Surface marker analysis method and surface marker analysis system

Abstract

To provide a surface marker analysis method and a surface marker analysis system that enable analysis of surface markers on vesicles.SOLUTION: According to an embodiment, a surface marker analysis method includes first to fifth steps. In the first step, a target vesicle is captured using a base plate. In the second step, a surface marker probe is bound to a surface marker of the target vesicle captured by the base plate. In the third step, a cluster plate is arranged at a site opposite the base plate, and a portion of the surface marker probe is captured by the cluster plate. In the fourth step, the portion of the surface marker probe captured by the cluster plate is amplified on the cluster plate. In the fifth step, the amplified portion of the surface marker probe is detected, and the surface marker is analyzed based on the amplified product.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] FIELD OF THE INVENTION An embodiment of the present invention relates to a surface marker analysis method and a surface marker analysis system. [Background technology]

[0002] Extracellular vesicles (EVVs) have attracted attention as biomarkers for use in early detection of diseases and recurrence monitoring. There is a need to analyze surface markers of vesicles such as EVVs. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-144722 Summary of the Invention [Problem to be solved by the invention]

[0004] An embodiment of the present invention aims to provide a surface marker analysis method and a surface marker analysis system capable of analyzing surface markers of vesicles. [Means for solving the problem]

[0005] According to an embodiment of the present invention, a surface marker analysis method includes a first step, a second step, a third step, a fourth step, and a fifth step. In the first step, target vesicles are captured by a base plate. In the second step, surface marker probes are bound to the surface markers of the target vesicles captured by the base plate. In the third step, a cluster plate is placed opposite the base plate, and a portion of the surface marker probes is captured by the cluster plate. In the fourth step, the portion captured by the cluster plate is amplified in the cluster plate. In the fifth step, an amplified product of the portion is detected, and the surface marker is analyzed based on the amplified product.

[0006] According to an embodiment of the present invention, a surface marker analysis system includes a base plate, surface marker probes, a cluster plate, a detection unit, and a control unit. The base plate is capable of capturing target vesicles. The surface marker probes are capable of binding to surface markers of the target vesicles. The cluster plate is positioned opposite the base plate. The cluster plate is capable of capturing and amplifying a portion of the surface marker probes. The detection unit detects the amplified product of the portion. The control unit analyzes the surface markers based on the amplified product. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a flowchart showing an example of a surface marker analysis method according to an embodiment. [Figure 2] FIG. 2 is an explanatory diagram showing the first step of the surface marker analysis method according to the embodiment. [Figure 3] FIG. 10 is an explanatory diagram showing a second step of the surface marker analysis method according to the embodiment. [Figure 4] FIG. 2 is an explanatory diagram showing the surface state of a target vesicle in the second step of the surface marker analysis method according to the embodiment. [Figure 5] FIG. 10 is an explanatory diagram showing a third step of the surface marker analysis method according to the embodiment. [Figure 6] FIG. 10 is an explanatory diagram showing the surface state of a target vesicle in the third step of the surface marker analysis method according to the embodiment. [Figure 7] 7(a) and 7(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 8] 8(a) and 8(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 9] 9(a) and 9(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 10]10(a) and 10(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 11] 11(a) and 11(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 12] 12(a) and 12(b) are explanatory views showing the fourth step of the surface marker analysis method according to the embodiment. [Figure 13] FIG. 10 is an explanatory diagram showing the fifth step of the surface marker analysis method according to the embodiment. [Figure 14] FIG. 10 is an explanatory diagram showing a sixth step of the surface marker analysis method according to the embodiment. [Figure 15] FIG. 1 is an explanatory diagram illustrating a surface marker analysis system according to an embodiment. [Figure 16] 16(a) and 16(b) are explanatory diagrams showing a base substrate and a cluster substrate of a surface marker analysis system according to an embodiment. [Figure 17] FIG. 1 is an explanatory diagram illustrating a surface marker analysis system according to an embodiment. [Figure 18] 18(a) and 18(b) are explanatory diagrams showing an example of a spacer arm of the surface marker analysis system according to the embodiment. [Figure 19] 19(a) to 19(c) are explanatory diagrams showing alignment in the third step of the surface marker analysis method according to the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are schematic or conceptual, and the relationship between the thickness and width of each part, the size ratio between parts, etc. are not necessarily the same as those in reality. Even when the same part is shown, the dimensions and ratios may be different depending on the drawing. In this specification and in each drawing, elements similar to those previously described with reference to the previous drawings are designated by the same reference numerals, and detailed descriptions thereof will be omitted where appropriate.

[0009] (Surface marker analysis method) FIG. 1 is a flowchart showing an example of a surface marker analysis method according to an embodiment. FIG. 2 is an explanatory diagram showing the first step of the surface marker analysis method according to the embodiment. FIG. 3 is an explanatory diagram showing the second step of the surface marker analysis method according to the embodiment. FIG. 4 is an explanatory diagram showing the surface state of a target vesicle in the second step of the surface marker analysis method according to the embodiment. FIG. 5 is an explanatory diagram showing the third step of the surface marker analysis method according to the embodiment. FIG. 6 is an explanatory diagram showing the surface state of a target vesicle in the third step of the surface marker analysis method according to the embodiment. Figures 7(a), 7(b), 8(a), 8(b), 9(a), 9(b), 10(a), 10(b), 11(a), 11(b), 12(a), and 12(b) are explanatory diagrams showing the fourth step of the surface marker analysis method according to the embodiment. FIG. 13 is an explanatory diagram showing the fifth step of the surface marker analysis method according to the embodiment. FIG. 14 is an explanatory diagram showing the sixth step of the surface marker analysis method according to the embodiment. 19(a) to 19(c) are explanatory diagrams showing alignment in the third step of the surface marker analysis method according to the embodiment.

[0010] The surface marker analysis method according to the embodiment is a method for analyzing the presence or absence of expression and the expression level of a surface marker 3 located on the surface of a target vesicle 1. The target vesicle 1 is, for example, a virus or an extracellular vesicle. The target vesicle 1 is preferably, for example, an extracellular vesicle. The target vesicle 1 has, for example, a lipid bilayer membrane 2 that covers the encapsulated material, and a surface marker 3 located on the surface of the lipid bilayer membrane 2. The surface marker 3, for example, penetrates the lipid bilayer membrane 2, or pierces and adheres to the lipid bilayer membrane 2. The surface marker 3 includes, for example, at least one of a sugar chain, a protein (polypeptide), and a nucleic acid.

[0011] As shown in FIG. 1, the surface marker analysis method according to the embodiment includes a first step, a second step, a third step, a fourth step, and a fifth step.

[0012] In the first step, target vesicles 1 are captured by a base plate 10. In the first step, for example, a sample liquid 5 containing target vesicles 1 is applied to the base plate 10, thereby capturing the target vesicles 1 by the base plate 10. As shown in FIG. 2, the base plate 10 includes, for example, a base substrate 11 and a vesicle capture probe 12. The base substrate 11 is, for example, a plate-shaped member. The vesicle capture probe 12 is immobilized on the base substrate 11. For example, multiple vesicle capture probes 12 are provided on one side of the base substrate 11. The vesicle capture probe 12 is capable of capturing the target vesicles 1. The vesicle capture probe 12 is, for example, annexin V. In the first step, for example, the target vesicles 1 are captured by the vesicle capture probe 12 on the base plate 10.

[0013] The base substrate 11 includes, for example, a first substrate 11a and a plurality of first electrodes 11b. The plurality of first electrodes 11b are arranged on the surface of the first substrate 11a so as to be spaced apart from one another (see FIG. 16(a)). The vesicle capture probes 12 are immobilized on the surface of each of the plurality of first electrodes 11b.

[0014] The plurality of first electrodes 11b each contain, for example, any of gold, silver, and copper. The vesicle capture probe 12 has, for example, a sulfur atom and is immobilized on the surface of each of the plurality of first electrodes 11b via the sulfur atom. The sulfur atom is, for example, part of a thiol group, a sulfide bond, a disulfide bond, etc. In other words, immobilization via a sulfur atom means immobilization via a thiol group, a sulfide bond, a disulfide bond, etc. The sulfur atom may be, for example, a thiol group from which a hydrogen atom has been removed (dehydrogenated). Hereinafter, the sulfur atom when bonding via a sulfur atom may be the same as described above.

[0015] In the second step, a surface marker probe 20 is bound to the surface marker 3 of the target vesicle 1 captured by the base plate 10. The second step is performed after the first step. As shown in Figures 3 and 4, the surface marker probe 20 includes, for example, a first portion 21 and a second portion 22. The first portion 21 is capable of binding to the surface marker 3. The second portion 22 is capable of binding to the first portion 21. In the second step, for example, the first portion 21 of the surface marker probe 20, to which the first portion 21 and the second portion 22 are bound, is bound to the surface marker 3 of the target vesicle 1 captured by the vesicle capture probe 12.

[0016] The first portion 21 includes, for example, a surface marker-binding portion 21a and a first binding portion 21b. The surface marker-binding portion 21a is capable of specifically binding to a surface marker 3. The surface marker-binding portion 21a includes, for example, at least one of a nucleic acid, a protein (polypeptide), and a sugar chain. The first binding portion 21b includes a first base sequence 21b1. In the example of FIG. 4, the first binding portion 21b further includes a connecting sequence 21b2 located between the surface marker-binding portion 21a and the first base sequence 21b1. The connecting sequence 21b2 is optional.

[0017] The second portion 22 includes, for example, a second binding portion 22a and an amplification portion 22b. The second binding portion 22a includes a second base sequence 22a1. The second base sequence 22a1 is a sequence complementary to the first base sequence 21b1. The second base sequence 22a1 forms a complementary strand with the first base sequence 21b1, thereby binding the second binding portion 22a (second portion 22) to the first binding portion 21b (first portion 21). In the example of FIG. 4, the second binding portion 22a further includes a connecting sequence 22a2 located between the second base sequence 22a1 and the amplification portion 22b. The connecting sequence 22a2 is optional. The amplification portion 22b includes a third base sequence 22b1, a fourth base sequence 22b2, and a fifth base sequence 22b3. The third base sequence 22b1 is a sequence complementary to the base sequence (sixth base sequence 33a) of the first primer 33 described below. The fourth base sequence 22b2 is a sequence for identifying the surface marker 3. In other words, the fourth base sequence 22b2 is a sequence that differs for each surface marker binding portion 21a. Because the surface marker binding portion 21a specifically binds to the surface marker 3, the fourth base sequence 22b2 is a sequence that differs for each surface marker 3. The fourth base sequence 22b2 is a so-called encoding sequence. The fifth base sequence 22b3 is the same sequence as the base sequence of the second primer 34 described below. In the second step, for example, the surface marker binding portion 21a of the first portion 21 of the surface marker probe 20 is bound to the surface marker 3 of the target vesicle 1 captured by the vesicle capture probe 12.

[0018] In the third step, the cluster plate 30 is positioned opposite the base plate 10, and a portion of the surface marker probe 20 is captured by the cluster plate 30. This step is performed after the second step. As shown in FIGS. 5 and 6, the cluster plate 30 includes, for example, a cluster substrate 31, a spacer arm 32, a first primer 33, and a second primer 34. The spacer arm 32 is capable of capturing the second portion 22 of the surface marker probe 20. The length of the spacer arm 32 is, for example, 100 nm or more. The length of the spacer arm 32 is preferably, for example, approximately 300 nm. The first primer 33 and the second primer 34 are capable of amplifying the second portion 22 (amplification portion 22b) of the surface marker probe 20. The spacer arm 32, the first primer 33, and the second primer 34 are each immobilized on the cluster substrate 31. In the third step, the second portion 22 of the surface marker probe 20 is captured by, for example, the spacer arm 32 of the cluster plate 30 .

[0019] The cluster substrate 31 includes a second substrate 31a and a plurality of second electrodes 31b. The plurality of second electrodes 31b are arranged on the surface of the second substrate 31a so as to be spaced apart from one another (see FIG. 16(b)). A spacer arm 32, a first primer 33, and a second primer 34 are immobilized on the surface of each of the plurality of second electrodes 31b. The distance between each of the plurality of second electrodes 31b is preferably longer than the length of the spacer arm 32. The distance between each of the plurality of second electrodes 31b is preferably about 300 nm.

[0020] Each of the second electrodes 31b includes, for example, gold, silver, or copper. The spacer arm 32, the first primer 33, and the second primer 34 each have, for example, a sulfur atom and are immobilized on the surface of each of the second electrodes 31b via the sulfur atom.

[0021] The spacer arm 32 includes a base 32a and a connecting portion 32b. The base 32a is immobilized on the cluster substrate 31 (the plurality of second electrodes 31b). The base 32a preferably includes, for example, polyethylene glycol (PEG). The base 32a may include a nucleic acid such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). The connecting portion 32b is capable of binding to the second base sequence 22a1. The connecting portion 32b preferably includes a peptide nucleic acid (PNA). The connecting portion 32b may include a nucleic acid such as DNA or RNA. The connecting portion 32b has a base sequence complementary to the second base sequence 22a1. In the third step, for example, the connecting portion 32b of the spacer arm 32 undergoes strand exchange with the first base sequence 21b1 bound to the second base sequence 22a1, thereby capturing the second portion 22 of the surface marker probe 20. Here, if the connector 32b of the spacer arm 32 is PNA and the second base sequence 22a1 and the first base sequence 21b1 are DNA (or RNA), the binding strength of a PNA-DNA (or PNA-RNA) double strand is stronger than the binding strength of a DNA (or RNA) double strand, and the strand exchange described above proceeds easily. Furthermore, a DNA (or RNA) double strand is more susceptible to the influence of salt concentration than a PNA-DNA (or PNA-RNA) double strand. At low salt concentrations, the binding strength of a DNA (or RNA) double strand decreases, whereas the binding strength of a PNA-DNA (or PNA-RNA) double strand does not decrease even at low salt concentrations. Therefore, the strand exchange described above proceeds more easily if the salt concentration is reduced by diluting the sample solution 5.

[0022] In the third step, when the cluster plate 30 is positioned opposite the base plate 10, it is preferable to use an alignment mark (alignment mark 11c described below) on the base substrate 11 and an alignment mark (alignment mark 31c described below) on the cluster substrate 31 to horizontally position the cluster plate 30 relative to the base plate 10. Furthermore, when positioning the cluster plate 30, it is desirable to use a device equipped with a high-precision alignment mechanism. For example, it is known that a hybrid bonding device used for stacking semiconductor wafers can achieve a horizontal positioning error of 150 nm or less. When positioning the cluster plate 30, it is desirable to use a device with alignment precision comparable to that of the present device.

[0023] In the third step, the sample liquid 5 is applied to the base plate 10. Therefore, if the alignment mark is read through the free surface of the sample liquid 5 as shown in FIG. 19(a), the correct position may not be detected due to refraction of light. Therefore, it is desirable to bring the cluster plate 30 close to the alignment mark and read the alignment mark while the sample liquid 5 is in contact with the cluster plate 30, as shown in FIG. 19(b). This method can be used when at least one of the base plate 10 and the cluster plate 30 is transparent. Alternatively, it is desirable to read the alignment mark after the sample liquid 5 is applied to the base plate 10, leaving the portion where the alignment mark is formed, as shown in FIG. 19(c). This method can be used when alignment is performed by inserting a camera with two fields of view, one above and one below, between the base plate 10 and the cluster plate 30.

[0024] When the cluster plate 30 is positioned horizontally relative to the base plate 10 using the alignment marks, it is preferable that the centers of the multiple second electrodes 31b of the cluster substrate 31 each overlap in the vertical direction with the centers of the multiple first electrodes 11b of the base substrate 11.

[0025] Furthermore, in the third step, when the cluster plate 30 is positioned opposite the base plate 10, it is preferable to position the cluster plate 30 in the vertical direction relative to the base plate 10 using multiple protrusions (multiple protrusions 15 described below) provided on at least one of the base substrate 11 and the cluster substrate 31.

[0026] In the fourth step, a portion of the surface marker probes 20 captured by the cluster plate 30 is amplified on the cluster plate 30. This step is performed after the cluster plate 30 is separated from the base plate 10 following the third step. In this step, the surface of the cluster plate 30 is coated with an aqueous solution having a salt concentration (100 mM or higher) similar to that of physiological saline. In this step, the amplified portion 22b of the second portion 22 of the surface marker probe 20 captured on the spacer arm 32 is amplified using, for example, a first primer 33 and a second primer 34. The first primer 33 includes a sixth base sequence 33a complementary to the third base sequence 22b1. The second primer 34 includes a fifth base sequence 22b3. In this step, the second portion 22 (amplified portion 22b) is amplified by bridge PCR (polymerase chain reaction) using, for example, the first primer 33 and the second primer 34.

[0027] Below, with reference to Figures 7(a), 7(b), 8(a), 8(b), 9(a), 9(b), 10(a), 10(b), 11(a), 11(b), 12(a), and 12(b), we will explain a method for amplifying the second portion 22 in the cluster plate 30 by bridge PCR using the first primer 33 and the second primer 34.

[0028] As shown in FIG. 7(a), the connecting portion 32b of the spacer arm 32 binds to the second base sequence 22a1 of the second portion 22 of the surface marker probe 20, thereby capturing the second portion 22b. Here, for example, a salt concentration of 100 mM or more, similar to that of physiological saline, is set, and polymerase and nucleotide monomers are added to perform one cycle of PCR amplification reaction. First, when the temperature is lowered to approximately 60°C or less, as shown in FIG. 7(b), the third base sequence 22b1 of the amplified portion 22b of the second portion 22 forms a complementary strand with the first primer 33 (sixth base sequence 33a). Further, when the temperature is raised to approximately 73°C, as shown in FIG. 8(a), a first complementary strand 25 is formed, which includes the first primer 33, a seventh base sequence 25a complementary to the fourth base sequence 22b2, and an eighth base sequence 25b complementary to the fifth base sequence 22b3. Immediately after replication, the first complementary strand 25 forms a double strand with the second portion 22 (amplified portion 22b). Next, when the temperature is raised to about 95°C, the double strand is dissolved, and the first complementary strand 25 and the second portion 22 (amplified portion 22b) are dissociated, as shown in Figure 8(b).

[0029] As shown in Figure 9(a), when the base 32a of the spacer arm 32 is cleaved with a restriction enzyme or the like that cleaves in a sequence-specific manner and then washed, the spacer arm 32 is removed along with the second portion 22. Here, since a restriction enzyme cleaves double-stranded nucleic acids in a sequence-specific manner, when a restriction enzyme is used, the site to be cleaved in the base 32a of the spacer arm 32 is made into a nucleic acid. Then, nucleic acid having a sequence complementary to the sequence at the site to be cleaved is added, and the temperature is lowered to approximately 60°C or below, forming a double-stranded strand at the site to be cleaved, and cleavage with the restriction enzyme is then performed. Alternatively, the site to be cleaved in the base 32a of the spacer arm 32 may be made into a peptide and cleaved using a protease.

[0030] Next, PCR amplification reactions are repeated multiple times. First, a high-salt aqueous solution containing polymerase and nucleotide monomers is used. The temperature is maintained at approximately 60°C or below. As shown in FIG. 9(b), the eighth base sequence 25b of the first complementary strand 25 forms a complementary strand with the second primer 34 (the fifth base sequence 22b3). Furthermore, as shown in FIG. 10(a), the second complementary strand 26 is formed. The second complementary strand 26 includes the second primer 34, the ninth base sequence 26a complementary to the seventh base sequence 25a, and the tenth base sequence 26b complementary to the sixth base sequence 33a. The ninth base sequence 26a and the tenth base sequence 26b are identical to the fourth base sequence 22b2 and the third base sequence 22b1, respectively. The second complementary strand 26 immediately after replication forms a double strand with the first complementary strand 25. Next, when the temperature is raised to about 95° C., the double strand is dissolved, and the first complementary strand 25 and the second complementary strand 26 are dissociated, as shown in FIG. 10(b).

[0031] Next, when the temperature is lowered to approximately 60°C or below, the eighth base sequence 25b of the first complementary strand 25 forms a complementary strand with the second primer 34 (the fifth base sequence 22b3), as shown in Figure 11(a). Furthermore, the tenth base sequence 26b of the second complementary strand 26 forms a complementary strand with the first primer 33 (the sixth base sequence 33a). When the temperature is further raised to approximately 73°C, the first complementary strand 25 and the second complementary strand 26 are formed, as shown in Figure 11(b). By repeating this process, the first complementary strand 25 and the second complementary strand 26 can be amplified, as shown in Figure 12(a). When the second complementary strand 26 is cleaved with a restriction enzyme or the like and washed, the second complementary strand 26 is removed, leaving the first complementary strand 25, as shown in Figure 12(b). Alternatively, the first complementary strand 25 may be removed, leaving the second complementary strand 26.

[0032] In this way, bridge PCR using the first primer 33 and the second primer 34 can amplify the first complementary strand 25, which is the complementary strand of the second portion 22, and the second complementary strand 26, which is the complementary strand of the first complementary strand 25, on the cluster plate 30.

[0033] In the fifth step, a partial amplification product of the surface marker probe 20 is detected, and the surface marker 3 is analyzed based on the amplification product. This step is performed after the fourth step. In this step, a partial amplification product of the surface marker probe 20 is first detected. As shown in FIG. 13, the amplification product is detected, for example, by a detection unit 60. The detection unit 60 is, for example, a fluorescent camera. A fluorescent probe containing a sequence complementary to the first complementary strand 25 is bound to the first complementary strand 25, and the excess fluorescent probe is washed away. Then, the fluorescence is detected by a fluorescent camera. Here, the first complementary strand 25 is detected with strong fluorescence intensity because it has been sufficiently amplified by PCR. Furthermore, the first complementary strand 25 corresponds to a probe that specifically binds to the surface marker. Therefore, the detection of fluorescence by the fluorescent probe containing a sequence complementary to the first complementary strand 25 indicates that the target vesicle 1 located opposite the site expressed the surface marker corresponding to the first complementary strand 25. The detection unit 60 outputs the detection result to the control unit 65 .

[0034] In the fifth step, the surface marker 3 is analyzed based on the amplification product of a portion of the surface marker probe 20. As shown in FIG. 13, the analysis of the surface marker 3 is performed, for example, by the control unit 65. The control unit 65 analyzes the presence or absence of expression of the surface marker 3 based on the detection result input from the detection unit 60 (i.e., the fluorescence indicating the presence of the first complementary strand 25). Because the fourth base sequence 22b2 of the surface marker probe 20 differs for each surface marker 3, the presence or absence of expression can be analyzed for each surface marker 3. This makes it possible to determine, for example, the surface markers expressed by vesicles contained in a sample on a single vesicle basis. Furthermore, it is also possible to identify target vesicles 1 expressing a desired combination of surface markers 3.

[0035] In the above explanation, the fluorescence observation in the fifth step was performed after the fourth step, but the fluorescence observation may be performed in real time during the fourth step. In that case, when proceeding with the procedures of the above-mentioned Figures 9(b), 10(a), 10(b), 11(a), 11(b), 12(a), and 12(b), SYBR (registered trademark) Green I or TaqMan TM By adding a fluorescent probe such as a probe and observing the fluorescence, it is possible to estimate the expression level of the surface marker from the PCR cycle number at which the fluorescence intensity is amplified.

[0036] The surface marker analysis method according to the embodiment may further include a sixth step. In the sixth step, target vesicles 1 are collected based on the analysis results of the surface markers 3. The sixth step is performed after the fifth step. As shown in FIG. 14, the first electrodes 11b of the base substrate 11 are connected to a voltage application circuit 55 via a switch circuit 50. By applying a voltage to the first electrodes 11b from the voltage application circuit 55, the vesicle capture probes 12 immobilized on the surfaces of the first electrodes 11b via sulfur atoms are dissociated from the first electrodes 11b. This allows the target vesicles 1 to be collected in the collection device 40 together with the vesicle capture probes 12. The sixth step can be omitted.

[0037] (Surface marker analysis system) FIG. 15 is an explanatory diagram illustrating a surface marker analysis system according to an embodiment. 16(a) and 16(b) are explanatory diagrams showing a base substrate and a cluster substrate of a surface marker analysis system according to an embodiment. FIG. 17 is an explanatory diagram illustrating a surface marker analysis system according to an embodiment.

[0038] The surface marker analysis system 100 according to the embodiment is a system that analyzes the presence or absence and expression level of a surface marker 3 located on the surface of a target vesicle 1. The surface marker analysis method according to the embodiment described above is performed, for example, by the surface marker analysis system 100 according to the embodiment.

[0039] 15, the surface marker analysis system 100 comprises a base plate 10, a surface marker probe 20, a cluster plate 30, a detection unit 60, and a control unit 65. Here, the base plate 10 and the cluster plate 30 cannot be reused and are therefore single-use replacement parts.

[0040] 2 , the base plate 10 is capable of capturing a target vesicle 1. The base plate 10 includes a base substrate 11 and a vesicle capture probe 12. The vesicle capture probe 12 is immobilized on the base substrate 11. The vesicle capture probe 12 is capable of capturing a target vesicle 1.

[0041] 2 and 16(a), the base substrate 11 includes a first substrate 11a and a plurality of first electrodes 11b. The plurality of first electrodes 11b are arranged spaced apart from one another on the surface of the first substrate 11a. The vesicle capture probes 12 are immobilized on the surface of each of the plurality of first electrodes 11b.

[0042] Each of the first electrodes 11b includes, for example, gold, silver, or copper. The vesicle capture probes 12 are immobilized on the surface of each of the first electrodes 11b via, for example, sulfur atoms.

[0043] 3 and 4, the surface marker probe 20 is capable of binding to the surface marker 3 of the target vesicle 1. The surface marker probe 20 includes a first portion 21 and a second portion 22. The first portion 21 is capable of binding to the surface marker 3. The second portion 22 is capable of binding to the first portion 21.

[0044] The first portion 21 includes a surface marker-binding portion 21a and a first binding portion 21b. The surface marker-binding portion 21a is capable of binding to the surface marker 3. The first binding portion 21b includes a first base sequence 21b1.

[0045] The second portion 22 includes a second binding portion 22a and an amplification portion 22b. The second binding portion 22a includes a second base sequence 22a1. The second base sequence 22a1 is a sequence complementary to the first base sequence 21b1. The amplification portion 22b includes a third base sequence 22b1, a fourth base sequence 22b2, and a fifth base sequence 22b3. The fourth base sequence 22b2 is a sequence for identifying the surface marker 3.

[0046] As shown in Figures 5 and 6, the cluster plate 30 is positioned opposite the base plate 10. The cluster plate 30 is capable of capturing and amplifying a portion of the surface marker probe 20. The cluster plate 30 includes a cluster substrate 31, a spacer arm 32, a first primer 33, and a second primer 34. The spacer arm 32 is capable of capturing a portion of the surface marker probe 20. The first primer 33 includes a sixth base sequence 33a complementary to the third base sequence 22b1. The second primer 34 includes a fifth base sequence 22b3. The first primer 33 and the second primer 34 are capable of amplifying the second portion 22 (amplified portion 22b) of the surface marker probe 20. The spacer arm 32, the first primer 33, and the second primer 34 are each immobilized on the cluster substrate 31.

[0047] 5, 6, and 16(b), the cluster substrate 31 includes a second substrate 31a and a plurality of second electrodes 31b. The plurality of second electrodes 31b are arranged on the surface of the second substrate 31a so as to be spaced apart from one another. A spacer arm 32, a first primer 33, and a second primer 34 are immobilized on the surface of each of the plurality of second electrodes 31b.

[0048] Each of the second electrodes 31b includes, for example, gold, silver, or copper. The spacer arm 32, the first primer 33, and the second primer 34 are immobilized on the surface of each of the second electrodes 31b via, for example, a sulfur atom.

[0049] As shown in Figure 6, the spacer arm 32 includes a base 32a and a connecting portion 32b. The base 32a is immobilized on the cluster substrate 31 (the plurality of second electrodes 31b). The base 32a preferably contains, for example, polyethylene glycol (PEG). The connecting portion 32b is capable of binding to the second base sequence 22a1. The connecting portion 32b preferably contains peptide nucleic acid (PNA). The connecting portion 32b captures the second portion 22 of the surface marker probe 20, for example, by strand exchange with the first base sequence 21b1 bound to the second base sequence 22a1.

[0050] 16(a), the distance P1 between the first electrodes 11b is preferably, for example, about 600 nm. As shown in FIGS. 16(a) and 17, the diameter R1 of the first electrodes 11b in plan view is preferably about 100 nm.

[0051] As shown in FIG. 16(b), the distance P2 between the multiple second electrodes 31b is preferably longer than the length of the spacer arm 32, for example. The length of the spacer arm 32 is, for example, 100 nm or more. The length of the spacer arm 32 is preferably, for example, about 300 nm. The distance P2 between the multiple second electrodes 31b is preferably, for example, about 300 nm. As shown in FIGS. 16(b) and 17, the diameter R2 of the multiple second electrodes 31b in a plan view is preferably about 400 nm.

[0052] As shown in Fig. 16(a), the base substrate 11 has alignment marks 11c for horizontally positioning the cluster plate 30 relative to the base plate 10. As shown in Fig. 16(b), the cluster substrate 31 has alignment marks 31c for horizontally positioning the cluster plate 30 relative to the base plate 10. When the cluster plate 30 is horizontally positioned relative to the base plate 10 by the alignment marks 11c and 31c, the centers of the multiple second electrodes 31b preferably overlap with the centers of the multiple first electrodes 11b in the up-down direction, for example. At least one of the first substrate 11a and the second substrate 31a is preferably light-transmitting.

[0053] 15, at least one of the base substrate 11 and the cluster substrate 31 has a plurality of protrusions 15 for determining the vertical position of the cluster plate 30 relative to the base plate 10. The height of the plurality of protrusions 15 is preferably, for example, about 200 nm. The plurality of protrusions 15 are preferably provided at intervals that prevent the cluster substrate 31 or the base substrate 11 from warping, for example.

[0054] The detection unit 60 detects the amplification product of a part of the surface marker probe 20. The detection unit 60 is, for example, a fluorescent camera. The detection unit 60 outputs the detection result to the control unit 65.

[0055] The control unit 65 analyzes the surface marker 3 based on the amplification product of a portion of the surface marker probe 20. The control unit 65 analyzes the presence or absence and expression level of the surface marker 3 based on the detection results input from the detection unit 60.

[0056] The surface marker analysis system 100 may further include a switch circuit 50 and a voltage application circuit 55. The voltage application circuit 55 is connected to the plurality of first electrodes 11b of the base substrate 11 via the switch circuit 50. The voltage application circuit 55 applies a voltage to the plurality of first electrodes 11b, thereby dissociating the vesicle capture probes 12 immobilized on the surfaces of the plurality of first electrodes 11b via sulfur atoms from the plurality of first electrodes 11b. This allows the target vesicles 1 captured by the vesicle capture probes 12 to be dissociated from the base plate 10. The switch circuit 50 may be incorporated into the base substrate 11. In this case, it is preferable that the base substrate 11 is made of a semiconductor such as silicon, and the switch circuit 50 is a semiconductor circuit.

[0057] The surface marker analysis system 100 may further include a collection device 40. The collection device 40 collects the target vesicles 1.

[0058] 18(a) and 18(b) are explanatory diagrams showing an example of a spacer arm of the surface marker analysis system according to the embodiment. As shown in Figures 18(a) and 18(b), the base 32a of the spacer arm 32 may be DNA capable of forming a hairpin structure. The base 32a has a first strand 32x and a second strand 32y. The second strand 32y includes a sequence complementary to the first strand 32x. As shown in Figure 18(a), when a complementary strand 35 complementary to the first strand 32x is present, the first strand 32x forms a double strand with the complementary strand 35. As shown in Figure 18(b), when the complementary strand 35 is not present, the first strand 32x forms a double strand (hairpin structure) with the second strand 32y. For example, when the spacer arm 32 captures the second portion 22 of the surface marker probe 20, a double strand is formed between the first strand 32x and the complementary strand 35, as shown in Figure 18(a). When the second portion 22 of the surface marker probe 20 is amplified using the first primer 33 and the second primer 34, a double strand (hairpin structure) is formed from the first strand 32x and the second strand 32y, as shown in Figure 18(b).

[0059] This brings the second portion 22 of the surface marker probe 20 captured by the spacer arm 32 into close proximity to the base where the spacer arm is immobilized. As a result, the product of the subsequent bridge PCR is amplified only near the point where the spacer arm 32 is immobilized. Using this method, the distance P2 between the multiple second electrodes 31b of the cluster substrate 31 shown in Figure 16(b) can be shortened regardless of the length of the spacer arm 32. The distance P1 between the multiple first electrodes 11b of the base substrate 11 shown in Figure 16(a) also becomes shorter accordingly, allowing for an increased number of multiple first electrodes 11b that can be arranged per unit area. This allows for more efficient analysis.

[0060] To change the double-stranded state of the first strand 32x and the complementary strand 35 shown in Figure 18(a) to the double-stranded state (hairpin structure) of the first strand 32x and the second strand 32y shown in Figure 18(b), it is necessary to dissociate and remove the complementary strand 35. To do this, the salt concentration is first lowered to dissociate the double-stranded state of the first strand 32x and the complementary strand 35, the released complementary strand 35 is washed, and then the salt concentration is increased again to form the double-stranded state (hairpin structure) of the first strand 32x and the second strand 32y. In this case, the second portion 22 of the surface marker probe 20 is not released because it is captured by a PNA-DNA bond whose binding strength is not reduced even at low salt concentrations.

[0061] The binding strength of the second strand 32y to the first strand 32x is preferably lower than the binding strength of the complementary strand 35 to the first strand 32x. This makes the non-hairpin structure shown in Figure 18(a) more stable than the hairpin structure shown in Figure 18(b). For example, by mismatching part of the base sequence of the second strand 32y with the base sequence of the first strand 32x, the binding strength of the second strand 32y to the first strand 32x can be reduced.

[0062] The effects of the surface marker analysis method and the surface marker analysis system 100 according to the embodiment will be described below.

[0063] Exosomes are a type of extracellular vesicle found in the body, tiny vesicles with a diameter of approximately 100 nm. Extracellular vesicles have a lipid bilayer structure containing nucleic acids and other molecules inside, and are known to move between distant organs, transmitting information and transporting substances. The lipid bilayer contains surface markers such as proteins, glycoproteins, and sugar chains, and observations have also suggested that cfDNA (cell-free DNA) is attached to the periphery. These vesicles are thought to contain information about the cells from which they were produced and the cells to which they migrate.

[0064] Extracellular vesicles have been reported to be deeply involved in cancer metastasis. They have also been reported to be involved in the transport of causative substances in non-cancer diseases, such as dementia (e.g., Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies), neurodegenerative diseases, and renal disorders. Therefore, extracellular vesicles have attracted attention as biomarkers for use in early detection of disease and recurrence monitoring. Furthermore, because much remains unknown about their structure and function, they have also attracted attention as a research subject, and the development of analytical methods for extracellular vesicle surface markers is desired.

[0065] One possible method for analyzing surface markers on extracellular vesicles is to bind a surface marker probe to the surface marker, amplify a portion of the surface marker probe by PCR, and then analyze the amplified DNA (amplicon) by real-time PCR. However, this method requires the recovery of a portion of the surface marker probe as a liquid, which requires submicron-sized microchannels and a large number of dispensing containers, resulting in increased costs.

[0066] In contrast, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, a portion of the surface marker probes 20 is captured by the cluster plate 30, and the portion of the surface marker probes 20 captured by the cluster plate 30 is amplified in the cluster plate 30. This allows a portion of the surface marker probes 20 to be amplified without forming a submicron-sized microchannel. Furthermore, because the expression state of the surface marker 3 can be analyzed at the single vesicle level, desired fractions can be collected based on the expression state of the surface marker 3 and sent for downstream analysis. Furthermore, because a portion of the surface marker probes 20 is amplified and detected, surface markers 3 with low expression levels can be detected with high sensitivity.

[0067] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, target vesicles 1 are captured by vesicle capture probes 12 on a base plate 10, a first portion 21 of a surface marker probe 20 is bound to a surface marker 3, and a second portion 22 of the surface marker probe 20 is captured and amplified by spacer arms 32 on a cluster plate 30. This makes it easier to capture the second portion 22 of the surface marker probe 20 on the cluster plate 30 and amplify it in the cluster plate 30.

[0068] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, the connecting portion 32b of the spacer arm 32 captures the second portion 22 (second base sequence 22a1) of the surface marker probe 20 by strand exchange with the first portion 21 (first base sequence 21b1) bound to the second portion 22. This makes it easier to capture the second portion 22 of the surface marker probe 20 on the cluster plate 30 and amplify it on the cluster plate 30.

[0069] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, the connector 32b of the spacer arm 32 contains a peptide nucleic acid. The bond between a peptide nucleic acid and a nucleic acid is more stable than the bond between a nucleic acid and a nucleic acid. Therefore, when the connector 32b of the spacer arm 32 contains a peptide nucleic acid, the bond between the second portion 22 (second base sequence 22a1) and the first portion 21 (first base sequence 21b1) of the surface marker probe 20 is more likely to undergo strand exchange with the bond between the connector 32b of the spacer arm 32 and the second portion 22 (second base sequence 22a1). Furthermore, strand exchange is more likely to occur without temperature cycling or changes in salt concentration. This reduces damage to the target vesicle 1 caused by the strand exchange process.

[0070] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, the cluster plate 30 is positioned horizontally relative to the base plate 10 using an alignment mark 11c provided on the base substrate 11 and an alignment mark 31c provided on the cluster substrate 31. This prevents misalignment of the horizontal positions of the first electrode 11b of the base plate 10 (base substrate 11) and the second electrode 31b of the cluster plate 30 (cluster substrate 31). This prevents unintended amplification of the surface marker 3 of a target vesicle 1 captured at an adjacent position. Furthermore, the cluster plate 30 is positioned vertically relative to the base plate 10 using multiple protrusions provided on at least one of the base substrate 11 and the cluster substrate 31. This prevents the gap between the base plate 10 and the cluster plate 30 from becoming too wide or too narrow. This makes it easier for the cluster plate 30 to capture the second portion 22 of the surface marker probe 20.

[0071] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, each of the multiple first electrodes 11b of the base substrate 11 contains one of gold, silver, and copper. The vesicle capture probe 12 is immobilized on the surface of each of the multiple first electrodes 11b via sulfur atoms. This eliminates the need for fine printing of the solution containing the vesicle capture probe 12, simplifying the immobilization process. Meanwhile, the multiple first electrodes 11b can be easily patterned using a semiconductor process. Furthermore, by applying a voltage to a first electrode at a desired position among the multiple first electrodes 11b, the vesicle capture probe 12 at the desired position can be easily dissociated from the multiple first electrodes 11b.

[0072] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, each of the multiple second electrodes 31b of the cluster substrate 31 contains any one of gold, silver, and copper. The spacer arm 32, the first primer 33, and the second primer 34 are immobilized on the surface of each of the multiple second electrodes 31b via sulfur atoms. This allows the spacer arm 32, the first primer 33, and the second primer 34 to be easily immobilized on the multiple second electrodes 31b.

[0073] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, the length of the spacer arm 32 is, for example, 100 nm or more. This allows the spacer arm 32 to easily capture the surface marker probe 20 bound to the surface marker 3 below the target vesicle 1 when the diameter of the target vesicle is approximately 100 nm.

[0074] Furthermore, in the surface marker analysis method and surface marker analysis system 100 according to the embodiment, for example, the base 32a of the spacer arm 32 contains polyethylene glycol. Polyethylene glycol is a hydrophilic, flexible, linear polymer that can move freely in an aqueous solution. A synthesis method for controlling the length has also been established.

[0075] Embodiments may include the following features.

[0076] (Configuration 1) a first step of capturing target vesicles with a baseplate; a second step of binding a surface marker probe to the surface marker of the target vesicle captured by the base plate; a third step of placing a cluster plate opposite the base plate and capturing some of the surface marker probes with the cluster plate; a fourth step of amplifying the portion captured by the cluster plate in the cluster plate; a fifth step of detecting the part of the amplified products and analyzing the surface markers based on the amplified products; A surface marker analysis method comprising:

[0077] (Configuration 2) A surface marker analysis method according to claim 1, further comprising a sixth step of recovering the target vesicles based on the results of the surface marker analysis.

[0078] (Configuration 3) In the first step, the target vesicles are captured by the vesicle capture probes of the base plate, the vesicle capture probes being immobilized on the base substrate and capable of capturing the target vesicles; In the second step, the first portion of the surface marker probe, which comprises a first portion capable of binding to the surface marker and a second portion capable of binding to the first portion, is allowed to bind to the surface marker of the target vesicle captured by the vesicle capture probe; In the third step, the second portion of the surface marker probe is captured by the spacer arm of the cluster plate, which includes a cluster substrate, a spacer arm immobilized on the cluster substrate, a first primer immobilized on the cluster substrate, and a second primer immobilized on the cluster substrate; 3. The surface marker analysis method according to claim 1, wherein in the fourth step, the second portion captured by the spacer arm is amplified using the first primer and the second primer.

[0079] (Configuration 4) In the second step, the surface marker of the target vesicle captured by the vesicle capture probe is bound to the surface marker of the surface marker probe, the surface marker binding portion of the surface marker probe comprising: a first portion comprising a surface marker binding portion capable of binding to the surface marker and a first binding portion comprising a first base sequence; a second binding portion comprising a second base sequence complementary to the first base sequence; and an amplification portion comprising a third base sequence, a fourth base sequence for identifying the surface marker, and a fifth base sequence; In the third step, the spacer arm includes a base portion immobilized on the cluster substrate and a connecting portion capable of binding to the second base sequence, and the connecting portion undergoes strand exchange with the first base sequence bound to the second base sequence, thereby capturing the second portion of the surface marker probe; A surface marker analysis method according to configuration 3, wherein in the fourth step, the second portion captured at the connection portion is amplified using the first primer including a sixth base sequence complementary to the third base sequence and the second primer including the fifth base sequence.

[0080] (Configuration 5) 5. The surface marker analysis method according to claim 4, wherein the connecting portion comprises a peptide nucleic acid.

[0081] (Configuration 6) the base substrate includes a first substrate and a plurality of first electrodes arranged on a surface of the first substrate so as to be spaced apart from one another; the vesicle capture probe is immobilized on a surface of each of the plurality of first electrodes; the cluster substrate includes a second substrate and a plurality of second electrodes arranged on a surface of the second substrate so as to be spaced apart from one another; the spacer arm, the first primer, and the second primer are immobilized on a surface of each of the plurality of second electrodes; the distances between the second electrodes are each longer than the length of the spacer arms; the base substrate and the cluster substrate have alignment marks for horizontally positioning the cluster plate relative to the base plate; At least one of the base substrate and the cluster substrate has a plurality of protrusions for determining the vertical position of the cluster plate relative to the base plate, A surface marker analysis method described in any one of configurations 3 to 5, wherein when the cluster plate is horizontally positioned relative to the base plate by the alignment marks, the centers of the multiple second electrodes each overlap with the centers of the multiple first electrodes in the vertical direction.

[0082] (Configuration 7) each of the plurality of first electrodes includes any one of gold, silver, and copper; A surface marker analysis method according to configuration 6, wherein the vesicle capture probe is immobilized on the surface of each of the plurality of first electrodes via a sulfur atom.

[0083] (Configuration 8) each of the plurality of second electrodes includes any one of gold, silver, and copper; A surface marker analysis method described in configuration 6 or 7, wherein the spacer arm, the first primer, and the second primer are immobilized on the surface of each of the plurality of second electrodes via a group sulfur atom.

[0084] (Configuration 9) 9. The surface marker analysis method according to any one of aspects 3 to 8, wherein the length of the spacer arm is 100 nm or more.

[0085] (Configuration 10) 6. The surface marker analysis method according to claim 4 or 5, wherein the base comprises polyethylene glycol.

[0086] (Configuration 11) a base plate capable of capturing target vesicles; a surface marker probe capable of binding to a surface marker of the target vesicle; a cluster plate disposed opposite the base plate and capable of capturing and amplifying a portion of the surface marker probes; a detection unit for detecting the part of the amplified products; a control unit that analyzes the surface marker based on the amplification product; A surface marker analysis system comprising:

[0087] (Configuration 12) 12. A surface marker analysis system according to claim 11, further comprising a collection device for collecting the target vesicles.

[0088] (Configuration 13) the base plate includes a base substrate and a vesicle capture probe immobilized on the base substrate and capable of capturing the target vesicles; the surface marker probe comprises a first portion capable of binding to the surface marker and a second portion capable of binding to the first portion; the cluster plate includes a cluster substrate, a spacer arm immobilized on the cluster substrate, a first primer immobilized on the cluster substrate, and a second primer immobilized on the cluster substrate; 13. The surface marker analysis system of claim 11, wherein the first primer and the second primer are capable of amplifying the second portion.

[0089] (Configuration 14) The first portion is a surface marker binding moiety capable of binding to the surface marker; a first binding portion including a first base sequence; Including, The second portion is a second binding portion including a second base sequence complementary to the first base sequence; an amplification unit including a third base sequence, a fourth base sequence for identifying the surface marker, and a fifth base sequence; Including, The spacer arm is a base immobilized on the cluster substrate; a connector capable of binding to the second base sequence; Including, the first primer comprises a sixth base sequence complementary to the third base sequence; the second primer comprises the fifth base sequence, 14. The surface marker analysis system of claim 13, wherein the connecting portion captures the second portion by strand exchange with the first base sequence bound to the second base sequence.

[0090] (Configuration 15) 15. The surface marker analysis system of claim 14, wherein the connecting portion comprises a peptide nucleic acid.

[0091] (Configuration 16) the base substrate includes a first substrate and a plurality of first electrodes arranged on a surface of the first substrate so as to be spaced apart from one another; the vesicle capture probe is immobilized on a surface of each of the plurality of first electrodes; the cluster substrate includes a second substrate and a plurality of second electrodes arranged on a surface of the second substrate so as to be spaced apart from one another; the spacer arm, the first primer, and the second primer are immobilized on a surface of each of the plurality of second electrodes; the distances between the second electrodes are each longer than the length of the spacer arms; the base substrate and the cluster substrate have alignment marks for horizontally positioning the cluster plate relative to the base plate; At least one of the base substrate and the cluster substrate has a plurality of protrusions for determining the vertical position of the cluster plate relative to the base plate, A surface marker analysis system described in any one of configurations 13 to 15, wherein when the cluster plate is horizontally positioned relative to the base plate by the alignment marks, the centers of the multiple second electrodes each overlap with the centers of the multiple first electrodes in the vertical direction.

[0092] (Configuration 17) each of the plurality of first electrodes includes any one of gold, silver, and copper; A surface marker analysis system as described in Aspect 16, wherein the vesicle capture probe is immobilized on the surface of each of the plurality of first electrodes via a sulfur atom.

[0093] (Configuration 18) each of the plurality of second electrodes includes any one of gold, silver, and copper; A surface marker analysis system according to aspect 16 or 17, wherein the spacer arm, the first primer, and the second primer are immobilized on the surface of each of the plurality of second electrodes via a sulfur atom.

[0094] (Configuration 19) 19. The surface marker analysis system according to any one of aspects 13 to 18, wherein the length of the spacer arm is 100 nm or more.

[0095] (Configuration 20) 16. The surface marker analysis system of claim 14 or 15, wherein the base comprises polyethylene glycol.

[0096] As described above, according to the embodiments, a surface marker analysis method and a surface marker analysis system capable of analyzing surface markers of vesicles can be provided.

[0097] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0098] 1: Targeted vesicles 2: Lipid bilayer membrane 3: Surface marker 5: Sample solution 10: Base plate 11: Base material 11a: First base 11b: 1st electrode 11c: Alignment mark 12: Vesicle capture probe 15: Protrusion 20: Surface marker probe 21: Part 1 21a: surface marker binding site 21b: 1st joint 21b1: First base sequence 21b2: Connection array 22:Second part 22a:Second joint 22a1: Second base sequence 22a2: Connection array 22b: Amplification section 22b1: Third base sequence 22b2: 4th base sequence 22b3: 5th base sequence 25: First complementary strand 25a: 7th base sequence 25b: 8th base sequence 26: Second complementary strand 26a: 9th base sequence 26b: 10th base sequence 30: Cluster Plate 31: Cluster substrate 31a: Second base 31b: 2nd electrode 31c: Alignment mark 32: Spacer arm 32a: base 32b: Connection 32x: 1st strand 32y: 2nd strand 33: First primer 33a: 6th base sequence 34: Second primer 35: Complementary strand 40: Recovery device 50: Switch circuit 55: Voltage application circuit 60:Detection unit 65: Control unit 100: Surface marker analysis system

Claims

1. a first step of capturing target vesicles with a baseplate; a second step of binding a surface marker probe to the surface marker of the target vesicle captured by the base plate; a third step of placing a cluster plate opposite the base plate and capturing some of the surface marker probes with the cluster plate; a fourth step of amplifying the portion captured by the cluster plate in the cluster plate; a fifth step of detecting the part of the amplified products and analyzing the surface markers based on the amplified products; A surface marker analysis method comprising:

2. The surface marker analysis method according to claim 1 , further comprising a sixth step of recovering the target vesicles based on the results of the surface marker analysis.

3. In the first step, the target vesicles are captured by the vesicle capture probes of the base plate, the vesicle capture probes being immobilized on the base substrate and capable of capturing the target vesicles; In the second step, the first portion of the surface marker probe, which comprises a first portion capable of binding to the surface marker and a second portion capable of binding to the first portion, is allowed to bind to the surface marker of the target vesicle captured by the vesicle capture probe; In the third step, the second portion of the surface marker probe is captured by the spacer arm of the cluster plate, which includes a cluster substrate, a spacer arm immobilized on the cluster substrate, a first primer immobilized on the cluster substrate, and a second primer immobilized on the cluster substrate; 2. The surface marker analysis method according to claim 1, wherein in the fourth step, the second portion captured by the spacer arm is amplified by the first primer and the second primer.

4. In the second step, the surface marker of the target vesicle captured by the vesicle capture probe is bound to the surface marker of the surface marker probe, the surface marker binding portion of the surface marker probe comprising: a first portion comprising a surface marker binding portion capable of binding to the surface marker and a first binding portion comprising a first base sequence; a second binding portion comprising a second base sequence complementary to the first base sequence; and an amplification portion comprising a third base sequence, a fourth base sequence for identifying the surface marker, and a fifth base sequence; In the third step, the spacer arm includes a base portion immobilized on the cluster substrate and a connecting portion capable of binding to the second base sequence, and the connecting portion undergoes strand exchange with the first base sequence bound to the second base sequence, thereby capturing the second portion of the surface marker probe; The surface marker analysis method of claim 3, wherein in the fourth step, the second portion captured at the connection portion is amplified using the first primer containing a sixth base sequence complementary to the third base sequence and the second primer containing the fifth base sequence.

5. The surface marker analysis method according to claim 4 , wherein the connecting portion comprises a peptide nucleic acid.

6. the base substrate includes a first substrate and a plurality of first electrodes arranged on a surface of the first substrate so as to be spaced apart from one another; the vesicle capture probe is immobilized on a surface of each of the plurality of first electrodes; the cluster substrate includes a second substrate and a plurality of second electrodes disposed on a surface of the second substrate so as to be spaced apart from one another; the spacer arm, the first primer, and the second primer are immobilized on a surface of each of the plurality of second electrodes; the distances between the second electrodes are each longer than the length of the spacer arms; the base substrate and the cluster substrate have alignment marks for horizontally positioning the cluster plate relative to the base plate; At least one of the base substrate and the cluster substrate has a plurality of protrusions for determining the vertical position of the cluster plate relative to the base plate, The surface marker analysis method described in claim 3, wherein when the cluster plate is horizontally positioned relative to the base plate by the alignment marks, the centers of the multiple second electrodes each overlap with the centers of the multiple first electrodes in the vertical direction.

7. each of the plurality of first electrodes includes any one of gold, silver, and copper; The surface marker analysis method according to claim 6 , wherein the vesicle capture probe is immobilized on the surface of each of the plurality of first electrodes via a sulfur atom.

8. each of the plurality of second electrodes includes any one of gold, silver, and copper; 7. The surface marker analysis method according to claim 6, wherein the spacer arm, the first primer, and the second primer are immobilized on the surface of each of the second electrodes via a sulfur atom.

9. The surface marker analysis method according to claim 3 , wherein the length of the spacer arm is 100 nm or more.

10. The surface marker analysis method according to claim 4 , wherein the base comprises polyethylene glycol.

11. a base plate capable of capturing target vesicles; a surface marker probe capable of binding to a surface marker of the target vesicle; a cluster plate disposed opposite the base plate and capable of capturing and amplifying a portion of the surface marker probes; a detection unit for detecting the part of the amplified products; a control unit that analyzes the surface marker based on the amplification product; A surface marker analysis system comprising:

12. The surface marker analysis system of claim 11 , further comprising a collection device for collecting the target vesicles.

13. the base plate includes a base substrate and a vesicle capture probe immobilized on the base substrate and capable of capturing the target vesicles; the surface marker probe comprises a first portion capable of binding to the surface marker and a second portion capable of binding to the first portion; the cluster plate includes a cluster substrate, a spacer arm immobilized on the cluster substrate, a first primer immobilized on the cluster substrate, and a second primer immobilized on the cluster substrate; the spacer arm is capable of capturing the second moiety; The surface marker analysis system according to claim 11 , wherein the first primer and the second primer are capable of amplifying the second portion.

14. The first portion is a surface marker binding moiety capable of binding to the surface marker; a first binding portion including a first base sequence; Including, The second portion is a second binding portion including a second base sequence complementary to the first base sequence; an amplification unit including a third base sequence, a fourth base sequence for identifying the surface marker, and a fifth base sequence; Including, The spacer arm is a base immobilized on the cluster substrate; a connector capable of binding to the second base sequence; Including, the first primer comprises a sixth base sequence complementary to the third base sequence; the second primer comprises the fifth base sequence, The surface marker analysis system according to claim 13 , wherein the connecting portion captures the second portion by strand exchange with the first base sequence bound to the second base sequence.

15. The surface marker analysis system according to claim 14 , wherein the connecting portion comprises a peptide nucleic acid.

16. the base substrate includes a first substrate and a plurality of first electrodes arranged on a surface of the first substrate so as to be spaced apart from one another; the vesicle capture probe is immobilized on a surface of each of the plurality of first electrodes; the cluster substrate includes a second substrate and a plurality of second electrodes disposed on a surface of the second substrate so as to be spaced apart from one another; the spacer arm, the first primer, and the second primer are immobilized on a surface of each of the plurality of second electrodes; the distances between the second electrodes are each longer than the length of the spacer arms; the base substrate and the cluster substrate have alignment marks for horizontally positioning the cluster plate relative to the base plate; At least one of the base substrate and the cluster substrate has a plurality of protrusions for determining the vertical position of the cluster plate relative to the base plate, The surface marker analysis system of claim 13, wherein when the cluster plate is horizontally positioned relative to the base plate by the alignment marks, the centers of the multiple second electrodes each overlap with the centers of the multiple first electrodes in the vertical direction.

17. each of the plurality of first electrodes includes any one of gold, silver, and copper; The surface marker analysis system of claim 16, wherein the vesicle capture probe is immobilized on the surface of each of the plurality of first electrodes via a sulfur atom.

18. each of the plurality of second electrodes includes any one of gold, silver, and copper; The surface marker analysis system according to claim 16, wherein the spacer arm, the first primer, and the second primer are immobilized on the surface of each of the plurality of second electrodes via a sulfur atom.

19. The surface marker analysis system according to claim 13 , wherein the length of the spacer arm is 100 nm or more.

20. The surface marker analysis system of claim 14 , wherein the base comprises polyethylene glycol.

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