Method for detecting multiple kinds of target molecules

Abstract

This method is used for detecting multiple kinds of target molecules in a sample on the same well array. The method includes a step (a) for binding multiple kinds of target molecules to a target molecule capture carrier and separating the target molecule capture carrier from the sample, a step (b) for suspending the separated target molecule capture carrier in a reaction liquid and introducing the suspended target molecule capture carrier into the wells of a well array, a step (c) for sealing the wells using a sealing liquid, a step (d) for pretreating the multiple kinds of target molecules in the wells, and a step (e) for detecting each of the multiple kinds of target molecules in the wells through Invasive Cleavage Assay (ICA). The multiple kinds of target molecules include at least a nucleic acid. The pretreatment includes dissociation of the nucleic acid from the target molecule capture carrier or transfer of the nucleic acid to a complementary strand. The reaction liquid is used in order to enable the pretreatment and the ICA to be performed without interfering with each other.

Description

Method for detecting multiple types of target molecules

[0001] The present invention relates to a method for detecting multiple types of target molecules. This application claims priority to Japanese Patent Application No. 2024-092616, filed on June 6, 2024, the contents of which are incorporated herein by reference.

[0002] Quantitative detection of target molecules in biological samples has been used for early detection of diseases and prediction of medication effects. Conventionally, protein quantification has been performed by enzyme-linked immunosorbent assay (ELISA) or the like, and nucleic acid quantification has been performed by real-time PCR or the like.

[0003] In recent years, there has been an increasing need for more accurate detection of target molecules for purposes such as earlier detection of diseases. Techniques for accurately detecting target molecules include those described in, for example, Patent Document 1, Patent Document 2, and Non-Patent Document 1, in which enzyme reactions are carried out in a large number of microcompartments. These techniques are called digital measurement.

[0004] In digital measurement, a sample solution is divided into an extremely large number of minute solutions. The signal from each minute solution is then binarized, and the presence or absence of target molecules is determined, and the number of target molecules is measured. Digital measurement can significantly improve detection sensitivity and quantitativeness compared to conventional methods such as ELISA and real-time PCR.

[0005] In digital PCR, a mixture of PCR reaction reagents and nucleic acids is diluted so that each microdroplet contains zero or one template nucleic acid. In digital PCR, a small volume of each microdroplet is preferable to increase the sensitivity of nucleic acid amplification and to simultaneously amplify multiple microdroplets. For example, Patent Document 3 discloses a microarray-shaped reaction vessel formed so that each well has a volume of 6 nL (nanoliters). Patent Document 1 also discloses a method for introducing a sample into a microarray having multiple wells, each 3 μm deep and 5 μm in diameter, formed in a flow channel. The sample is then introduced into each well by flowing the sample through the flow channel, and then excess reagent in the flow channel is pushed out with oil.

[0006] Meanwhile, techniques for measuring multiple types of target molecules have been developed. For example, Patent Document 4 describes a method for measuring protein and gene expression in cells using a barcode sequence nucleic acid-modified antibody and a target gene-binding barcode particle.

[0007] Furthermore, Patent Document 5 describes a method for detecting surface target molecules present on the surface of a structure and internal target molecules present inside the structure.

[0008] Japanese Patent No. 6183471 Special Publication No. 2014-503831 International Publication No. 2013-151135 Japanese Patent No. 7091348 International Publication No. 2020-235607

[0009] Kim SH, et al., Large-scale femtoliter droplet array for digital counting of single biomolecules, Lab on a Chip, 12 (23), 4986-4991, 2012.

[0010] However, when measuring multiple types of target molecules, such as nucleic acids and proteins, the separation conditions are different, making it difficult to extract or separate them simultaneously. For example, the conditions for extracting nucleic acids may cause proteins to be denatured and lost. Furthermore, different measurement methods and measurement devices are required for each type of target molecule, making it difficult to simultaneously measure multiple types of target molecules.

[0011] An object of the present invention is to provide a technique for detecting multiple types of target molecules on the same device.

[0012] The present invention includes the following aspects: [1] A method for detecting multiple types of target molecules in a sample on the same well array, comprising: (a) a step of binding the multiple types of target molecules to target molecule capture carriers and separating the target molecule capture carriers to which the target molecules are bound from the sample; (b) a step of suspending the separated target molecule capture carriers in a reaction solution and introducing the resulting suspension into wells of the well array; (c) a step of sealing the wells with a sealing solution; (d) a step of pretreating the multiple types of target molecules in the wells; and (e) a step of detecting each of the multiple types of target molecules in the wells by Invasive Cleavage Assay (ICA), wherein the multiple types of target molecules include at least nucleic acids, the pretreatment includes dissociating the nucleic acids from the target molecule capture carriers or transcribing the nucleic acids into complementary strands, and the reaction solution is for carrying out the pretreatment and the ICA without mutually inhibiting each other. [2] The method according to [1], wherein the target molecule capture carrier is a magnetic particle, and the separation is performed by magnetism. [3] The method according to [1] or [2], wherein the target molecule capture carrier comprises a solid-phase reversible immobilization (SPRI) particle. [4] The method according to any one of [1] to [3], wherein the volume of the well is 10 fL to 100 pL. [5] The method according to any one of [1] to [4], wherein the reaction solution comprises 5 to 25 mM magnesium ions. [6] The method according to any one of [1] to [5], wherein the reaction solution comprises 0.01 to 0.05 mM ethylenediaminetetraacetic acid. [7] The method according to any one of [1] to [6], wherein the reaction solution comprises 10 to 50 mM trishydroxymethylaminomethane. [8] The method according to any one of [1] to [7], wherein the pH of the reaction solution at 25°C is 7.5 to 8.5. [9] The method according to any one of [1] to [8], wherein the target molecule capture carrier includes a first target molecule capture carrier and a second target molecule capture carrier, the multiple types of target molecules include a first target molecule and a second target molecule, the first target molecule binds to the first target molecule capture carrier, and the second target molecule binds to the second target molecule capture carrier.

[10] The method according to any one of [1] to [9], wherein the plurality of types of target molecules comprises proteins.

[11] The method according to any one of [1] to

[10] , wherein the sample comprises cells, and further comprises a step of lysing the cells prior to the separating step (a).

[12] The method according to any one of [1] to

[10] , wherein the plurality of types of target molecules comprise HER2 protein and ErbB2 gene.

[13] A method for detecting a plurality of types of target molecules in a sample on the same well array, comprising the steps of: (a) binding the plurality of types of target molecules to a target molecule capture carrier and separating the target molecule-bound target molecule capture carrier from the sample; (b) suspending the separated target molecule capture carrier in a reaction solution and introducing it into wells of the well array; (c) sealing the wells with a sealing liquid; (d) pretreating the plurality of types of target molecules in the wells; and (e) invasive cleavage of the plurality of types of target molecules in the wells. and step (e) detecting each of the target molecules by Indirect Cadence Assay (ICA), wherein the multiple types of target molecules include at least nucleic acids, the pretreatment includes dissociating the nucleic acids from the target molecule capture carrier or transcribing the nucleic acids into complementary strands, the reaction solution contains 5 to 25 mM magnesium ions, 0.01 to 0.05 mM ethylenediaminetetraacetic acid, and 10 to 50 mM trishydroxymethylaminomethane, the pH of the reaction solution at 25°C is 7.5 to 8.5, and the target molecules are HER2 protein and ErbB2 gene.

[0013] According to the present invention, it is possible to provide a technique for detecting multiple types of target molecules on the same device.

[0014] FIG. 1 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 2 is a schematic cross-sectional view illustrating a method for detecting a target molecule. FIG. 3 is a schematic cross-sectional view illustrating a method for detecting a target molecule. FIG. 4 is a schematic cross-sectional view showing an example of a fluidic device. FIG. 5 is a schematic cross-sectional view illustrating a method for detecting a target molecule. FIG. 6 is a schematic cross-sectional view illustrating a method for detecting a target molecule. FIG. 7 is a schematic view illustrating an example of Invasive Cleavage Assay (ICA). FIG. 8 is an image showing the results of Experimental Example 1.

[0015] In this specification, for example, a numerical range expressed as "1 to 2" means a numerical range including the numbers 1 and 2 before and after "1" as the lower and upper limits.

[0016] 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.

[0017] In one embodiment, the present invention provides a method for detecting multiple types of target molecules in a sample on the same well array, comprising the steps of: (a) binding the multiple types of target molecules to target molecule capture carriers and separating the target molecule-bound target molecule capture carriers from the sample; (b) suspending the separated target molecule capture carriers in a reaction solution and introducing them into wells of the well array; (c) sealing the wells with a sealing solution; (d) pretreating the multiple types of target molecules in the wells; and (e) detecting the multiple types of target molecules in the wells by Invasive Cleavage Assay (ICA), wherein the multiple types of target molecules include at least nucleic acids, the pretreatment includes dissociating the nucleic acids from the target molecule capture carriers or transcribing the nucleic acids into complementary strands, and the reaction solution is designed to perform the pretreatment and the ICA without mutually inhibiting each other. This method allows multiple types of target molecules to be detected on the same device.

[0018] The sample is not particularly limited as long as it contains multiple types of target molecules, and examples include serum, urine, exosomes, cells, tissues, whole blood, etc. The sample may be, for example, cells themselves, a cell lysate, or a cell population containing multiple cells.

[0019] Examples of target molecules include proteins, nucleic acids, and metabolites. Examples of nucleic acids include DNA and RNA. Multiple types of target molecules mean, for example, that the target molecules are a combination of multiple types, such as a combination of protein and DNA, a combination of protein and RNA, a combination of protein, DNA, and RNA, a combination of DNA and RNA, a combination of multiple types of DNA with different sequences, a combination of multiple types of RNA with different sequences, a combination of one or multiple types of DNA and one or multiple types of RNA, or a combination of multiple types of proteins. Multiple types of DNA may be, for example, multiple types of DNA with different sequences. Similarly, multiple types of RNA may be, for example, multiple types of RNA with different sequences. Multiple types of target molecules include at least nucleic acids.

[0020] In step (a), multiple types of target molecules in a sample are bound to a target molecule capture carrier, and the target molecule capture carrier to which the target molecules are bound is separated from the sample. When the sample is a structure such as an exosome or a cell, it is preferable to disrupt the structure in advance to expose the target molecules contained within the structure.

[0021] For example, when multiple types of target molecules include proteins and nucleic acids, the target molecule capture carrier may include an antibody-modified carrier against the protein and a nucleic acid-binding carrier that binds to the nucleic acid. In this case, the nucleic acid modified onto the target molecule capture carrier has a sequence complementary to the target nucleic acid. In addition, in this case, multiple types of target molecules are separated by binding to multiple types of target molecule capture carriers, respectively. For example, when the target molecules include first and second target molecules, the target molecule capture carrier may include a first target molecule capture carrier that binds to the first target molecule and a second target molecule capture carrier that binds to the second target molecule.

[0022] Alternatively, when the multiple types of target molecules include proteins and RNAs, the nucleic acid modified into the target molecule capture carrier may have a sequence corresponding to the Poly A sequence.

[0023] Alternatively, one type of target molecule capture carrier may be modified with an antibody against a protein and a probe against a nucleic acid, and one target molecule capture carrier may capture and separate proteins and nucleic acids in a sample. Here, the number of proteins and / or nucleic acids is not limited to one. For example, one type of target molecule capture carrier may be modified with an antibody against one or more types of proteins and / or a probe against one or more types of nucleic acids, and one target molecule capture carrier may capture and separate one or more types of proteins and / or one or more types of nucleic acids in a sample.

[0024] Alternatively, for example, one type of target molecule capture carrier may be modified with an antibody against exosomes, an antibody against proteins, and a probe against nucleic acids, and in step (a), the target molecule capture carrier may first capture and recover exosomes from a sample, and then a separation and extraction operation may be performed to extract proteins and nucleic acids in the exosomes, and the extracted proteins and nucleic acids may be captured and separated by the target molecule capture carrier. In other words, when the target molecules include first to third target molecules, the target molecule capture carrier may be modified with first to third probes corresponding to the first to third target molecules, respectively.

[0025] The target molecule capture carrier is preferably a magnetic particle, and separation is preferably performed by magnetism, which allows multiple types of target molecules to be simultaneously separated by the same method.

[0026] When the capture carrier includes a nucleic acid-binding carrier, the nucleic acid-binding carrier may be a solid-phase reversible immobilization (SPRI) particle. SPRI particles have a structure in which the core is covered with a thin film of magnetic iron, and can be easily recovered by magnetism. The surface of the magnetic particle is coated with carboxyl groups, and can bind to DNA / RNA in the presence of salt.

[0027] The target molecule capture carrier is, for example, Invitrogen ＴＭ Dynabeads ＴＭ It may be a series. ＴＭ Dynabeads ＴＭThe series is made of magnetizable materials (γFe ２ O ３ and Fe ３ O ４ These beads have a uniform particle size and consist of a core of a high molecular weight polymer with a uniform distribution of ) coated with a hydrophilic polymer. The probes to be modified can be changed depending on the application.

[0028] The target molecule capture carrier has at least one of a carboxyl group, an amino group, an epoxy group, and a tosyl group on the particle surface in order to modify an antibody or a nucleic acid.

[0029] The surface of the target molecule capture carrier may be either a hydrophilic surface or a hydrophobic surface.

[0030] Subsequently, in step (b), the separated target molecule capturing carriers are suspended in a reaction solution and introduced into the wells of the well array.

[0031] The reaction solution used here is capable of carrying out the pretreatment described below and is also intended for carrying out Invasive Cleavage Assay (ICA). When the target molecule contains a protein, the reaction solution must also not inhibit the detection of the protein by ICA. In other words, the reaction solution is adjusted so as not to inhibit both the pretreatment and ICA.

[0032] When the target molecule includes a protein, it is preferable to nucleic acid-label the protein captured on the target molecule capture carrier before step (b). Nucleic acid labeling makes it possible to detect the protein by ICA. Nucleic acid labeling can be performed, for example, by binding an antibody against the target molecule, the protein, labeled with a nucleic acid to the protein captured on the target molecule capture carrier.

[0033] Subsequently, in step (c), the wells are sealed with a sealing liquid. As a result, each well of the well array becomes an independent reaction space. Here, it is preferable that one well contains zero or one target molecule. This enables digital measurement.

[0034] Subsequently, in step (d), the multiple types of target molecules in the wells are pretreated. Pretreatment can be performed by incubating the target molecule capture carrier in the above-mentioned reaction solution. Pretreatment can also involve dissociating the captured nucleic acid from the target molecule capture carrier. Dissociating the nucleic acid from the target molecule capture carrier makes detection easier.

[0035] Alternatively, the pretreatment may involve transcription of the captured nucleic acid into a complementary strand. For example, the nucleic acid captured by the target molecule capture carrier may be RNA, and the pretreatment may involve reverse transcribing the captured RNA into cDNA using a reverse transcriptase. Reverse transcription of RNA into cDNA facilitates detection. In this case, the reaction solution contains a reverse transcriptase and nucleoside triphosphates (NTPs). The reaction solution may further contain a primer for reverse transcription.

[0036] Subsequently, in step (e), the multiple types of target molecules in the wells are each detected by Invasive Cleavage Assay (ICA), which will be described later.

[0037] When one target molecule capturing carrier captures multiple types of target molecules in a sample, ICA may be detected in multiple colors, and each target molecule may be detected as a fluorescent signal of a different wavelength.

[0038] The above method allows simultaneous separation of multiple types of target molecules that require different separation conditions and are difficult to extract or separate simultaneously, and also allows simultaneous measurement of multiple types of target molecules that have traditionally been measured using different measurement methods and measurement devices.

[0039] Fig. 1 is a schematic cross-sectional view showing an example of a fluidic device in which the method of this embodiment can be suitably carried out. As shown in Fig. 1, the fluidic device 200 includes a substrate 210 and a lid member 220 disposed opposite the substrate 210. The lid member 220 has a convex portion 221, and the tip of the convex portion 221 contacts the substrate 210. In the fluidic device 200, a well array 240 is integrally molded with the substrate 210 on one side of the substrate 210 and faces the lid member 220. The well array 240 has a plurality of wells 241. The lid member 220 may be welded or glued to the substrate 210.

[0040] The wells 241 are open on the surface of the substrate 210. There are no particular limitations on the shape, size, or arrangement of the wells 241, but the wells 241 are preferably microwells with small volumes. For example, the volume of one well 241 may be approximately 10 fL to 100 pL. In the fluidic device 200, a plurality of wells 241 of the same shape and size constitute a well array 240. The term "same shape and size" means that the wells have the same shape and capacity to the extent required for digital measurement, and variations within the range of manufacturing errors are acceptable.

[0041] The diameter of well 241 may be, for example, about 1 to 10 μm, and the depth of well 241 may be, for example, about 1 to 10 μm. The arrangement of wells 241 is not particularly limited, and may be, for example, arranged in a triangular lattice pattern, a square lattice pattern, or randomly arranged.

[0042] In the fluidic device 200, the presence of the convex portion 221 forms a space between the well array 240 and the lid member 220. This space constitutes a flow channel 230. The flow channel 230 functions as a path for transporting liquids such as a reaction solution in which a target molecule capture carrier is suspended, and a sealing liquid, which will be described later. The shape, structure, capacity, etc. of the flow channel 230 are not particularly limited, but the height of the flow channel 230 (the distance between the surface of the substrate 210 and the surface of the lid member 220 facing the substrate 210) may be, for example, 500 μm or less, for example, 300 μm or less, for example, 200 μm or less, or for example, 100 μm or less.

[0043] The convex portion 221 may be molded integrally with the lid member 220. The lid member 220 can be formed into a plate shape having the convex portion 221, for example, by molding a thermoplastic resin fluid using a molding die. Furthermore, the lid member 220 may be formed with an inlet port 222 and an outlet port 223 for a reagent.

[0044] When the lid member 220 has a protrusion 221, the lid member 220 and the substrate 210 are overlapped so that the protrusion 221 contacts the surface of the substrate 210 where the well 241 opens. As a result, the space between the lid member 220 and the substrate 210 becomes the flow path 230. The lid member 220 and the substrate 210 may be welded by laser welding or the like.

[0045] The fluidic device used in the method of this embodiment is not limited to the above-described fluidic device 200. Fig. 4 is a schematic cross-sectional view showing an example of a fluidic device. As shown in Fig. 4, the fluidic device 500 includes a substrate 210 and a wall member 510. In the fluidic device 500, a well array 240 is integrally formed with the substrate 210 on one side of the substrate 210. The well array 240 has a plurality of wells 241. The fluidic device 500 differs from the above-described fluidic device 200 mainly in that the fluidic device 500 does not include a cover member 220.

[0046] In the above-described fluidic device 200, the cover member 220 and the protrusion 221 are integrally molded. However, the cover member 220 and the protrusion 221 may be molded as separate bodies.

[0047] Furthermore, in the above-described fluidic device 200 and fluidic device 500, the well array 240 is integrally molded with the substrate 210 on one side of the substrate 210. However, the well array does not have to be integrally molded with the substrate 210. For example, the well array 240 molded separately from the fluidic device may be disposed on the substrate 210 of the fluidic device. Alternatively, a resin layer may be laminated on the surface of the substrate 210, and the well array may be formed in the resin layer by etching or the like.

[0048] In the fluidic device, the substrate 210 is formed using, for example, a resin. The type of resin is not particularly limited, but a resin that is resistant to reagents and sealing liquid is preferable. Furthermore, when the signal to be detected is fluorescence, a resin with low autofluorescence is preferable. Examples of the resin include, but are not limited to, cycloolefin polymer, cycloolefin copolymer, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesin, and amorphous fluororesin.

[0049] A plurality of wells 241 may be formed on one surface in the thickness direction of the substrate 210. Methods for forming wells using resin include injection molding, thermal imprinting, and photoimprinting.

[0050] Alternatively, for example, a fluororesin may be laminated on the substrate 210 and processed by etching or the like to form a well array. As the fluororesin, for example, CYTOP (registered trademark) (Asahi Glass) or the like can be used.

[0051] Furthermore, when the fluidic device has a lid member 220, the material of the lid member 220 is preferably a resin with low autofluorescence, and may be, for example, a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0052] Furthermore, the cover member 220 may be made of a material that does not transmit light of wavelengths close to the wavelength detected during fluorescence observation of the signal, or may be made of a material that is completely opaque to light. For example, the cover member 220 may be made of a thermoplastic resin to which carbon, metal particles, or the like are added.

[0053] Next, the method of this embodiment will be described with reference to FIGS. 1 to 3, taking the case where the fluidic device 200 is used as an example.

[0054] 1, a reaction solution L210 containing a target molecule capture carrier suspended therein is introduced through an inlet port 222 of the fluidic device 200 and sent to a flow path 230. The reaction solution L210 is capable of carrying out the above-described pretreatment and is also used for performing ICA. Furthermore, when the target molecule contains a protein, the reaction solution L210 must also not inhibit the detection of the protein by ICA.

[0055] The reaction solution L210 sent to the channel 230 comes into contact with the well array 240. Then, the reaction solution L210 is contained inside the wells 241. As a result, the target molecules and the reaction solution are introduced into the wells 241.

[0056] Although there is no particular limitation on the number of target molecules introduced into one well 241, preferably one or less, i.e., zero or one target molecule, is introduced into one well 241. This allows target molecules to be detected one by one, i.e., digital measurement becomes possible. Furthermore, it is not necessary to introduce target molecules into all wells of the well array.

[0057] Alternatively, one or less target molecule capture carriers may be introduced into one well 241. Here, if multiple types of target molecules are captured on one target molecule capture carrier, multiple types of target molecules may be contained in one well 241. In this case, ICA may be detected in multiple colors, and each target molecule may be detected as a fluorescent signal of a different wavelength.

[0058] After the reaction solution L210 is introduced into the wells 241, a step of sealing the openings of the wells 241 is performed. Specifically, as shown in FIG. 2 , a sealing solution L220 is sent from an inlet port 222 of the lid member 220 to a flow path 230 between the substrate 210 and the lid member 220. The sealing solution L220 sent to the flow path 230 comes into contact with the well array 240. The sealing solution L220 then flushes out and replaces the reagent solution L210 sent to the flow path 230 that is not contained in the wells 241. As a result, the sealing solution L220 individually seals each of the multiple wells 241 that contain the reaction solution L210 containing the target substance, and the wells 241 become independent reaction spaces (also referred to as microcompartments 242). When the flow path 230 is filled with the sealing solution L220, excess sealing solution L220 is discharged from a discharge port 223. FIG. 3 shows a state in which all of the wells 241 of the well array 240 have been sealed with sealing liquid L220, forming sealed wells (i.e., microcompartments) 242.

[0059] The sealing liquid is a liquid that can individually seal the liquids introduced into the multiple wells 241 so as not to mix with each other, thereby forming droplets (also referred to as microdroplets), and is preferably an oily solution, more preferably an oil. Examples of oils that can be used include fluorine-based oils, silicone-based oils, hydrocarbon-based oils, and mixtures thereof. More specifically, products such as "FC-40" manufactured by Sigma and "KF-96" manufactured by Shin-Etsu Chemical Co., Ltd. can be used. FC-40 (CAS number: 86508-42-1) is a fluorinated aliphatic compound with a specific gravity of 1.85 g / mL at 25°C.

[0060] Here, an Invasive Cleavage Assay (ICA) will be described. Fig. 7 is a schematic diagram illustrating an example of ICA. In the example of Fig. 7, ICA detects double-stranded nucleic acid 150 formed by hybridization of at least a portion of a first single-stranded nucleic acid fragment 141 and at least a portion of a second single-stranded nucleic acid fragment 171.

[0061] First, an allele probe (also called a flap probe) is hybridized to the first single-stranded nucleic acid fragment 141 or the second single-stranded nucleic acid fragment 171. The allele probe is a single-stranded nucleic acid fragment that hybridizes to the DNA to be detected to form a flap site. In the example of FIG. 7 , the allele probe 810 hybridizes to the second single-stranded nucleic acid fragment 171. As a result, a first flap site 811 is formed.

[0062] Subsequently, when a flap endonuclease (FEN) is reacted with the first flap site 811, the first flap site 811 is cleaved to generate a nucleic acid fragment 811. A flap endonuclease is an endonuclease that recognizes a specific DNA higher-order structure (also called a flap structure) rather than a DNA sequence, and has the activity of cleaving the flap strand.

[0063] Nucleic acid fragment 811 then hybridizes to a fluorescent substrate (also called a FRET cassette, nucleic acid fragment 820 ) to form a second flap site 821 .

[0064] In the example of Figure 7, a fluorescent substance F is bound to the 5' end of nucleic acid fragment 820, and a quencher Q is bound to a few bases 3' from the 5' end of nucleic acid fragment 820. Subsequently, when FEN is reacted with second flap site 821, second flap site 821 is cleaved and nucleic acid fragment 821 is generated. As a result, fluorescent substance F is separated from quencher Q and a fluorescent signal is generated. By detecting this fluorescent signal, the formation of double-stranded nucleic acid 150 can be detected.

[0065] The reaction liquid L210 may be a liquid generally used in biochemical analyses using fluidic devices, and is preferably an aqueous solution. The reaction liquid L210 may also contain a surfactant or the like to facilitate sealing the liquid in the wells.

[0066] When ICA is used to detect the formation of double-stranded nucleic acid 150, if double-stranded nucleic acid 150 is present, an isothermal enzymatic reaction causes fluorescent substance F to be released from quencher Q, emitting a predetermined fluorescent signal in response to excitation light.

[0067] The formation of double-stranded nucleic acid 150 can be detected by any known appropriate method selected depending on the type of signal to be detected. For example, when observing a fluorescent signal, excitation light corresponding to the fluorescent substance is irradiated onto well 242, and the fluorescence emitted by the fluorescent substance is observed. For example, as shown in Figure 3, a predetermined reaction is carried out in sealed well 242, and the generated signal is observed. In Figure 3, well 242R is a well in which a signal was detected, and well 242 is a well in which a signal was not detected.

[0068] Next, with reference to FIGS. 4 to 6 , the method of this embodiment will be described using a fluidic device 500 as an example. First, as shown in FIG. 4 , a reaction solution L210 is introduced into the fluidic device 500. The reaction solution L210 is capable of carrying out the above-described pretreatment and is also used for performing ICA. Furthermore, if the target molecule contains a protein, the reaction solution L210 must also not inhibit the detection of the protein by ICA. The concentration of the target molecule in the reaction solution L210 may be adjusted to a concentration such that one or less target molecule molecules are introduced into each well 241. Alternatively, the concentration of the target molecule capture carrier in the reaction solution L210 may be adjusted to a concentration such that one or less target molecule capture carriers are introduced into each well 241.

[0069] 5, a sealing liquid L220 is introduced into the fluidic device 500. The specific gravity of the sealing liquid L220 is greater than that of the mixed liquid L210. Therefore, the sealing liquid L220 sinks below the mixed liquid L210 that is not contained in the wells 241, and comes into contact with the well array 240. The sealing liquid L220 then individually seals each of the multiple wells 241 that contain the reaction liquid L210 containing the target molecules, forming independent reaction spaces (i.e., microcompartments 242).

[0070] Subsequently, a predetermined reaction is carried out in well 242, and the generated signal is observed, as shown in Fig. 6. In Fig. 6, well 242R is a well in which a signal was detected, and well 242 is a well in which a signal was not detected.

[0071] In the method of this embodiment, the reaction solution must be capable of undergoing the above-mentioned pretreatment, be capable of performing ICA, and, if the target molecule contains a protein, not inhibit detection of the protein by ICA.

[0072] As described later in the Examples, the inventors have found that the above condition is met when the magnesium ion concentration in the reaction solution is 5 to 25 mM, i.e., when the magnesium ion concentration relative to the total volume of the reaction solution is 5 to 25 mM. The inventors have also found that the above condition is met when the ethylenediaminetetraacetic acid concentration in the reaction solution is 0.01 to 0.05 mM, i.e., when the ethylenediaminetetraacetic acid concentration relative to the total volume of the reaction solution is 0.01 to 0.05 mM. The inventors have also found that the above condition is met when the tris(hydroxymethyl)aminomethane concentration in the reaction solution is 10 to 50 mM, i.e., when the tris(hydroxymethyl)aminomethane concentration relative to the total volume of the reaction solution is 10 to 50 mM. The inventors have also found that the above condition is met when the pH of the reaction solution at 25°C is 7.5 to 8.5.

[0073] The reaction solution further contains an allele probe, an ICA oligo, a FRET cassette, and a flap endonuclease for performing ICA. Furthermore, if the nucleic acid captured by the target molecule capture carrier is RNA and the RNA is reverse transcribed into cDNA in the pretreatment, the reaction solution further contains a reverse transcriptase and nucleoside triphosphates (NTPs). The reaction solution may further contain a primer for reverse transcription.

[0074] An ICA oligo is a single-stranded nucleic acid fragment that hybridizes to a target molecule, such as a protein or nucleic acid. The ICA oligo may hybridize directly to the target molecule, such as a nucleic acid, or may hybridize to a nucleic acid modified with an antibody against the target molecule, such as a protein.

[0075] The allele probe, FRET cassette, and flap endonuclease are the same as those described above.

[0076] As an example, we will explain the case where a type of target molecule capture carrier is used, exosomes are used as a sample, and proteins and RNA are detected as target molecules.

[0077] The target molecule capture carrier used here has magnetic particles whose surfaces are modified with antibodies against exosomes, antibodies against proteins, and RNA-capturing nucleic acids.

[0078] In step (a), a sample of a structure such as cells, tissue, or whole blood is disrupted to expose the exosomes contained in the structure. Next, the exosomes are captured using a target molecule capture carrier, and the target molecule capture carrier with the captured exosomes is recovered by magnetic separation.

[0079] The target molecules, proteins and RNA, are extracted from the captured exosomes using an extraction solution such as a solution containing a surfactant.The proteins and RNA are then captured using a target molecule capture carrier, and the target molecule capture carrier with the captured proteins and RNA is recovered by magnetic separation.

[0080] To nucleic acid-label the proteins contained in the recovered target molecule capture carriers, a sandwich reaction is carried out using an antibody against the protein that has been labeled with nucleic acid, and the target molecule capture carriers after the sandwich reaction are then recovered by magnetic separation.

[0081] In step (b), the recovered target molecule capture carrier, an ICA reaction solution for protein detection, an ICA reaction solution for RNA detection, and a reaction solution containing reverse transcriptase and nucleoside triphosphate (NTP) are mixed and introduced into wells of a well array.

[0082] In step (c), the wells are sealed with a sealing liquid, so that each well in the well array becomes an independent reaction space.

[0083] In step (d), the target molecule, RNA, is reverse transcribed into cDNA using reverse transcriptase.

[0084] In step (e), protein and RNA in the well are each detected by an ICA reaction. More specifically, protein is detected by detecting nucleic acid labeled to protein by sandwich reaction using an ICA reaction. RNA is detected by detecting cDNA generated by reverse transcription using an ICA reaction. In this case, the fluorescent signal detected by the ICA reaction for cDNA and the fluorescent signal detected by the ICA reaction for nucleic acid labeled to protein are each set to different wavelengths. This allows protein and RNA to be detected simultaneously.

[0085] The above method makes it possible to simultaneously separate proteins and RNAs, which require different separation conditions and are difficult to extract or separate simultaneously.

[0086] As another example, we will explain the case where multiple types of target molecule capture carriers are used, exosomes are used as samples, and proteins and RNA are detected as target molecules.

[0087] The target molecule capture carriers used here are SPRI particles as nucleic acid binding carriers, and magnetic particles modified with antibodies against proteins on their surfaces as protein capture carriers.

[0088] In step (a), a sample of a structure such as cells, tissue, or whole blood is first disrupted to expose the exosomes contained in the structure. Next, the exosomes are captured using a target molecule capture carrier, and the target molecule capture carrier with the captured exosomes is recovered by magnetic separation. Exosome capture may be performed using a protein capture carrier that has been modified with an antibody for capturing exosomes. Alternatively, a separate capture carrier for capturing exosomes may be used.

[0089] The target molecules, protein and RNA, are extracted from the captured exosomes using an extraction solution such as a solution containing a surfactant.The protein and RNA are then captured using a protein capture carrier and SPRI particles, respectively, and the protein capture carrier with the captured protein and the SPRI particles with the captured RNA are recovered by magnetic separation.

[0090] To nucleic acid-label the proteins contained in the recovered protein capture carriers, a sandwich reaction is carried out using a nucleic acid-labeled antibody against the protein, and the protein capture carriers after the sandwich reaction are then recovered by magnetic separation.

[0091] In step (b), the recovered protein capture carrier and SPRI particles are mixed with an ICA reaction solution for protein detection, an ICA reaction solution for RNA detection, and a reaction solution containing reverse transcriptase and nucleoside triphosphate (NTP), and the mixture is introduced into wells of a well array.

[0092] In step (c), the wells are sealed with a sealing liquid, so that each well in the well array becomes an independent reaction space.

[0093] In step (d), the target molecule, RNA, is reverse transcribed into cDNA using reverse transcriptase.

[0094] In step (e), protein and RNA in the well are each detected by an ICA reaction. More specifically, protein is detected by detecting nucleic acid labeled to protein by sandwich reaction using an ICA reaction. RNA is detected by detecting cDNA generated by reverse transcription using an ICA reaction. In this case, the fluorescent signal detected by the ICA reaction for cDNA and the fluorescent signal detected by the ICA reaction for nucleic acid labeled to protein are each set to different wavelengths. This allows protein and RNA to be detected simultaneously.

[0095] The above method makes it possible to simultaneously separate proteins and RNAs, which require different separation conditions and are difficult to extract or separate simultaneously.

[0096] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0097] In this example, two resin components were used to fabricate the experimental fluidic device: a cycloolefin (COP) base material formed by injection molding and a COP lid material (colored with added carbon black, with inlet and outlet ports). The wells (i.e., microcompartments) formed in the base material had a diameter of 5 or 10 μm and were large enough to detect signals via the Invader reaction within a few minutes. The base material was bonded to the lid material to form an internal space (i.e., a flow channel) of the fluidic device with a height of 100 μm.

[0098] Example 1 (Detection of Protein and DNA) Serum to which protein and DNA had been added was used as a sample, and the protein and DNA in the sample were detected.

[0099] <<Sample Preparation>> Samples were prepared by adding protein and DNA to serum. HER2 protein was used as the protein. ErbB2 gene fragment (base sequence shown in SEQ ID NO: 1) was used as the DNA. For comparison, serum without added protein or DNA was also used.

[0100] <<Preparation of anti-HER2 antibody-modified magnetic particles>> Anti-HER2 antibody-modified magnetic particles were prepared by binding anti-HER2 antibody (BAF1129, R&D Systems) to magnetic particles (Magnosphere MS300 / Carboxyl, StreptAvidin, MBL).

[0101] <Preparation of nucleic acid-labeled anti-HER2 antibody> A DNA fragment for detection (the base sequence of which is shown in SEQ ID NO: 2) was bound to an anti-HER2 antibody (Mab1129, R&D Systems) to prepare a nucleic acid-labeled anti-HER2 antibody. The DNA fragment was bound using a commercially available kit (trade name "oYo-Link （Ｒ） Oligo Custom (ssDNA), manufactured by Alfathera, was used.

[0102] <<Separation of Proteins and DNA in a Sample>> Anti-HER2 antibody-modified magnetic particles and magnetic particles for nucleic acid isolation (SPRI magnetic particles, MiniMax cfDNA isolation, Beckman Coulter) were added to the sample and mixed. As a result, HER2 protein in the sample was captured by the anti-HER2 antibody-modified magnetic particles. Furthermore, DNA (ErbB2 gene fragment) in the sample was captured by the SPRI magnetic particles. Subsequently, the anti-HER2 antibody-modified magnetic particles and SPRI magnetic particles were collected using a magnet stand (Takara Bio Inc.).

[0103] <<Reaction of nucleic acid-labeled anti-HER2 antibody>> Next, the recovered anti-HER2 antibody-modified magnetic particles and SPRI magnetic particles were reacted with a nucleic acid-labeled anti-HER2 antibody. As a result, the nucleic acid-labeled anti-HER2 antibody bound to the HER2 protein captured by the anti-HER2 antibody-modified magnetic particles. Next, each magnetic particle was washed and recovered using a magnet stand (Takara Bio Inc.).

[0104] <<Suspension with Reaction Solution>> Each recovered magnetic particle was suspended in a reaction solution having the composition shown in Table 1 below to prepare a reaction mixture. This reaction solution has the effect of eluting the nucleic acid captured from the SPRI magnetic particles, and can detect the nucleic acid (SEQ ID NO: 2) and ErbB2 gene fragment (SEQ ID NO: 1) bound to the nucleic acid-labeled anti-HER2 antibody by ICA reaction. In Table 1, the allele probe and ICA oligo were obtained from FASMAC Corporation. The FRET cassette was obtained from Nippon Bioservice Co., Ltd. TKO-FEN was obtained from Nippon Gene Co., Ltd.

[0105]

[0106] <<Feeding of Reaction Mixture>> The reaction mixture was fed into a fluidic device having a well array and introduced into each well. Subsequently, FC-40 (Sigma-Aldrich) was fed as a sealing liquid to seal each well. This resulted in sealing one or less HER2 protein or ErbB2 gene fragment per well.

[0107] <<ICA>> The above fluidic device was placed on a hot plate (Taitec Co., Ltd.) and reacted at 66°C for 25 minutes. This resulted in recognition of the ErbB2 gene fragment by the ErbB2 detection allele probe and ErbB2 detection ICA oligo, cleavage of the allele probe by TKO-FEN, binding of the released allele probe fragment to the ErbB2 detection FRET cassette, cleavage of the ErbB2 detection FRET by TKO-FEN, and generation of a fluorescent signal from Redmond Red. Similarly, recognition of the HER2 gene fragment by the HER2 detection allele probe and HER2 detection ICA oligo, cleavage of the allele probe by TKO-FEN, binding of the released allele probe fragment to the HER2 detection FRET cassette, cleavage of the HER2 detection FRET by TKO-FEN, and generation of a fluorescent signal from Alexa488 occurred.

[0108] Bright-field and Fluorescence Observation of Wells After heating at 66°C for 25 minutes, bright-field and fluorescence images of each well in the fluidic device were taken using a 10x objective lens on a BZ-710 fluorescence microscope (KEYENCE). Figure 8 shows the microscopic images taken. The top row shows the results for serum to which no protein or DNA was added (negative control), and the bottom row shows the results for serum to which protein and DNA was added. The left image is a bright-field image, the center image is an image detecting Alexa488 fluorescence, which indicates the presence of HER2 protein, and the right image is an image detecting Redmond Red fluorescence, which indicates the presence of ErbB2 gene fragments.

[0109] As a result, the number of Alexa 488-positive wells present within the field of view in the negative control was 89, whereas the number of Alexa 488-positive wells present within the field of view in the serum to which protein and DNA had been added was 659. Furthermore, the number of Redmond Red-positive wells present within the field of view in the negative control was 4, whereas the number of Redmond Red-positive wells present within the field of view in the serum to which protein and DNA had been added was 550. These results demonstrate that this experimental example enabled the detection of protein and DNA in serum on the same device.

[0110] [Example 2] (Study of reaction solution) The composition of a reaction solution that has the function of eluting nucleic acids captured from SPRI magnetic particles and also performs an ICA reaction was studied. Reaction solutions with various compositions shown in Table 2 below were prepared. The pH in Table 2 is the value measured at 25°C. 144 types of reaction solutions were prepared. The experiment was performed with n=2.

[0111]

[0112] Each reaction solution was dispensed into a 96-well plate at 20 μL / well, and the ICA reaction was measured using a LightCycler LC480 (Roche). The temperature of the LightCycler was kept constant at 65°C.

[0113] Subsequently, the reaction solution was evaluated according to the following evaluation criteria to see whether it maintained the function of detecting DNA by ICA reaction.

[0114] (Evaluation criteria) Signal brightness 400 or more Noise brightness 250 or less Signal:Noise ratio (S / N) 2 or more

[0115] As a result, all of the reaction solutions tested met the evaluation criteria. These results demonstrate that a reaction solution with a magnesium ion concentration of 5 to 25 mM, an EDTA concentration of 0.01 to 0.05 mM, a trishydroxymethylaminomethane concentration of 10 to 50 mM, and a pH range of 7.5 to 8.5 has the effect of eluting nucleic acids captured from SPRI magnetic particles and is also capable of carrying out an ICA reaction.

[0116] In another aspect, the present invention includes the following:

[14] A method for detecting a target molecule, comprising: binding multiple types of target molecules in a sample to a target molecule capture carrier; separating the target molecule capture carriers bound to the target molecules from the sample; suspending the separated target molecule capture carriers in a reaction solution; introducing the reaction solution into wells of a well array; sealing the wells with a sealing solution; pretreating the multiple types of target molecules in the wells; and detecting each of the multiple types of target molecules in the wells by invasive cleavage assay, wherein the reaction solution is adjusted so as not to inhibit the pretreatment and the invasive cleavage assay.

[15] The method for detecting a target molecule according to

[14] , wherein the target molecule capture carriers are magnetic particles and the separation is performed by magnetism.

[16] The method for detecting a target molecule according to

[14] or

[15] , wherein the target molecule capture carrier comprises solid-phase reversible immobilization particles.

[17] The method for detecting a target molecule according to any one of

[14] to

[16] , wherein the volume of the well is 10 fL to 100 pL.

[18] The method for detecting a target molecule according to any one of

[14] to

[17] , wherein the reaction solution comprises 5 to 25 mM magnesium ions relative to the total volume of the reaction solution.

[19] The method for detecting a target molecule according to any one of claims

[14] to

[18] , wherein the reaction solution comprises 0.01 to 0.05 mM ethylenediaminetetraacetic acid relative to the total volume of the reaction solution.

[20] The method for detecting a target molecule according to any one of

[14] to

[19] , wherein the reaction solution comprises 10 to 50 mM trishydroxymethylaminomethane relative to the total volume of the reaction solution.

[21] The method for detecting a target molecule according to any one of

[14] to

[20] , wherein the pH of the reaction solution is 7.5 to 8.5 at 25°C.

[22] The method for detecting a target molecule according to any one of

[14] to

[121] , wherein the target molecule capture carrier comprises a first target molecule capture carrier and a second target molecule capture carrier, the multiple types of target molecules comprise a first target molecule and a second target molecule, the first target molecule binds to the first target molecule capture carrier, and the second target molecule binds to the second target molecule capture carrier.

[23] The method for detecting a target molecule according to any one of

[14] to

[22] , wherein the multiple types of target molecules comprise a first target molecule and a second target molecule, and the target molecule capture carrier binds to the first target molecule and the second target molecule.

[24] The method according to any one of

[14] to

[23] , wherein the multiple types of target molecules comprise proteins.

[25] The method according to any one of

[14] to

[24] , wherein the sample contains cells, and further comprises a step of lysing the cells prior to the separation step (a).

[26] The method according to any one of

[14] to

[25] , wherein the multiple types of target molecules include HER2 protein and ErbB2 gene.

[0117] According to the present invention, it is possible to provide a technique for detecting multiple types of target molecules on the same device.

[0118] 141, 171...single-stranded nucleic acid fragment, 150...double-stranded nucleic acid (double-stranded nucleic acid region), 200, 500...fluidic device, 210...substrate, 220...lid member, 221...protrusion, 222...inlet port, 223...outlet port, 230...flow path, 241, 242, 242R...well (microcompartment), 240...well array, 510...wall member, 810...allele probe, 811, 821...flap region (nucleic acid fragment), 820...nucleic acid fragment.

Claims

1. A method for detecting multiple types of target molecules in a sample on the same well array, comprising: (a) a step of binding the multiple types of target molecules to target molecule capture carriers and separating the target molecule capture carriers to which the target molecules are bound from the sample; (b) a step of suspending the separated target molecule capture carriers in a reaction solution and introducing the resulting suspension into wells of the well array; (c) a step of sealing the wells with a sealing solution; (d) a step of pretreating the multiple types of target molecules in the wells; and (e) a step of detecting each of the multiple types of target molecules in the wells by Invasive Cleavage Assay (ICA), wherein the multiple types of target molecules include at least nucleic acids, the pretreatment includes dissociating the nucleic acids from the target molecule capture carriers or transcribing the nucleic acids into complementary strands, and the reaction solution is designed to allow the pretreatment and the ICA to be carried out without mutual inhibition.

2. The method according to claim 1, wherein the target molecule capture carrier is a magnetic particle, and the separation is performed magnetically.

3. The method of claim 1 or 2, wherein the target molecule capture carrier comprises a solid-phase reversible immobilization (SPRI) particle.

4. The method of claim 1 or 2, wherein the volume of the well is between 10 fL and 100 pL.

5. The method according to claim 1 or 2, wherein the reaction solution contains 5 to 25 mM magnesium ions.

6. The method according to claim 1 or 2, wherein the reaction solution contains 0.01 to 0.05 mM ethylenediaminetetraacetic acid.

7. The method according to claim 1 or 2, wherein the reaction solution contains 10 to 50 mM trishydroxymethylaminomethane.

8. The method according to claim 1 or 2, wherein the pH of the reaction solution at 25°C is 7.5 to 8.

5.

9. The method according to claim 1 or 2, wherein the target molecule capture carrier includes a first target molecule capture carrier and a second target molecule capture carrier, the multiple types of target molecules include a first target molecule and a second target molecule, the first target molecule binds to the first target molecule capture carrier, and the second target molecule binds to the second target molecule capture carrier.

10. The method of claim 1 or 2, wherein the plurality of target molecules includes proteins.

11. The method of claim 1 or 2, wherein the sample contains cells, and further comprising the step of lysing the cells prior to the separating step (a).

12. The method of claim 1 or 2, wherein the multiple types of target molecules include HER2 protein and ErbB2 gene.

13. A method for detecting multiple types of target molecules in a sample on the same well array, comprising: (a) a step of binding the multiple types of target molecules to target molecule capture carriers and separating the target molecule capture carriers to which the target molecules are bound from the sample; (b) a step of suspending the separated target molecule capture carriers in a reaction solution and introducing the resulting suspension into wells of the well array; (c) a step of sealing the wells with a sealing solution; (d) a step of pretreating the multiple types of target molecules in the wells; and (e) a step of detecting each of the multiple types of target molecules in the wells by Invasive Cleavage Assay (ICA), wherein the multiple types of target molecules include at least nucleic acids, and the pretreatment includes dissociating the nucleic acids from the target molecule capture carriers or transcribing the nucleic acids into complementary strands, The reaction solution contains 5 to 25 mM magnesium ions, 0.01 to 0.05 mM ethylenediaminetetraacetic acid, and 10 to 50 mM trishydroxymethylaminomethane, the pH of the reaction solution at 25°C is 7.5 to 8.5, and the target molecules are HER2 protein and ErbB2 gene.

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