Detecting kit, detecting device, and detecting method for detecting trace amount of heavy metal in sample

Abstract

The objective of the present invention is to provide a detecting kit, a detecting device, and a detection method employing the same, which enable trace amounts of heavy metals contained in water, soil, foodstuffs, and food products, etc., to be detected rapidly, with high sensitivity, and with heavy metal selectivity, using a simple method at or around a site such as a water source, agricultural land, or a food product factory, for example. Provided is a detecting kit for detecting a trace amount of a heavy metal in a sample, the kit containing a DNA aptamer that is labeled with a fluorescent molecule and that binds specifically to the heavy metal. The heavy metal in the sample may be selected from cadmium, mercury, and arsenic. The detection may be performed by visual observation, fluorescence analysis, or fluorescence polarization analysis.

Description

Detection kit, detection device, and detection method for detecting trace amounts of heavy metals in a sample

[0001] The present invention relates to a detection kit, a detection device, and a detection method for detecting trace amounts of heavy metals in a sample.

[0002] Problems caused by heavy metals in the water and soil at construction sites and agricultural land have long been known. For example, when heavy metals are present in the water or soil of rice or vegetable farms, the heavy metals accumulate in the cultivated crops, such as rice and vegetables. By consuming such crops or drinking groundwater containing heavy metals, the heavy metals are absorbed into the human body. Similar problems arise from pesticides and impurities in food ingredients and foods.

[0003] When ingested, heavy metals can have serious adverse health effects. Heavy metals such as cadmium, mercury, and arsenic can cause acute or chronic symptoms in the human body, including developmental disorders in children, problems during pregnancy, damage to the liver, kidneys, and intestines, anemia, and cancer. Cadmium, mercury, and arsenic are consistently listed among the World Health Organization's top 10 chemicals of public health concern. Cadmium and related compounds are currently listed in Group A of the IARC classification of human carcinogens.

[0004] Many conventional methods for testing heavy metals in water or soil require a long time for detection or use expensive equipment that requires careful handling when detecting heavy metals. Patent Document 1 discloses a method for analyzing the content of harmful substances using an X-ray fluorescence analyzer to measure the content of harmful substances such as heavy metals eluted from soil. Patent Document 2 discloses a method for adding a chelating agent to a test solution obtained from soil to form a chelate complex, which is then subjected to X-ray fluorescence analysis.

[0005] Cadmium can be detected by several instrumental analytical techniques, such as atomic absorption spectrometry and inductively coupled plasma analysis, but these techniques are known to require relatively high initial and running costs (Non-Patent Documents 1 and 2). Furthermore, most conventional detection methods require large, expensive equipment and trained operators.

[0006] Japanese Patent No. 4647405 Japanese Patent Laid-Open No. 2004-294329

[0007] Chen K, Mou P, Zhu A, Chen P, Chen J, Gao G, Wang X, Feng X, Yu C (2023) Environmental Monitoring and Assessment 195. Pyle SM, Nocerino JM, Deming SN, Palasota JA, Palasota JM, Miller EL, Hillman DC, Kuharic CA, Cole WH, Fitzpatrick PM, Watson MA, Nichols KD (1996) Environmental Science & Technology 30:204-213.

[0008] To address the above-mentioned problems, if the presence of heavy metals in water or soil could be detected quickly and easily near water sources, farmland, or food factories, it would be possible to take measures such as quickly cleaning the contaminated water or soil or stopping shipments. For example, if the concentration of heavy metals in water were lower (up to 10 －１０ Although a simple method for detecting heavy metals in trace amounts (mol / L) has been desired, it has not yet been achieved. There is a strong demand for a system that can quickly, inexpensively, and easily detect trace amounts of heavy metals in the environment, such as water and soil.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a detection kit, detection device, and detection method that can detect the presence of trace amounts of heavy metals in water, soil, ingredients, and food using a simple method near water sources, farmland, and food factories with high sensitivity and in a short period of time.

[0010] In order to solve the above problems, the detection kit, detection device, and detection method of the present invention for detecting trace amounts of heavy metals in a sample employ the following means.

[0011] A first aspect of the present invention provides a detection kit for detecting trace amounts of heavy metals in a sample, which contains a DNA aptamer that is labeled with a fluorescent molecule and selectively binds to a specific heavy metal.

[0012] In the first aspect, the heavy metal may be at least one selected from the group consisting of cadmium, mercury, and arsenic.

[0013] In the first aspect, detection may be performed visually, by fluorescence analysis, or by fluorescence polarization analysis.

[0014] In the first aspect, the sample may be water, soil, foodstuffs or food.

[0015] In the first aspect, the DNA aptamer may be labeled with two fluorescent molecules.

[0016] In the first aspect, the buffer may further comprise a MOPS buffer, a HEPES buffer, or a sodium bicarbonate-carbonate buffer.

[0017] A second aspect of the present invention provides a detection device that includes a DNA aptamer that is labeled with a fluorescent molecule and that selectively binds to a specific heavy metal, and a fluorescence measurement unit that measures the fluorescence intensity of the fluorescent molecule of the DNA aptamer, and that detects trace amounts of heavy metals in a sample by measuring the fluorescence intensity or fluorescence polarization intensity derived from the fluorescent molecule that labels the DNA aptamer that has bound to the specific heavy metal.

[0018] In the second aspect, the heavy metal may be at least one selected from the group consisting of cadmium, mercury, and arsenic.

[0019] In the second embodiment, the heavy metals may be detected by fluorescence analysis or fluorescence polarization analysis.

[0020] In the second aspect, the sample may be water, soil, foodstuffs or food.

[0021] A third aspect of the present invention provides a detection method for detecting trace amounts of heavy metals in a sample, comprising the steps of: mixing a solution in which a DNA aptamer that is labeled with a fluorescent molecule and that specifically binds to a specific heavy metal is dissolved with a liquid containing a sample that may contain the specific heavy metal to obtain a mixed solution; measuring the fluorescence intensity or fluorescence polarization intensity derived from the fluorescent molecule of the DNA aptamer contained in the mixed solution; and detecting the heavy metal based on the measurement results.

[0022] In the third aspect, the step of measuring the fluorescence polarization intensity may further include a step of analyzing the concentration of heavy metals.

[0023] The detection kit and detection method of the present invention enable rapid and simple detection of trace amounts of heavy metals on the subnanomolar order without requiring advanced detection technology. By providing an easily usable detection device, the detection device of the present invention can be placed near sites where water quality testing is required, such as farms, wells, water purification plants, poultry farms, and food factories, and the presence or absence of heavy metals in a sample can be easily detected within a short time after sample collection.

[0024]

[0033] FIG. 1 is a conceptual diagram of a method for detecting trace amounts of heavy metals by fluorescence polarization using a DNA aptamer of the present invention.

[0034] FIG. 1 is a conceptual diagram of a method for detecting trace amounts of heavy metals using FRET when a DNA aptamer having an FITC group at one end and a TAMRA group at the other end is used.

[0035] FIG. 1 is a diagram showing an example of a heavy metal detection member used in the heavy metal detection method of the present invention.

[0036] FIG. 2 is an image of heavy metal detection using the heavy metal detection member of the present invention.

[0037] FIG. 3 is a diagram showing an example of a heavy metal detection device used in the heavy metal detection method of the present invention.

[0038] FIG. 4 is a graph comparing the fluorescence intensities of aptamers 1 to 12 dissolved in MOPS buffer and HEPES buffer. For each aptamer, the left bar shows the results when MOPS buffer was used, and the right bar shows the results when HEPES buffer was used.

[0039] FIG. 5 is a graph showing the relationship between the final cadmium concentration of a solution obtained by mixing aptamers 1 to 12 with a cadmium solution and the fluorescence intensity of the mixed solution. 1 is a graph showing the buffer dependency of fluorescence intensity for solutions obtained by mixing a cadmium solution with a MOPS buffer or a HEPES buffer solution for each of aptamers 1, 5, and 6. For each aptamer, the bar on the left shows the results when a MOPS buffer was used, and the bar on the right shows the results when a HEPES buffer was used. This graph shows the relationship between the pH of the buffer used to dissolve aptamer 1 or aptamer 4 and the fluorescence polarization degree of a mixture of the dissolved buffer and a cadmium solution. This graph shows the relationship between the final cadmium concentration of a solution obtained by mixing a buffer solution and a cadmium solution for each of aptamers 1 to 12 and the fluorescence polarization degree of the mixed solution. This graph shows the buffer dependency of fluorescence polarization degree for a mixture of a cadmium solution and a MOPS buffer or a HEPES buffer solution for each of aptamers 1 to 12. For each aptamer, the bar on the left shows the results when a MOPS buffer was used, and the bar on the right shows the results when a HEPES buffer was used. This graph shows the relationship between the concentration of the cadmium-containing solution to be mixed and the fluorescence polarization degree of the mixed solution of the aptamer and cadmium solution in sodium bicarbonate-carbonate buffer (pH 10) using buffer solutions of aptamer 1 and aptamer 4.14A shows a graph illustrating the relationship between the final cadmium concentration of solutions obtained by mixing solutions of aptamers 1 to 12 with a cadmium solution dissolved in well water and the fluorescence polarization index of the mixed solutions. 14B shows a graph illustrating the relationship between the fluorescence polarization index at various temperatures for mixed solutions obtained by mixing MOPS buffer solutions of aptamers 1, 6, 7, and 11 with a cadmium ion-containing solution. 14C shows photographs taken under room light of filters onto which a cadmium ion solution or a blank solution was dropped, followed by the addition of an aptamer solution of the present invention. 14B shows a filter onto which a blank solution not containing cadmium ions was dropped, and 14C shows a filter onto which a cadmium ion-containing solution (1 mM) was dropped. 14C shows photographs of the filters (a) and (b) photographed in FIG. 14A taken under blue light through an orange filter.

[0025] An embodiment of a detection kit, a detection device, and a detection method for detecting trace amounts of heavy metals in a sample according to the present invention will be described below.

[0026] This embodiment provides a novel approach for rapid and sensitive detection of heavy metals contained in water, soil, ingredients, food, etc., utilizing changes in the fluorescence intensity or fluorescence polarization intensity of a fluorescent molecule (fluorophore) chemically bound to an aptamer that occur when the aptamer binds to the heavy metal. The ability to more rapidly detect trace amounts of heavy metals contained in water, soil, ingredients, food, etc. makes it possible to reduce human exposure to heavy metals.

[0027] One of the features of the heavy metal detection method according to this embodiment is that it combines fluorescence polarization and aptamers. Aiming to establish a simple and rapid method for detecting and quantifying heavy metals contained in environmental water, drinking water, soil, and the like, the inventors investigated various heavy metal sensor molecules and heavy metal detection protocols. In the process, the inventors discovered that by using a fluorescently labeled aptamer that selectively binds to highly toxic heavy metals such as cadmium, mercury, and arsenic as a sensor molecule and an existing fluorescence polarization analyzer, it is possible to detect and measure these heavy metals in samples such as environmental water, drinking water, and soil.

[0028] Aptamers are artificially synthesized DNA, RNA, or peptides of medium molecular size (10 to 100 bases), or derivatives thereof, that can selectively and specifically bind to target substances by appropriately designing their sequences. In this embodiment, a DNA aptamer consisting of a single-stranded DNA modified with a fluorescent molecule is used as a probe for capturing heavy metals. DNA aptamers can be obtained in a relatively short period of time using search (selection) techniques such as the SELEX method. Furthermore, DNA aptamers can be produced in large quantities at relatively low cost by chemical synthesis.

[0029] Antibodies, which are composed of proteins, have traditionally been used as molecules that bind to target substances. DNA aptamers, despite being approximately 1 / 10 or less of the molecular weight of antibodies, exhibit high target selectivity comparable to that of antibodies. DNA aptamers, which are composed of single-stranded DNA, are generally more stable compounds than antibodies, which are composed of proteins. For example, dried DNA aptamers do not require refrigeration or freezing for storage and can be stored for a long period of time. This is one of their major advantages over antibodies.

[0030] An aptamer-based heavy metal detection method using fluorescence polarization measurement will be described with reference to Figure 1. Fluorescence polarization is a method for analyzing the interaction between a fluorescent molecule (including "a molecule bound to a fluorescent molecule" herein) and a target substance by measuring fluorescence polarization using a fluorescence polarimeter.

[0031] Fluorescence polarization refers to the degree to which a fluorescent molecule rotates between excitation and emission of fluorescence. When a three-dimensional structure is formed, the degree of freedom of rotation of the fluorescent molecule decreases, depending on the three-dimensional structure, and the rotation of the fluorescent molecule slows down. At this time, the fluorescence polarization increases. In this way, the fluorescent molecule's movement becomes limited due to the influence of the surrounding three-dimensional structure, or it becomes immobilized by adhering to a large amount of target substance. As a result, the rotation of the fluorescent molecule is strictly restricted, and the fluorescence polarization increases. Measuring fluorescence polarization allows for rapid analysis of the interaction between a fluorescent molecule and a target substance, even with a small sample volume.

[0032] FIG. 1A shows a conceptual diagram of a method for detecting trace amounts of heavy metals by fluorescence polarization using a DNA aptamer according to this embodiment. The DNA aptamer used in this embodiment has a fluorescent molecule bound to at least one of the 3'-end and 5'-end of single-stranded DNA. The fluorescent molecule is generally bound to the DNA aptamer via a linker. Hereinafter, a DNA aptamer modified with a fluorescent molecule will also be referred to as a "fluorescently labeled DNA aptamer." The target substances of the DNA aptamer according to this embodiment are heavy metals contained in environmental water, drinking water, soil, etc.

[0033] In an environment where the target heavy metal is not present, the fluorescently labeled DNA aptamer 1 labeled with the fluorescent molecule 2 exists as a single strand. At this time, in the solution in which the fluorescently labeled DNA aptamer 1 is dissolved, the fluorescent molecule 2 bound to the DNA aptamer 1 rotates freely. This corresponds to the state before heavy metal binding, shown at the base of the arrow in Figure 1A. At this time, the degree of fluorescence polarization of the solution is small.

[0034] When this fluorescently labeled DNA aptamer comes into contact with a heavy metal target substance in solution, its three-dimensional structure changes so that a portion of the single strand surrounds the heavy metal and forms a loop. Meanwhile, the portion of the DNA aptamer sequence that is not directly involved in binding to the heavy metal forms a double strand, resulting in a stem-loop structure (also known as a hairpin structure) as a whole. The DNA aptamer with this stem-loop structure forms a three-dimensional structure and is stabilized as a whole. The arrow in Figure 1A shows an image of a fluorescently labeled DNA aptamer 1' that has bound to a heavy metal 10 and formed a stem-loop structure. The double-stranded stem portion contributes to stabilizing the three-dimensional structure after binding to the target substance (heavy metal in this embodiment).

[0035] The DNA aptamer that has incorporated the heavy metal 10 and formed a three-dimensional structure has a rigid structure. Therefore, the rotational movement of the fluorescent molecules that label the DNA aptamer is restricted and reduced. As a result, the fluorescence polarization increases.

[0036] In principle, fluorescence polarization analysis does not take into account the concentration of fluorescent molecules in the solution, so changes in the concentration of fluorescent molecules have only a slight effect on the polarization value. However, it should be noted that fluorescence polarization is affected by the sample matrix, so it is necessary to confirm the chemical components contained in the sample to be measured in advance.

[0037] In the heavy metal detection method according to this embodiment, the degree of binding of the heavy metal to the fluorescently labeled DNA aptamer is detected by measuring the degree of change in the degree of fluorescence polarization (ΔmP). Fluorescence polarization analysis has high detection sensitivity, making it possible to detect changes in the degree of fluorescence polarization even in extremely small amounts of sample.

[0038] Therefore, by using the fluorescently labeled DNA aptamer of this embodiment, heavy metals can be detected at subnanomolar levels. The inventors have investigated fluorescently labeled DNA aptamers with various sequences and have developed a fluorescently labeled DNA aptamer-based heavy metal detection kit for detecting various heavy metals.

[0039] According to the heavy metal detection method of this embodiment, the degree of change in the fluorescence polarization of the sample solution is detected and measured. Therefore, there is no need to separate and purify the fluorescently labeled DNA aptamer bound to the heavy metal from the fluorescently labeled DNA aptamer not bound to the heavy metal. This makes it possible to shorten the preparation time of the sample solution used for measurement, and also shorten the overall detection time. This allows for rapid measurement and detection of water or soil samples to be measured on-site or in the surrounding area. Furthermore, the heavy metal detection method of this embodiment can be extended not only to the detection of heavy metals in water or soil, but also to the detection of pesticides and the like in ingredients and foods.

[0040] Examples of fluorescent molecules that can be bound to the DNA aptamer of this embodiment include commonly used fluorescent molecules such as fluorescein, fluorescein isothiocyanate (FITC), Alexa Fluor® 488, Alexa Fluor® 514, Texas Red™, and Cy3 groups. These fluorescent molecules can serve as donor fluorescent molecules. FIG. 1A illustrates a DNA aptamer to which an FITC group is applied as the donor fluorescent molecule.

[0041] When a heavy metal binds to a fluorescent molecule bound to a DNA aptamer, the three-dimensional structure of the aptamer changes as described above, restricting the rotation of the fluorescent molecule. This restriction of rotation increases the degree of fluorescence polarization, and by measuring the degree of increase in fluorescence polarization, it is possible to detect whether or not the fluorescent molecule has bound to a heavy metal.

[0042] As shown in the example of Figure 1A, in this embodiment, a DNA aptamer bound to only one fluorescent molecule can also be used as a detection probe using fluorescence polarization. However, in addition to the donor fluorescent molecule, another fluorescent molecule serving as a FRET acceptor can also be bound to the DNA aptamer. Figure 1B shows an example of detection using such a fluorescently labeled DNA aptamer. Examples of acceptor fluorescent molecules include commonly used fluorescent molecules such as 5-carboxytetramethylrhodamine (TAMRA) group, Cy5 group, Cy5.5 group, ROX group, Alexa Fluor (registered trademark) 555, and Alexa Fluor (registered trademark) 647.

[0043] As in the above example, a fluorescent molecule that can serve as a donor and a fluorescent molecule that can serve as an acceptor are bound to appropriate positions within a single DNA aptamer molecule. When a heavy metal, which is a target substance, binds to this DNA aptamer, a change in the three-dimensional structure occurs, and the distance between the donor fluorescent molecule and the acceptor fluorescent molecule decreases.

[0044] When light having the excitation wavelength of the donor fluorescent molecule is irradiated in this state, fluorescence resonance energy transfer (FRET) occurs between the two fluorescent molecules of one DNA aptamer molecule, and energy is transferred to the acceptor fluorescent molecule by excitation of the donor fluorescent molecule. As a result, the acceptor fluorescent molecule exhibits fluorescence. Therefore, the binding of heavy metals to the fluorescently labeled DNA aptamer can be detected as a change in the degree of fluorescence polarization or an increase in fluorescence intensity at the detection wavelength of the acceptor fluorescent molecule.

[0045] In the example of Figure 1B, a donor fluorescent molecule 12 (FITC in this example) and another acceptor fluorescent molecule 13 (TAMRA in this example) are bound to appropriate positions on the DNA aptamer (one at each end of the DNA strand in this example) to one molecule of DNA aptamer 11. When a heavy metal, which is the target substance, approaches the fluorescently labeled DNA aptamer sufficiently, the three-dimensional structure of aptamer 11 changes, bringing FITC and TAMRA closer in distance.

[0046] At this time, in the fluorescently labeled DNA aptamer 11' to which the heavy metal target substance is bound, FRET occurs between the donor FITC molecule and the acceptor TAMRA molecule, increasing the fluorescence intensity from the TAMRA molecule. In the example of Figure 1, by irradiating with light of the excitation wavelength of FITC, the donor fluorescent molecule 12, and measuring the change in fluorescence intensity of the wavelength from the TAMRA molecule, the acceptor fluorescent molecule 13, it is possible to detect whether or not a heavy metal is present in the measurement system.

[0047] It is also possible to bind only the acceptor fluorescent molecule described above to the DNA aptamer of this embodiment and use it as a fluorescence polarization detection probe.

[0048] Furthermore, the DNA aptamer of this embodiment may be bound not only to a fluorescent molecule but also to a quencher molecule that does not emit fluorescence itself but quenches the fluorescence from other molecules. Examples of quencher molecules include commonly used molecules such as DABCYL, Tide Quencher, QSY7, and black hole quencher (BHQ).

[0049] An aptamer is synthesized in which a fluorescent molecule that can serve as a donor and a quencher molecule are bound via linkers to appropriate positions within a single DNA aptamer molecule. In the example shown in Figure 1B, the quencher molecule is bound instead of the acceptor fluorescent molecule 13. When a target substance binds to this DNA aptamer, the three-dimensional structure changes, bringing the fluorescent molecule and quencher molecule closer in distance.

[0050] In this state, when light having the excitation wavelength of the donor fluorescent molecule is irradiated, the fluorescent molecule functions as a donor, and FRET occurs between the donor fluorescent molecule and the quencher molecule. As a result, the fluorescence intensity at the detection wavelength of the donor fluorescent molecule decreases. In this way, the presence or absence of binding of a heavy metal to the fluorescently labeled DNA aptamer can be detected as a decrease in the fluorescence intensity at the detection wavelength of the donor fluorescent molecule.

[0051] Thus, the fluorescently modified DNA aptamer used in this embodiment can be a useful tool that can be used to detect heavy metal ions in a sample, such as in a system that detects changes in fluorescence polarization accompanying structural changes upon binding to a target substance, a system that detects changes in fluorescence intensity, a system that detects the ON-OFF state of fluorescence, or a colorimetric detection tool.

[0052] By chemically modifying any position in the DNA aptamer sequence with a fluorescent molecule, the DNA aptamer can be used as a detection probe using fluorescence. This degree of freedom in modification is also an advantage of DNA aptamers that antibodies do not have. Furthermore, DNA aptamers can be designed to undergo much larger changes in three-dimensional structure and mobility than antibodies upon binding to a target substance.

[0053] Furthermore, when a DNA aptamer in which both a donor fluorescent functional group and an acceptor fluorescent functional group are bound in one molecule is used, the wavelength of fluorescence from the acceptor fluorescent molecule can be used for detection, as described above. This allows target substance detection to be performed in a short time without the need for a purification step, even if a fluorescently labeled DNA aptamer that is not bound to the target substance and a fluorescently labeled DNA aptamer that is bound to the target substance are mixed in the measurement system.

[0054] Furthermore, in the detection systems of Figures 1A and 1B, when an FITC group is used as the donor fluorescent molecule, instead of measuring fluorescence polarization, it is also possible to measure the degree to which the fluorescence intensity decreases by measuring the fluorescence wavelength derived from the FITC group.

[0055] 1A and 1B illustrate an image in which only one heavy metal is bound to the DNA aptamer, but the number of heavy metal ions bound to the DNA aptamer is not limited to one, and depending on the design of the DNA aptamer, multiple heavy metal ions may bind to the DNA aptamer depending on the concentration of heavy metal ions in the measurement system. When multiple heavy metal ions bind to the DNA aptamer, it is thought that the three-dimensional structure will change even more significantly.

[0056] In the heavy metal detection method according to this embodiment, the buffer solution used for measurement is preferably 3-(N-morpholino)propanesulfonic acid buffer (hereinafter referred to as MOPS buffer), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid buffer (hereinafter referred to as HEPES buffer), or sodium bicarbonate-carbonate buffer. It is more preferable to use a sodium bicarbonate-carbonate buffer with a pH of around 10.

[0057] As will be described in detail later, in the experimental example, when MOPS buffer was used in the detection system, the aminomethylfluorescein derivative (FAM)-labeled aptamer showed higher fluorescence intensity than in other buffer systems. Furthermore, the pH of the buffer affects the degree of fluorescence polarization.

[0058] In this embodiment, a test kit containing the above-described fluorescently labeled DNA aptamer can be provided. For example, the kit may include a fluorescently labeled DNA aptamer powdered by freeze-drying as a reagent, and MOPS buffer, HEPES buffer, or sodium bicarbonate-carbonate buffer. If necessary, a detection microplate or filter may be added to the set.

[0059] In the experimental examples described below, examples of heavy metal detection on a microplate and on a filter using fluorescently labeled DNA aptamers are described. In addition to microplates and filters, detection components capable of measuring fluorescence polarization can also be used as tools for detection. Such detection components may be added to the detection kit of this embodiment.

[0060] An example of a detection member is shown in Figure 2. The detection member 30 shown in Figure 2 has patches 41 to 45, each impregnated with a test reagent of different concentrations and dried, attached to a paper-based base 31. As an example, five patches are attached, each 10 times the reagent concentration. A blank patch 50 containing no test reagent is also placed on the base 31. The test member is portable in size so that testing can be performed on-site at the water resource or soil to be tested, such as a water source or agricultural land. The test reagent in this example is the fluorescently labeled DNA aptamer described above.

[0061] An example of how to use the detection element described above will be described. A test solution containing a sample such as water or soil that may contain the heavy metal to be detected is prepared, and the solution is dripped onto patches of various concentrations. If the solution contains the substance to be detected, the color change of the patch caused by FRET or quenching of the fluorescently labeled DNA aptamer contained in the patch is observed. If the color change of the patch cannot be clearly observed, an LED light irradiation device 60 is used to auxiliary irradiate the detection element 30 with LED light 61 of a predetermined wavelength (e.g., 480 nm when the fluorescently labeled DNA aptamer has an FITC group), as shown in FIG. 3, and the color change is observed. The LED light irradiation device 60 can be portable.

[0062] By using such a detection element, it is possible to more easily and quickly detect the presence or absence of heavy metals at or near food factories, water sources, construction sites, farmland, and other locations where the objects to be inspected, such as water, soil, ingredients, and food, are present.

[0063] If the test substance cannot be easily detected using the above-mentioned detection member and portable LED light irradiation device, the detection member is subjected to fluorescence polarization measurement to measure the concentration of the test substance. Before performing the fluorescence polarization measurement, a calibration curve for the test substance at the concentration of each test reagent impregnated into the patch is prepared.

[0064] In this embodiment, a commonly used fluorescence spectrometer and a fluorescence polarization analyzer can be used as the fluorescence detection device.

[0065] 4 shows an example of a fluorescence polarization detector used for fluorescence polarization measurement. The above-described detection member 30 (including a fluorescently labeled DNA aptamer having an FITC group) is irradiated with light having a wavelength of 480 nm from a fluorescence polarization detector 70. Images of the irradiated light are captured with a sensor-equipped camera, and the captured image data is analyzed using analysis software to quantitatively determine the concentration of the test substance.

[0066] Although Figure 4 shows an example of detection using the detection member 30 of Figure 2, it is also possible to quantitatively detect the target substance in a sample dropped onto other members such as a microplate or a filter using a similar fluorescence polarization detection device.

[0067] In this way, by using fluorescently labeled DNA aptamers designed and created according to the substance to be detected as a detection kit, it is possible to detect the presence or absence of target substances such as heavy metals and analyze their concentrations quickly and easily at low cost.

[0068] According to the detection method of this embodiment, the presence or absence of heavy metals can be quickly confirmed at the site where the test object, such as water or soil, is present. Furthermore, it is also possible to quantify the content of heavy metals as needed. Therefore, compared to conventional methods, heavy metals can be detected simply and at low cost without requiring advanced technology.

[0069] In the experimental examples described below, cadmium is used as the target heavy metal substance, but the heavy metals that can be detected in this embodiment are not limited to cadmium. Other target substances that can be detected in the detection method of this embodiment include mercury and arsenic.

[0070] The following reagents were used in the experimental examples herein. A 1000 μM stock solution of cadmium ions was freshly prepared before each experiment using cadmium chloride 2.5 hydrate (Fujifilm Wako Pure Chemical Industries, Ltd., Japan). Fluorescently labeled DNA aptamers were synthesized by FASMAC Co. Ltd. (Japan). Fluorescence polarization assays and fluorescence intensity measurements were recorded using a TECAN Spark 10 at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0071] Experimental Example 1: Confirmation of sequence dependency of fluorescently labeled DNA aptamers in cadmium ion detection Fluorescently labeled DNA aptamers with various sequences were used to confirm the sequence dependency of heavy metal binding ability. The sequences of the aptamers used in this experiment are summarized in Table 1. All of the aptamers listed in Table 1 have 32 to 38 bases and have a FAM group bound to the 5'-end or 3'-end. Aptamers with the sequences set forth in SEQ ID NOS: 1 to 12 are hereinafter referred to as Aptamer 1 to Aptamer 12, respectively.

[0072]

[0073] Aptamer 1 is known in the literature. １ The results for Aptamer 1 correspond to the comparative example in Experimental Example 1. Aptamer 2 has the same base sequence as SEQ ID NO: 1, but the FAM group is bound to the 5'-end instead of the 3'-end. Aptamer 3 is a known aptamer. ２ and has a sequence in which a total of three bases have been substituted with other bases from the sequence set forth in SEQ ID NO: 1, five bases have been added to the 5'-end, and the 3'-end has been shortened by five bases. Aptamer 4 has a sequence in which three more bases have been added to the 3'-end of the sequence set forth in SEQ ID NO: 3.

[0074] Aptamer 5 has the same base sequence as SEQ ID NO: 1, with a FAM group bound to the 5'-end and a 5-biotin group bound to the 3'-end. Aptamer 6 has the same base sequence as SEQ ID NO: 1, with a FAM group bound to the 5'-end and a TAMRA group bound to the 3'-end.

[0075] Aptamers 7 and 8 have a 5-biotin group bound to the 5'-end of the aptamer having the sequences set forth in SEQ ID NOs: 4 and 3, respectively. Aptamer 9 has five bases at the 3'-end of the sequence set forth in SEQ ID NO: 2 substituted with other bases and has a biotin group bound to it. Aptamer 10 has a total of three bases substituted with other bases from the sequence set forth in SEQ ID NO: 9.

[0076] Aptamers 11 and 12 share a common base portion but have a different base sequence from aptamers 1 to 10. SEQ ID NO: 11 and 12 differ in that the FAM group is attached to the 3'-end (SEQ ID NO: 11) or the 5'-end (SEQ ID NO: 12).

[0077] [Experimental Example 2] Relationship between Fluorescence Intensity of Aptamers and Buffer 10 μL of a solution (0.5 μM) of each of the 12 types of aptamers listed in Table 1 was dissolved in 230 μL of 10 mM alkaline (pH 9.0) 3-(N-morpholino)propanesulfonic acid buffer (hereinafter referred to as MOPS buffer), and the fluorescence intensity derived from the FAM group bound to the aptamer was measured. Similarly, 10 μL of a solution (0.5 μM) of each of the 12 types of aptamers listed in Table 1 was dissolved in 230 μL of 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid buffer (hereinafter referred to as HEPES buffer), and the fluorescence intensity derived from the FAM group bound to the aptamer was measured.

[0078] Figure 5 shows a graph comparing the fluorescence intensities of aptamers 1 to 12 dissolved in MOPS buffer and HEPES buffer. It was found that depending on the base sequence, there was a large difference in fluorescence intensity between the two buffers. This suggests that the interaction between MOPS and FAM differs significantly depending on the base sequence.

[0079] (Relationship between Cadmium Ion Concentration and Fluorescence Intensity) Next, an experiment was conducted using aptamers 1 to 12 to confirm the relationship between the concentration of the cadmium-containing solution to be mixed and the fluorescence intensity of the solution after mixing. 10 μL of each of aptamers 1 to 12 (0.5 μM) was dissolved in 230 μL of 10 mM MOPS buffer or HEPES buffer. A cadmium chloride solution of a predetermined concentration dissolved in double-distilled water (hereinafter referred to as DDW) was added to the resulting solution and mixed. After standing at room temperature for 20 minutes, the fluorescence intensity was measured at 515 nm.

[0080] The results are shown in Figure 6. The vertical axis represents relative fluorescence intensity, and the horizontal axis represents the final concentration of cadmium ions. For each aptamer, the fluorescence intensity at 0 μM cadmium ions was set to 1, and the change in fluorescence intensity due to the added concentration of cadmium ions was taken as the relative fluorescence intensity. Aptamer 5 showed an increase in fluorescence intensity with increasing cadmium ion concentration, but the other aptamers showed a decrease of 10% or more, particularly Aptamer 1, which showed a decrease of 60% or more, and Aptamer 6, which showed a decrease of 90% or more. In Aptamer 1 and Aptamer 6, it is believed that the binding of cadmium ions to the aptamer changed the three-dimensional structure of the aptamer, affecting the fluorescence intensity derived from the FAM group.

[0081] To confirm the buffer dependency of aptamers 1, 5, and 6, aptamers 1, 5, and 6 were dissolved in 230 μL of 10 mM MOPS buffer or HEPES buffer. Cadmium chloride solution of a predetermined concentration dissolved in DDW was added to the resulting solution and mixed. After standing at room temperature for 20 minutes, the fluorescence intensity was measured at 515 nm. The results are shown in Figure 7. The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of the aptamer used. The fluorescence intensity in Figure 7 is shown relative to the fluorescence intensity of the aptamer solution without added cadmium, which is set to 1. While the difference between buffers was small for aptamers 1 and 5, the fluorescence intensity of aptamer 6 varied by more than twofold depending on the buffer.

[0082] (Relationship between buffer pH and fluorescence polarization index) An experiment was conducted using aptamer 1 and aptamer 4 to confirm the effect of buffer pH on fluorescence polarization index. 10 μL of aptamer 1 or aptamer 4 (0.5 μM) was dissolved in 230 μL of 10 mM MOPS buffer. A cadmium chloride solution of a predetermined concentration dissolved in DDW or well water was added to the resulting solution and mixed. After allowing to stand at room temperature for 20 minutes, the fluorescence intensity was measured at 515 nm. Well water collected from a well near the inventor's experimental location was used.

[0083] The results are shown in Figure 8. The vertical axis represents the fluorescence polarization (ΔmP value), and the horizontal axis represents the final concentration of cadmium ions. In Figure 8, (a) shows the results using a DDW solution of aptamer 1 and cadmium chloride, (b) shows the well water solution of aptamer 1 and cadmium chloride, (c) shows the DDW solution of aptamer 1 and cadmium chloride, and (d) shows the well water solution of aptamer 4 and cadmium chloride. The pH comparison results for aptamer 1 showed the highest ΔmP value at an acidic pH of 3.8. Similarly, the pH comparison results for aptamer 4 showed the highest ΔmP value at an acidic pH of 3.8. In the results using aptamer 1, the fluorescence polarization tends to decrease compared to distilled water samples on the alkaline side due to the influence of salts in the well water, but the influence of salts is not significant on the acidic side. With aptamer 4, the polarization tends to decrease overall in well water samples.

[0084] Experimental Example 3: Relationship between Cadmium Concentration and Fluorescence Polarization Index Aptamer 1 to 12 were used to confirm the relationship between the concentration of the cadmium-containing solution to be mixed and the fluorescence polarization index of the resulting solution. A cadmium chloride solution of a predetermined concentration dissolved in DDW was added to each of the solutions obtained by dissolving 10 μL of each of aptamers 1 to 12 (0.5 μM) in 230 μL of 10 mM MOPS buffer, and then mixed. After allowing to stand at room temperature for 20 minutes, the fluorescence polarization index was measured at 515 nm. The fluorescence polarization index was measured using a TECAN Spark.

[0085] The results are shown in Figure 9. The vertical axis represents the fluorescence polarization index, and the horizontal axis represents the final concentration of cadmium ions. For all aptamers, the fluorescence polarization index increased with increasing cadmium concentration. As expected, the increase in fluorescence polarization index (ΔmP) is thought to be due to the restriction of the free rotation of the FAM group and the structural change of the aptamer, which is an oligonucleotide, accompanied by an increase in molecular weight due to the binding of a large number of cadmium atoms to the aptamer.

[0086] To confirm the buffer dependence of the fluorescence polarization index of mixtures of aptamers 1 to 12 with cadmium solution, aptamers 1, 5, and 6 were dissolved in 230 μL of 10 mM MOPS buffer, and a cadmium chloride solution of a predetermined concentration dissolved in DDW was added to the resulting solution. The fluorescence polarization index of the resulting mixture was measured. The results are shown in Figure 10. This is thought to be due to the interaction between the MOPS molecule and the FAM group, which enhanced the polarization effect of the aptamer. Compared to when HEPES buffer was used, when MOPS buffer was used, all aptamers showed higher ΔmP values ​​than when HEPES buffer was used.

[0087] Additionally, an experiment was conducted to confirm the relationship between the concentration of the cadmium-containing solution to be mixed and the degree of fluorescence polarization using a pH 10 sodium bicarbonate-carbonate buffer solution for each of aptamer 1 and aptamer 6. 10 μL of aptamer 1 or aptamer 6 (0.5 μM) was dissolved in 230 μL of 10 mM sodium bicarbonate-carbonate buffer. A cadmium chloride solution of a predetermined concentration dissolved in DDW or tap water was added to the resulting solution and mixed. After allowing to stand at room temperature for 20 minutes, the fluorescence intensity was measured at 515 nm.

[0088] The results are shown in Figure 11. For aptamer 1, no increase or decrease in fluorescence intensity was observed in DDW even when the cadmium concentration was changed, but slight fluctuations were observed in tap water. For aptamer 4, the degree of fluorescence polarization increased as the cadmium concentration increased in both DDW and tap water.

[0089] Experimental Example 4: Effect of Cadmium Solution Dissolved in Well Water on Fluorescence Polarization. In actual farms and construction sites, heavy metals are not dissolved in distilled water, but in water containing other metals and their salts. To confirm the effect of such an environment on the fluorescence polarization of samples, a sample was prepared by dissolving cadmium chloride in well water instead of DDW, and the sample was then bound to an aptamer and measured for fluorescence polarization. The well water used was collected from a well near the inventor's experimental site. The conductivity of the well water used was 0.3 mS / cm, which is higher than the conductivity of typical river water in Japan (0.06 mS / cm). The experiment was conducted under the same conditions as Experimental Example 3, except that well water instead of DDW was used to prepare the cadmium chloride solution.

[0090] The results are shown in Figure 12. The vertical axis represents the fluorescence polarization index, and the horizontal axis represents the final concentration of cadmium ions. For all aptamers, the fluorescence polarization index increased with increasing cadmium concentration. Only Aptamer 1 exhibited a higher fluorescence polarization index than when DDW solution was used, while all other aptamers exhibited a lower fluorescence polarization index than when DDW solution was used. The types of aptamers with higher fluorescence polarization index also showed a different trend from when DDW solution was used. These results are thought to be due to the fact that metals other than cadmium or their salts contained in well water bound to the aptamer, changing the structure of the aptamer and affecting the behavior of the FAM group.

[0091] [Experimental Example 5] Relationship between temperature and fluorescence polarization index The effect of various temperatures on the fluorescence polarization index was confirmed for mixed solutions of MOPS buffer solution and cadmium ion-containing solution for each of aptamers 1, 6, 7, and 11. After allowing the mixed solutions to stand at a predetermined temperature for 30 minutes, the fluorescence polarization index of each solution was measured.

[0092] The results are shown in Figure 13. The vertical axis represents the ΔmP value, and the horizontal axis represents the temperature. Aptamers 6, 7, and 11 showed no significant difference in the fluorescence polarization index between 30°C and 60°C, yielding stable values. However, a decrease in the fluorescence polarization index was observed above 70°C. In contrast, when aptamer 1 was used, a tendency for the fluorescence polarization index to decrease over the temperature range from 30°C to 90°C was observed. It was confirmed that the aptamer used in this example could be measured at room temperature and properly detected. In other words, by using the aptamer of this example, the presence or absence of heavy metals can be easily detected at sites such as water sources, farmland, and food factories without the need for large-scale equipment.

[0093] [Experimental Example 6] Detection of cadmium ions on a filter While Experimental Example 1 was a detection experiment in a microplate, Experimental Example 2 attempted to detect heavy metals on a filter.

[0094] As the aptamer, aptamer 6 having the sequence set forth in SEQ ID NO: 6 in Table 1 above was used.

[0095] 5 μL of aptamer (1 μM) was added to a membrane filter and air-dried. After drying, 1 μL of MOPS buffer containing cadmium was added to the filter impregnated with the aptamer. After addition, the filter was irradiated with a blue LED and observed through an orange filter.

[0096] 14A shows photographs taken under room light of filters to which a cadmium ion solution or a blank solution was dropped, followed by the addition of the aptamer solution of the present embodiment. The right (b) shows a filter to which a cadmium ion-containing solution (1 mM) was dropped, and the left (a) shows a filter to which a blank solution not containing cadmium ions was dropped.

[0097] Photographs of filters (a) and (b) photographed in Figure 14A taken under blue light through an orange filter are shown in Figure 14B. Compared to filter (a), stronger light with a wavelength around 560 nm was detected from filter (b). This wavelength is the wavelength of fluorescence derived from the TAMRA group possessed by aptamer 6. From these results, it was confirmed that no structural change occurred in the aptamer on filter (a) onto which the blank solution was dropped, whereas FRET occurred due to a change in the three-dimensional structure of the aptamer on filter (b) onto which the cadmium ion-containing solution was dropped, i.e., cadmium ions were trapped by the aptamer.

[0098] This demonstrates that the presence of cadmium ions in an aqueous solution can be detected by using the fluorescence polarization detection kit of this experimental example.

[0099] In all of the above experimental examples, measurements were performed using cadmium as the heavy metal. The results demonstrated that the method of this experimental example can detect the presence or absence of cadmium in a sample and its concentration. Compared to conventional methods, the method of this experimental example can detect cadmium in a sample simply and quickly without requiring advanced analytical skills.

[0100] An appropriately designed aptamer selectively binds to a target heavy metal. Therefore, by selecting and preparing aptamers capable of selectively binding to other heavy metals in advance, it is expected that other heavy metals can also be detected easily and quickly using a method similar to that of this experimental example. Furthermore, aptamers are not limited to selectively binding to heavy metals; they can also be designed to bind to specific compounds. Therefore, the aptamer and detection method of this embodiment are not limited to the detection of heavy metals, and can also be used to detect pesticides and impurities in ingredients and foods, for example.

[0101] (References) 1. Liu Y, Zhang D, Ding J, Hayat K, Yang X, Zhan X, Zhang D, Lu Y, Zhou P (2021) A Facile Aptasensor for Instantaneous Determination of Cadmium Ions Based on Fluorescence Amplification Effect of MOPS on FAM-Labeled Aptamer. Biosensors 11::133 2. Pavadai R, Perumal P (2022) Versatile sensing platform of an innovative copper oxide-assisted Cu-phenolic coordination nanosheet-mediated fluorophore-tagged GT-rich SSA-based fluorescence ON-OFF biosensor for the subsequent detection of CD ２＋ and S ２－ ions. New Journal of Chemistry 46::3431-3447.

[0102] REFERENCE SIGNS LIST 1 Fluorescence-labeled DNA aptamer 1' Fluorescence-labeled DNA aptamer forming a stem-loop structure 2 Fluorescent molecule 10 Heavy metal 11 Fluorescence-labeled DNA aptamer 12 Donor fluorescent molecule 13 Acceptor fluorescent molecule 30 Detection member 31 Base 41, 42, 43, 44, 45 Patch 50 Blank patch 60 LED light irradiation device 61 LED light 70 Fluorescence polarization detection device

Claims

1. A detection kit for detecting trace amounts of heavy metals in a sample, which contains a DNA aptamer that is labeled with a fluorescent molecule and selectively binds to a specific heavy metal.

2. The detection kit according to claim 1, wherein the heavy metal is at least one selected from the group consisting of cadmium, mercury, and arsenic.

3. The detection kit of claim 1, wherein detection is performed visually, by fluorescence analysis, or by fluorescence polarization analysis.

4. The detection kit according to claim 1, wherein the sample is water, soil, foodstuff or food.

5. The detection kit according to claim 1, wherein the DNA aptamer is labeled with two fluorescent molecules.

6. The detection kit according to claim 1, further comprising a MOPS buffer, a HEPES buffer, or a sodium bicarbonate-carbonate buffer.

7. A detection device comprising: a DNA aptamer that is labeled with a fluorescent molecule and selectively binds to a specific heavy metal; and a fluorescence measurement unit that measures the fluorescence intensity of the fluorescent molecule of the DNA aptamer, and detects trace amounts of heavy metals in a sample by measuring the fluorescence intensity or fluorescence polarization intensity derived from the fluorescent molecule that labels the DNA aptamer that has bound to the specific heavy metal.

8. The detection device according to claim 7, wherein the heavy metal is at least one selected from the group consisting of cadmium, mercury, and arsenic.

9. The detection device according to claim 7, which detects heavy metals by fluorescence analysis or fluorescence polarization analysis.

10. The detection device according to claim 7, wherein the sample is water, soil, foodstuff or food.

11. A method for detecting trace amounts of heavy metals in a sample, comprising the steps of: mixing a solution in which a DNA aptamer that is labeled with a fluorescent molecule and selectively binds to a specific heavy metal is dissolved with a liquid containing a sample that may contain the specific heavy metal to obtain a mixed solution; measuring the fluorescence intensity or fluorescence polarization intensity derived from the fluorescent molecule of the DNA aptamer contained in the mixed solution; and detecting the specific heavy metal based on the results of the measurement.

12. The detection method according to claim 11, further comprising a step of measuring the concentration of heavy metals in the step of measuring the fluorescence polarization intensity.