Sample solution separation method, sample solution separation device, and sample solution separation apparatus

Abstract

In order to achieve a highly robust sample solution separation technique using a liquid photocurable resin in a microdevice for analyzing a biological sample, the present invention proposes a method for separating a sample solution in a microdevice for analyzing a biological sample, comprising a step for introducing a sample solution into a plurality of microcells of the microdevice for biological sample analysis, and a step for introducing a liquid photocurable resin into a flow path of the microdevice for biological sample analysis and covering openings of the plurality of microcells with the liquid photocurable resin to separate the plurality of microcells into which the sample solution has been introduced. And a step for irradiating the microdevice for biological sample analysis, into which the liquid photocurable resin has been introduced, with light to cure the liquid photocurable resin (referring to Figure 2).

Description

Technical Field

[0001] The present disclosure relates to a sample solution separation method, a sample solution separation device (micro device) and a sample solution separation apparatus. Background Art

[0002] In the past, PCR and real-time PCR were used in genetic testing. In these technologies, when the object of measurement (nucleic acid) is in trace amounts, there is a problem of low measurement accuracy. In order to solve this problem, digital PCR technology has received attention in recent years. In digital PCR technology, a sample containing the nucleic acid of the detection object is separated into multiple micro-regions, and PCR is performed on each micro-region. Then, the fluorescence intensity is used to distinguish the segments including the nucleic acid of the detection object and the segments not including the nucleic acid of the detection object, thereby distinguishing the types of nucleic acids present in each micro-region. For example, in Patent Document 1, as a DNA detection method using digital PCR, a DNA detection method is disclosed that measures the melting temperature of DNA and a fluorescently labeled probe in a droplet containing DNA and a fluorescently labeled probe hybridized with the DNA.

[0003] In micro devices for PCR, ultraviolet curing resin is often used as one of the components. For example, Patent Document 2 discloses an example of using ultraviolet curing resin as an adhesive between the cover and the substrate of a micro device in a microfluidic chip that can easily dispense a small amount of sample. In addition, Patent Document 3 discloses that in a reaction liquid container for promoting a nucleic acid amplification reaction including a PCR reaction or an LCR reaction, the container is sealed by introducing ultraviolet curing resin into the flow path in the container.

[0004] In microfluidic devices in digital PCR, oil is often used to separate samples into micro areas. Patent Document 4 discloses that in a microarray device for separating a small amount of samples containing nucleic acids, after the sample is introduced into the hole, the hole is covered with a hydrophobic substance such as mineral oil or silicone oil, thereby performing solution separation. Since the sample in the hole is hydrophilic and the oil is hydrophobic, the relationship between water and oil can be used to separate the sample solution to each hole without mixing.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application No. 2018-108063

[0008] Patent Document 2: Japanese Patent Application Publication No. 2011-163946

[0009] Patent Document 3: International Publication No. 2008 / 146754

[0010] Patent Document 4: U.S. Patent No. 9518299 Summary of the invention

[0011] Problems to be solved by the invention

[0012] When using a biological sample analysis microdevice for PCR or digital PCR, it is important that the biological sample solution enclosed in the hole is not affected by the heating during thermal cycling and does not leak. Currently, oil, which is a hydrophobic substance, is often used as a material for sealing the biological sample solution in the hole.

[0013] However, if the biological sample solution in the hole is heated to about 95°C during thermal cycling, bubbles will be generated in the hole, and the solution in the hole will sometimes push away the covering oil and leak out of the hole. This will cause contamination of the surrounding holes, so it is necessary to avoid such a situation. Therefore, liquid photocurable resin can be expected as a sealing material instead of oil.

[0014] However, even if they are all called liquid photocurable resins, there are various limitations on the liquid photocurable resins that can be used as microdevices for biological sample analysis. For example, the viscosity of the photocurable resin and the nature of PCR inhibition are very important. The former is important in terms of easy introduction into microfluidic devices, and the latter is important in terms of biological samples being unaffected by the liquid photocurable resin and performing PCR.

[0015] In view of such circumstances, the present disclosure proposes a sample solution separation technology with high robustness (high robustness means that the sample solution in the pores (micro-areas) will not leak) using a liquid photocurable resin in a micro-device for biological sample analysis.

[0016] Methods for solving problems

[0017] In order to solve the above-mentioned problems, the present disclosure proposes a sample solution separation method, which is a sample solution separation method in a microdevice for biological sample analysis, comprising: a process of introducing a sample solution into a plurality of microregions possessed by the microdevice for biological sample analysis, a process of introducing a liquid photocurable resin into a flow path of the microdevice for biological sample analysis and covering the openings of the plurality of microregions with the liquid photocurable resin to thereby separate the plurality of microregions into which the sample solution has been introduced, and a process of irradiating the microdevice for biological sample analysis into which the liquid photocurable resin has been introduced with light to thereby cure the liquid photocurable resin.

[0018] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. In addition, the present disclosure is achieved and realized by the combination of elements and a plurality of elements, the following detailed description and the appended claims.

[0019] The description in this specification is merely a typical example and does not limit the claims or application examples of the present disclosure in any sense.

[0020] Effects of the Invention

[0021] According to the technology disclosed in the present disclosure, in a biological sample analysis microdevice, it is possible to achieve highly robust sample solution separation (high robustness means that the sample solution in the holes (microdomains) does not leak) using a liquid photocurable resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing an overview of the curing process of a liquid photocurable resin.

[0023] Figure 2 The diagram shows a method of introducing a liquid photocurable resin into a micro device and changes in properties of the liquid photocurable resin caused by light irradiation after the introduction.

[0024] Figure 3 This is a diagram showing a configuration example of a biological sample analysis microdevice according to Example 1.

[0025] Figure 4 It means to Figure 3 The micro-device shown is a diagram of a state where a liquid photocurable resin is introduced.

[0026] Figure 5 This is a diagram for explaining the results of digital PCR performed on a microdevice to which a highly robust sample solution separation method is applied.

[0027] Figure 6 This is a diagram showing a configuration example of a biological sample analysis micro-device according to Example 2 in which the flow channel and the orifice are directly connected without other flow channels connecting them.

[0028] Figure 7 This is a diagram showing the results of digital PCR measurement after introducing a sample solution and a liquid photocurable resin into the micro-device for biological sample analysis of Example 2.

[0029] Figure 8 This is a diagram showing a configuration example of a microdevice according to Example 3.

[0030] Fig. 9 This is a diagram showing the results of digital PCR measurement performed after introducing a sample solution and a liquid photocurable resin into the micro-device for biological sample analysis of Example 3.

[0031] Fig.10 This is a diagram for explaining that if the viscosity of the liquid photocurable resin is too high, the liquid photocurable resin cannot be introduced into the flow path of the micro device.

[0032] Fig.11 This is a diagram for explaining the relationship between the liquid specific gravity of a liquid photocurable resin and a sample solution.

[0033] Fig.12 This is a table showing the curing method, viscosity, liquid specific gravity, and device incorporation availability of commercially available liquid photocurable resins sold by multiple manufacturers.

[0034] Fig.13 The diagram shows a scheme for evaluating the presence or absence of PCR inhibition of commercially available liquid photocurable resins (upper diagram) and the results of real-time PCR measurement (lower diagram).

[0035] Fig.14 This is a diagram showing the results of real-time PCR measurement performed to evaluate the presence or absence of PCR inhibition in a hydrophilic liquid photocurable resin.

[0036] Fig.15 This is a diagram showing the results of real-time PCR measurement performed to evaluate the presence or absence of PCR inhibition in a hydrophobic liquid photocurable resin.

[0037] Fig.16 It is a diagram showing a schematic configuration example of the sample solution separation device 1600 .

[0038] Fig.17 This is a diagram showing a schematic cross-sectional structure example of an improved microdevice. DETAILED DESCRIPTION

[0039] The embodiments of the present disclosure propose a highly robust sample solution separation technology using a liquid photocurable resin in a biological sample analysis microdevice, and propose a sample solution separation method in which the liquid photocurable resin does not adversely affect PCR and digital PCR.

[0040] Hereinafter, embodiments and implementation methods of the present disclosure will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements are sometimes shown with the same number. It should be noted that the accompanying drawings show specific implementation methods and installation examples that follow the principles of the present disclosure, but these are for the purpose of understanding the present disclosure and are by no means intended to limit the interpretation of the present disclosure.

[0041] In the present embodiment, those skilled in the art have described in sufficient detail in order to implement the present disclosure, but other installations and methods are also possible, and it is necessary to understand that the composition and structure can be changed and various elements can be replaced without departing from the scope and spirit of the technical idea of ​​the present disclosure. Therefore, the following description is not limited to this for interpretation.

[0042] <Example of Sample Solution Separation>

[0043] (Example 1)

[0044] Reference Figures 1 to 5 , Example 1 is described. Example 1 shows the outline of a liquid photocurable resin and that the introduction of the liquid photocurable resin into a microdevice enables highly robust separation of a sample solution.

[0045] (i) Curing process of liquid photocurable resin

[0046] Figure 1 1 is a diagram showing an overview of the curing process of a liquid photocurable resin. In order to cure the liquid photocurable resin, a light source 1 is required. First, a coating layer 3 is formed on a substrate 2 (preparation). The coating layer 3 contains a liquid photocurable resin 4 and a photoinitiator 5. At this time, the coating layer 3 is in a liquid state, and the solution moves due to tilting, etc. When the coating layer 3 is irradiated with light from the light source 1, the liquid photocurable resin 4 and the photoinitiator 5 initiate a photopolymerization reaction. Through the photopolymerization reaction, the liquid photocurable resin 4 is cross-linked between resins to form a polymer structure 6 based on the liquid photocurable resin 4.

[0047] At this time, in order to promote the photopolymerization reaction, the light source 1 preferably irradiates light near the absorption wavelength of the photoinitiator 5. Therefore, it is suitable to use light with a wavelength of 250 to 600 nm. However, the nucleic acid to be measured may be destroyed at a wavelength in the ultraviolet region. Therefore, it is preferred to use a wavelength that does not destroy the nucleic acid. If the condition of not destroying the nucleic acid is also considered, light with a wavelength of 350 to 600 nm can be used. As suitable light sources, lasers, LEDs, mercury lamps, deuterium lamps, tungsten lamps, xenon lamps, halogen lamps, etc. can be listed.

[0048] (ii) Robust sample solution separation

[0049] Figure 2 This is a diagram showing a method for introducing a liquid photocurable resin into a microdevice and changes in the properties of the liquid photocurable resin caused by light irradiation after introduction. Figure 2 In FIG. 1 , the state after the sample solution is introduced into the micro-device is first shown. The sample solution is introduced into each hole (not limited to the shape of the hole, as long as it is a micro-domain) from the inlet portion 106 of the micro-device.

[0050] according to Figure 2After the sample solution is introduced, the microdevice has an outlet portion 100 (it may also be a state where the remaining sample solution is accumulated), a substrate 101, a hole 102 into which the sample solution is introduced, and a cover 103 of the substrate. A liquid photocurable resin 105 is introduced into the flow path of the microdevice from an inlet portion 106 of the microdevice using a pressure applying device 104. At this time, each sample solution in the hole is sealed by the introduced liquid photocurable resin. It should be noted that the introduction of the liquid photocurable resin can be started in a state where the sample solution remains in the flow path portion of the microdevice, or the introduction of the liquid photocurable resin can be started after the sample solution is discharged from the flow path portion.

[0051] Next, the introduced liquid photocurable resin is irradiated with light from a light source 107. This promotes the photopolymerization reaction. Then, the introduced liquid photocurable resin 105 is modified into a solid photocurable resin 108, whereby the sealing of each solution in the hole is changed from being sealed with a liquid material to being sealed with a solid material. By sealing with a solid instead of a liquid, it can be expected that the robustness of the sample solution in the hole will be improved. It should be noted that, as examples of the pressure applying device 104, vacuum pumps such as syringe pumps, diaphragm pumps, and rotary pumps, compressors, etc. can be cited. In addition, as the material of the substrate 101 and the cover 103, glass, cycloolefin polymers, cycloolefin copolymers, resins such as polypropylene, semiconductors such as silicon, metals such as copper and zinc, etc. can be used.

[0052] (iii) Examples of microdevice shapes (structures)

[0053] Figure 3 This is a diagram showing an example of the configuration of a biological sample analysis microdevice according to Example 1. This microdevice is configured such that a flow channel and an aperture are separated. Figure 3 A plurality of branch eyelet groups (left) and an enlarged configuration of a portion thereof (right) are shown.

[0054] The micro device comprises an inlet 200, a main flow path 201, a plurality of holes 202 (micro-regions), and a sub-flow path 203 connecting the main flow path 201 and the holes 202. Figure 3 As shown, a plurality of holes 202 are arranged in an extended manner on the XY plane. The sample solution is introduced from the inlet 200, through the main flow path 201 and each sub-flow path 203, and then introduced into the holes 202. The size of the holes 202 is extended from nanometers to millimeters, and the shape can be a quadrilateral, a circle, a hexagon, etc. In addition, the number of holes 202 is at least one and at most 3 million or less.

[0055] (iv) Introduction of liquid photocurable resin into microdevices

[0056] Figure 4 It means to Figure 3The micro-device shown is a diagram of a state where a liquid photocurable resin is introduced. Figure 4 The left picture shows the process of introducing liquid light-curable resin. Figure 4 The right figure shows the introduced liquid photocurable resin after being solidified by light irradiation.

[0057] Introduced into the main flow path portion 201 of the micro device (refer to Figure 3 ) squeezes out the sample solution 303 originally present in the main flow path portion 201. For the hole 300 into which the sample solution 303 is introduced, there is an interface 301 between the sample solution 303 on the main flow path portion 201 and the liquid photocurable resin 304. In the area where the liquid photocurable resin 304 has been introduced into the main flow path portion 201, the interface 302 between the sample solution 303 in the hole 202 and the liquid photocurable resin 304 in the main flow path portion 201 is observed. On the other hand, in the area where the liquid photocurable resin 304 has not been introduced, the sample solution 303 on the flow path is observed.

[0058] In the main channel section 201, if the main channel section 201 in which the sample solution 303 is replaced by the liquid photocurable resin 304 is irradiated with light, the liquid photocurable resin 304 in the main channel section 201 becomes the solid photocurable resin 305. At this time, the interface 302 between the sample solution 303 in the hole 202 and the liquid photocurable resin 304 in the main channel section 201 becomes the interface 306 between the sample solution 303 in the hole 202 and the solid photocurable resin 305 in the main channel section 201. That is, this means that the sample solution 303 in each hole 202 is sealed by a solid substance instead of a liquid substance. As a result, compared with the sealing by a liquid substance as in the conventional example, it is possible to perform a sample separation with high robustness.

[0059] (v) PCR assay results

[0060] Below, use Figure 5 The results of digital PCR performed on a microdevice to which a highly robust sample solution separation method is applied are described. By using the microdevice of Example 1 and a liquid photocurable resin, it is possible to perform highly robust sample solution separation and measurement accompanied by heating such as digital PCR.

[0061] Figure 5 The left figure shows the state after the sample solution and the photocurable resin are introduced into the microdevice for biological sample analysis in which the flow channel and the hole are separated. Figure 5 The right figure of FIG. 1 shows the result of digital PCR measurement using the microdevice. As described above, after the sample solution 401 is separated by the solid photocurable resin 400, the solid photocurable resin 400 is present in the main flow path portion 201 (see FIG. 2 ). Figure 3), the sample solution 401 is present in the well 202. In digital PCR, the microdevice is subjected to PCR by a heating medium such as a thermal cycler. Thus, in each well, PCR is promoted in the wells where nucleic acid is present, and PCR does not occur in the wells where nucleic acid is not present. By pre-introducing a fluorescent labeled probe into the sample solution, only the wells where PCR has occurred emit light during fluorescence measurement.

[0062] exist Figure 5 In the right figure, the luminous hole 402 and the non-luminous hole 403 are observed. Therefore, from this fluorescence observation result, it can be seen that digital PCR can be implemented in a microdevice using a solid photocurable resin. By the way, other experiments have shown that even if a liquid medium such as oil is introduced into the flow path and PCR is performed in the same way, the result of digital PCR cannot be obtained, and the connection with the adjacent holes and the dispersion of the solution in the flow path can be observed. Therefore, this result represents the result obtained by sealing the hole into which the sample solution is introduced with a solid photocurable resin.

[0063] (Example 2)

[0064] Reference Figure 6 and Figure 7 , Example 2 is described. Example 2 describes the results of digital PCR measurement after introducing a sample solution and a liquid photocurable resin into a biological sample analysis microdevice connected to a flow path and an orifice. According to Example 2, as in Example 1, it is possible to perform a measurement accompanied by heating such as digital PCR and sample solution separation with high robustness.

[0065] (i) Microdevice Configuration Example

[0066] Figure 6 This is a diagram showing a configuration example of a biological sample analysis micro-device according to Example 2 in which the flow channel and the orifice are directly connected without other flow channels connecting them. Figure 6 An example of the top surface structure of the micro device (upper figure) and an example of the cross-sectional structure of the micro device (lower figure) are shown.

[0067] The microdevice of Example 2 includes an aperture 500, a flow path 501, and a substrate 502. In Example 2, the aperture 500 and the flow path 501 are connected in a series, which is slightly different from the structure of the microdevice of Example 1. It should be noted that the side and bottom portions of the aperture (micro-region) 500 and the flow path portion 501 may also be subjected to a hydrophilic treatment or a hydrophobic treatment. Specifically, the side and bottom portions of the aperture 500 may be subjected to a hydrophilic treatment, the flow path 501 may be subjected to a hydrophobic treatment, or both may be subjected to a hydrophilic treatment.

[0068] (ii) PCR results

[0069] Figure 7 This is a diagram showing the results of digital PCR measurement after introducing a sample solution and a liquid photocurable resin into the micro-device for biological sample analysis of Example 2. Figure 7 The upper figure shows a state where a sample solution 602 introduced into a hole 601 of a micro device is sealed with a solid photocurable resin 600 . Figure 7 The figure below shows the results of actually implementing PCR using the microdevice.

[0070] The solid photocurable resin 600 seals the sample solution 602 in the hole 601. In addition, the flow path 604 and the hole 601 are formed by processing the substrate 603. The result of implementing digital PCR on the microdevice is that the luminous hole 605 and the non-luminous hole 606 are observed in the same way as in Example 1. It can be seen that the presence or absence of nucleic acid to be measured can be confirmed by fluorescence measurement. In addition, in the fluorescence measurement, since the solid photocurable resin 600 does not emit fluorescence, it is observed to be darker. In addition, the substrate 603 is also observed to be darker. This indicates that the autofluorescence of the solid photocurable resin 600 and the substrate 603 is low. In the fluorescence measurement, the fluorescence from outside the measurement object will enhance the background intensity, making it difficult to perform high-precision measurement. Therefore, the substrate and liquid photocurable resin used are preferably substances that show low autofluorescence. As a material with low autofluorescence in the substrate, for example, cycloolefin polymer (COP), cycloolefin copolymer (COC), polydimethylsiloxane (PDMS), metals such as gold, silver, copper, and zinc can be cited. In order to make the solid photocurable resin 600 exhibit low autofluorescence, it is possible to have almost no π-conjugated systems such as polycyclic aromatic hydrocarbons but to have cycloalkanes, normal alkanes, isoalkanes, etc. in the molecular skeleton of the solid photocurable resin 600 .

[0071] (Example 3)

[0072] Reference Figure 8 and Fig. 9 , Example 3 is described. Example 3 describes the results of digital PCR measurement after introducing a sample solution and a liquid photocurable resin into a biological sample analysis microdevice having through holes. According to Example 3, as in Examples 1 and 2, it is possible to perform a measurement accompanied by heating such as digital PCR and sample solution separation with high robustness.

[0073] (i) Microdevice Configuration Example

[0074] Figure 8 1 is a diagram showing a configuration example of a microdevice of Example 3. The microdevice has a space formed on the upper surface and the bottom surface of a substrate 705, that is, a space 706 that becomes a flow path (may be referred to as flow path 706), and has a through hole 704 that penetrates the substrate 705 sandwiched by the space 706. Figure 8The left figure is a microscope image showing the structure of the upper surface of the microdevice. Figure 8 The right figure of is a diagram showing an example of a cross-sectional structure of a micro device.

[0075] like Figure 8 As shown in the left figure, the microdevice has an inlet 700, an area 701 where a through hole is present, a port 702 for folding back the liquid photocurable resin from the upper surface to the lower surface, and an outlet 703. In addition, when focusing on the area 701 where a through hole 704 is present, the through hole 704 is formed by opening a through hole on a substrate 705, and there are spaces (flow paths) 706 on its upper and lower surfaces, respectively. It should be noted that the side portions or the spaces (flow path portions) 706 of the through hole (micro-area) 704 may also be subjected to a hydrophilic treatment or a hydrophobic treatment. Specifically, the side portions of the through hole 704 may be subjected to a hydrophilic treatment, the space 706 may be subjected to a hydrophobic treatment, or both may be subjected to a hydrophilic treatment.

[0076] (ii) Introduction of photocurable resin

[0077] Next, the flow of the liquid photocurable resin in the microdevice of Example 3 is described. When the sample solution is introduced from the inlet 700 and injected into the through-hole eyelet 704, the sample solution is retained in the through-hole eyelet 704 due to surface tension. After that, the liquid photocurable resin is introduced from the inlet 700 in the same manner as the sample solution. The introduced liquid photocurable resin passes through the space 706, and first, the liquid photocurable resin seals the upper surface (opening) of the through-hole eyelet. After sealing the upper surface, the liquid photocurable resin passes through the lower surface of the space 706 through the port 702 for folding the photocurable resin from the upper surface to the lower surface. Thus, the liquid photocurable resin seals the lower surface (opening) of the through-hole eyelet 704. Excess liquid photocurable resin is discharged from the outlet 703. It should be noted that as the material of the substrate 705 having the through-hole formed therein, for example, semiconductors such as silicon, glass, cycloolefin polymers, resins such as polypropylene, metals such as gold, silver, copper, zinc, etc. can be cited.

[0078] (iii) PCR assay results

[0079] Fig. 9 This is a diagram showing the results of digital PCR measurement performed after introducing a sample solution and a liquid photocurable resin into the micro-device for biological sample analysis of Example 3. Fig. 9 The upper figure is a cross-sectional view showing the state after the sample solution and liquid photocurable resin are introduced into the microdevice and photocured. Fig. 9 The figure below shows the results of digital PCR measurement performed in the microdevice.

[0080] In the micro device, each through hole 704 (see Figure 8 ) is injected and maintained with a sample solution 801. The upper and lower surfaces of the through hole into which the sample solution 801 is injected are sealed with a solid photocurable resin 802. The microdevice is subjected to digital PCR measurement, and the result is as follows: Fig. 9 As shown, the well 803 that emits light and the well 804 that does not emit light are observed. Although repeated, it shows that the nucleic acid to be measured is introduced into the well that emits light. Since there are wells 803 that emit light and adjacent wells that do not emit light, it can be understood that the sample solution 801 does not contaminate the surrounding wells.

[0081] Therefore, it can be seen from Example 3 that the sample solution can be separated with high robustness. From this result, it can be seen that in the method combining the present device and the liquid photocurable resin, the sample solution can be separated with high robustness, and further, the digital PCR measurement can be performed with heating operations (such as melting curve analysis, etc.) and fluorescence measurement.

[0082] It should be noted that, when the opening of the hole 500 (Example 2) and the through hole hole 704 (Example 3) filled with the sample solution is sealed with a solid photocurable resin (the liquid photocurable resin is irradiated with light to change it into a solid photocurable resin) as in Example 2 and Example 3, the refractive index of the solid photocurable resin is preferably close to the refractive index of the surrounding material (resin, solution). This is because, in the optical measurement using the micro device, it has the effect of preventing the light emitted from the sample solution from being lost due to the solid photocurable resin. For example, in the case of a sample solution with water as the medium, the refractive index of the sample solution is 1.3, so the refractive index of the photocurable resin can be greater than 1 and less than 2. In addition, the closer the value is to 1.3, the more beautiful the microscope image with less loss can be obtained. In addition, when the material of the substrate 800 or the substrate 705 is a resin such as polypropylene, the refractive index is about 1.5. Therefore, the refractive index of the solid photocurable resin is about 1.5, which is the best, and can be set to at least 1 or more and less than 2.5.

[0083] <Conditions of Liquid Photocurable Resin>

[0084] Reference Figures 10 to 12 , the conditions of the best liquid photocurable resin that matches the shape of the flow cell are described. By using a liquid photocurable resin that meets the conditions of the present disclosure, the liquid photocurable resin can be introduced into the flow path in the microdevice. That is, not all commercially available liquid photocurable resins can be used, and the specifications of the liquid photocurable resin need to be confirmed according to the shape of the microdevice and used separately.

[0085] (i) Viscosity of liquid photocurable resin

[0086] Fig.10 This is a diagram for explaining that when the viscosity of the liquid photocurable resin is too high, the liquid photocurable resin is not introduced into the flow path of the microdevice. The viscosity condition of the liquid photocurable resin is applicable to all microdevices having the configurations shown in the above-mentioned embodiments 1 to 3. However, here, the viscosity condition of the liquid photocurable resin is explained using the microdevice having the configuration described in embodiment 1.

[0087] The micro device includes: an outlet portion 900 where the remaining sample solution accumulates, a substrate 901, at least one hole 902 into which the sample solution is introduced, and a cover 903 of the substrate. A liquid photocurable resin 905 is introduced into the micro device from an inlet portion 906 of the micro device by a pressure applying device 904. At this time, if the viscosity of the liquid photocurable resin 905 is too high, it is difficult to introduce the liquid photocurable resin into the flow path. Therefore, in the case where the viscosity of the liquid photocurable resin is high, as a countermeasure, a relatively strong pressure can be applied from the pressure applying device 904 to introduce the photocurable resin. However, if excessive pressure is applied, there is a possibility that the bonding of the manufactured micro device will be peeled off and damaged.

[0088] Therefore, the relationship between flow rate, pressure and viscosity is explained with reference to the following formula (1) and formula (2). Assume that a liquid photocurable resin is successfully introduced into a microdevice under certain conditions. If the successfully introduced liquid photocurable resin must be changed to a liquid photocurable resin with a viscosity four times its viscosity, and the same flow rate is applied to the microdevice, the pressure applied to the microdevice and the liquid photocurable resin is 16 times the usual pressure. It can be seen that the higher the viscosity of the liquid photocurable resin, the more proportional to the square of the pressure required for introduction. In addition, it can also be understood that the higher the viscosity, the more the flow rate decreases in a relationship of 1 / 2 of the difference between the required pressure and the atmospheric pressure (i.e., the differential pressure). Based on these circumstances, it can be seen that the viscosity of the liquid photocurable resin 905 needs to be at least 500 mPa·s or less.

[0089] (Flow)∝(Differential Pressure) 1 / 2 …(1)

[0090] (Flow rate)∝1 / viscosity···(2)

[0091] (ii) Specific gravity of liquid photocurable resin

[0092] Fig.11 is a diagram for explaining the relationship between the liquid specific gravity of the liquid photocurable resin and the sample solution. If the liquid specific gravity of the liquid photocurable resin is too high, it is difficult to introduce the liquid photocurable resin into the through hole or blind hole. It should be noted that the liquid specific gravity conditions of the liquid photocurable resin described below are applicable to the microdevices shown in Examples 2 and 3 (in the case of Example 1, as shown in Figure 3 As shown, the sample is introduced in the XY plane direction, so the separation process is not affected by the liquid specific gravity of the liquid photocurable resin).

[0093] The liquid specific gravity of the sample solution 1001 in the hole is close to 1. Therefore, when the liquid photocurable resin 1000 with a high liquid specific gravity is introduced into the micro-devices shown in Examples 1 to 3, Fig.11 As shown, the sample solution is squeezed out of the hole. The extruded sample solution 1002 is squeezed in different directions depending on the shape of the hole. For example, when the shape of the hole is a blind hole (the case of Example 2), the sample solution 1002 is squeezed upward. When the shape of the hole is a through hole (the case of Example 3), the sample solution is squeezed below the hole. The sample solution is squeezed out, and on the other hand, a liquid photocurable resin with a high liquid specific gravity is injected into the hole. Therefore, it is necessary to reduce the liquid specific gravity of the liquid photocurable resin used. The value of the liquid specific gravity of the liquid photocurable resin is determined according to the relationship with the liquid specific gravity of the sample solution, for example, it can be set to the liquid specific gravity of the sample solution + 0.2g / cm 3 Therefore, when the liquid specific gravity of the sample solution is 1.0g / cm 3 When the liquid photocurable resin has a liquid specific gravity of 1.2 g / cm 3 the following.

[0094] (iii) Summary of viscosity and liquid specific gravity conditions (taking commercially available liquid photocurable resin as an example)

[0095] It has been described above that viscosity and liquid specific gravity are important when introducing a liquid photocurable resin into a microdevice. Here, the viscosity and liquid specific gravity conditions of various commercially available liquid photocurable resins are examined.

[0096] Fig.12 This is a table showing the curing method, viscosity, liquid specific gravity, and whether it can be introduced into the device for commercially available liquid photocurable resins sold by multiple manufacturers. Liquid photocurable resins are sometimes also used in 3D printers, etc., and most of them usually have high viscosity. On the other hand, there are many liquid photocurable resins that meet the conditions for liquid specific gravity. If it is shown whether the liquid photocurable resins of each company can be introduced into micro devices, then agents A, E, H, and I can be introduced into the device. However, from the perspective of viscosity, agents B, C, and D are difficult to import. In addition, from the perspective of viscosity, agent F can be imported, but the curing method has not only ultraviolet curing but also moisture curing. If moisture curing is given, the liquid photocurable resin will solidify at the moment of contact with the sample solution in the flow path. Therefore, in liquid photocurable resins with moisture curing properties, the resin will not spread to the outlet, so it is not suitable.

[0097] In summary, it can be understood that not all commercially available liquid photocurable resins can be used. Therefore, it is known that the liquid photocurable resin needs to be selected according to the above conditions.

[0098] (iv) Regarding hydrophilic liquid photocurable resin and hydrophobic liquid photocurable resin

[0099] Next, the conditions of the liquid photocurable resin that inhibits PCR while performing PCR and digital PCR are described. By using the conditions of the liquid photocurable resin examined here, the liquid photocurable resin introduced into the microdevice can perform PCR measurement and digital PCR measurement without PCR inhibition. That is, as with the conditions of the viscosity and liquid specific gravity described above, not all commercially available liquid photocurable resins can be used, and it is necessary to select a liquid photocurable resin that does not inhibit PCR.

[0100] Fig.13 The figure shows the scheme for evaluating the presence or absence of PCR inhibition of commercially available liquid photocurable resins (upper figure) and the results of real-time PCR measurement (lower figure). Fig.13 In the figure above, the scheme for evaluating PCR inhibition by real-time PCR is first described. Liquid photocurable resin 1100 and sample solution 1101 are introduced into a PCR tube and mixed. As a result, since the liquid photocurable resin 1100 and the sample solution 1101 are in contact with each other, PCR inhibition caused by changes in enzyme salt concentration, pH, and adsorption of nucleic acids may occur at this time. After this scheme, light irradiation is performed, and the liquid photocurable resin 1100 is converted into a solid photocurable resin 1102. Then, finally, the process of real-time PCR measurement is performed.

[0101] Fig.13 The figure below shows the results of real-time PCR using a commercially available liquid photocurable resin based on the present scheme. The horizontal axis of the curve represents the number of PCR cycles, and the vertical axis represents the fluorescence intensity. In this curve, the black line is the positive control, which is the result of real-time PCR performed using only the sample solution without introducing a liquid photocurable resin. Therefore, when the sample solution into which the liquid photocurable resin has been introduced also shows the same curve shape as the positive control, it can be said that the PCR is not inhibited by the liquid photocurable resin (PCR inhibition does not occur). However, in the real-time PCR measurement results of the samples mixed with agents A to H respectively, the same curve shape as the positive control is not shown, and the curve is flat. Therefore, it can be seen that all commercially available liquid photocurable resins will cause PCR inhibition. It should be noted that the labeling of agents A to H is the same as that of the sample solution. Fig.12 Therefore, from this result, it can be seen that even a liquid photocurable resin that meets the viscosity and liquid specific gravity conditions for device introduction cannot be used due to PCR inhibition. Fig.12 The commercially available liquid photocurable resin shown below causes PCR inhibition. The conditions for a liquid photocurable resin that is less likely to cause PCR inhibition are described below.

[0102] Fig.14 This is a graph showing the results of real-time PCR measurement in order to evaluate the presence or absence of PCR inhibition on a hydrophilic liquid photocurable resin. Here, hydrophilicity means that the liquid photocurable resin has high solubility in water, and hydrophobicity means that the liquid photocurable resin has low solubility in water. Therefore, a hydrophilic liquid photocurable resin means that it has high compatibility with water, and a hydrophobic liquid photocurable resin means that it has low compatibility with water (poorly soluble or difficult to mix). Refer to Fig.14 It can be seen that the characteristics of each hydrophilic liquid photocurable resin are not the same as Fig.13 Therefore, from the results of real-time PCR measurement using a hydrophilic liquid photocurable resin, it can be seen that although PCR inhibition does not occur like commercially available liquid photocurable resins, even hydrophilic liquid photocurable resins tend to inhibit PCR.

[0103] Fig.15 This is a graph showing the results of real-time PCR measurement performed to evaluate the presence or absence of PCR inhibition in a hydrophobic liquid photocurable resin. Fig.15 It can be seen that in the real-time PCR measurement results when the hydrophobic liquid photocurable resin is used, the influence of PCR inhibition is lower than that of the commercially available liquid photocurable resin and the hydrophilic liquid photocurable resin. In addition, the shape of the real-time PCR measurement results also shows a shape that is roughly the same as the black line of the positive control. Therefore, it can be seen that the use of the hydrophobic liquid photocurable resin is effective in real-time PCR measurement and digital PCR measurement.

[0104] In short, in order to avoid (not produce) PCR inhibition, it is better to use a hydrophobic liquid photocurable resin rather than a hydrophilic liquid photocurable resin. As a condition for a hydrophobic (poorly soluble) liquid photocurable resin, it is important that the solubility in water is less than 100 g / L. This is an indicator of compatibility with water. If it is less than 100 g / L, it can be said to be poorly soluble. As the molecular structure of the resin, in order to show poor solubility in water, for example, a resin in which no hydroxyl group or the like is present in the molecular skeleton can be used. Among them, if the molecular skeleton of the liquid photocurable resin is focused on, it is easy to show hydrophobic properties when it is composed of cycloalkanes or normal alkanes or isoalkanes with a carbon number of 6 or more. Therefore, a liquid photocurable resin having such a structure can be used. In fact, in the microdevices in which digital PCR is implemented in Examples 1 to 3, a hydrophobic liquid photocurable resin can be used as a liquid photocurable resin. Therefore, a hydrophobic liquid photocurable resin can be used for real-time PCR and can also be used for digital PCR. In addition, from the viewpoint of viscosity, the molecules of the liquid photocurable resin may be molecules mainly composed of monomers instead of oligomers.

[0105] Based on the above, regarding liquid photocurable resin, in the case of the structure of the microdevice as shown in Examples 2 and 3, the important things are viscosity, liquid specific gravity and hydrophobicity (poorly soluble), but in the case of the structure of the microdevice as shown in Example 1, the important things are viscosity and hydrophobicity.

[0106] <Sample Solution Separation Device>

[0107] Here, the outline of the biological sample analysis micro-device and the sample solution separation device using the liquid photocurable resin is described. By using the sample solution separation device, the sample solution can be separated using the liquid photocurable resin on the device.

[0108] Fig.16 1 is a diagram showing a schematic configuration example of a sample solution separation device 1600. The sample solution separation device 1600 includes a sample setting portion 1200, a biological sample analysis micro-device 1201 placed on the sample setting portion 1200, a photocurable resin reservoir 1202 for holding a liquid photocurable resin, a pressure applying device 1203, and a light source portion 1204 in a light-shielding environment. The biological sample analysis micro-device 1201 is connected to the pressure applying device 1203 via the photocurable resin reservoir 1202. In the sample solution separation device 1600, pressure is applied by the pressure applying device 1203, so that liquid photocurable resin is introduced from the photocurable resin reservoir 1202 into the biological sample analysis micro-device 1201.

[0109] When all the holes in the biological sample analysis micro-device 1201 are sealed with the liquid photocurable resin, the light source unit 1204 irradiates light to the biological sample analysis micro-device 1201. The liquid photocurable resin in the biological sample analysis micro-device 1201 is completely solidified by the light, and a highly robust sample solution separation can be performed. At this time, the light source unit 1204 or the sample setting unit 1200 may also have a driving mechanism, and the position-selective solidification of the liquid photocurable resin can be achieved by the driving mechanism.

[0110] In addition, in the sample solution separation device 1600, liquid photocurable resin needs to be stored in the device, so an environment that blocks light from the outside (light-shielding environment) is required. However, if a light-shielding environment is difficult, the photocurable resin storage unit 1202 can be replaced by a black container.

[0111] It should be noted that, according to the sample solution separation device 1600, the sample solution can be separated, but by further improving the sample setting part 1200 and the light source part 1204, it can also be effectively used as various derivative devices. For example, by adding a heat source part such as a thermal cycler to the sample setting part 1200, PCR or digital PCR can be performed in the micro-device 1201 for biological sample analysis after the sample solution is separated. In addition, the heat source part can also be used to heat the micro-device 1201 for biological sample analysis in the range from room temperature to about 90°C while introducing the sample solution or liquid photocurable resin. As a result, the viscosity of the liquid photocurable resin, which is the above-mentioned subject, can be slightly reduced. In addition, by adding a plurality of wavelengths of excitation light sources, excitation light filters, fluorescence filters, dichroic mirrors, focusing lenses, CCDs, CMOS cameras and other light detectors to the light source part 1204, fluorescence measurement of the micro-device 1201 for biological sample analysis after PCR can be performed.

[0112] <About Improved Microdevices>

[0113] Improvements of the biological sample analysis microdevices of Examples 1 to 3 are described. Fig.17 1 is a diagram showing a schematic cross-sectional configuration example of an improved microdevice. The improved microdevice relates to a sample solution separation package 1700 having a liquid photocurable resin. By using the sample solution separation package, the user does not need to prepare a liquid photocurable resin for separating the sample solution, and can separate the sample solution using the liquid photocurable resin simply by directly connecting the microdevice to a pressure applying device or the like.

[0114] like Fig.16As shown, the sample solution separation package (improved microdevice) 1700 has a substrate 1300, an inlet 1301 for introducing a sample solution, an outlet 1302 for discharging the sample solution and liquid photocurable resin, a cover 1303, a hole 1304 for introducing a sample solution, a photocurable resin storage portion 1305 and a pressure application hole 1306.

[0115] As in the case of Examples 1 to 3, the sample solution is introduced from the inlet 1301 and introduced into the eyelet 1304. After the sample solution is introduced into the eyelet 1304, pressure is applied from the pressure application hole 1306 using an external device (pressure applying device), and the liquid photocurable resin retained in the photocurable resin reservoir 1305 is introduced into the flow path 1307 in the package body, thereby sealing each eyelet 1304. Then, the last is a scheme of photocuring by an external light source. It should be noted that, as described above (refer to Fig.16 ), it is preferred that the surroundings of the photocurable resin storage portion 1305 within the sample solution separation package (improved microdevice) 1700 be set as a light-shielding environment.

[0116] <Conclusion>

[0117] (i) According to the present embodiment, a highly robust sample solution separation technology is proposed in which the sample solution introduced into the multiple holes (micro-areas) of a microdevice for digital PCR or real-time PCR does not leak out of the holes. Specifically, first, the sample solution is introduced into the multiple holes (micro-areas) of the microdevice for biological sample analysis, and then, the liquid photocurable resin is introduced into the flow path of the microdevice for biological sample analysis. Thus, the liquid photocurable resin covers the openings of the multiple micro-areas, so that the sample solutions retained in the multiple micro-areas are separated. Then, the microdevice for biological sample analysis into which the liquid photocurable resin has been introduced is irradiated with light to cure the liquid photocurable resin. Thus, the separation of the sample solutions retained in each micro-area becomes reliable.

[0118] The morphology of the above-mentioned multiple micro-regions is various. For example, in the case of Example 1, Figure 3 As shown in the figure, the microdevice has a main flow path connected from the inlet to the outlet and a plurality of sub-flow paths connected from the main flow path to each micro-region. In this case, first, the sample solution flows into each of the plurality of micro-regions through the inlet, the main flow path and the sub-flow paths. Then, the liquid photocurable resin flows from the inlet into the main flow path, and the plurality of micro-regions are separated. In the case of Example 2, as shown in Figure 6 As shown, the microdevice has a plurality of holes (microdomains) 500 formed on a substrate and open on one surface, and a flow path 501 formed in a manner connecting the openings of the holes. In addition, in the case of Example 3, the microdevice has a plurality of through holes formed on the substrate, and has these through holes as microdomains. That is, Figure 8As shown, the micro-device has a plurality of through holes (micro-domains) 704 formed in a substrate and a flow path 706 formed in a manner connecting the upper and lower openings of each through hole. In this case, a sample solution is flowed into the open holes, and then a liquid photocurable resin is flowed into the flow path 501 and the flow path 706. Thus, the openings of the open holes (Example 2: the openings on the upper surface, Example 3: the upper and lower openings of the through holes) are covered.

[0119] (ii) According to this embodiment, the specific gravity of the liquid photocurable resin used for sample solution separation can be set to the specific gravity of the sample solution + 0.2 g / cm 3 As follows. This can prevent the sample solution from mixing with the liquid photocurable resin in the microdevice or the liquid photocurable resin from entering the holes (microregions) and squeezing out the sample solution held and contained in the holes (microregions). It should be noted that the liquid specific gravity condition of the liquid photocurable resin is applicable when a microdevice having the structure of the above-mentioned embodiment 2 or embodiment 3 is used to perform sample solution separation processing.

[0120] In addition, the viscosity of the liquid photocurable resin is preferably 500 mPa·s or less. Thus, even without applying a high pressure to the extent that the microdevice is destroyed, it can be smoothly introduced into the microdevice. It should be noted that, if the viscosity is taken into consideration, the liquid photocurable resin may not be an oligomer but a resin mainly composed of monomers.

[0121] In addition, the liquid photocurable resin is poorly soluble in water, and when expressed as solubility in water, the solubility in water is less than 100 g / L. In addition, the liquid photocurable resin can be composed of a material having cycloalkanes, normal alkanes or isoalkanes with a carbon number of 6 or more in the molecular skeleton. Thus, the liquid photocurable resin can be made hydrophobic, which can prevent the occurrence of PCR inhibition.

[0122] The refractive index of the liquid photocurable resin can be set to be greater than 1 and less than 2.5. Thus, the light emitted from the sample solution can be prevented from being lost by the photocurable resin. In addition, the liquid photocurable resin has low autofluorescence characteristics for light in the wavelength range of 400nm to 700nm. Thus, the background intensity caused by autofluorescence can be suppressed during PCR measurement, and high-precision PCR measurement can be performed.

[0123] The wavelength of light emitted from the light source to cure the liquid photocurable resin is light in the wavelength range of 250nm to 600nm. By irradiating light near the absorption wavelength of the photoinitiator contained in the liquid photocurable resin, the photopolymerization reaction can be promoted.

[0124] (iii) If Fig.16As shown, the present embodiment also proposes a microdevice having a new structure. Specifically, the microdevice is a sample solution separation device for biological sample analysis, which comprises a substrate, an inlet and an outlet formed on the substrate, a plurality of holes (micro-regions) formed in the substrate portion between the inlet and the outlet, and a photocurable resin storage portion formed on the substrate. In addition, a pressure application hole for applying pressure to discharge the accumulated liquid photocurable resin from the photocurable resin storage portion is formed in the photocurable resin storage portion. In this way, by accumulating the liquid photocurable resin in the microdevice, the user does not need to specially prepare the liquid photocurable resin, and an easy-to-use microdevice can be provided.

[0125] (iv) The technology disclosed in the present invention is not limited to the above-mentioned embodiments, and includes various modified examples. For example, the above-mentioned embodiments are examples that are described in detail in order to easily explain the technology disclosed in the present invention, and are not limited to all the structures described. In addition, a part of the structure of a certain embodiment can be replaced with the structure of other embodiments, and in addition, the structure of other embodiments can be added to the structure of a certain embodiment. Furthermore, with respect to a part of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0126] Explanation of symbols

[0127] 1.107.1204: Light source (part),

[0128] 2, 101, 502, 603, 705, 800, 901, 1300: base material,

[0129] 3: coating layer,

[0130] 4, 105, 304, 905, 1100: liquid light-curing resin,

[0131] 5: Photoinitiator,

[0132] 6: polymer structure,

[0133] 100, 703, 900, 1302: Export department,

[0134] 102, 202, 300, 500, 601, 902, 1304: eyelets,

[0135] 103, 903, 1303: cover,

[0136] 104, 904, 1203: pressure applying device,

[0137] 106, 200, 700, 906, 1301: Entrance Department,

[0138] 108, 305, 400, 600, 802, 1102: solid light-curing resin,

[0139] 201: Main road section,

[0140] 203: sub-flow path part,

[0141] 501, 604, 1307: flow path (part),

[0142] 301: Interface between the sample solution on the flow path and the liquid photocurable resin,

[0143] 302: Interface between the sample solution in the hole and the liquid photocurable resin in the flow path,

[0144] 303, 401, 602, 801, 1101: sample solution,

[0145] 306: the interface between the sample solution in the hole and the solid photocurable resin in the flow path,

[0146] 402, 605, 803: glowing eyes,

[0147] 403, 606, 804: non-luminous holes,

[0148] 701: The area where the through hole is located,

[0149] 702: A port for folding the light-curing resin from the upper surface to the lower surface.

[0150] 704: Through hole eyelet,

[0151] 706: flow path (space),

[0152] 1000: Liquid light-curing resin with high liquid specific gravity.

[0153] 1001: sample solution in the hole,

[0154] 1002: squeezed sample solution,

[0155] 1200: Sample setting department,

[0156] 1201: Microdevices for biological sample analysis,

[0157] 1202, 1305: light curing resin storage unit,

[0158] 1306: Pressure application hole,

[0159] 1600: Sample solution separation device,

[0160] 1700: Sample solution separation package (improved micro device).

Claims

1. A method for separating a sample solution in a microdevice for biological sample analysis, comprising: A step of introducing a sample solution into a plurality of micro-regions of the biological sample analysis micro-device; A step of introducing a liquid photocurable resin into a flow path of the biological sample analysis microdevice, covering the openings of the plurality of microdomains with the liquid photocurable resin, thereby separating the plurality of microdomains into which the sample solution has been introduced; as well as and a step of irradiating the biological sample analysis micro-device into which the liquid photocurable resin has been introduced with light to cure the liquid photocurable resin.

2. The sample solution separation method according to claim 1, wherein: The biological sample analysis microdevice is used for digital PCR or real-time PCR.

3. The sample solution separation method according to claim 1, wherein: The plurality of micro-areas are through-holes formed in a base material of the biological sample analysis micro-device.

4. The sample solution separation method according to claim 1, wherein: Each of the plurality of micro-regions is at least one open hole in a first surface and a second surface opposite to the first surface, The step of introducing the sample solution includes the step of allowing the sample solution to flow into the open hole. The step of isolating the plurality of micro-domains includes flowing the sample solution into the open wells, and then flowing the liquid photocurable resin into a flow path of the biological sample analysis micro-device to cover the openings of the open wells.

5. The sample solution separation method according to claim 3 or 4, wherein: The specific gravity of the liquid photocurable resin is the specific gravity of the sample solution + 0.2 g / cm 3 the following.

6. The sample solution separation method according to claim 1, wherein: The biological sample analysis micro-device comprises a main flow path connected from an inlet to an outlet and a plurality of sub-flow paths connected from the main flow path to each micro-region. The step of introducing the sample solution includes the step of allowing the sample solution to flow into each of the plurality of micro-regions via the inlet, the main flow path, and the sub-flow path. The step of separating the plurality of micro-domains includes the step of separating the plurality of micro-domains by flowing the liquid photocurable resin from the inlet into the main channel after flowing the sample solution into the plurality of micro-domains.

7. The sample solution separation method according to claim 1, wherein: The viscosity of the liquid photocurable resin is 500 mPa·s or less.

8. The sample solution separation method according to claim 7, wherein: The liquid photocurable resin is composed of a monomer material.

9. The sample solution separation method according to claim 1, wherein: The liquid photocurable resin is poorly soluble in water.

10. The sample solution separation method according to claim 9, wherein: The solubility of the liquid photocurable resin in water is less than 100 g / L.

11. The sample solution separation method according to claim 9, wherein: The liquid photocurable resin is composed of a hydrophobic material having cycloalkane, normal alkane or isoalkane having 6 or more carbon atoms in its molecular skeleton.

12. The sample solution separation method according to claim 1, wherein: The liquid photocurable resin has a refractive index of 1 or more and 2.5 or less.

13. The sample solution separation method according to claim 1, wherein: The liquid photocurable resin has a low autofluorescence characteristic to light in a wavelength range of 400 to 700 nm.

14. The sample solution separation method according to claim 1, wherein: The light for curing the liquid photocurable resin is light having a wavelength in the range of 250 to 600 nm.

15. A sample solution separation device, which is a sample solution separation device for biological sample analysis, comprising: Base material; an inlet portion, formed on the substrate, for introducing a sample solution; an outlet portion formed on the substrate for discharging at least excess sample solution; A plurality of micro-areas formed on the substrate for holding the introduced sample solution; as well as a photocurable resin storage portion formed on the substrate and used to store liquid photocurable resin; The photo-curable resin reservoir has a pressure application hole formed therein for applying pressure to discharge the stored liquid photo-curable resin from the photo-curable resin reservoir.

16. A sample solution separation device for separating a sample solution introduced into a plurality of micro-regions of a micro-device for biological sample analysis, comprising: a device placement portion on which the biological sample analysis microdevice is placed; a photocurable resin storage unit storing a liquid photocurable resin, wherein the liquid photocurable resin is introduced into the biological sample analysis micro-device after the sample solution is introduced into the plurality of micro-regions; a pressure applying device for applying pressure to the photocurable resin storage portion to introduce the liquid photocurable resin into the biological sample analysis micro-device; as well as A light irradiation unit irradiates light onto the liquid photocurable resin introduced into the biological sample analysis micro-device.

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