200 is an information processing device with an integrated sensor that can be attached to a toilet or the like from which urine or feces can be collected as a specimen. For example, when a user defecates in the toilet, the integrated sensor automatically measures the excrement as a specimen, and the information processing functional unit processes the measurement results, which are physical quantities.

[0045] Alternatively, the analysis system 200 may be a cloud server that implements information processing functions and a device equipped with a sensor that is communicatively connected to the cloud server. Such a device may be a terminal device owned by the user, such as a smartphone, tablet, or PC. For example, the user measures a sample using a sensor attached to the terminal device and transmits the measurement results, which are physical quantities, to the cloud server. In the cloud server, an information processing function unit processes the received measurement results.

[0046] The sensors in the above two examples are the first acquisition unit 151 and the second acquisition unit 152. In other words, the first acquisition unit 151 and the second acquisition unit 152 are modules or analytical instruments for acquiring physical quantities. Note that, although the first acquisition unit 151 and the second acquisition unit 152 are described here as functionally separate components, the first acquisition unit 151 and the second acquisition unit 152 are implemented by the same sensor. The sensor performs the functions of each functional unit in a time-division manner, such as functioning as the first acquisition unit 151 at one time and as the second acquisition unit 152 at another time. This is preferable because it reduces the difference in measurement values ​​between the sensors compared to a configuration using two sensors. However, the first acquisition unit 151 and the second acquisition unit 152 may each be implemented by a separate sensor.

[0047] Furthermore, although details will be described later in the Examples section, the sensors of the first acquisition unit 151 and the second acquisition unit 152 can be any existing sensor (module or analytical equipment), such as a two-dimensional imaging device such as a hyperspectral camera, a spectrophotometer, a potential sensor, or a calorimetric device, as long as it can capture changes in physical quantities that occur before and after the bonding of substances.

[0048] The analytical device 100 includes a communication unit 101, a processor 102, and a memory 103. The analytical device 100 is an information processing device that processes measurement results received from a first acquisition unit 151 and a second acquisition unit 152 via the communication unit 101. The analytical device 100 realizes a measurement result processing function by executing a predetermined program stored in the memory 103 with the processor 102. The analytical device 100 may also be realized by a microcomputer or the like.

[0049] In this embodiment, the program stored in memory 103 may perform an analysis of the target substance in a sample based on the difference between a first signal of the sample after a reaction between the sample and a first test substance containing an aptamer that binds to the target substance and a second signal of the sample after a reaction between the sample and a second test substance containing a DNA assembly consisting of one or more types of DNA sequences. The communication unit 101 is a communication module connected to the first acquisition unit 151 and the second acquisition unit 152 and acquires measurement results from the first acquisition unit 151 and the second acquisition unit 152. Communication between the communication unit 101 and the first acquisition unit 151 and the second acquisition unit 152 may be wireless or wired. The communication method between the communication unit 101 and the first acquisition unit 151 and the second acquisition unit 152 is also not particularly limited. In addition, a local communication network or a wide area communication network may be interposed between the communication unit 101 and the first acquisition unit 151 and the second acquisition unit 152, or another device such as a gateway may be interposed between the communication unit 101 and the first acquisition unit 151 and the second acquisition unit 152.

[0050] The processor 102 is a logic circuit made up of semiconductors. The processor 102 reads out a program stored in the memory 103 from the memory 103 and executes it, and also reads out various data related to the processing of the program from the memory 103 and stores it therein.

[0051] The memory 103 is a storage device for storing information, and is realized by a semiconductor memory, a magnetic memory, an optical memory, or the like.

[0052] Next, the concept of analysis according to this embodiment will be described with reference to Figures 2 and 3. Figures 2 and 3 are diagrams showing the concept of analysis according to this embodiment.

[0053] 2 and 3, in the analysis according to the present embodiment, a DNA aptamer is used to qualitatively or quantitatively analyze a molecule (target substance) to which the DNA aptamer can specifically bind. That is, in the analysis according to the present embodiment, the binding amount of the DNA aptamer is measured as a physical quantity, and the presence or absence of the target substance in the system or the amount of the target substance is quantified.

[0054] A target substance is a molecule to which a DNA aptamer specifically binds, and can be, for example, proteins, lipids, sugars, nucleic acids, and other metabolic products derived from living organisms, various artificially synthesized chemicals such as pesticides, pollutants produced in the environment such as dioxins, viruses, microorganisms, and bacteria.

[0055] An example of a target substance is copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt, a phthalocyanine derivative compound. In the case of this phthalocyanine derivative compound, a DNA aptamer (K) with the base sequence GGG(TTAGGG)3 is D =4.2×10 -5 In the case of this phthalocyanine derivative compound, T 21 DNA assembly of the base sequence of AGAAGAGAAAGA and its complementary strand, and DNA assembly of λDNA (all KD Another example of a target substance is thrombin. In the case of thrombin, a DNA aptamer (K) with the base sequence GGTTGGTGTGGTTGG can be used. D =2.0×10 -7 In the case of thrombin, N 60 A DNA assembly (K D (: a very small value at the detection limit) can be used.

[0056] As shown in Figure 2, under ideal conditions where only the target substance is present in the system, when a sample (corresponding to the specimen described above) that may contain the target substance is added to the DNA aptamer, a specific bond is formed between the DNA aptamer and the target substance. The DNA aptamer is then divided into two types: one that has formed a specific bond and one that remains free. Because these two types behave differently, a signal can be obtained as a measurement result by distinguishing one from the other and measuring the amount or presence or absence of the other.

[0057] On the other hand, as shown in Figure 3(a), in an actual system, non-target substances exist in addition to target substances. Therefore, when a sample (corresponding to the specimen described above) that may contain both target and non-target substances is added to a location where a DNA aptamer is present, specific binding occurs between the DNA aptamer and the target substance, and non-specific binding occurs between the DNA aptamer and the non-target substance. DNA aptamers are divided into three types: DNA aptamers that have formed specific binding, DNA aptamers that have formed non-specific binding, and DNA aptamers that remain free. Of these three types, what needs to be measured separately is the amount or presence or absence of DNA aptamers that have formed specific binding, or the total amount or presence or absence of DNA aptamers that have formed non-specific binding and DNA aptamers that remain free. However, DNA aptamers that have formed nonspecific bonds and DNA aptamers that have formed specific bonds behave similarly, making it difficult to distinguish and measure only the amount or presence or absence of DNA aptamers that have formed specific bonds, or to distinguish and measure the total amount or presence or absence of DNA aptamers that have formed nonspecific bonds and DNA aptamers that are still free.

[0058] Therefore, in this embodiment, after the reaction shown in Figure 3(a), the total amount or presence of DNA aptamers that have formed specific bonds and DNA aptamers that have formed non-specific bonds is measured to obtain a first signal as the measurement result. Alternatively, the amount or presence of DNA aptamers that remain free is measured to obtain a first signal as the measurement result. Furthermore, after the reaction shown in Figure 3(b), the amount or presence of DNA derived from the DNA assembly that has formed non-specific bonds is measured to obtain a second signal as the measurement result.

[0059] In (b) of Figure 3, instead of DNA aptamers, a sample that may contain both target substances and non-target substances, as in (a) of Figure 3, is added to the DNA assembly. For example, two samples with approximately the same composition, obtained by aliquoting equal amounts from the same sample for (a) of Figure 3 and (b) of Figure 3, may be used. More preferably, a sample obtained by aliquoting multiple times for (a) of Figure 3 and a sample obtained by aliquoting multiple times for (b) of Figure 3 may be used. Averaging the measurement results is advantageous because it reduces variations in the amount of aliquots taken and the composition of each aliquot.

[0060] The DNA assemblies used in Figure 3(b) have low binding affinity to the target substance, in other words, the dissociation constant (K D ) is a DNA with a relatively large base sequence and consists of DNA with one or more types of base sequence. In contrast, DNA aptamers, which have specific binding function, have a high binding affinity to the target substance; in other words, the dissociation constant (K D ) is a DNA with a relatively small base sequence. When a sample containing both target and non-target substances is added to a DNA aggregate, the DNA aggregate and the target substance will not bond, or will only bond to a negligible extent.

[0061] The DNA aggregate and the non-target substance only bind nonspecifically, similar to the binding between a DNA aptamer and a non-target substance. In other words, after the reaction shown in Figure 3(b), the DNA can be roughly divided into two types: DNA that has formed nonspecific bonds and DNA that remains free. Because these two types behave differently, it is possible to distinguish one from the other and measure their quantity or presence / absence. As a result, the second signal indicates a measurement result measuring the quantity or presence / absence of DNA derived from the DNA aggregate that has formed nonspecific bonds. In other words, the second signal is a measurement result close to the quantity or presence / absence of DNA aptamers that have formed nonspecific bonds. Alternatively, the second signal indicates a measurement result measuring the quantity or presence / absence of DNA that remains free. In other words, the second signal is a measurement result close to the quantity or presence / absence of DNA aptamers that remain free.

[0062] The first and second signals obtained as measurement results by the measurement as described above are transmitted to the analysis device 100. The analysis device 100 calculates the difference between the received first and second signals.

[0063] This calculation yields the difference between the measurement result of the total amount or presence or absence of DNA aptamers that have formed specific bonds and DNA aptamers that have formed nonspecific bonds, and the measurement result of the amount or presence or absence of DNA derived from the DNA assembly that has formed nonspecific bonds. Since this difference is the difference between the measurement result of the total amount or presence or absence of DNA aptamers that have formed specific bonds and DNA aptamers that have formed nonspecific bonds, and the measurement result of the amount or near-presence or absence of DNA aptamers that have formed nonspecific bonds, a numerical value close to the amount or presence or absence of DNA aptamers that have formed specific bonds is calculated.

[0064] Alternatively, what is obtained by calculation is the difference between the measurement result close to the amount or presence of DNA aptamers still in a free state in the reaction shown in (b) of Figure 3 and the measurement result of the amount or presence of DNA aptamers still in a free state in the reaction shown in (a) of Figure 3. If it is assumed that the amount of DNA in the original DNA aptamer is the same as the amount of DNA in the original DNA assembly, then the measurement result of the amount or presence of DNA aptamers that have formed nonspecific bonds should be approximately the same as the measurement result of the amount or presence of DNA derived from the DNA assembly that has formed nonspecific bonds, and this difference corresponds to the amount or presence of DNA aptamers that have formed specific bonds.

[0065] In this manner, in this embodiment, two tests using a DNA aptamer and a DNA assembly are performed, and the difference between the two signals obtained in each test is calculated to determine the amount or presence of a DNA aptamer that has formed a specific bond of interest. Furthermore, since the amount or presence of a DNA aptamer that has formed a specific bond corresponds to the amount or presence of a target substance, the above calculations enable more accurate quantitative or qualitative analysis of the target substance.

[0066] The dissociation constant will be explained below with reference to Figs. 4 and 5. Figs. 4 and 5 are diagrams for explaining the binding dissociation constants of the DNA aptamer and DNA assembly of this embodiment. In Fig. 4, the horizontal axis shows the concentration of the target substance, and the vertical axis shows the proportion of DNA (DNA constituting the DNA aptamer or DNA assembly) bound to the target substance. The dissociation constant of DNA bound to the target substance is K D =10 -9 When M, 10 -8 When M, 10 -7 When M, 10 -6 When M, 10 -5 In addition, in Fig. 5, the graphs are shown for the cases where the concentration of the target substance is 10 -8 The specific values ​​of the proportion of DNA bound to the target substance under each dissociation constant condition when M (corresponding to the two-dot chain line in Figure 4) are summarized in a table.

[0067] As described above, the dissociation constant between the DNA constituting the DNA assembly and the target substance must be larger than the dissociation constant between the DNA aptamer and the target substance. This is because, when the number of bonds between the DNA constituting the DNA assembly and the target substance is sufficiently negligible compared to the number of bonds between the DNA aptamer and the target substance, the amount or presence of a DNA aptamer that has formed a specific bond can be calculated based on the difference described above.

[0068] For example, as shown in FIGS. 4 and 5, when the dissociation constant between the DNA aptamer and the target substance is 10 -9 M, 10% of the binding number is a dissociation constant that is two orders of magnitude larger, i.e., 100 times larger, 10 -7 M. In addition, when the dissociation constant between the DNA aptamer and the target substance is 10 -9 M, the dissociation constant that is 1% of the binding number is three orders of magnitude larger, i.e., 10 -6 This is the case when M. In this way, the dissociation constant between the DNA constituting the DNA assembly and the target substance is preferably, for example, 100 times or more, more preferably 1000 times or more, the dissociation constant between the DNA aptamer and the target substance.

[0069] [Analysis method] Next, the analysis method according to this embodiment will be described with reference to Fig. 6. Fig. 6 is a flowchart showing the analysis method according to this embodiment.

[0070] As shown in Figure 6, first, a sample is obtained and reacted with a first test substance (S101). The first test substance is, for example, a DNA solution containing a DNA aptamer. The sample solution is added to a reaction system (disposable tube, cuvette, etc.) containing a DNA solution with an adjusted concentration of the DNA aptamer, and the mixture is stirred to homogenize the mixture, allowing the reaction to proceed.

[0071] After a certain time has elapsed since the reaction, the next step is to measure and obtain the first signal of the sample (mixture of DNA solution and sample solution) after the reaction (S102). Note that the certain time may be the time until the binding between the target substance and DNA reaches equilibrium, a time previously determined by reaction kinetic analysis, or an excessive time such as several hours.

[0072] Next, the same sample as in step S101 is reacted with a second test substance (S103). The second test substance is, for example, a DNA solution containing a DNA assembly. The sample solution is added to a reaction system (disposable tube, cuvette, etc.) containing a DNA solution in which the DNA concentration of the DNA assembly has been adjusted, and the mixture is stirred to homogenize the mixture, allowing the reaction to proceed.

[0073] After a certain time has elapsed since the reaction, the next step is performed, and a second signal of the sample (mixture of DNA solution and sample solution) after the reaction is measured and obtained (S104). The sufficient time here may be the same as the sufficient time in step S101, or it may be different. The order of steps S101 to S104 may be reversed, or steps may be performed simultaneously in parallel. In particular, from the viewpoint of requiring sufficient time for the reaction, it is preferable that step S103 be performed at least partially overlapping with step S101.

[0074] Then, based on the difference between the acquired first signal and the acquired second signal, the amount or presence of DNA aptamer bound to the target substance is calculated, and a qualitative or quantitative analysis of the target substance is performed based on the calculation results (S105).

[0075] [Example 1] Hereinafter, some specific examples of realizing the above-described embodiment will be described based on examples. Figures 7 to 9 are diagrams for explaining Example 1 of the embodiment.

[0076] In Example 1 shown in Figures 7 to 9, the first and second test substances contain gold nanoparticles in addition to DNA aptamers or DNA assemblies, and an example is described in which the first and second signals are obtained using these gold nanoparticles. As shown in Figure 7, the DNA aptamers are present in the solution by adsorption onto the surface of the gold nanoparticles. As a result, the surfaces of the gold nanoparticles are charged by the negative charges of the DNA aptamers, causing electrostatic repulsion between the gold nanoparticles. In other words, the gold nanoparticles are present in a dispersed state.

[0077] When a sample containing both target and non-target substances is added, ideally, as shown in the figure, the non-target substances do not participate in the reaction, and the DNA aptamer detaches from the surface of the gold nanoparticles in proportion to the amount of target substance, specifically binding to the target substance. The gold nanoparticles from which the DNA aptamer has been detached have a correspondingly smaller negative charge, weakening the electrostatic repulsion. Therefore, by adding a salt such as NaCl, the gold nanoparticles approach each other and aggregate.

[0078] However, in reality, some non-target substances, for example, have a positive charge, which causes them to bind nonspecifically to the DNA aptamer and relieve the electrostatic repulsion between the gold nanoparticles. In such cases, aggregation of the gold nanoparticles can occur even without the target substance, as shown in Figure 8.

[0079] Therefore, as in the above embodiment, a solution containing a DNA aptamer and gold nanoparticles is prepared as the first test substance and reacted with the sample (for example, FIG. 9(a)). Furthermore, a solution containing a DNA assembly and gold nanoparticles is prepared as the second test substance and reacted with the sample (for example, FIG. 9(b)). In the reaction between the first test substance and the sample, the DNA aptamer binds specifically to the target substance and also nonspecifically to the non-target substance, resulting in aggregation of gold nanoparticles corresponding to both. An example of an absorption spectrum measured using a spectrophotometer is shown as the first signal to be measured.

[0080] 9(a), the first signal obtained by reacting the first test substance with the sample exhibits a significant decrease in absorbance at a specific wavelength within the 500-550 nm wavelength range (e.g., a wavelength showing high absorbance within that range, such as 520 nm) compared to the absorption spectrum of the first test substance alone before the reaction (the dashed-dotted line in the spectrum of the first signal), indicating absorbance a. Furthermore, the first signal obtained by reacting the first test substance with the sample exhibits a significant increase in absorbance at a specific wavelength within the 600-800 nm wavelength range (e.g., a wavelength showing high absorbance within that range, such as 680 nm) compared to the absorption spectrum of the first test substance alone before the reaction, indicating absorbance b.

[0081] On the other hand, in the reaction between the second test substance and the sample, the DNA aggregates do not bind to the target substance but bind nonspecifically to the non-target substance, resulting in the aggregation of gold nanoparticles corresponding to this. An example of the absorption spectrum measured using a spectrophotometer is shown as the second signal.

[0082] As shown in Figure 9(b), the second signal obtained by reacting the second test substance with the sample shows a certain decrease in absorbance at a specific wavelength within the wavelength range of 500 to 550 nm compared to the absorption spectrum of the second test substance alone before the reaction (the dashed-dotted line in the spectrum of the second signal), indicating absorbance c. Furthermore, the second signal obtained by reacting the second test substance with the sample shows a slight increase in absorbance at a specific wavelength within the wavelength range of 600 to 800 nm compared to the absorption spectrum of the second test substance alone before the reaction, indicating absorbance d.

[0083] The difference between the first and second signals is calculated as the difference spectrum between the solid line spectrum of the first signal and the solid line spectrum of the second signal, and the amount or presence of DNA aptamer specifically bound to the target substance is calculated based on the absorbance at a specific wavelength within the wavelength range of 500 to 550 nm and the absorbance at a specific wavelength within the wavelength range of 600 to 800 nm. An analytical result indicating the presence of the target substance can then be obtained that corresponds to the calculated amount or presence of DNA aptamer specifically bound to the target substance.

[0084] Furthermore, the difference between the first signal and the second signal may be calculated by calculating the amount or presence of a DNA aptamer that has specifically bound to a target substance based on the absorbance of the first signal at a specific wavelength within the wavelength range of 500 to 550 nm and the absorbance of the first signal at a specific wavelength within the wavelength range of 600 to 800 nm, or by calculating the amount or presence of a DNA aptamer that has specifically bound to a target substance based on either the absorbance of the first signal at a specific wavelength within the wavelength range of 500 to 550 nm or the absorbance of the first signal at a specific wavelength within the wavelength range of 600 to 800 nm.

[0085] Furthermore, when using absorbance at specific wavelengths, the ratio of absorbance at two specific wavelengths, i.e., b / a and c / d, may be used. The difference in such absorbance ratios can also be used to calculate the amount or presence of DNA aptamers specifically bound to a target substance. Furthermore, the difference between the absorption spectra before and after the reaction can be compared between the first and second signals. Since the dot-dash spectra have nearly identical shapes, similar results can be obtained. In order to align the pre-reaction signals between the first and second signals, it is important to align the concentration of the aptamer contained in the first test substance with the concentration of the DNA constituting the DNA aggregate contained in the second test substance, as well as the number of bases in the DNA aptamer with the number of bases in the DNA constituting the DNA aggregate. In other words, it is recommended to combine DNA aptamers of the same chain length with the DNA constituting the DNA aggregate.

[0086] [Example 2] 10 and 11 are diagrams for explaining Example 2 of the present embodiment.

[0087] 10 and 11, the reaction with the first test substance and the second test substance is carried out on a substrate, and therefore, the DNA aptamer of the first test substance and the DNA assembly of the second test substance are provided in a state of being immobilized on the substrate.

[0088] A sample containing both target and non-target substances is added to the mixture. In this example, the target and non-target substances are molecules that can be fluorescently labeled (bound with a fluorescent dye), such as proteins. Proteins may be fluorescently labeled with organic dyes, such as fluorescein and its derivatives, or biological fluorescent molecules, such as green fluorescent protein, or with quantum dots, whose emitted fluorescence properties can be designed.

[0089] When a sample containing both target and non-target substances is added, ideally, the non-target substances are fluorescently labeled but do not participate in the reaction, as shown in the figure. The DNA aptamer captures the fluorescently labeled target substance, and the concentration of the fluorescent dye on the substrate increases according to the amount of the target substance. The non-target substances are then removed from the substrate through a washing process.

[0090] However, in reality, non-target substances may bind to DNA aptamers through non-specific binding, and may remain on the substrate even after the washing process, as shown in Figure 11(a).

[0091] Therefore, as in the above embodiment, the DNA aptamer of the first test substance is reacted with the sample on a substrate on which the DNA aptamer is immobilized (for example, (a) of Figure 11). Also, the DNA assembly of the second test substance is reacted with the sample on a substrate on which the DNA assembly is immobilized (for example, (b) of Figure 11). In the reaction between the DNA aptamer and the sample, the DNA aptamer specifically binds to the target substance and also nonspecifically binds to the non-target substance, so that fluorescent dyes corresponding to both remain on the substrate. The first signal to be measured may be the fluorescence emission intensity measured using a fluorescence spectrophotometer.

[0092] On the other hand, in the reaction between the DNA aggregates and the sample, the DNA aggregates do not bind to the target substance but bind nonspecifically to the non-target substance, so the corresponding fluorescent dye remains on the substrate. The second signal to be measured can be the fluorescence intensity measured using a fluorescence spectrophotometer.

[0093] The amount or presence of DNA aptamer specifically bound to the target substance is calculated based on the difference between the first and second signals, which indicate the fluorescence emission intensity on the substrate obtained as described above. An analytical result indicating the presence of the target substance can then be obtained, which corresponds to the calculated result of the amount or presence of DNA aptamer specifically bound to the target substance.

[0094] In the above example, the substrate may be made of any material that can immobilize DNA so that it does not detach from the substrate during the washing process. For example, when a gold substrate is used, DNA can be immobilized on the gold substrate by reacting DNA with thiolated ends. Even when a substrate other than gold is used, DNA can be immobilized by reacting thiolated DNA as long as a thin gold film can be formed on the substrate. Substrates other than gold may include, for example, inorganic materials such as quartz, glass, silica, and ceramics; resins such as polystyrene, polycarbonate, and cycloolefin polymer; natural materials including rubber materials such as hydrogel, agarose, cellulose, and isoprene; and metal materials such as iron, alumina, and silver. Furthermore, by using DNA modified with amino groups at the ends and a substrate with carboxyl groups on the surface, DNA can be immobilized on the substrate through a condensation reaction between the two functional groups. Substrates with carboxyl groups on the surface include glass substrates, plastic substrates, etc., modified with carboxyl groups. Furthermore, when a glass substrate or plastic substrate modified with amino groups and DNA modified with carboxyl groups are used, DNA can be immobilized on the substrate through a condensation reaction between the two functional groups. As another example, if a glass substrate with the protein avidin immobilized on its surface is prepared and reacted with DNA modified at its ends with biotin, DNA can be immobilized on the glass substrate through a binding reaction between avidin and biotin.

[0095] On the other hand, in the example using the substrate shown in Figures 10 and 11, electrochemical labels can be used instead of fluorescent labels. That is, in Figures 10 and 11, target and non-target substances can be labeled with electrochemically detectable substances instead of fluorescent labels, thereby electrochemically obtaining the first and second signals. In this case, the substrate can be made of conductive materials such as gold, glassy carbon, boron-doped diamond, or tin-doped indium tin oxide (ITO), which can immobilize DNA so that it does not detach during the washing process. Examples of electrochemical labeling substances include ferrocene. In this case, the first and second signals correspond to electrochemical signals, such as current values, that are obtained by converting the presence and / or concentration of the electrochemical labeling substance.

[0096] In addition, in an example using a substrate such as those shown in FIGS. 10 and 11, thermal energy generated in the reaction between DNA and the target substance and non-target substance may be detected and used as the first and second signals. In this case, the substrate is made of a material capable of transmitting thermal energy, i.e., a thermally conductive material. However, the bond between the DNA and the substrate material may be formed in any manner as long as the thermal energy can be detected and a signal can be obtained. Another substance that mediates the bond between the DNA and the substrate material may be present between them. Furthermore, examples of detecting thermal energy to obtain the first and second signals are not limited to those using substrates such as those shown in FIGS. 10 and 11. Even if DNA is not immobilized on a substrate or the like, if the reaction system, i.e., the vessel containing the reaction system containing DNA and the sample, is made of a thermally conductive material (e.g., gold, as mentioned above, or an alloy known as Hastelloy (registered trademark)), it is possible to detect the thermal energy transmitted through the vessel and obtain the first and second signals.

[0097] In Examples 1 and 2, the sample may not contain a non-target substance that non-specifically binds to the DNA aptamer, but in that case, the DNA aptamer does not non-specifically bind to the non-target substance in the reaction between the first test substance and the sample, and the DNA ensemble does not non-specifically bind to the non-target substance in the reaction between the second test substance and the sample. Therefore, even when this method is performed in a case where a non-target substance that non-specifically binds to the DNA aptamer is not contained, highly accurate analysis is possible, just as when a non-target substance that non-specifically binds to the DNA aptamer is contained.

[0098] While the analytical method, analytical device, and analytical system according to the present disclosure have been described above based on the embodiments, the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications conceivable by those skilled in the art to the embodiments and other forms constructed by combining some of the components of the embodiments are also included within the scope of the present disclosure. [Industrial Applicability]

[0099] The analytical methods and the like of the present disclosure are useful for qualitative or quantitative analysis of target substances. [Explanation of symbols]

[0100] 100 Analyzer 101 Communications Department 102 processors 103 memory 151 First acquisition part 152 Second acquisition part 200 Analysis System

Claims

1. An analytical method for analyzing a target substance in a sample that may contain the target substance, comprising: reacting the sample with a first test substance containing an aptamer that binds to the target substance, and acquiring a first signal from the sample after the reaction; Reacting the sample with a second test substance containing a DNA assembly consisting of DNA of one or more types of sequences, and acquiring a second signal of the sample after the reaction; performing an analysis of the target substance in the sample based on a difference between the first signal and the second signal; The dissociation constant between the DNA assembly and the target substance is larger than the dissociation constant between the aptamer and the target substance. Analysis method.

2. The dissociation constant between the DNA assembly and the target substance is 100 times or more the dissociation constant between the aptamer and the target substance. The analytical method according to claim 1 .

3. The dissociation constant between the DNA assembly and the target substance is 1000 times or more greater than the dissociation constant between the aptamer and the target substance. The analytical method according to claim 2.

4. the aptamer is DNA, The number of bases in the DNA of the aptamer is equal to the number of bases in the DNA constituting the DNA assembly. The analytical method according to claim 1 .

5. The concentration of the aptamer contained in the first test substance is equal to the concentration of DNA constituting the DNA assembly contained in the second test substance. The analytical method according to claim 1 .

6. each of the first test substance and the second test substance comprises gold nanoparticles; The first signal and the second signal include at least one of absorbance at a specific wavelength within a wavelength range of 500 to 550 nm and absorbance at a specific wavelength within a wavelength range of 600 to 800 nm. The analytical method according to any one of claims 1 to 5.

7. the aptamer is immobilized on a first substrate; DNA constituting the DNA assembly is immobilized on a second substrate; the first signal is the intensity of fluorescence detected by a fluorescent label from the sample after the reaction on the first substrate; The second signal is the intensity of fluorescence detected by a fluorescent label from the sample after the reaction on the second substrate. The analytical method according to any one of claims 1 to 5.

8. the aptamer is immobilized on a first substrate; DNA constituting the DNA assembly is immobilized on a second substrate; the first signal is the intensity of an electrochemical signal detected by an electrochemical label of the sample after the reaction on the first substrate; The second signal is the intensity of an electrochemical signal detected by an electrochemical label of the sample after the reaction on the second substrate. The analytical method according to any one of claims 1 to 5.

9. An analytical device for analyzing a target substance in a sample, the analytical device using the sample that may contain the target substance, a processor; a memory; The processor uses the memory to: an analysis of the target substance in the sample based on a difference between a first signal of the sample after a reaction when the sample is reacted with a first test substance including an aptamer that binds to the target substance, and a second signal of the sample after a reaction when the sample is reacted with a second test substance including a DNA assembly consisting of DNA of one or more types of sequences; The dissociation constant between the DNA assembly and the target substance is larger than the dissociation constant between the aptamer and the target substance. Analyzer.

10. The analysis device according to claim 9 ; a first acquisition unit that acquires the first signal; a second acquisition unit that acquires the second signal; Analysis system.

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