Analysis pretreatment method

Abstract

To fix samples contained in respective droplets in mutually separated states without performing operation on each individual droplet.SOLUTION: A pretreatment method for analysis of samples, comprising a step of preparing an emulsion 7 including an oil 6A and first and second droplets 501, 502 present in the oil 6A and accommodating first and second samples, respectively, a step of placing an aggregate 5A of the first and second droplets 501, 502 in the emulsion 7 on a substrate 1, and a step of evaporating an oil 6C and water on the substrate 1 to separate first and second evaporation residues 511, 512 respectively containing the first and second samples from each other.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to pre-analysis methods, and more particularly to pre-analysis methods for analyzing samples in droplets. [Background technology]

[0002] One of the cell culture methods is to culture cells in droplets of water containing cells and encapsulated in a culture medium dispersed in oil. In this method, droplets are used as independent culture vessels separated by oil.

[0003] Non-Patent Document 1 discloses a technique for regularly arranging droplets one by one on a slide glass using a three-axis micromanipulator. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sara E. Bell et al., “Droplet Microfluidics with MALDI-MS Detection: The Effects of Oil Phases in GABA Analysis”, ACS Measurement Science, 2021, 1, 3, pp. 147-156., https: / / doi.org / 10.1021 / acsmeasuresciau.1c00017 Summary of the Invention [Problem to be solved by the invention]

[0005] It is conceivable to use the technique of Non-Patent Document 1 as a pretreatment method for sample analysis, but there is room for improvement in throughput.

[0006] It is an object of the present invention to provide a high-throughput method for immobilizing samples in droplets while keeping them separate from each other. [Means for solving the problem]

[0007] An analytical pretreatment method according to one aspect of the present disclosure is a pretreatment method for sample analysis, and includes the steps of preparing an emulsion containing oil and first and second droplets present in the oil and containing first and second samples, respectively; placing a collection of the first and second droplets in the emulsion on a substrate; and evaporating the oil and water on the substrate to separate the first and second evaporation residues containing the first and second samples, respectively, from each other. [Effects of the Invention]

[0008] The pre-analysis method according to the present disclosure can provide a high-throughput method for immobilizing samples in each droplet in a state where the samples are separated from each other. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a schematic configuration diagram of an analysis system according to an embodiment. [Figure 2] FIG. 10 is a diagram for explaining pre-processing according to the embodiment. [Figure 3] FIG. 10 is a diagram for explaining pre-processing according to the embodiment. [Figure 4] 1 is a flowchart illustrating an example of a preprocessing method according to an embodiment. [Figure 5] FIG. 1 shows droplets during drying with different concentrations of surfactant. [Figure 6] FIG. 1 shows mass spectra obtained using surfactants at different concentrations. [Figure 7] FIG. 1 shows evaporation residue under different humidity conditions. [Figure 8] FIG. 10 is a diagram for explaining pre-processing according to a modified example. [Figure 9]10 is a flowchart illustrating an example of a preprocessing method according to a modified example. [Figure 10] FIG. 1 is a diagram illustrating sample collection in an example. [Figure 11] FIG. 1 is a diagram illustrating analysis results in an example. [Figure 12] FIG. 1 is a diagram illustrating analysis results in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.

[0011] [Analysis system configuration] FIG. 1 is a schematic diagram of an analysis system 100 according to this embodiment.

[0012] The analysis system 100 includes an experimental device 8 and a control device 9. The experimental device 8 includes a pre-processing device 81 and an analysis device .

[0013] The pretreatment device 81 performs a pretreatment method for analyzing a sample according to this embodiment. In this specification, the pretreatment method for analyzing a sample is also referred to as an "analysis pretreatment method."

[0014] The analytical device 82 analyzes the sample pretreated by the pretreatment device 81. In one embodiment, the analytical device 82 is a mass spectrometer. In a more specific example, the analytical device 82 is a mass spectrometer that performs MALDI analysis.

[0015] The control device 9 controls the pretreatment device 81 and the analytical device 82. The control device 9 may monitor the process and / or results of the pretreatment by the pretreatment device 81, and may analyze the process and / or results of the analysis by the analytical device 82.

[0016] The control device 9 includes a processor 90 , a memory 91 , an input device 92 , and a display device 93 .

[0017] The processor 90 includes, for example, a CPU (Central Processing Unit). The processor 90 loads a program stored in the memory 91 into a RAM or the like and executes the program.

[0018] The memory 91 includes, for example, a read-only memory (ROM), a random access memory (RAM), and a non-volatile memory. The program stored in the ROM is a program in which the processing procedures of the control device 9 are written. The non-volatile memory stores data sent from the pre-processing device 81 and / or the analytical device 82. The memory 91 may include a hard disk drive instead of or in addition to the non-volatile memory.

[0019] The input device 92 is a device for inputting user instructions to the control device 9. For example, the input device 92 includes a keyboard and a pointing device such as a mouse.

[0020] The display device 93 includes a liquid crystal display, etc. The display device 93 may display the process and / or results of the pretreatment by the pretreatment device 81, or the process and / or results of the analysis by the analysis device 82.

[0021] [Pretreatment method according to the embodiment] The pre-processing method according to the embodiment may be performed by the control device 9 controlling the pre-processing device 81, or may be performed manually by a user.

[0022] 2 and 3 are diagrams illustrating a pretreatment process according to an embodiment. Referring to FIG. 2, the oil 6 may be referred to as "a first oil 6A before being placed on the substrate 1 and in which droplets 5 are dispersed," "a second oil 6B pre-placed on the substrate 1," or "a third oil 6C after being placed on the substrate 1 and in which droplets 5 are dispersed." Note that, in this specification, "droplets 5 dispersed in the oil 6" does not necessarily mean that the droplets 5 in the oil 6 are spaced apart from one another; at least some of the droplets 5 may be in contact with one another. FIGS. 2A to 2C show common steps in the pretreatment process according to an embodiment. FIGS. 3D to 3E show steps specific to the pretreatment process for MALDI analysis. FIGS. 3F to 3G show steps for MALDI analysis and MALDI imaging. In other words, FIGS. 2 and 3A to 3E show a pretreatment process for MALDI analysis, which is an example of a pretreatment process according to an embodiment.

[0023] Fig. 4 is a flowchart showing an example of a pre-processing method according to an embodiment. Each step (hereinafter also referred to as "ST") in Fig. 4 may be performed by the control device 9 controlling the pre-processing device 81, or may be performed manually by an analyst. Below, the pre-processing according to an embodiment will be described along Fig. 4, with reference to Figs. 2 and 3 as appropriate.

[0024] In ST1 of Fig. 4, an emulsion 7 is prepared. The emulsion 7 includes oil 6 and droplets 5 present in the oil 6, each containing a sample. More specifically, the emulsion 7 includes oil 6 and first and second droplets 501 and 502 present in the oil 6 and containing first and second samples, respectively.

[0025] In one embodiment, the sample contains cells and / or cell-derived materials. Cells are composed of metabolites such as proteins, lipids, nucleic acids, and carbohydrates. In this specification, cell-derived materials typically include metabolites excreted from cells, but may also include parts of disrupted cells. Therefore, by analyzing the components contained in the droplet 5, it is possible to analyze the cells themselves (e.g., bacterial cells) or metabolites inside and outside the cells. In a typical example, the droplet 5 also contains a cell culture medium containing the sample. In this configuration, the droplet 5 can be used as an incubator for specific cells.

[0026] The emulsion 7 is a water-in-oil emulsion, and is produced by, for example, using a microfluidic device to generate droplets 5 containing a culture medium containing cells in oil 6.

[0027] When droplets 5 are generated, each droplet 5 is preferably adjusted to contain one or fewer cells. In this manner, each droplet 5 contains a sample derived from a single cell. In other words, each droplet 5 contains only one type of cell and a substance derived from that cell. This makes analysis of each sample easier than when each droplet 5 contains multiple types of cells, and is particularly much easier than when each droplet 5 contains a large number of cells.

[0028] For example, even if a droplet 5 is found to have a high rate of increase of a certain metabolite, if the droplet 5 contains multiple types of cells, it is difficult to identify the specific type of cell that has a high rate of increase of the metabolite. Furthermore, in this case, the droplet 5 contains as many genes related to the production of the metabolite as there are cell types, making it difficult to identify the gene responsible for the high rate of increase of the metabolite. These difficulties are greater when the droplet 5 contains multiple types of cells than when it contains only two or three types of cells. Therefore, it is preferable to contain as few types of cells as possible in each droplet 5, and more specifically, to configure each droplet 5 so that it contains only one type of cell. Furthermore, it is preferable to fix the sample under conditions that prevent multiple droplets 5 from fusing together during fixation. Note that, as used herein, "gene" refers to a DNA (deoxyribonucleic acid) sequence and / or an RNA (ribonucleic acid) sequence.

[0029] In one embodiment, the first and second samples contained in the first and second droplets 501 and 502 are samples of the same type. More specifically, the first and second samples contained in the first and second droplets 501 and 502 are samples derived from the same type of cell. With this configuration, by analyzing the first and second samples, respectively, it is possible to analyze how multiple types of samples derived from the same type of cell change. Similarly, if the first and second samples are samples from the same strain, it is possible to analyze how multiple types of samples derived from the same strain of cell change.

[0030] In ST2, an assembly 5A of droplets 5 in emulsion 7 is placed on substrate 1. More specifically, an assembly 5A of first and second droplets 501 and 502 in emulsion 7 is placed on the substrate. Here, "assembly 5A of first and second droplets 501 and 502" refers to an assembly 5A of multiple droplets including first and second droplets 501 and 502. Substrate 1 has surface 11 on which assembly 5A of droplets 5 is placed. Forming surface 11 into a planar shape makes it easy to simultaneously analyze multiple droplets 5 on surface 11. In one embodiment, substrate 1 is a substrate having two opposing flat surfaces, surface 11 and surface 12.

[0031] As a more specific example, the substrate 1 is a sample plate for mass spectrometry using the MALDI method. In this specification, mass spectrometry using the MALDI method is also referred to as "MALDI analysis," and a sample plate for MALDI analysis is also referred to as "MALDI plate." When a MALDI plate is used as the substrate 1, MALDI analysis can be easily performed by performing the pretreatment method according to this embodiment on the sample on the substrate 1 and then moving the substrate 1 to the mass spectrometer 1. In particular, by performing MALDI imaging, which will be described later, analysis of each sample on the substrate 1 can be performed with good throughput.

[0032] In this specification, a plane parallel to the base 1 is sometimes referred to as the XY plane, and a direction perpendicular to the XY plane is sometimes referred to as the Z axis direction. Furthermore, the positive direction of the Z axis (the direction from surface 12 toward surface 11 of the base 1) is sometimes referred to as "upward," and the negative direction of the Z axis (the direction from surface 11 toward surface 12 of the base 1) is sometimes referred to as "downward."

[0033] In this specification, the assembly 5A includes a plurality of droplets 5. In other words, the assembly 5A includes a group of droplets 5. The assembly 5A corresponds to one example of an "assembly."

[0034] In one embodiment of ST2, the aggregate 5A is dropped onto the surface 11. In another embodiment, the aggregate 5A may be scooped up with a spoon and placed on the surface 11. The aggregate 5A may also be placed on the surface 11 using a dropper or the like whose tip touches the surface 11.

[0035] In one embodiment, droplets 5 are formed in oil with a surfactant concentration of approximately 2%. Droplets 5 become unstable in oils with lower surfactant concentrations. Therefore, when considering immobilization using lower surfactant concentrations, a low-concentration second oil 6B is pre-applied to the portion of surface 11 where aggregates 5A will be placed, and droplets 5 in oil with a surfactant concentration of approximately 2% are then dropped onto the second oil 6B. This allows droplets 5 to be stably maintained until just before immobilization. As described above, second oil 6B may be pre-applied to the portion of surface 11 where aggregates 5A will be placed. However, in other embodiments, second oil 6B may not be pre-applied to the portion of surface 11 where aggregates 5A will be dropped. This configuration eliminates the need to pre-apply second oil 6B to surface 11.

[0036] In ST3, the oil 6 (the third oil 6C in FIG. 2) and water on the substrate 1 are evaporated, separating the evaporation residues 51 containing the respective samples. More specifically, the oil 6 and water on the substrate 1 are evaporated, separating the first and second evaporation residues 511 and 512 containing the first and second samples, respectively. The first evaporation residue 511 is the dried product of the first droplet 501, and the second evaporation residue 512 is the dried product of the second droplet 502. The water may be, for example, water contained in the culture medium in the droplet 5 and / or water contained in the cells. In the example of FIG. 2, the third oil 6C is a mixture of the first oil 6A and the second oil 6B. In an embodiment in which the second oil 6B is not previously disposed on the substrate 1, the third oil 6C is the same oil as the first oil 6A.

[0037] In ST3, the oil 6 and water are removed from the substrate 1, and the droplets 5 placed on the substrate 1 become evaporation residue 51. In this specification, drying the oil 6 and water from the emulsion 7 to generate evaporation residue 51 in a state in which the sample can be analyzed is also referred to as "fixation." However, evaporation residue 51 may contain oil 6, surfactant, and / or water to the extent that it does not interfere with sample analysis.

[0038] In this specification, a group of evaporation residues 51 including a plurality of evaporation residues 51 is referred to as an evaporation residue aggregate 51A.

[0039] In ST4, a matrix solution is deposited onto the sample on the substrate 1. More specifically, the matrix solution is deposited onto the first and second samples on the substrate 1. ST4 is performed after ST3.

[0040] A matrix solution is a solution containing a matrix substance. A matrix substance is a substance that easily absorbs laser light energy and ionizes compounds contained in an analytical sample. Examples of matrix substances include α-cyano-4-hydroxycinnamic acid (4-CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA).

[0041] In one embodiment of ST4, the matrix solution is sprayed onto the evaporation residue 51 on the MALDI plate. This spraying causes the matrix solution to be deposited onto the samples in the evaporation residue 51. With this configuration, even if the samples are irregularly distributed on the MALDI plate, the matrix solution can be easily deposited onto each sample.

[0042] 3 can be subjected to MALDI analysis. In one embodiment, a laser beam is scanned over the entire evaporation residue assembly 51A on the substrate 1 to perform MALDI analysis to simultaneously obtain analysis results for the entire evaporation residue assembly 51A, and then MALDI imaging is performed to image the results of the MALDI analysis.

[0043] The mesh pattern on the substrate 1 in Figure 3(F) represents the traces of laser irradiation during MALDI analysis (see Figure 9). In addition, in MALDI imaging, coloring (brightness in Figure 3(G)) indicates the strength of signal intensity. This makes it possible to express the distribution of signal intensity on the substrate 1 as an image. This allows the distribution of substances corresponding to a specific m / z to be visually understood as a distribution of colors indicating the signal intensity at that m / z.

[0044] As described above, MALDI imaging makes it possible to simultaneously obtain mass spectrometry results for each sample placed at each position on the substrate 1. This makes it possible to analyze the components contained in each sample with good throughput.

[0045] As shown in FIG. 3, after spraying the matrix solution, methanol (MeOH) may be sprayed before MALDI analysis is performed.

[0046] As described above, according to the embodiment, the process of isolating the droplets 5 and the process of placing each droplet 5 at a predetermined position on the substrate 1 are eliminated, and samples derived from each droplet 5 can be immobilized on the substrate 1 without being mixed with each other. In other words, a high-throughput method can be provided for immobilizing samples in each droplet while keeping them separate from each other. Therefore, the throughput of immobilizing samples in each droplet 5 in the emulsion 7 can be improved compared to conventional methods. This also improves the overall throughput of analyzing the samples contained in each droplet 5 in the emulsion 7.

[0047] In the process of Figure 4, the sample contained in droplet 5 may be a microorganism, and the process of Figure 4 may further include a step of culturing the microorganism in emulsion 7. More specifically, in the process of Figure 4, the first and second samples contained in droplets 501 and 502 may be microorganisms, and the process of Figure 4 may further include a step of culturing the microorganism in emulsion 7. With this configuration, the microorganism itself in droplet 5 and / or a predetermined metabolite produced by the microorganism can be grown to a sufficient amount before being subjected to analysis. The sufficient amount is, for example, an amount suitable for analysis.

[0048] In one embodiment, the aggregate 5A is produced in the microfluidic device, then cultured in a predetermined container for a predetermined period of time, and then placed on the substrate 1. In another embodiment, the aggregate 5A may be produced in the microfluidic device, then immediately placed on the substrate 1, and cultured on the substrate 1. Furthermore, if a culture step before analysis is not required, the aggregate 5A may be produced in the microfluidic device, then immediately placed on the substrate 1, and then analyzed.

[0049] Furthermore, samples on the substrate 1 can be managed according to their positions (e.g., XY coordinates) on the substrate 1. Since the traceability of each sample is ensured in this way, it is also possible to perform other analyses, such as genetic analysis, on any sample on the substrate 1 that has been focused on by MALDI analysis (see the modified example described below).

[0050] In one embodiment, the volatility of the oil 6 is equal to or higher than that of water. This configuration allows the oil 6 to evaporate at a rate equal to or higher than that of water in ST3. As a result, the shape of the droplets 5 can be prevented from being damaged by the oil 6 during water evaporation. Furthermore, since the oil 6 has also completely evaporated by the time the water has completely evaporated, the remaining oil 6 does not damage the shape of the droplets 5. The "oil more volatile than water" described above is, for example, fluorine oil. Fluorine oil is sometimes referred to as fluorine-based oil by those skilled in the art.

[0051] Furthermore, if an oil having a higher specific gravity than water is used as the oil 6, the droplets 5 will rise to the surface of the emulsion 7 in ST3, and the emulsion 7 can be dried while the droplets 5 remain in a single phase without overlapping each other. As a result, by using an oil having a lower specific gravity than water, the droplets 5 are less likely to fuse together during drying than when the emulsion is dried with the droplets submerged in the emulsion. From this perspective, it is preferable to use a fluorinated oil having a higher specific gravity than water as the oil 6.

[0052] Next, the environmental conditions for performing ST3 (the fixation step) will be described in detail. The inventors have investigated various conditions and found that the concentration of the surfactant in the oil 6 and the humidity around the substrate 1 are important for preventing the droplets 5 from fusing together. Therefore, in this specification, the environmental conditions for preventing the droplets 5 from fusing together include a concentration condition in which the concentration of the surfactant in the oil 6 on the substrate 1 during fixation (ST3) is within a predetermined concentration range, and / or a humidity condition in which the humidity around the substrate 1 during fixation (ST3) is within a predetermined humidity range. By adjusting the concentration condition and / or humidity condition as described below, an analyzable evaporation residue 51 and / or an evaporation residue 51 suitable for analysis can be generated.

[0053] In ST3, the temperature around the base 1 is preferably room temperature, which may be 20°C to 25°C, for example.

[0054] [Surfactant concentration conditions] The surfactant used in this embodiment is preferably one that is easily mixed with the fluorine oil and that can stably hold the droplets 5 when mixed with the fluorine oil. From this perspective, a fluorine-based surfactant is preferably used in this embodiment. Examples of commercially available fluorine-based surfactants that can be used include 008-FluoroSurfactant (RAN Biotechnologies) and Pico-Surf (registered trademark, Sphere Fluidics).

[0055] The concentration of the surfactant is not limited thereto, but is, for example, 1% by mass or more and 4.5% by mass or less, and preferably 1% by mass or more and 2% by mass or less. By configuring in this manner, an analyzable evaporation residue 51 and / or an evaporation residue 51 suitable for analysis is generated, as exemplified below.

[0056] In this specification, when the concentration of a surfactant in the oil 6 is simply expressed as "%", it means mass % (w / w%) unless otherwise specified.

[0057] Fig. 5 shows droplets 5 being dried when surfactants of different concentrations are used. Fig. 5 shows droplets 5 being dried under concentration conditions 1, 2, 4, and 5. In this specification, concentration conditions 1 to 5 correspond to the following conditions: the amount of first oil 6A in which droplets 5 are dispersed before being placed on substrate 1 is 1 μL, the surfactant concentration in first oil 6A is 2%, the amount of second oil 6B placed on substrate 1 in advance is 5 μL, and the surfactant concentrations in second oil 6B are 0%, 0.1%, 1%, 2%, and 5%, respectively.

[0058] The concentration of the third oil 6C after the droplet 5 is placed on the substrate 1 is {(amount of the first oil) × (surfactant concentration in the first oil) + (amount of the second oil) × (surfactant concentration in the second oil)} / {(amount of the first oil) + (amount of the second oil)}. Therefore, under concentration conditions 1 to 5, the concentrations of the surfactant in the third oil 6C are 0.33%, 0.42%, 1.17%, 2.00%, and 4.50%, respectively.

[0059] Other environmental conditions included a humidity of approximately 30% and room temperature for the substrate 1. 2% (w / w) 008-FluoroSurfactant (RAN Biotechnologies) was used as the first oil 6A. 2% (w / w) 008-FluoroSurfactant (RAN Biotechnologies) diluted appropriately with fluorine oil was used as the second oil 6B for concentration conditions 2 to 5. Fluorine oil was used as the second oil 6B for concentration condition 1.

[0060] Referring to FIG. 5, it can be seen that the higher the surfactant concentration in the oil 6 (third oil 6C) on the substrate 1, the less overlapping the droplets 5 are in the XY plane and the greater the distance between the droplets 5. Furthermore, while the droplets 5 sometimes fused under concentration conditions 1 and 2, they did not fuse under concentration conditions 4 and 5. Furthermore, under concentration condition 3, the droplets 5 did not fuse. Therefore, in terms of preventing the droplets 5 from fusing, it is preferable that the surfactant concentration in the oil 6 on the substrate 1 be higher than that under concentration condition 3 (approximately 1%). Furthermore, in terms of increasing the distance between the droplets 5, a higher surfactant concentration in the oil 6 on the substrate 1 is preferable. For example, referring to FIG. 5, concentration condition 5 is preferable because the gaps between the droplets 5 are larger than those under concentration condition 4.

[0061] Figure 6 shows mass spectra obtained using surfactants at different concentrations. Figure 6 shows mass spectra of samples pretreated under concentration conditions 3 to 5. The upper panel of Figure 6 shows the mass spectrum near m / z = 808.118, which corresponds to acetyl-CoA. The lower panel of Figure 6 shows the mass spectrum near m / z = 191.019, which corresponds to citric acid. Referring to Figure 6, in the mass spectrum under concentration condition 5, peaks near m / z = 808.118 and m / z = 191.019 are detected, but their intensities are relatively weak. In particular, the peak near m / z = 808.118 is not particularly clear compared to the surrounding signal intensities. On the other hand, in the mass spectra under concentration conditions 3 and 4, the peaks near m / z = 808.118 and m / z = 191.019 are clearly detected. Furthermore, in the mass spectrum under concentration condition 3, higher peaks are detected than in the mass spectrum under concentration condition 4. 6, it is preferable that the concentration of surfactant in the oil 6 on the substrate 1 is low. This is thought to be because when the concentration of surfactant in the oil 6 on the substrate 1 is high, the amount of surfactant contained in the evaporation residue 51 also increases, which inhibits mass spectrometry. For example, it is thought that phenomena such as the laser light being incident on the surfactant and preventing it from reaching the sample and / or inhibiting ionization after the laser light has reached the sample occur.

[0062] To summarize the above results, under concentration conditions 3 and 4, the fusion of droplets 5 on the substrate 1 can be suppressed and a clear peak is observed in the mass spectrum. Therefore, the surfactant concentration in oil 6 on the substrate 1 is preferably about 1 to 2%, which corresponds to concentration conditions 3 and 4.

[0063] [Humidity conditions] The humidity around the substrate 1 during fixation is, but is not limited to, 75% or less relative humidity, preferably 50% or less. This relative humidity is, for example, the relative humidity of the atmosphere in a closed system (inside a box). This humidity is adjusted using a humidity adjustment unit such as the AS ONE Humidity Adjustment Unit (Dehumidifier) ​​STU-1. This humidity is measured, for example, using an A&D Thermo-Hygrometer with External Sensor (with internal and external temperature and humidity sensors) AD5648A. More specifically, if the coffee ring effect does not affect subsequent applications or analyses, a humidity of 75% or less is sufficient. On the other hand, for applications or analyses where a more uniform evaporation residue 51 is desired, a humidity of 50% or less is preferable, and even lower humidity is even more preferable. The coffee ring effect generally refers to the phenomenon in which particles within a droplet migrate to the periphery of the droplet as it dries, accumulate at the periphery, and form a ring-shaped residue. The coffee ring effect in this embodiment refers to the phenomenon in which, when the droplet 5 is dried, a substance (such as a sample) in the droplet 5 moves to the periphery of the droplet and dries to form a rim around the periphery. With the above configuration, an analyzable evaporation residue 51 and / or an evaporation residue 51 suitable for analysis is generated, as exemplified below.

[0064] FIG. 7 shows the evaporation residue under different humidity conditions. The droplets 5 in FIG. 7 are filled with water containing dissolved food coloring in order to confirm the positions of the droplets 5. With this configuration, the positions on the substrate 1 where the droplets 5 are fixed are colored. Therefore, the distribution of the positions where the droplets 5 are fixed can be confirmed based on the distribution of the coloring on the substrate 1.

[0065] FIG. 7 shows the evaporation residue 51 when dried under humidity conditions 1 to 4. In this specification, humidity conditions 1 to 4 correspond to conditions where the humidity around the substrate 1 is 30%, 40%, 50%, and approximately 75%, respectively. Under humidity conditions 1 to 3, the humidity was adjusted using an AS ONE humidity adjustment unit (dehumidifier) ​​STU-1. Under humidity condition 4, the humidity around the substrate 1 was not adjusted, resulting in a humidity of approximately 75%.

[0066] Other environmental conditions included room temperature. The amount of first oil 6A in which droplets 5 were dispersed before being placed on substrate 1 was 1 μL, and the surfactant concentration in first oil 6A was 1%, while the amount of second oil 6B placed on substrate 1 in advance was 5 μL, and the surfactant concentration in second oil 6B was 1%. The first oil 6A and second oil 6B were prepared by diluting 2% (w / w) 008-FluoroSurfactant (RAN Biotechnologies) with fluorine oil.

[0067] 7, the lower the humidity during fixing, the more clearly independent evaporation residues 51 are formed. On the other hand, the higher the humidity, the less clear the boundaries between the evaporation residues 51 become.

[0068] As a result, the lower the humidity during fixation, the easier it is to identify the positions corresponding to the evaporation residues 51. Furthermore, when the humidity is low, it can also be confirmed that the droplets 5 do not fuse together during fixation.

[0069] Furthermore, the lower the humidity during fixation, the faster the water in the droplets 5 dries, suppressing the coffee ring effect and allowing the droplets 5 to be fixed in a uniformly spread state. Referring to Figure 7, the lower the humidity during fixation, the higher the percentage of the evaporation residue 51 that is uniformly colored both inside and outside, and the higher the percentage of the peripheral area that is colored over the entire circumference rather than just a portion. This indicates that the lower the humidity during fixation, the more uniformly the contents of the droplets 5 are distributed.

[0070] [Pretreatment method according to modified example] Genetic analysis can also be performed on samples prepared by the pretreatment method according to the embodiment. A modified example of pretreatment for such genetic analysis will be described below. Genetic analysis corresponds to an example of "analysis of a sample."

[0071] The pre-processing method according to the modified example may be performed by the control device 9 controlling the pre-processing device 81, or may be performed manually by the user.

[0072] Figure 8 is a diagram for explaining pre-processing according to a modified example. In the modified example, the steps in Figures 2(A) to (C) and 3(D) to (E) are the same as those in the embodiment, so they are omitted from Figure 8. Figure 8(J) shows steps specific to pre-processing for genetic analysis. Figure 8(K) shows steps for genetic analysis.

[0073] Fig. 9 is a flowchart showing an example of a preprocessing method according to a modified example. Each step in Fig. 9 may be performed by the control device 9 controlling the preprocessing device 81, or may be performed manually by an analyst. Below, the preprocessing according to the modified example will be described along Fig. 9, with appropriate reference to Fig. 8.

[0074] ST1 to ST4 in FIG. 9 correspond to ST1 to ST4 in FIG.

[0075] In ST5, MALDI analysis is performed on the sample prepared in ST4 so that it can be analyzed by MALDI.

[0076] In ST6, the sample is collected from the substrate 1. Specifically, for example, a predetermined evaporation residue is scraped off from the substrate 1 using a capillary. In one embodiment, the predetermined evaporation residue is found by MALDI analysis to contain a predetermined substance in a larger amount than other evaporation residues.

[0077] In ST7, the collected sample is prepared for genetic analysis. More specifically, at least one of the first and second samples on the substrate 1 is prepared for genetic analysis. One example of preparation for genetic analysis includes mixing the collected sample with a PCR (polymerase chain reaction) reaction solution. The PCR reaction solution contains substances necessary for PCR (primers for amplifying a predetermined gene sequence, heat-resistant DNA polymerase, etc.).

[0078] The sample that has undergone the pretreatment shown in Figure 8 can be subjected to genetic analysis. For example, the sample can be subjected to size separation by electrophoresis or sequence analysis.

[0079] In the example of Figure 8, genetic analysis can be performed on samples that have undergone MALDI analysis. This allows multiple types of biological characteristics (e.g., proteins and genes) to be revealed for samples contained in a given evaporation residue. For example, it is possible to analyze genes contained in a given evaporation residue that contains a higher amount of a given substance than other evaporation residues. This allows the search for genes responsible for the production of a higher amount of the given substance.

[0080] In another embodiment of the modified example, ST4 to ST5 may not be performed, and ST6 and ST7 may be performed after ST1 to ST3. In other words, the samples (FIG. 2(C)) fixed in a state separated from each other up to ST3 may be collected using a capillary or the like and subjected to genetic analysis.

[0081] According to a variant, it is possible to genetically analyze at least one of the samples in each fixed droplet in a high-throughput manner.

[0082] [Example] An example of an embodiment and a modification will be described below.

[0083] Figure 10 is a diagram illustrating sample recovery in an example. The left and right figures in Figure 10 are photographs of the MALDI plate before and after recovery, respectively, taken from the side of the substrate 1 where the sample is not fixed (surface 12 side). The left figure shows multiple evaporation residues (e.g., evaporation residues indicated by reference numeral 34) on the MALDI plate, as well as traces of laser irradiation during MALDI analysis. The right figure is a photograph of the evaporation residues indicated by reference numeral 34 in the left figure after they have been recovered with the capillary indicated by reference numeral 32. The right figure shows traces 36 where the evaporation residues indicated by reference numeral 34 have been scraped off with the capillary.

[0084] In this example, the pretreatment shown in Figure 8 was performed on Escherichia coli (E. coli). Specifically, multiple droplets were created from a bacterial culture of E. coli, and the multiple droplets were immobilized on a substrate. MALDI analysis was performed on the evaporation residue obtained on the substrate, and the evaporation residue was then collected, prepared, and PCR was performed. 16S rRNA gene universal primers were used as PCR primers. More specifically, 8F (also known as 27F) was used as the forward primer, and 1492R was used as the reverse primer. If the PCR product is electrophoresed, a band of approximately 1,500 bp (theoretically 1,483 bp) is expected to be detected if the sample contains E. coli 16S rRNA. Actual electrophoresis results are shown in Figures 11 and 12.

[0085] Figure 11 is a photograph of a gel in which PCR products were electrophoresed, and Figure 12 is a table explaining the samples corresponding to each lane of the gel.

[0086] Referring to Figures 11 and 12, lane 1 shows the results of electrophoresis of size markers. Perfect DNA (registered trademark) Markers, 0.5-12 kbp (Novagen) were used as size markers. The bands indicated by symbols 381, 382, ​​and 383 correspond to 2000 bp, 1500 bp, and 1000 bp, respectively. In Figure 12, the range of approximately 1500 bp corresponding to 16S rRNA is indicated by box 39.

[0087] Lane 3 shows the results of genetic analysis of one droplet of the evaporation residue that was collected and prepared. Since no bands are visible in lane 3, it is believed that the evaporation residue collected in lane 3 was originally a droplet that did not contain E. coli.

[0088] Lane 4 shows the results of genetic analysis of multiple droplets of evaporation residue that were collected and prepared. A band corresponding to approximately 1500 bp was detected in lane 4. This suggests that at least one of the droplets corresponding to the multiple evaporation residues contained E. coli.

[0089] Lane 5 shows the results of genetic analysis of a wide range of evaporation residues that were collected and prepared together. A band corresponding to approximately 1500 bp is detected in lane 5, and is stronger than that in lane 4. Therefore, it is believed that at least one of the multiple droplets corresponding to the wide range of evaporation residues contained E. coli.

[0090] Lane 6 shows the results of electrophoresis of a control E. coli culture solution subjected to PCR, in which a band containing approximately 1500 bp was detected.

[0091] As described above, according to this example, even after MALDI analysis was performed, DNA derived from E. coli was detected from the evaporation residue fixed on the substrate 1. This demonstrates that the genetic information of the cells contained in the droplets can be analyzed from the sample fixed on the substrate 1 by the pretreatment methods according to the embodiments and modifications.

[0092] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0093] (Item 1) An analytical pretreatment method according to one embodiment is a pretreatment method for analyzing a sample, and includes the steps of preparing an emulsion containing oil and first and second droplets present in the oil and containing first and second samples, respectively; placing a collection of the first and second droplets in the emulsion on a substrate; and evaporating the oil and water on the substrate to separate the first and second evaporation residues containing the first and second samples, respectively, from each other.

[0094] The pre-analysis method described in item 1 can provide a high-throughput method for immobilizing samples in each droplet in a state where the samples are separated from each other.

[0095] (Item 2) In the analysis pretreatment method according to item 1, the first and second samples are samples of the same type.

[0096] According to the analytical pretreatment method described in item 2, by analyzing the first and second samples, it is possible to analyze how each of a plurality of types of samples derived from the same type of cells changes.

[0097] (Item 3) In the analysis pretreatment method according to item 1 or 2, the first and second samples are microorganisms, and the analysis pretreatment method further includes a step of culturing the microorganisms in an emulsion.

[0098] According to the analytical pretreatment method described in paragraph 3, the microorganisms themselves in the droplets and / or the specified metabolites produced by the microorganisms can be grown to a sufficient amount before being subjected to analysis.

[0099] (4) In the pre-analysis method according to any one of paragraphs 1 to 3, the environmental conditions under which the evaporation step is carried out include a concentration condition in which the concentration of the surfactant in the oil on the substrate is within a predetermined concentration range, and / or a humidity condition in which the humidity around the substrate is within a predetermined humidity range.

[0100] According to the pre-analysis method described in Section 4, an analyzable evaporation residue 51 and / or an evaporation residue 51 suitable for analysis can be generated.

[0101] (Item 5) In the analysis pretreatment method according to item 4, the concentration range is 1% by mass or more and 4.5% by mass or less.

[0102] According to the pre-analysis method described in Section 5, an evaporation residue 51 that can be analyzed is produced.

[0103] (Item 6) In the pre-analysis method according to item 4 or 5, the humidity range is 75% or less.

[0104] According to the pre-analysis method described in Section 6, an evaporation residue 51 that can be analyzed is produced.

[0105] (Item 7) In the analysis pretreatment method according to any one of items 1 to 6, the volatility of the oil is equal to or higher than the volatility of water.

[0106] According to the analysis pretreatment method described in paragraph 7, oil can be evaporated at a rate equal to or faster than that of water. As a result, the shape of the droplets can be prevented from being damaged by the oil during water evaporation. Furthermore, since the oil has completely evaporated by the time the water has completely evaporated, the remaining oil does not damage the shape of the droplets.

[0107] (Item 8) In the analysis pretreatment method according to any one of Items 1 to 7, the sample contains cells and / or cell-derived substances.

[0108] According to the analytical pretreatment method described in item 8, by analyzing the components contained in the droplets, it is possible to analyze the cells themselves (e.g., bacterial cells) or metabolites inside and outside the cells.

[0109] (Item 9) In the analysis pretreatment method according to any one of items 1 to 8, the analysis is mass spectrometry using the MALDI (Matrix-Assisted Laser Desorption) method, and the substrate is a sample plate for mass spectrometry using the MALDI method.

[0110] According to the analysis pretreatment method described in item 9, components in a sample can be analyzed by performing MALDI analysis on the sample that has been subjected to the analysis pretreatment method. In particular, by performing MALDI imaging, each sample on a substrate can be analyzed with high throughput.

[0111] (Item 10) The pre-analysis method according to item 9 further comprises the step of depositing a matrix solution onto the first and second samples on the substrate.

[0112] According to the pre-analysis method described in item 10, even if the samples are irregularly distributed on the MALDI plate, the matrix solution can be easily vapor-deposited onto each sample.

[0113] (Item 11) The analysis pretreatment method according to any one of items 1 to 10, further comprising the step of preparing at least one of the first and second samples on the substrate for genetic analysis.

[0114] According to the pre-analysis method described in item 11, it is possible to perform genetic analysis on at least one of the samples in each fixed droplet with high throughput.

[0115] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0116] 5 Droplet, 5A Aggregate, 6 Oil, 6A First Oil, 6B Second Oil, 6C Third Oil, 7 Emulsion, 8 Experimental Apparatus, 9 Control Device, 11, 12 Surface, 36 Trace, 51 Evaporation Residue, 51A Evaporation Residue Aggregate, 81 Pretreatment Device, 82 Analytical Device, 90 Processor, 91 Memory, 92 Input Device, 93 Display Device, 100 Analytical System, 501 First Droplet, 502 Second Droplet, 511 Second Evaporation Residue, 512 Second Evaporation Residue.

Claims

1. A method for pre-treating a sample for analysis, comprising: providing an emulsion comprising oil and first and second droplets present in the oil and containing first and second samples, respectively; disposing a collection of the first and second droplets in the emulsion on a substrate; allowing the oil and water on the substrate to evaporate and separating first and second evaporation residues containing the first and second samples, respectively.

2. 2. The analysis pretreatment method according to claim 1, wherein the first and second samples are samples of the same type.

3. the first and second samples are microorganisms; The analysis pretreatment method according to claim 1 or 2, further comprising the step of culturing the microorganism in the emulsion.

4. 3. The analysis pretreatment method according to claim 1, wherein the environmental conditions under which the evaporation step is carried out include a concentration condition in which the concentration of the surfactant in the oil on the substrate is within a predetermined concentration range, and / or a humidity condition in which the humidity around the substrate is within a predetermined humidity range.

5. The analysis pretreatment method according to claim 4 , wherein the concentration range is from 1% by mass to 4.5% by mass.

6. The analysis pretreatment method according to claim 4 , wherein the humidity range is 75% or less.

7. The analysis pretreatment method according to claim 1 , wherein the volatility of the oil is equal to or higher than the volatility of the water.

8. The analysis pretreatment method according to claim 1 or 2, wherein the sample contains cells and / or cell-derived substances.

9. The analysis is mass spectrometry using a MALDI (Matrix-Assisted Laser Desorption) method, 3. The analysis pretreatment method according to claim 1, wherein the substrate is a sample plate for mass spectrometry using the MALDI method.

10. 10. The analysis pretreatment method according to claim 9, further comprising the step of depositing a matrix solution onto the first and second samples on the substrate after the evaporating step.

11. 3. The method for pre-analysis according to claim 1, further comprising the step of preparing at least one of the first and second samples on the substrate for genetic analysis after the evaporation step.

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