Method for detecting target substance, fluidic device and kit

Abstract

This method for detecting a target substance in a sample comprises the steps of: introducing a sample and a reagent that generates a fluorescent substance in the presence of the target substance into each well of a well array; sealing each well of the well array with a sealing liquid, so that each well forms an independent reaction space; replacing the sealing liquid with a water-absorbing organic solvent, thereby dehydrating the contents of the wells, reducing the volume thereof, and generating the fluorescent substance when the target substance is present in the contents; and emitting excitation light to the fluorescent substance to detect fluorescent light generated in each well of the well array. Detection of fluorescent light in each well indicates the presence of the target substance in the well.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a target substance, a fluidic device, and a kit. This application claims priority to Japanese Patent Application No. 2020-169092, filed on October 6, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, studies have been conducted to detect and monitor diseases based on enzyme activity. Such diagnostic methods are called Activity-Based Diagnostics (ABDx) (see, for example, Non-Patent Document 1).

[0003] Diseases that can be diagnosed using ABDx include infectious diseases such as viral infections and non-infectious diseases such as cancer. For example, viral infections can be detected by detecting viral nucleic acids as markers. Cancer can also be detected by detecting uncontrolled enzyme activity such as cathepsin in cancer tissues as a disease marker.

[0004] It is also known that free DNA (cell-free DNA, cfDNA) released from cells upon cell death exists in the blood. The cfDNA of cancer patients also contains circulating tumor DNA (ctDNA), which is DNA derived from cancer cells.

[0005] In addition, various cells secrete membrane vesicles called exosomes, and exosomes are known to be found in biological samples such as saliva, blood, urine, amniotic fluid, and malignant ascites, as well as in the supernatants of cultured cells. Exosomes contain various proteins, lipids, microRNA, DNA, etc., derived from the cells that secreted them.

[0006] In recent years, research has been conducted into the application of microRNA (miRNA), DNA, etc. contained in membrane vesicles such as cfDNA and exosomes to the early detection of cancer and various other diseases, predicting the effectiveness of anticancer drugs, diagnosing disease predisposition, and diagnosing hereditary diseases.

[0007] Thus, there is a need for technology that can detect viral nucleic acids, enzymes, cfDNA, ctDNA, miRNA, and other target substances with high sensitivity.

[0008] Incidentally, Patent Document 1 describes the detection of enzyme activity at the single molecule level using a microchamber with a size on the order of femtoliters. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-309405 [Non-patent literature]

[0010] [Non-Patent Document 1] Soleimany AP and Bhatia SN, Activity-Based Diagnostics: An Emerging Paradigm for Disease Detection and Monitoring, Trends Mol Med, 26 (5), 450-468, 2020. Summary of the Invention [Problem to be solved by the invention]

[0011] As with the microchamber described in Patent Document 1, the detection time for an enzymatic reaction can be shortened by miniaturizing the volume of the reaction space in which the enzymatic reaction occurs. However, miniaturizing the volume of the reaction space can reduce the proportion of target substances captured in the reaction space, which can result in a decrease in detection sensitivity. Therefore, an object of the present invention is to provide a technology that can detect target substances with high sensitivity. [Means for solving the problem]

[0012] The present invention includes the following aspects. [1] A method for detecting a target substance in a sample, comprising the steps of: introducing the sample and a reagent that generates a fluorescent substance when the target substance is present into each well of a well array; sealing each well of the well array with a sealing liquid so that each well forms an independent reaction space; replacing the sealing liquid with a water-absorbing organic solvent, thereby dehydrating the contents of the well, reducing its volume, and generating the fluorescent substance when the target substance is present in the contents; and irradiating the fluorescent substance with excitation light and detecting the generated fluorescence for each well of the well array, wherein detection of fluorescence in the well indicates the presence of the target substance in the well. [2] The method according to [1], wherein each well of the well array has a first well and a second well that is located at the bottom of the first well and has a smaller volume than the first well, and when the volume of the contents of the first well becomes smaller, the contents accumulate in the second well. [3] The method according to [2], wherein the ratio of the volume of the first well to the volume of the second well (volume of the first well:volume of the second well) is 10:1 to 1,000,000:1. [4] The method according to [2] or [3], wherein the volume of the first well is 1 to 1,000 pL and the volume of the second well is 0.1 to 1,000 fL. [5] The method according to any one of [2] to [4], wherein 0 or 1 target substance is introduced into each of the first wells. [6] The method according to any one of [1] to [5], wherein the sealing liquid is a fluorine-based liquid, a mineral oil, or a linear or branched, saturated or unsaturated hydrocarbon having 7 to 17 carbon atoms. [7] The method according to any one of [1] to [6], wherein the water-absorbing organic solvent is a linear or branched, saturated or unsaturated aliphatic alcohol having 4 to 11 carbon atoms. [8] A fluidic device comprising: a substrate having a well array on its surface, the well array having a plurality of wells, each well having a first well and a second well located at the bottom of the first well and having a smaller capacity than the first well; a lid member located opposite the well array; and a spacer that separates the substrate and the lid member, wherein the space between the well array and the lid member forms a flow path through which a fluid flows. [9] A kit for detecting a target substance, comprising the fluidic device according to [8], a reagent that generates a fluorescent substance in the presence of a target substance, a sealing liquid, and a water-absorbent organic solvent. [Effects of the Invention]

[0013] According to the present invention, a technique can be provided that can detect a target substance with high sensitivity. [Brief explanation of the drawings]

[0014] [Figure 1] 1(a) and 1(b) are schematic diagrams illustrating an example of a method for detecting a target substance. [Figure 2] 1(a) is a schematic diagram illustrating an example of a well array having first and second wells, and FIG. 1(b) is a cross-sectional view taken along line bb' in FIG. 1(a). [Figure 3] 1(a) is a schematic diagram illustrating an example of a well array having first and second wells, and FIG. 1(b) is a cross-sectional view taken along line bb' in FIG. 1(a). [Figure 4] 1(a) to 1(f) are schematic cross-sectional views illustrating the steps of manufacturing a well array. [Figure 5] 1(a) and 1(b) are schematic cross-sectional views illustrating each step in the manufacture of a well array. [Figure 6] 1(a) is a top view showing an example of a fluidic device, and FIG. 1(b) is a cross-sectional view taken along line bb' in FIG. [Figure 7] 1(a) to 1(d) are schematic cross-sectional views illustrating an example of a procedure for carrying out a method for detecting a target substance using a fluidic device. [Figure 8] 1 is a micrograph of a well array. [Figure 9] Photographs (a) to (d) show the results of Experimental Example 1. [Figure 10] 2(a) and 2(b) are graphs showing the results of Experimental Example 1. [Figure 11] 1 is a graph showing the results of Experimental Example 1. [Figure 12] 10 is a graph showing the results of Experimental Example 2. [Figure 13] 10 is a graph showing the results of Experimental Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and redundant explanations will be omitted. The dimensional ratios in the drawings may be exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0016] [Method for detecting target substances] In one embodiment, the present invention provides a method for detecting a target substance in a sample, comprising the steps of: introducing the sample and a reagent that produces a fluorescent substance in the presence of the target substance into each well of a well array; sealing each well of the well array with a sealing liquid so that each well forms an independent reaction space; replacing the sealing liquid with a water-absorbing organic solvent, thereby dehydrating the contents of the well, reducing its volume, and producing the fluorescent substance in the presence of the target substance in the contents; and irradiating the fluorescent substance with excitation light and detecting the generated fluorescence for each well of the well array, wherein detection of fluorescence in the well indicates the presence of the target substance in the well.

[0017] As will be described later in the Examples, the method of this embodiment allows for highly sensitive detection of a target substance.

[0018] (sample) The sample is not particularly limited, and examples thereof include biological samples such as saliva, blood, urine, amniotic fluid, malignant ascites, pharyngeal swabs, and nasal swabs, as well as supernatants of cultured cells.

[0019] (Target substance) The target substance is not particularly limited, and examples thereof include enzymes, single-stranded nucleic acid fragments, double-stranded nucleic acid fragments, etc. The nucleic acid fragment may be a viral nucleic acid, cfDNA, ctDNA, miRNA, etc. The enzyme may be any enzyme, and examples thereof include main protease of coronavirus, alkaline phosphatase, and proteases derived from living organisms. Examples of proteases derived from living organisms include serine proteases, cysteine ​​proteases, and cathepsins.

[0020] (A reagent that produces a fluorescent substance in the presence of a target substance) When the target substance is an enzyme, an example of a reagent (detection reagent) that generates a fluorescent substance in the presence of the target substance is a fluorescent substrate corresponding to the enzyme, which generates a fluorescent substance through an enzymatic reaction.

[0021] More specifically, when the enzyme is a peptidase, examples include peptide substrates labeled with a fluorescent substance and a quencher. Fluorescence is detected when such peptide substrates are cleaved by the peptidase. Furthermore, when the enzyme is a phosphatase, examples include fluorescent substances quenched by the addition of a phosphate group. Fluorescence is detected when the phosphate group is removed from such substances.

[0022] In addition, among the CRISPR / Cas family proteins that have been applied to genome editing in recent years, Cas12 and Cas13 have been shown to form a ternary complex with gRNA and target nucleic acid, and when they cleave the target nucleic acid, they express the activity of cleaving the surrounding DNA or RNA.

[0023] This reaction can be used to generate fluorescent substances using single-stranded or double-stranded nucleic acid fragments as target substances (target nucleic acids). Specifically, the Cas12 protein can be used to detect double-stranded DNA fragments as target substances. The Cas13 protein can be used to detect single-stranded RNA fragments or single-stranded DNA fragments as target substances.

[0024] Figures 1(a) and (b) are schematic diagrams illustrating this reaction. Figures 1(a) and (b) illustrate the case where the CRISPR / Cas family protein is the Cas12a protein.

[0025] First, as shown in Figure 1(a), when a Cas12a protein 110 and a gRNA 120 are brought into contact with each other, they bind to form a binary complex 130. The gRNA 120 has a base sequence complementary to a target nucleic acid fragment 140 (target substance) in part.

[0026] Subsequently, when the target nucleic acid fragment 140 in the sample comes into contact with the binary complex 130, the Cas12a protein 110, the gRNA 120, and the target nucleic acid fragment 140 form a ternary complex 100. At this stage, the Cas12a protein 110 does not express nuclease activity, and therefore the substrate nucleic acid fragment 150 is not cleaved. In the example of Figures 1(a) and (b), the substrate nucleic acid fragment 150 is a single-stranded DNA fragment labeled with a fluorescent substance F and a quencher Q. Irradiating the substrate nucleic acid fragment 150 with excitation light does not generate fluorescence.

[0027] Once the ternary complex 100 is formed, the Cas12a protein 110 cleaves the target site of the target nucleic acid fragment 140. In FIG. 1(a), the target site of the target nucleic acid fragment 140 is indicated by an arrowhead. FIG. 1(b) is a schematic diagram showing the ternary complex 100' in which the target site of the target nucleic acid fragment 140 has been cleaved. As shown in FIG. 1(b), the ternary complex 100' exhibits nuclease activity. The ternary complex 100' then cleaves the substrate nucleic acid fragment 150 present around the ternary complex 100'. As a result, the fluorescent substance F of the substrate nucleic acid fragment 150 separates from the quencher Q. Fluorescence can be detected by irradiating the fluorescent substance F separated from the quencher Q with excitation light. If fluorescence is detected, it can be determined that the target nucleic acid fragment 140 was present in the sample.

[0028] In this case, it can be said that the reagents that produce a fluorescent substance in the presence of a target substance are the CRISPR / Cas family protein 110, the gRNA 120, and the substrate nucleic acid fragment 150.

[0029] The sample, the CRISPR / Cas family protein 110, the gRNA 120, and the substrate nucleic acid fragment 150 may be mixed and contacted in any order.

[0030] For example, first, a CRISPR / Cas family protein 110 and a gRNA 120 may be contacted to form a binary complex 130, and then the sample may be contacted. In this case, if a target nucleic acid fragment 140 is present in the sample, the target nucleic acid fragment 140 binds to the binary complex 130, forming a ternary complex 100. Thereafter, a substrate nucleic acid fragment 150 may be contacted.

[0031] Alternatively, after the binary complex 130 is formed, the target nucleic acid fragment 140 and the substrate nucleic acid fragment 150 may be contacted simultaneously.

[0032] Alternatively, the CRISPR / Cas family protein 110, the gRNA 120, and the sample may be contacted simultaneously. Even in this case, if the target nucleic acid fragment 140 is present in the sample, a ternary complex 100 is eventually formed. Thereafter, the substrate nucleic acid fragment 150 may be contacted.

[0033] Alternatively, the sample, CRISPR / Cas family protein 110, gRNA 120, and substrate nucleic acid fragment 150 may be simultaneously contacted. Even in this case, if target nucleic acid fragment 140 is present in the sample, ternary complex 100 is eventually formed, and when the target site of target nucleic acid fragment 140 is cleaved in ternary complex 100, it is converted to ternary complex 100′, which expresses nuclease activity and cleaves substrate nucleic acid fragment 150.

[0034] gRNA In the method of this embodiment, the guide RNA (gRNA) is not particularly limited as long as it can be used with the CRISPR / Cas family protein used, and may be a complex of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), a single gRNA (sgRNA) combining tracrRNA and crRNA, or crRNA alone.

[0035] When the CRISPR / Cas family protein used is a Cas12a protein, the crRNA can have, for example, the following base sequence: First, the base sequence obtained by removing the protospacer adjacent motif (PAM) sequence from the target base sequence is used as the spacer base sequence. Next, a base sequence is designed in which a scaffold sequence is linked to the 3' end of the spacer base sequence, and the complementary strand is used as the crRNA base sequence.

[0036] For example, if the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-GCCAAGCGCACCTAATTTCC-3'" (SEQ ID NO: 1), the base sequence of the crRNA for the Cas12a protein can be "5'-AAUUUCUACUAAGUGUAGAUGGAAAUUAGGUGCGCUUGGC-3'" (SEQ ID NO: 2).

[0037] When the CRISPR / Cas family protein used is the Cas13a protein, the crRNA can have, for example, the following base sequence: First, a base sequence is designed in which a scaffold sequence is linked to the 3' end of a base sequence complementary to the target base sequence, and the complementary strand is used as the base sequence of the crRNA.

[0038] For example, if the target nucleotide sequence is "5'-AUGGAUUACUUGGUAGAACAGCAAUCUA-3'" (SEQ ID NO: 3), the nucleotide sequence of the crRNA for the Cas13a protein can be "5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU-3'" (SEQ ID NO: 4).

[0039] <CRISPR / Cas family proteins> Any CRISPR / Cas family protein can be used as long as it expresses nuclease activity after forming a ternary complex with the gRNA and the target nucleic acid fragment. More precisely, as described above, the nuclease activity is expressed after the ternary complex is formed and the CRISPR / Cas family protein cleaves the target nucleic acid fragment.

[0040] Such CRISPR / Cas family proteins include Cas12 protein, Cas13 protein, etc. As used herein, the Cas12 protein and Cas13 protein may be Cas12 protein, Cas13 protein, orthologs of these proteins, modified forms of these proteins, etc.

[0041] More specific examples of CRISPR / Cas family proteins that can be used in the method of this embodiment include the Cas12a protein derived from Lachnospiraceae bacterium ND2006 (LbCas12a, UniProtKB accession number: A0A182DWE3), the Cas12a protein derived from Acidaminococcus sp. (AsCas12a, UniProtKB accession number: U2UMQ6), the Cas12a protein derived from Francisella tularensis subsp. novicida (FnCas12a, UniProtKB accession number: A0Q7Q2), the Cas12b protein derived from Alicyclobacillus acidoterrestris (AaCas12b, UniProtKB accession number: T0D7A2), and the Cas12b protein derived from Leptotrichia Cas13a protein from L. wadei (LwaCas13a, NCBI accession number: WP_021746774.1), Cas13a protein from Lachnospiraceae bacterium NK4A179 (LbaCas13a, NCBI accession number: WP_022785443.1), Cas13a protein from Leptotrichia buccalis C-1013-b (LbuCas13a, NCBI accession number: WP_015770004.1), Cas13b protein from Bergeyella zoohelcum (BzoCas13b, NCBI accession number: WP_002664492), Cas13b protein from Prevotella intermedia (PinCas13b, NCBI accession number: WP_036860899), and Prevotella buccae (PbuCas13b, NCBI accession number: WP_004343973), Cas13b protein from Alistipes sp. ZOR0009 (AspCas13b, NCBI accession number: WP_047447901), Prevotella sp.Cas13b protein from MA2016 (PsmCas13b, NCBI accession number: WP_036929175), Cas13b protein from Riemerella anatipestifer (RanCas13b, NCBI accession number: WP_004919755), Cas13b protein from Prevotella aurantiaca (PauCas13b, NCBI accession number: WP_025000926), Cas13b protein from Prevotella saccharolytica (PsaCas13b, NCBI accession number: WP_051522484), Cas13b protein from Prevotella intermedia (Pin2Cas13b, NCBI accession number: WP_061868553), and Capnocytophaga Cas13b protein from Enterococcus canimorsus (CcaCas13b, NCBI accession number: WP_013997271), Cas13b protein from Porphyromonas gulae (PguCas13b, NCBI accession number: WP_039434803), Cas13b protein from Prevotella sp. P5-125 (PspCas13b, NCBI accession number: WP_044065294), Cas13b protein from Porphyromonas gingivalis (PigCas13b, NCBI accession number: WP_053444417), Cas13b protein from Prevotella intermedia (Pin3Cas13b, NCBI accession number: WP_050955369), and Examples include the Csm6 protein derived from Lactobacillus italicus (EiCsm6, NCBI accession number: WP_007208953.1), the Csm6 protein derived from Lactobacillus salivarius (LsCsm6, NCBI accession number: WP_081509150.1), and the Csm6 protein derived from Thermus thermophilus (TtCsm6, NCBI accession number: WP_011229148.1).

[0042] In the method of this embodiment, the CRISPR / Cas family protein may be a mutant of the above-mentioned Cas family protein, such as a mutant that exhibits increased nuclease activity after forming a ternary complex.

[0043] 《Substrate nucleic acid fragment》 The substrate nucleic acid fragment is labeled with a fluorescent substance and a quencher, and when it is cleaved by the nuclease activity of the ternary complex and the fluorescent substance is separated from the quencher, it emits fluorescence when irradiated with excitation light.

[0044] The substrate nucleic acid fragment may be appropriately selected depending on the substrate specificity of the CRISPR / Cas family protein used. For example, the Cas12 protein cleaves single-stranded DNA as a substrate. Therefore, when using the Cas12 protein, it is recommended to use single-stranded DNA as the substrate nucleic acid fragment. Furthermore, the Cas13 protein cleaves single-stranded RNA as a substrate. Therefore, when using the Cas13 protein, it is recommended to use single-stranded RNA as the substrate nucleic acid fragment.

[0045] The combination of fluorescent substance and quencher is such that they can quench the fluorescence of the fluorescent substance when placed close to each other. For example, when FAM, HEX, or the like is used as the fluorescent substance, Iowa Black FQ (IDT) or TAMRA, or the like can be used as the quencher.

[0046] (Well Array) In the method of this embodiment, each well of the well array may have a first well and a second well located at the bottom of the first well and having a smaller volume than the first well, and when the volume of the contents of the first well becomes smaller, the contents may accumulate in the second well.

[0047] 2(a) and 2(b) are schematic diagrams illustrating an example of a well array having a first well and a second well, where Fig. 2(a) is a top view and Fig. 2(b) is a cross-sectional view taken along line bb' in Fig. 2(a).

[0048] 2(a) and (b), well array 200 is formed on one surface of substrate 210. Each well of well array 200 has a first well 220 and a second well 230 that is located at the bottom of well 220 and has a smaller volume than well 220. As will be described later, when the volume of the contents of first well 220 becomes smaller, the contents accumulate in second well 230.

[0049] That is, by capturing the target substance in the large-capacity well 220 and detecting it in the small-capacity well 230, the target substance can be detected with high sensitivity and the time required for detection can be shortened.

[0050] The ratio of the volumes of the first well 220 and the second well 230 (volume of the well 220:volume of the well 230) may be 10:1 to 1,000,000:1.

[0051] Alternatively, the volume of the first well 220 may be 1 to 1,000 pL, and the volume of the second well 230 may be 0.1 to 1,000 fL.

[0052] Figures 3(a) and (b) are schematic diagrams illustrating another example of a well array having first and second wells, where Figure 3(a) is a top view and Figure 3(b) is a cross-sectional view taken along line bb' in Figure 3(a).

[0053] 3(a) and (b), well array 300 is formed on one surface of substrate 210. As in well array 300, multiple second wells 230 may be arranged per first well 220. Even in this case, when the volume of the contents of first well 220 becomes small, the contents accumulate in second well 230.

[0054] There are no particular limitations on the shape of the first well 220 and the second well 230, and they may be, for example, cylindrical, polyhedrons formed by multiple faces (for example, rectangular parallelepipeds, hexagonal prisms, octagonal prisms, etc.), or the like.

[0055] It is preferable that the plurality of first wells 220 are all the same shape and size, and it is also preferable that the plurality of second wells 230 are all the same shape and size. Here, "same shape and size" means that they have the same shape and capacity to the extent required for digital measurement, and variations within the range of manufacturing errors are acceptable.

[0056] (Manufacturing method of well array) Using well array 300 as an example, an example of a method for manufacturing a well array having first and second wells will be described.

[0057] 4(a) to (f) and 5(a) and (b) are schematic cross-sectional views illustrating each step in the manufacture of well array 300. First, as shown in FIG. 4(a), film 400 is laminated on the surface of substrate 210.

[0058] Examples of materials for the substrate 210 include glass, resin, etc. Examples of resin include polyethylene, polypropylene, polystyrene, polycarbonate, cyclic polyolefin, acrylic, etc. Examples of materials for the film 400 include fluororesin, cyclic polyolefin, silicone resin, etc.

[0059] Next, as shown in Figure 4(c), a resist film 410 is laminated on the surface of the film 400. Next, using a mask with a well array pattern, the resist film 410 is exposed to active energy rays using an exposure machine. Next, the resist film 410 is developed with a developer, and as shown in Figure 4(d), the resist film 410 in the areas where wells will be formed is removed.

[0060] Subsequently, as shown in FIG. 4( e ), the film 400 masked with the resist film 410 is etched to form a second well 230 in the film 400 .

[0061] Subsequently, as shown in FIG. 4(f), the substrate is washed to remove the resist film 410, thereby obtaining an array of wells 230.

[0062] 5(a), a resist film 410 is again laminated on the array of wells 230 obtained in FIG. 4(f). As the resist film 410, a sheet-type resist can be preferably used.

[0063] 5(b), a mask having a well array pattern is used to irradiate the resist film 410 with active energy rays using an exposure machine. The resist film 410 is then developed with a developer to remove the portions of the resist film 410 where the first wells 220 will be formed. As a result, a well array 300 having first wells 220 and second wells 230 is obtained.

[0064] In the method of this embodiment, it is preferable that 0 or 1 target substance be introduced per first well. By introducing 0 or 1 target substance per first well, digital measurement can be performed. In other words, the number of wells from which fluorescence is detected can be correlated with the number of target substance molecules in the sample.

[0065] (Sealing liquid) The sealing liquid is preferably immiscible with water. "Immiscible with water" means that when the sealing liquid and water are thoroughly mixed and then allowed to stand, they separate into an aqueous phase and an organic phase. Furthermore, the sealing liquid preferably has low water absorption. "Low water absorption" means that when the sealing liquid is mixed with an equal volume of water at 20°C and allowed to stand, and the aqueous phase and organic phase separate, the volume change of the organic layer is 1% or less.

[0066] The sealing liquid may be a substance having a boiling point of about 100°C or higher and being liquid at room temperature. Specific sealing liquids include fluorine-based liquids such as FC-40, FC-43, FC-770, FC-72, and FC-3283 (all manufactured by 3M), and Fomblin (registered trademark) oil (Solvay), mineral oil (Sigma-Aldrich), and linear or branched, saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms. These may be used alone or in combination of two or more.

[0067] Examples of linear or branched saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms include heptane (C7H 16 ), octane (C8H 18 ), Nonane (C9H 20 ), Decane (C 10 H 22 ), Undecane (C 11 H 24 ), dodecane (C 12 H 26 ), tridecane (C 13 H 28 ), tetradecane (C 14 H 30 ), pentadecane (C 15 H 32 ), hexadecane (C 16 H 34 ), heptadecane (C 17 H 36 ), heptene (C7H 14 ), Octene (C8H 16 ), Nonene (C9H 18 ), Decene (C 10 H 20 ), Undecene (C 11 H 22 ), Dodecene (C 12 H 24 ), tridecene (C 13 H 26 ), tetradecene (C 14 H 28 ), pentadecene (C 15 H 30 ), hexadecene (C 16 H 32 ), heptadecene (C 17 H34 ) and the like. These may be any isomers.

[0068] For example, isomers of octane include 1-octane, 2-methylheptane, 3-methylheptane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,3,3-trimethylpentane, etc. Furthermore, for example, isomers of octene include 1-octene, 2-methyl-1-heptene, 2,3-dimethyl-1-hexene, 2-ethyl-1-hexene, 2,3,3-trimethyl-1-butene, etc.

[0069] (Water-absorbing organic solvent) The water-absorbing organic solvent can be one that has a boiling point of about 100°C or higher, is liquid at room temperature, and is immiscible with water, and examples thereof include linear or branched, saturated or unsaturated aliphatic alcohols having 4 to 11 carbon atoms. "Immiscible with water" means that when water and the organic solvent are thoroughly mixed and then allowed to stand, the mixture separates into an aqueous phase and an organic phase. "Water-absorbing" means that the solvent dissolves water. The water-absorbing organic solvent may be a monohydric alcohol or a dihydric or higher alcohol.

[0070] A specific example of a water-absorbent organic solvent is butanol (CH 10 O), pentanol (CH 12 O), hexanol (CH 14 O), heptanol (CH 16 O), octanol (CH 18 O), nonanol (CH 20 O), decanol (C 10 H 22 O), undecanol (C 11 H 24 O), pentanediol (CH 12 O2). These may be any isomer. These may be used alone or in combination of two or more.

[0071] For example, octanol isomers include 1-octanol, isooctyl alcohol, 2-ethylhexanol, etc. Furthermore, pentanediol isomers include 1,5-pentanediol, 1,2-pentanediol, 2,3-pentanediol, etc.

[0072] As will be described later in the Examples, an increase in fluorescence intensity due to dehydration and concentration is observed, particularly when 1-heptanol, 1-octanol, or 1-nonanol is used, and target substances tend to be detected with high sensitivity.

[0073] [Fluid Devices] In one embodiment, the present invention provides a fluidic device comprising: a substrate having a well array disposed on its surface, the well array comprising a plurality of wells, each well having a first well and a second well located at the bottom of the first well and having a smaller volume than the first well; a cover member disposed opposite the well array; and a spacer that provides a gap between the substrate and the cover member, wherein the space between the well array and the cover member forms a flow path through which a fluid flows.

[0074] In the fluidic device of this embodiment, the substrate on which the well array is arranged is the same as that described above.

[0075] Fig. 6(a) is a top view showing an example of the fluidic device of this embodiment, and Fig. 6(b) is a cross-sectional view taken along line bb' in Fig. 6(a).

[0076] 6(a) and 6(b), the fluidic device 600 includes a substrate 210 having a well array 200 on its surface, the well array 200 including a plurality of wells, each of which has a first well 220 and a second well 230 that is located at the bottom of the first well 220 and has a smaller capacity than the first well 220, a spacer 610, and a cover member 620 having a liquid inlet 621. A space 630 between the substrate 210 and the cover member 620 functions as a flow path for the flow of a sample, a detection reagent, a sealing liquid, a water-absorbent organic solvent, etc.

[0077] The fluidic device of this embodiment can be suitably used in the above-mentioned method for detecting a target substance.

[0078] (Method for detecting target substances) Here, the method for detecting a target substance according to the above-described embodiment will be described in more detail with reference to FIGS. 7(a) to 7(d).

[0079] As described above, the method for detecting a target substance includes the steps of introducing the sample and a reagent that produces a fluorescent substance when the target substance is present into each well of a well array; sealing each well of the well array with a sealing liquid so that each well forms an independent reaction space; replacing the sealing liquid with a water-absorbing organic solvent, thereby dehydrating the contents of the well, reducing its volume, and producing the fluorescent substance when the target substance is present in the contents; and irradiating the fluorescent substance with excitation light and detecting the generated fluorescence for each well of the well array.

[0080] 7(a) to 7(d) are schematic cross-sectional views illustrating an example of a procedure for carrying out a method for detecting a target substance. In these figures, a single-stranded RNA fragment (tgRNA) is detected as the target substance. Cas13a protein, gRNA (crRNA), and a substrate nucleic acid fragment are used as reagents that generate a fluorescent substance in the presence of the target substance.

[0081] First, as shown in Figure 7(a), an assay solution 710 containing a mixture of a sample, Cas13a protein, crRNA, and a substrate nucleic acid fragment is introduced through a liquid inlet 621 of the fluidic device 600. As a result, as shown in Figure 7(a), the inside of the wells 220 and 230, and the space 630 between the substrate 210 and the cover member 620 are filled with the assay solution 710. That is, a sample and a reagent that generates a fluorescent substance in the presence of a target substance are introduced into each well of the well array.

[0082] Next, each well of the well array is sealed with a sealing liquid. Specifically, sealant 720 is introduced through liquid inlet 621. When sealant 720 is introduced, the opening of well 220 is sealed with sealant 720 while the inside of well 220 is filled with assay solution 710. As a result, each well forms an independent reaction space.

[0083] Next, as shown in Figure 7(b), the sealant 720 is replaced with a water-absorbent organic solvent 730. As a result, the contents of the well (assay solution 710) are dehydrated and their volume is reduced (concentrated), and the Cas13a-crRNA-tgRNA ternary complex cleaves the substrate nucleic acid fragment (Reporter) in the assay solution 710. As a result, the fluorescent substance F bound to the substrate nucleic acid fragment is separated from the quencher Q and begins to emit fluorescence when irradiated with excitation light. Detecting fluorescence in the well indicates the presence of the target substance in that well.

[0084] In addition, when the target substance is not present in the assay solution 710, the ternary complex of Cas13a-crRNA-tgRNA is not formed, so the substrate nucleic acid fragment is not cleaved and no fluorescence is generated.

[0085] Furthermore, when the amount of dehydration reaches a desired amount, the water-absorbent organic solvent 730 may be replaced with the sealant 720 again. This allows the dehydration of the contents of the well (assay solution 710) to be stopped. In other words, the method for detecting a target substance in a sample may further include a step of replacing the water-absorbent organic solvent with a sealant.

[0086] In this way, by capturing the target substance in the large-capacity well 220, the probability of capturing the target substance is improved, and by performing detection in the small-capacity well 230, the target substance can be detected with high sensitivity and the time required for detection is also shortened.

[0087] [Target substance detection kit] In one embodiment, the present invention provides a kit for detecting a target substance, comprising the above-described fluidic device, a reagent that generates a fluorescent substance in the presence of a target substance, a sealing liquid, and a water-absorbent organic solvent.

[0088] The kit of this embodiment can be used to suitably carry out the above-described target substance detection method. In the kit of this embodiment, the fluidic device is the same as that described above. The target substance, the reagent that generates a fluorescent substance in the presence of the target substance, the sealing liquid, and the water-absorbing organic solvent are also the same as those described above. [Example]

[0089] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0090] Materials and Methods (Preparation of Cas13a protein) Leptotrichia wadei Cas13a (LwCas13a) was expressed by transfection into Escherichia coli BL21(DE3) strain. The expression vector was a pET-based vector containing a 10xHis tag, maltose-binding protein (MBP), and a TEV protease cleavage site at the N-terminus. The expressed Cas13a protein was purified using Ni-NTA resin. Subsequently, after incubation with TEV protease overnight at 4°C, the protein was subjected to cation exchange chromatography using an MBPTrap HP column (GE Healthcare) connected to a HiTrap Heparin HP column (GE Healthcare), followed by gel filtration chromatography using a Superdex 200 column (GE Healthcare).

[0091] (Preparation of target nucleic acid fragments) The target nucleic acid fragment (single-stranded RNA fragment, SEQ ID NO: 5) was chemically synthesized by an outsourced supplier (IDT).

[0092] (gRNA preparation) A DNA fragment encoding gRNA (crRNA) was prepared by PCR amplification using overlapping primers containing a T7 promoter sequence, a 20-base target sequence, and a scaffold sequence as a template. The resulting DNA fragment was then subjected to in vitro transcription to prepare crRNA. The base sequence of the gRNA (crRNA) for Cas13a protein is shown in SEQ ID NO: 4.

[0093] (Preparation of Substrate Nucleic Acid Fragment) The substrate nucleic acid fragment (single-stranded RNA fragment) was chemically synthesized by an outsourced supplier (IDT). The 5' end of the substrate nucleic acid fragment was labeled with the fluorescent substance FAM, and the 3' end was labeled with the quencher Iowa Black FQ (IDT). The base sequence of the chemically synthesized substrate nucleic acid fragment (single-stranded RNA fragment) was "5'-(FAM)UUUUU(IABkFQ)-3'" (where "IABkFQ" stands for Iowa Black FQ).

[0094] (Preparation of Well Array A) Well array A was fabricated using the same procedures as those shown in Figures 4(a) to 4(f). First, as shown in Figure 4(a), glass substrate 210 was immersed in an 8 M potassium hydroxide solution for about 24 hours to form hydroxyl groups on the surface.

[0095] 4(b), a fluororesin (CYTOP, manufactured by AGC Corporation) was spin-coated on the surface of the glass substrate 210 to form a film 400. The spin-coating conditions were 1,000 rpm (revolutions per minute) for 30 seconds. Under these conditions, the film 400 had a thickness of approximately 1.8 μm.

[0096] Subsequently, the film 400 was adhered to the surface of the glass substrate 210 by baking on a hot plate at 180° C. for 1 hour, causing dehydration condensation between the silanol groups of the film 400 (CYTOP) and the hydroxyl groups on the glass surface.

[0097] Subsequently, as shown in FIG. 4( c ), a resist (product name “AZ-P4903” manufactured by AZ Electronic Materials) was spin-coated on the surface of the film 400 at 4000 rps for 60 seconds to form a resist film 410 .

[0098] Subsequently, the glass substrate 210 was baked on a hot plate at 110° C. for 1 hour to evaporate the organic solvent in the resist film 410, thereby adhering the resist film 410 to the surface of the film 400.

[0099] Next, as shown in Figure 4(d), using a mask with a well array pattern, the resist film 410 was exposed to ultraviolet light at 250 W for 14 seconds using an exposure machine (manufactured by Union Optical Co., Ltd.). The resist film 410 was then immersed in a developer (AZ developer, manufactured by AZ Electronic Materials Co., Ltd.) for 1.5 minutes for development. As a result, the resist film 410 in the areas where the wells would be formed was removed.

[0100] Next, as shown in FIG. 4(e), the film 400 masked with the resist film 410 was dry-etched for 30 minutes using a reactive ion etching device (manufactured by YAC) under conditions of O2 200 sccm, pressure 5 Pa, and power 50 W, thereby forming a well 230 in the film 400.

[0101] 4(f), the glass substrate 210 was immersed in acetone, washed with isopropanol, and then washed with pure water to remove the resist film 410, thereby obtaining an array (well array A) of wells 230. Well array A had cylindrical wells 230 with a diameter of 3.5 μm and a depth of 1.8 μm, arranged 1 cm apart. 2 The volume per well was 17 fL.

[0102] (Preparation of Well Array B) Well array B, which has first and second wells, was fabricated using the same procedures as those shown in Figures 4(a)-(f) and Figures 5(a) and (b). First, the well array obtained in the same manner as in Figures 4(a)-(f) was subjected to dry etching (O2 13 sccm, pressure 14 Pa, power 125 W) using a Reacte ion etching device (manufactured by Samco) for 5 seconds to hydrophilize the well array surface. Next, the well array was placed on a hot plate at 65°C, and a sheet-type resist (product name "SU-8 3020CF DFR Type-S", manufactured by KAYAKU Advanced Materials, Inc.) was adhered to the well array using a laminating roller to form a resist film 410, as shown in Figure 5(a).

[0103] Next, as shown in Figure 5(b), using a mask with the well array pattern, the resist film 410 was exposed to ultraviolet light for 20 seconds using an exposure machine (manufactured by Union Optical Co., Ltd.). The resist film 410 was then immersed in a developer (product name "SU8 developer", manufactured by KAYAKU Advanced Materials, Inc.) for 8 minutes for development. As a result, the resist film 410 in the areas where the wells would be formed was removed, and well array B having first wells 220 and second wells 230 was obtained.

[0104] Well array B has cylindrical wells 230 with a diameter of 3.5 μm and a depth of 1.8 μm, arranged on a 1 cm 2 The array contains 1,500,000 wells, each 1 cm long, with cylindrical wells 220 of 40 μm diameter and 20 μm depth. 2 The structure was a stack of 40,000 well arrays.

[0105] Well array B had a shape in which 12 to 18 wells 230 were arranged at the bottom of each well 220. Figure 8 is a micrograph of well array B that was fabricated.

[0106] (Fabrication of Fluidic Device A) 6, spacers 220 were placed on the above-described well array A, and then a glass plate 620 having a liquid inlet 621 formed therein was placed on top to fabricate fluidic device A. As a result, fluidic device A was obtained in which the space between well array A and glass plate 620 formed a flow path.

[0107] (Fabrication of fluidic device B) 6, spacers 220 were placed on the above-described well array B, and then a glass plate 620 having a liquid inlet 621 formed therein was placed on top to produce fluidic device B. As a result, fluidic device B was obtained in which the space between well array B and glass plate 620 formed a flow path.

[0108] [Experimental Example 1] (Study using Cas13a) The Cas13a protein, gRNA (SEQ ID NO: 4), and target nucleic acid fragment were mixed in buffer A having the composition shown in Table 1 below so that the final concentrations of the Cas13a protein, gRNA, and target nucleic acid fragment were 40 nM, 25 nM, and 30 pM, 3 pM, 0.3 pM, and 0 pM, respectively, to form a ternary complex. Hereinafter, this solution is referred to as the ternary complex solution.

[0109] [Table 1]

[0110] Four of the above-described fluidic devices A were prepared. In addition, a solution was prepared by dissolving the substrate nucleic acid fragment in the above-described buffer A to a final concentration of 10 μM.

[0111] Next, assay solutions were prepared by mixing the above-mentioned ternary complex solution and a solution of the substrate nucleic acid fragment, and immediately introduced into the liquid inlet of each fluidic device A. As a result, the assay solutions were introduced into each well of the well array.

[0112] Next, a sealant (hexadecane, Sigma-Aldrich) was introduced into each fluidic device A through the liquid inlet. As a result, the wells into which the assay solution had been introduced were sealed with the sealant, and each well became an independent reaction space. After a few minutes, the well array of each fluidic device A was observed under a fluorescence microscope.

[0113] Figure 9(a) is a representative fluorescence micrograph showing the results for an assay solution with a final target nucleic acid fragment concentration of 0 pM. Figure 9(b) is a representative fluorescence micrograph showing the results for an assay solution with a final target nucleic acid fragment concentration of 0.3 pM. Figure 9(c) is a representative fluorescence micrograph showing the results for an assay solution with a final target nucleic acid fragment concentration of 3 pM. Figure 9(d) is a representative fluorescence micrograph showing the results for an assay solution with a final target nucleic acid fragment concentration of 30 pM. The scale bar is 50 μm.

[0114] Figure 10(a) is a representative graph showing the number of wells that exhibited a given fluorescence intensity (relative value) based on a photograph of a well array into which each assay solution was introduced. Figure 10(b) is a graph similar to Figure 10(a) but enlarged and arranged in a row, showing the area corresponding to the area surrounded by the dotted line in Figure 10(a) for assay solutions with final concentrations of target nucleic acid fragments of 30 pM, 3 pM, 0.3 pM, and 0 pM, respectively.

[0115] As a result, it was revealed that the percentage of wells in which fluorescence was detected increased depending on the concentration of the target nucleic acid fragment.

[0116] Figure 11 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the target nucleic acid fragment. The vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the target nucleic acid fragment. The results showed that the detection sensitivity when using the fluidic device was approximately 56 fM.

[0117] [Experimental Example 2] (Concentration Study 1) An assay solution containing 150 fM alkaline phosphatase (Sigma-Aldrich) and 1 μM fluorescent substrate (sTG-phos) was prepared and introduced into the liquid inlet of the above-mentioned fluidic device B. The chemical formula of sTG-phos is shown in formula (1) below (see Sakamoto S., et al., Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples, Sci Adv. 6 (11), eaay0888, 2020). As a result, the assay solution was introduced into each well of the well array.

[0118] [ka]

[0119] Next, a sealant (hexadecane, Sigma-Aldrich) was introduced through the liquid inlet of fluidic device B. As a result, the wells into which the assay solution had been introduced were sealed with the sealant, and each well became an independent reaction space.

[0120] Next, a water-absorbent organic solvent was introduced through the liquid inlet of fluidic device B to replace the sealant. 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, and 1-nonanol were tested as water-absorbent organic solvents. As a result, the contents of the well were dehydrated, their volume decreased, and alkaline phosphatase in the contents reacted with sTG-phos to produce a fluorescent substance (sTG). The chemical formula of sTG is shown below in formula (2).

[0121] [ka]

[0122] Next, the fluorescence of sTG was detected over time. Figure 12 is a graph showing the results of measuring the change in fluorescence intensity (relative value) of sTG over time when various water-absorbent organic solvents were used. As a result, an increase in fluorescence intensity due to dehydration and concentration was observed, particularly when 1-heptanol, 1-octanol, and 1-nonanol were used.

[0123] [Experimental Example 3] (Concentration Study 2) An assay solution was prepared by mixing serially diluted alkaline phosphatase (Sigma-Aldrich) with 1 μM fluorescent substrate (sTG-phos), and introduced into the liquid inlet of the above-mentioned fluidic device B. As a result, the assay solution was introduced into each well of the well array.

[0124] Next, a sealant (hexadecane, Sigma-Aldrich) was introduced through the liquid inlet of fluidic device B. As a result, the wells into which the assay solution had been introduced were sealed with the sealant, and each well became an independent reaction space.

[0125] Next, 1-octanol was introduced through the fluid inlet of the fluidic device to replace the sealant, resulting in dehydration of the contents of the well, a decrease in volume, and the reaction between alkaline phosphatase and sTG-phos in the contents to produce a fluorescent substance (sTG).

[0126] Next, 2.5 minutes after the introduction of 1-octanol, the 1-octanol was replaced with a sealant (product name "Fomblin® Oil", Solvay). As a result, dehydration of the well contents stopped. The fluorescence of sTG was then detected. Figure 13 is a representative graph showing the percentage (%) of wells that exhibited a given fluorescence intensity (relative value) based on a photograph of the well array in which sTG fluorescence was detected. In Figure 13, the horizontal axis of the graph indicates the concentration of alkaline phosphatase (ALP).

[0127] As a result, it was revealed that the presence of approximately 80 aM alkaline phosphatase could be detected. In other words, it was revealed that the detection sensitivity when dehydration and concentration using 1-octanol was performed using fluidic device B was approximately 80 aM. [Industrial Applicability]

[0128] According to the present invention, a technique can be provided that can detect a target substance with high sensitivity. [Explanation of symbols]

[0129] 100,100'...ternary complex, 110...Cas12a protein, 120...gRNA, 130...binary complex, 140...target substance (target nucleic acid fragment), 150...substrate nucleic acid fragment, 200,300...well array, 210...substrate, 220...first well, 230...second well, 400,410...membrane, 600...fluidic device, 610...spacer, 621...liquid inlet, 620...lid member, 630...space, 710...assay solution, 720...sealant, 730...water-absorbent organic solvent, F...fluorescent substance, Q...quencher.

Claims

1. A method for detecting a target substance in a sample, comprising: introducing the sample and a reagent that generates a fluorescent substance in the presence of the target substance into each well of a well array; a step of sealing each well of the well array with a sealing liquid so that each well forms an independent reaction space; replacing the sealing liquid with a water-absorbent organic solvent, thereby dehydrating the contents of the well, reducing the volume, and generating the fluorescent substance if the target substance is present in the contents; irradiating the fluorescent substance with excitation light and detecting the emitted fluorescence for each well of the well array; wherein detection of fluorescence in the well indicates the presence of the target substance in the well.

2. 2. The method of claim 1, wherein each well of the well array has a first well and a second well located at the bottom of the first well and having a smaller volume than the first well, and when the volume of the contents of the first well becomes smaller, the contents accumulate in the second well.

3. 3. The method of claim 2, wherein the ratio of the volumes of the first well to the second well (volume of the first well:volume of the second well) is 10:1 to 1,000,000:

1.

4. 4. The method according to claim 2, wherein the volume of the first well is 1 to 1,000 pL and the volume of the second well is 0.1 to 1,000 fL.

5. The method according to any one of claims 2 to 4, wherein 0 or 1 target substance is introduced per each first well.

6. 6. The method according to claim 1, wherein the sealing liquid is a fluorine-based liquid, a mineral oil, or a linear or branched, saturated or unsaturated hydrocarbon having 7 to 17 carbon atoms.

7. The method according to any one of claims 1 to 6, wherein the water-absorbing organic solvent is a linear or branched, saturated or unsaturated aliphatic alcohol having 4 to 11 carbon atoms.

8. a substrate having a well array disposed on its surface, the well array including a plurality of wells, each of the wells having a first well and a second well disposed at the bottom of the first well and having a smaller volume than the first well; a cover member disposed opposite the well array; a spacer that separates the substrate and the lid member, a space between the well array and the cover member forms a flow path through which a fluid flows; A fluidic device for detecting a target substance in a sample, wherein the first well has a volume of 1 to 1,000 pL and the second well has a volume of 0.1 to 1,000 fL.

9. A kit for detecting a target substance, comprising: the fluidic device according to claim 8; a reagent that generates a fluorescent substance in the presence of a target substance; a sealing liquid; and a water-absorbent organic solvent.

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