WO2018134907A1 - Device and method for extracting multiple biomolecules from single cell - Google Patents

Abstract

In order to measure trace amounts of multiple kinds of biomolecules in a single cell, the present invention provides a device and method capable of differentiating biomolecules derived from a single cell and separating and extracting samples with high efficiency. Specifically, the present invention provides a single cell analysis device, which has a cell separating means and a reaction field disposed near or inside said separation means, the single cell analysis device being characterized in that: said reaction field has an area in which there are first carriers for trapping a first biomolecule extracted from a trapped cell and second carriers for trapping a second biomolecule different from the first biomolecule; the first carriers and/or the second carriers are provided with an identification tag sequence of the trapped cell; and the first carriers and the second carriers have different, mutually separable physical characteristics.

Description

Apparatus and method for extracting multiple biomolecules of the same cell

The present invention relates to an apparatus and method for simultaneously performing analysis such as genome analysis, gene expression analysis, gene sequence analysis, and protein analysis in a single cell for each single cell.

In single cell analysis, biomolecules in a single cell are individually extracted, and an appropriate sample is prepared to enable analysis, and then the sample is measured. This measurement includes genome sequence analysis, gene sequence analysis, gene expression analysis, protein analysis, and the like. That is, the single cell analysis cannot be directly measured because the amount of the sample is very small, and the analysis needs to be executed by combining sample preparation and measurement.

In single-cell analysis, when preparing the amount of sample required for measurement from a small amount of biomolecule (sample), which is a single cell, the sample loss is reduced to the limit and an appropriate sample amplification method is applied. It is important to. Here, the loss means that it is adsorbed on the wall surface of the container in the extraction / concentration process or amplification process of the biomolecule to be measured and lost without being used as a measurement sample. As a conventional technique for solving such a problem, for example, Patent Document 1 discloses that a cell is isolated by capturing a cell in a hole on a flat chip and a DNA probe fixed on the porous material surface immediately below the cell. By capturing the nucleic acid therein, mRNA that is a biomolecule to be measured is extracted. As described above, the cell capture position and the position for capturing the biomolecule are brought close to each other, and sample preparation for measurement is executed on the planar chip, thereby reducing sample loss. As another method, Patent Document 2 discloses that a single cell and a bead with a DNA probe for extracting (capturing) nucleic acid in the cell are confined in an emulsion droplet (a droplet in oil), A method for capturing / extracting the nucleic acid at high efficiency and preparing a sample for sequencing is disclosed. Even in this technology, by isolating the cells to be analyzed, capturing and extracting nucleic acids in the vicinity, and performing part of sample preparation for high-throughput DNA sequencers, Among nucleic acids, the efficiency of nucleic acids analyzed by this sequencer is increased, and sample loss is reduced. Therefore, 0.1 to 10 pg of mRNA (nucleic acid) in a single cell can be extracted for sequence analysis and quantification.

On the other hand, a method of first extracting a nucleic acid sample from a cell sample in a container and then re-extracting the nucleic acid to be measured is generally performed in this technical field. In this case, single cell analysis is generally not performed. This is because the amount of nucleic acid sample required for applying this method is 3 μg (after the genomic DNA is fragmented, it is extracted by the above method. Therefore, the genomic DNA amount can be compared with the mRNA amount. )), And there is a difference of about 6 digits compared to the case of the amount of nucleic acid of about 0.1 to 10 pg which is the amount of nucleic acid in the single cell analysis. Such differences include the process of elution of nucleic acid in the solution from the solid surface without sample preparation on the solid surface where the capture and extraction of nucleic acids from the cells were captured in the vicinity of the cells. This is because not only the ratio of the sample loss above becomes high, but also that the loss due to adsorption to the surface of the container becomes large even if it is moved to another container or the like in a solution state.

US Patent Application Publication No. US2015 / 0167063 US Patent Application Publication No. US2015 / 0376609

In single cell analysis, measurement of multiple types of biomolecules in the same cell, and measurement of biomolecules that require different sample processing, even for the same type of biomolecule, are very useful. For example, simultaneous analysis of genome mutation analysis or epigenome analysis and gene expression analysis reveals the relationship between genome and gene expression status and provides information for predicting the state of cells and tissues from genome information with high accuracy. . Similarly, by performing the gene expression analysis and the protein analysis at the same time, it can be expected to obtain information on the relationship between the genome information and the state of the cells that is more stable in time than the genome and the gene expression. Furthermore, antibodies in immune cells enable effective cell group repertoire analysis through sequence analysis of the variable region of the T cell receptor (Repertoire analysis) and gene expression analysis, which is useful information for selecting treatments such as cancer. Expected to give.

As described above, sample preparation devices for single-cell analysis so far have a small amount of measurement target, so sample processing is performed at or near the same time as cell capture and cell disruption. Since there is one cell, there is one reaction tank. Therefore, only one type of reaction treatment could be performed. That is, it was not possible to analyze a plurality of types of biomolecules for the same cell. On the other hand, if the sample is separated and the reaction vessel is divided into a plurality of samples, the sample is lost at the time of separation / extraction, so a large amount of sample is required before the separation, and a minute sample of a single cell cannot be handled.

In the present invention, there is provided a means capable of highly efficient sample separation / extraction so that a plurality of sample preparations necessary for a plurality of measurements can be performed even with a minute sample of a single cell. Objective.

As a result of intensive studies to solve the above problems, the present inventors have obtained knowledge that the above problems can be solved by a single cell analysis device having the following configuration and a single cell analysis method including the following steps. It came. That is, the present invention
Means for separating cells;
A single cell analysis device comprising a reaction field disposed in the vicinity of or within the separation means,
The reaction field includes a first carrier that captures a first biomolecule extracted from the captured cells, and a second carrier that captures a second biomolecule different from the first biomolecule. Have an area to
One or both of the first carrier and the second carrier comprise a tag sequence for identifying captured cells,
The first carrier and the second carrier relate to a single cell analysis device characterized by having different physical properties that can be separated from each other.

The present invention also provides the single cell analysis device, wherein the cell is captured by the separation means, and the first biomolecule and the second biomolecule extracted from the captured cell are respectively reacted in the reaction field with the first carrier and Including separately processing the first biomolecule and the second biomolecule by capturing on the second carrier and separating the first carrier and the second carrier based on differences in physical properties The present invention relates to a single cell analysis method.

With the device and method according to the present invention, biomolecules with different optimal sample preparation processes or measurement methods existing in a single cell are individually extracted without loss, and information on which single cell is derived is retained. By performing sample preparation individually and performing individual measurement, it is possible to measure the state of a complex biological tissue composed of a large number of cells. In particular, the simultaneous analysis of DNA genomic mutation analysis and mRNA gene expression analysis enables analysis of how genomic mutations affect gene expression for each single cell. It becomes possible to analyze.

Also, the same effect can be expected from simultaneous analysis of epigenome analysis and gene expression analysis, and gene expression analysis and protein analysis. Furthermore, as a medical application, the measurement of the above-mentioned plurality of biomolecules is a single analysis of two types of molecules, that is, an mRNA sequence analysis corresponding to a T cell receptor and an antibody and an mRNA expression analysis related to a cell state. What can be done for each cell is expected to lead to highly accurate cancer diagnosis because information on antigens related to cancer can be obtained from sequence analysis, and the activity state of cells can be obtained from gene expression analysis.

It is a figure which shows the chip | tip structure of Example 1 as a basic structural example of the device which concerns on this invention. 6 is a diagram illustrating a sample processing flow corresponding to Example 1. FIG. FIG. 3 is a diagram showing the first half of a sample processing flow of second beads (solid support) of Example 1. FIG. 4 is a diagram showing the second half of the sample processing flow of the second bead (solid carrier) of Example 1. FIG. 3 is a diagram showing a first half of a sample processing flow of first beads (solid support) of Example 1. FIG. 4 is a diagram showing the latter half of the sample processing flow of the first beads (solid support) of Example 1. It is a figure which shows the flow cell device structure of Example 2 as a structural example provided with the device which concerns on this invention. It is a figure which shows the device structure of Example 3 as another structural example of the device which concerns on this invention. 10 is a diagram illustrating a sample processing flow of Example 3. FIG. It is a figure which shows one Embodiment of the structure of the device which concerns on this invention. It is a figure which shows the chip | tip structure of Example 6 as a basic structural example of the device which concerns on this invention.

The present invention provides a device, apparatus, and method for preparing a plurality of types of biomolecule samples from each single cell in parallel and rapidly for a plurality of cells and analyzing each sample. Here, the sample is prepared so as to be able to distinguish between single cell and biomolecule types.

In the present invention, “preparation of a biomolecule sample” means that a biomolecule contained in a cell is extracted and separated from other cell components. In particular, a plurality of types of biomolecules can be distinguished. It means that the sample is prepared separately. In addition, “analysis of a sample” is to analyze a biomolecule related to the sample, specifically, to analyze the expression of the biomolecule in the sample (cell, tissue section, etc.) quantitatively, Analyzing the expression distribution of biomolecules in a sample, analyzing the presence or absence of biomolecules (eg, genomic DNA having a specific mutation) in a sample, and correlating data between a specific position in a sample and the amount of biomolecule expression It means getting.

The biomolecule to be analyzed in the present invention is not particularly limited as long as it is a biomolecule contained in a cell. Nucleic acid (eg, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genome) DNA, and fragments thereof), proteins (eg, enzymes, antibodies, etc.), low molecular compounds, and the like. In the present invention, a plurality of types, for example, two types of biomolecules are analyzed. For example, both the first biomolecule and the second biomolecule can be mRNA. Alternatively, one of the first biomolecule and the second biomolecule can be mRNA, and the other can be protein. Also, one of the first biomolecules can be genomic DNA and the other can be mRNA. The types of biomolecules to be analyzed can be set according to the purpose of analysis, such as two or more, for example, two, three, four, and five.

In the present invention, an individual reaction field for capturing a biomolecule is set for each individual cell, but the cell is separated / captured in a position near the reaction field (for example, an adjacent position) or in the reaction field. The reaction field is filled with a carrier for capturing the first measurement target biomolecule, and in addition, the second measurement target biomolecule can be separated from the support and captured. To be filled with another carrier. With such a configuration, it becomes possible to quickly and efficiently perform separation / capture of cells, extraction of biomolecules from the captured cells, and preparation of separate samples of a plurality of types of biomolecules.

In a first aspect, the present invention provides:
Means for separating cells;
A single cell analysis device comprising a reaction field disposed in the vicinity of or within the separation means,
The reaction field includes a first carrier that captures a first biomolecule extracted from the captured cells, and a second carrier that captures a second biomolecule different from the first biomolecule. Have an area to
One or both of the first carrier and the second carrier comprise a tag sequence for identifying captured cells,
The first carrier and the second carrier provide a single cell analysis device characterized by having different physical properties that can be separated from each other.

An example of a specific configuration for measuring a plurality of types, for example, two types of biomolecules (= performing two types of analysis) is to capture a plurality of cells in which the separation means and the reaction field are arranged in a plane. The substrate is provided with a hole, and the reaction tank is disposed immediately below the hole. Specifically, a reaction tank for allowing the cell extraction solution to pass therethrough is provided immediately below the hole in the substrate provided with a plurality of cell capturing (isolation) holes arranged in a plane, and the first measurement target living body is provided here. A first solid support on which a molecule for capturing a molecule and a first capture probe having a cell identification tag sequence are immobilized is filled, and in addition, the physical properties of the first solid support are different. A second capture probe for capturing the second biomolecule is fixed to another solid support, and the reaction vessel is filled.

A device including a substrate provided with a plurality of cell trapping holes arranged on a plane and a reaction vessel arranged immediately below the holes is also called a two-dimensional array or chip. Patent Document 1, International Patent Application Publication No. WO2014 / It is known in the art as described in US Pat.

For example, the substrate is not particularly limited as long as it is made of a material generally used in the technical field. Examples of the material include metals such as gold, chromium, platinum, titanium, and nickel; alloys such as stainless steel and duralumin; silicon; glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive. Glass materials such as reactive glass; plastics such as polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), dimethylpolysiloxane (PDMS), cyclic polyolefin, nylon, acrylic resin, fluorine resin; agarose Dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, and chitosan. The material used for the substrate is preferably a hydrophobic material, which can reduce adsorption of cells and reagents. Alternatively, the substrate may be surface-coated so that other substances (nucleic acid, protein, reagent, etc.) do not adsorb.

Also known is a method in which one cell capturing hole is provided on one surface of the substrate, a reaction tank is provided immediately below the hole, and the reaction tank is filled with a carrier for capturing biomolecules. The size of the cell trapping hole needs to be smaller than the size of the cell to be trapped and large enough to generate a flow toward the reaction vessel. For example, the thickness may be 5 to 10 μm, preferably 8 to 10 μm, but may be appropriately changed depending on the type of cells to be captured. The arrangement and interval of the cell trapping holes on the substrate can also be appropriately changed according to the type of cells to be trapped. Moreover, the reaction tank may be formed integrally with the cell trapping hole, or may be formed separately and connected by a flow path.

As another specific configuration example, the separation means and the reaction field are a flow path through which a nonpolar solvent flows and a droplet including a single cell formed in the nonpolar solvent. Specifically, using a droplet containing a single cell formed in a nonpolar solvent as a reaction field, a reagent for crushing the cell and the first biomolecule to be measured are captured in the droplet. In addition to this, a solid support on which a first capture probe having a molecule and a cell identification tag sequence is immobilized, and another solid support having physical properties different from those of the solid support are introduced.

A technique for extracting and capturing biological substances (particularly nucleic acids) in cells from droplets in a nonpolar solvent is known as so-called emulsion droplets, and is described in, for example, Patent Document 2.

As the nonpolar solvent, for example, mineral oil, silicone oil or the like is used, and emulsion droplets are formed in which single cells, reagents and carriers are sealed. Emulsion formation is well known in the art and can be done by any method. Nonpolar solvents and emulsion droplets are preferably thermally stable for biomolecule extraction and sample processing. The size of the emulsion droplet varies depending on the type and size of the cell to be analyzed, the type and size of the carrier to be used, and is about 100 μL or less, 50 μL or less, 10 μL or less, 5 μL or less, 1 μL or less, It can be set appropriately such as 500 pL or less.

In the present invention, in order to capture a plurality of types of biomolecules, a plurality of types of carriers having different physical properties that can be separated from each other are used. For example, when two types of biomolecules are captured, a first carrier and a second carrier for capturing each biomolecule are filled or introduced into the reaction field. When capturing three or more types of biomolecules, a carrier for capturing each of these biomolecules is filled or introduced into the reaction field.

Each carrier is different in at least one physical property selected from the group consisting of size, weight, specific gravity, magnetism and shape. Examples of the carrier having a different shape include beads, a porous structure, and a reaction vessel wall surface. Examples of the carrier having a different size and / or weight or specific gravity include beads having different sizes and / or weight or specific gravity. Examples of the carrier having different magnetism include magnetic beads and a magnetic porous structure. By using a combination of a plurality of types of carriers having at least one different physical property, it is possible to easily separate biomolecules captured by the carriers.

Considering the point of increasing the capture efficiency of biomolecules, it is preferable to use a material having a large surface area as the carrier. For example, it is preferable to adopt a structure filled with a large number of beads, a porous structure, a mesh structure, or the like. From the viewpoint of simplicity of operation, it is preferable to use beads as at least one of the carriers. When beads are used as the carrier, the beads can be produced from a resin material (such as polystyrene), oxide (such as glass), metal (such as iron), sepharose, and combinations thereof. Further, when magnetic beads are used as at least one of the carriers, the carrier can be separated quickly and easily.

The probe for capturing the biomolecule to be analyzed is fixed to the carrier. Such a probe can be designed to specifically bind to a biomolecule depending on the type of biomolecule to be analyzed. For example, when the biomolecule is mRNA, a DNA probe containing a poly T sequence can be used. A DNA probe containing a poly T sequence, that is, an oligo (dT) can be synthesized by a conventional method, and the degree of polymerization of the oligo (dT) is hybridized with the poly A sequence of mRNA, and the mRNA is oligo (dT). Any degree of polymerization that can be trapped on a fixed carrier. For example, it can be about 10 to 30 bases, 10 to 20 bases, 10 to 15 bases. When the biomolecule is mRNA, non-coding RNA (ncRNA), microRNA or genomic DNA, a DNA probe comprising a random sequence or a DNA probe having a sequence complementary to a specific target sequence can be used. Further, when the biomolecule is a protein or a low molecular compound, a molecule that specifically binds to the biomolecule, such as an antibody, a receptor, or an aptamer, can be used as a probe. Furthermore, when the biomolecule is a protein or a low molecular compound and uses a cell recognition tag sequence, a first binding molecule (such as an antibody or an aptamer) that specifically binds to the biomolecule, A first DNA probe bound to a binding molecule can be used. A second binding molecule that binds to the biomolecule in a sandwich state with the binding molecule (preferably a molecule of the same type as the binding molecule, such as an antibody or an aptamer), and a second binding When a second DNA probe bonded to a sex molecule is added and a target biomolecule exists, the DNA probe and the second DNA probe are ligated, and a ring probe specific to the biomolecule is It is formed. This method is called a proximity ligation method (Proximity 例 え ば Ligation Method) (for example, Malin Jarvius et al. Molecular & Cellular9Proteomics 6500 (9) p.1500, 2007) and is useful for the construction of DNA libraries corresponding to proteins.

The probe is fixed to the carrier by any method known in the art. For example, covalent bond, ionic bond, physical adsorption, biological bond (for example, binding of biotin and avidin or streptavidin, antigen and antibody, etc. The probe can be fixed by using a bond). It is also possible to fix the probe to the carrier via a spacer sequence. When a protein or a low molecular weight compound is processed as a biomolecule using the proximity ligation, the first binding molecule can be immobilized on another carrier.

Here, any one of the above carriers, for example, one or both of the first carrier and the second carrier is provided with a tag sequence for identifying captured cells. For example, a cell identification tag sequence can be introduced into a probe fixed to a carrier. In particular, in the case of a probe fixed to a carrier (eg, a bead) separated from the device, it is derived from any cell or position even after separation from the device using a cell identification tag sequence. Such information can be held, which is preferable. When there are three or more types of biomolecules to be measured, it is preferable to introduce a cell identification tag sequence into at least two types of carriers. Those skilled in the art can design cell identification tag sequences so that they can be distinguished according to the number of cells to be captured and the number of reaction fields.

The reason why the problem of the present invention is solved by the configuration as described above is as follows. That is, cell disruption is performed in the vicinity of the reaction field or in the reaction field, and by providing a carrier that captures all the measurement target biomolecules in the reaction field, the cell extract The biomolecule to be measured contained in is captured by a probe for capturing the biomolecule on the carrier before it touches the reaction vessel. Thereby, a biomolecule is hardly adsorbed on a wall surface such as a container and the sample is hardly lost. Next, by separating the captured biomolecules based on the physical properties of the carrier, that is, size, shape, specific gravity, magnetism, etc., the probability of adsorbing to the container wall surface can be reduced even during the separation. As a result, it is possible to separate an extremely small amount of a biomolecule to be measured in a single cell and perform optimum sample preparation individually for the separated sample.

In another aspect, the present invention provides a single cell analysis device according to the present invention, in which a cell is captured by a separation means, and a first biomolecule and a second biomolecule extracted from the captured cell are reacted. The first biomolecule and the second biomolecule are captured by the first carrier and the second carrier, respectively, in the field, and the first carrier and the second carrier are separated based on the difference in physical properties. A single cell analysis method is provided that includes processing samples individually.

The cell to be analyzed is not particularly limited as long as it is a biological sample containing cells. The living body from which the sample is derived is not particularly limited. When used in the device or method according to the present invention, the sample needs to be in a form in which the cells are separated from each other. Therefore, when the sample is a solid sample (for example, a tissue section), it is preferable to form a liquid sample by dissolving or suspending the solid sample in a solvent. Further, when the sample is a gas sample (for example, air, exhaled air, etc.), it is preferable to suspend the cells contained in the gas sample in a solvent to obtain a liquid sample. Sample preparation methods are routinely performed in the art and can be easily understood by those skilled in the art.

The sample cell is introduced into the single cell analysis device according to the present invention. A cell is captured by a separation means (for example, a cell capture hole or a nonpolar solvent), and a biomolecule is extracted from the captured cell. For example, cells can be lysed using a cell lysis reagent known in the art, and nucleic acids contained in the cells can be extracted. For example, using a proteolytic enzyme such as proteinase K, a chaotropic salt such as guanidine thiocyanate / guanidine hydrochloride, a surfactant such as Tween and SDS, or a commercially available cell lysis reagent such as a lysis solution, The contained nucleic acids, ie DNA and RNA, can be eluted. When capturing mRNA as a biomolecule, only RNA may be captured after degrading DNA with a DNA degrading enzyme (DNase).

∙ Capture multiple types of extracted biomolecules in a carrier with different physical properties in the reaction field. The carrier is then separated based on the difference in physical properties. For example, when using carriers having different sizes, they can be separated using a filter or the like. In the case of a carrier having a different weight or specific gravity, it can be separated using centrifugation, sedimentation or the like. In the case of a carrier having a different shape, it can be separated using washing, filtering, centrifugation or the like. In the case of a carrier having different magnetism, it can be separated using a magnet. The separation of the carrier can be performed by appropriately combining the above means depending on the difference in physical properties of the carrier used.

After separating the carrier, the biomolecules captured on each carrier are individually sampled. In sample processing, it is also possible to analyze which cell or position the processed sample is derived from using a cell identification tag sequence.

In the single cell analysis method according to the present invention, cell disruption is performed in the vicinity of the reaction field or in the reaction field, and all the biomolecules to be measured are captured in the reaction field. By providing the carrier, the biomolecule to be measured contained in the cell extract is captured by the probe for capturing the biomolecule on the carrier before touching the reaction container. Thereby, a biomolecule is hardly adsorbed on a wall surface such as a container and the sample is hardly lost. Next, by separating the captured biomolecules based on the physical properties of the carrier, that is, size, shape, specific gravity, magnetism, etc., the probability of adsorbing to the container wall surface can be reduced even during the separation. As a result, it is possible to separate a very small amount of a biomolecule to be measured in a single cell and to perform optimum sample preparation for each separated sample.

Such analysis can provide information on the distribution of various cells from the viewpoint of molecular biology in biological tissues, and the diversity of the cancer progression and associated immune responses at the individual level can also be analyzed by conventional genetic analysis. More detailed and accurate. Therefore, such research is expected to contribute to the development of new disease diagnostic methods and drug discovery, and in particular, to the study of selection of appropriate treatment methods for each individual.

Hereinafter, specific embodiments of the present invention will be described with reference to the drawings.

In this example, a planar substrate provided with a plurality of cell trapping holes arranged in a plane and a reaction vessel for allowing a cell extraction solution to pass therethrough are provided as reaction fields, and two kinds of beads are filled therein. This is an example of preparing a sample containing two types of biomolecules to be measured.

As a measurement target of two types of biomolecules, in the present embodiment, the same mRNA is used, but first sequence information of mRNA (first biomolecule) in a region where mutation or recombination occurs in the sequence is measured. The analysis and the second analysis for measuring the abundance (= expression level) of mRNA (second biomolecule) encoding a protein with almost no mutation and the like are performed for each single cell. The devices and methods that can be realized simultaneously for cells are described. As a specific example of the first analysis, here, the sequence analysis target part of the variable region of the immune cell is separated from the 3 ′ end (the degree of separation is separated from the length of the sequencing lead for performing the sequence analysis). In addition, the number of expression copies in a single cell is small and the amplification rate of the gene must be large, but it is not necessary to quantify the analysis, and only the sequence information is obtained. On the other hand, the second analysis is a quantitative analysis (so-called gene expression analysis) that counts the number of molecules for each gene. The sequence analysis of the 3 ′ end region of mRNA (sequence region close to the poly A sequence) and the gene sequence database The number of mRNA molecules for each gene is counted by mapping of.

FIG. 1 shows a configuration diagram of a reaction device which is a basic configuration of the present embodiment. 1A and 1B are a top view and a cross-sectional view of the device, respectively. The cell trapping holes (3) are arranged in a square lattice pattern at equal intervals on the flat substrate (1), and the reaction vessel (2) is placed immediately below the cell trapping holes. In this embodiment, the planar substrate is made of PDMS (polydimethylsiloxane), but resin materials such as polycarbonate, polypropylene, (cyclic) cycloolefin, semiconductor materials such as silicon, and inorganic materials such as glass and alumina, A metal material such as stainless steel may be used. In addition, the cell trapping hole (in the narrowest part) can be selected to have a diameter of about 0.1 μm to 100 μm and suitable for the size of the cell, but usually about 2 to 3 μm is preferable. Further, the interval between the cell trapping holes is preferably about 1 μm to 1 mm, and particularly about 100 μm. Further, the size (for example, diameter) of the reaction vessel is preferably about 1 μm to about 500 μm, particularly about 80 μm. The height of the reaction tank is preferably about 1 μm to about 1 mm, and particularly preferably about 100 μm.

In this example, the reaction tank (2) was filled with two types of beads having different diameters and physical properties as two types of solid carriers. First, in order to capture mRNA as a first biomolecule, a first DNA having a gene-specific sequence complementary to a fixed region in the vicinity of a variable region on a Sepharose bead having a diameter of 30 μm as a first solid carrier. Fix the probe. For the fixation of the DNA probe to the Sepharose beads, Streptavidin-fixed Sepharose beads are commercially available (for example, manufactured by GE Health Care). Fix it. Next, a second DNA probe having a poly-T sequence is immobilized in order to capture a gene, which is a second biomolecule, on magnetic beads having a diameter of 1 μm as a second solid support. For this immobilization, magnetic beads having streptavidin immobilized on the surface are commercially available (for example, manufactured by Dynal). As in the case of the first DNA probe, the 5 'end is modified with biotin, and the DNA probe can be immobilized by mixing the beads and the DNA molecule under the conditions according to the instructions. Of course, both may be fixed by different mechanisms.

These two kinds of beads (solid carriers 5 and 6) are mixed and filled in the reaction tank (2). Further, by attaching a bead-holding membrane (7) to the back surface of the substrate (1), the solution dispensed on the substrate (1) flows through the cell trapping hole toward the back surface of the substrate. It has a simple structure. A track etch membrane having a pore size of about 0.05 μm to 80 μm can be used as this membrane, but other porous membranes of other materials and structures may be used as long as they are hydrophilic porous membranes. In this example, a track etch membrane having a pore size of 0.8 μm was used.

Next, an overview of cell isolation and sample preparation methods will be described. The overall processing flow of sample processing is shown in FIG. A cell suspension to be measured is dropped on the substrate, a negative pressure (95 kPa) is applied to the back surface of the bead-holding membrane (7), and the cell solution becomes a cell trapping hole (3) and a reaction vessel (2). And flow through the membrane (7). During this process, the cells in the cell suspension stop in the form of plugging the cell trapping holes, and the holes blocked by the cells stop the flow of the solution, so the cells that have not yet been trapped have priority. The cells become trapped. This process can be repeated to capture cells at most cell capture holes. In this way, the cells (4) are captured on the substrate (1).

After all the cells in the solution are captured by aspirating the entire cell suspension, the cells are crushed to extract the molecules to be measured in the cells. For this purpose, a lysis solution is dispensed on the device, and a negative pressure (96 kPa) is applied to the back surface of the device in the same manner as in the case of the cell suspension so that the cell extract passes through the reaction vessel. Although the flow of the solution does not occur in the cell trapping hole blocked with cells, when the cells are crushed, the cell extraction solution flows through the reaction trap through the cell trapping hole. Subsequently, the mRNA to be measured is captured on different beads by the capture DNA probes on the two types of beads (the first solid support 5 and the second solid support 6).

Next, cDNA, which is a complementary strand of mRNA, is synthesized. Thereby, it is possible to prepare to amplify DNA having the same sequence as mRNA. An enzyme reagent for cDNA synthesis is dropped on the substrate (1) in the same manner as the lysis solution to weaken the application of pressure (about 0 to 5 kPa) and slow down the flow of the solution. The temperature of the device is raised to a temperature suitable for cDNA synthesis (50 ° C.) and reacted for an appropriate time (about 50 minutes).

After completing cDNA synthesis, remove the membrane (7), separate the two kinds of beads, and prepare two kinds of appropriate samples. Again, the sample solution is mixed and DNA sequencing is performed. Using the tag sequence information described later, the measurement data for each type of gene and each reaction tank is separated to obtain necessary data.

The separation method of the two types of beads utilized both the bead size and magnetic properties. That is, after removing the membrane, the obtained bead solution is dispensed into a separation tube (8) and centrifuged, so that a large sepharose bead (first bead) is formed on the upper part of the separation filter (9). The rest, beads with a small diameter settle at the bottom. Magnets were also used to assist in settling.

Next, the sample preparation process on two kinds of beads and the change due to the reaction of molecules will be described.
The reaction is described again because the change due to the molecular reaction differs from the part where the beads are not separated.

First, preparation of a sample for gene expression analysis on the second bead will be described. Steps 1 to 5 in FIG. 3 and FIG. 4 show examples of sample preparation methods up to nucleic acid amplification (PCR) after cDNA synthesis possible with this device.

The separation of the beads may be performed after capturing the mRNA before step 1 cDNA synthesis, but here, as shown in step 1, after the cDNA synthesis, the enzyme was thermally inactivated.

FIG. 3 (step 1) shows an enlarged view of the surface of the second bead (6) on which the DNA probe (31) is fixed. The DNA probe (31 (SEQ ID NO: 1)) immobilized on the beads includes a cell recognition tag sequence (302) for identifying the position of the reaction vessel. The 3 'end of the DNA probe (31) has a poly T sequence (301), and captures the mRNA by hybridizing with the poly A sequence at the 3' end of the mRNA.

The DNA probe for capturing mRNA (31 (SEQ ID NO: 1)) has a slightly more complicated sequence structure in this example, and as shown in FIG. 3 (step 1), it is used for PCR amplification from the 5 ′ end. It consists of a common sequence (303) (Forward direction (SEQ ID NO: 23)), a tag sequence for cell recognition (302) and a nucleic acid capture sequence (301). Here, a poly-T sequence was used as the nucleic acid capture sequence. The degree of polymerization of the poly T sequence may be any degree of polymerization that can hybridize with the poly A sequence of the mRNA and capture the mRNA on the beads to which the nucleic acid probe containing the poly T sequence is immobilized. For example, it can be about 10 to 30 bases, 10 to 20 bases, 10 to 15 bases. By introducing the common sequence for PCR amplification into the DNA probe (31), this sequence can be used as a common primer in the subsequent PCR amplification step. As for the cell recognition tag sequence, for example, when a random sequence of 5 bases is used, 4 ⁵ = 1024 positions or regions (for example, 4 ⁵ = 1024 single cells) can be recognized. That is, a cDNA library can be prepared from 1024 single cells at a time, and it is possible to recognize which cell is derived from the sequence data of the next-generation sequencer finally obtained. Furthermore, a tag sequence for molecular recognition (for example, 7 bases) may be introduced into the DNA probe (31) (for example, between (301) and (302)). As a result, since 4 ⁷ = 1.6 × 10 ⁵ molecules can be recognized, the amplification product having the same gene sequence derived from the same cell from the DNA sequence data of the amplification product obtained by the next-generation sequencer It becomes possible to recognize whether it originates. Details of these tag sequences are described in, for example, WO2014 / 141386.

In this example, a poly-T sequence was used as part of the capture DNA probe (31) to analyze mRNA. However, in order to perform microRNA or genome analysis, a random nucleic acid capture sequence was used instead of the poly-T sequence. A sequence or a sequence complementary to a part of the nucleic acid to be analyzed may be used.

Next, 1st cDNA strand (33) is synthesized using mRNA (32) captured by DNA probe (31) on the bead as a template. In this step, the void portion of the beads packed with a solution containing reverse transcriptase and a synthetic substrate is filled, and the temperature is slowly raised to 50 ° C. to carry out a complementary strand synthesis reaction for about 50 minutes. After completion of the reaction, the mixture is kept at 85 ° C. for 5 minutes, and the reverse transcriptase is thermally inactivated, and then the beads are separated.

To separate the beads, the membrane (7) was removed, and as shown in FIG. 1 (c), the upper side of the 5 μm pore filter tube containing PBS (phosphate buffered saline) buffer together with the substrate (1). Then, the beads are released from the substrate by stirring. Unnecessary chips are removed from the tube and centrifuged to collect large sepharose beads along with the solution. Magnetic beads having a diameter of 1 μm are collected on the lower side of the tube.

However, the magnetic beads may be recovered using a magnet without using a filter. In this case, the collection operation may be performed several times.

The collected Sepharose beads are collected again in a tube, and RNase enzyme is added to decompose and remove mRNA (32). A solution containing an alkali denaturant and a washing solution are added, the beads are precipitated at the bottom of the tube by centrifugation, and the supernatant is removed to remove residues and degradation products. A cDNA library array as shown in FIG. 5 (step 2) is constructed on the beads packed in the nucleic acid extraction part by the process so far, reflecting the positions of individual cells captured in the cell capture holes. .

Next, a primer (34) containing a plurality (up to several hundreds) of target gene-specific sequences (311) to which a common sequence for PCR amplification (Reverse (SEQ ID NO: 2)) (309) is added is added to the first cDNA strand (33 ) (FIG. 3 (step 2)), and a 2nd cDNA strand (35) is synthesized by a complementary strand extension reaction (FIG. 3 (step 3)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions. Thus, for a plurality of target genes, a common sequence for amplification (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) is provided at both ends, a cell recognition tag sequence, a molecular recognition tag sequence, and a gene-specific sequence Is synthesized in a double-stranded cDNA. In this embodiment, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ± 5 bases of 109 ± 8 bases upstream from the poly A tail of the target gene of OAZ1) were used as gene-specific sequences (SEQ ID NO: 3-22). This is to unify the product size to about 200 bases. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from a single molecule (emulsion PCR, etc.) Has an effect that can be used directly.

Second DNA probe (ATP5B gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATCCCTAACCCAAAAAGCTTCATT (SEQ ID NO: 3)
Second DNA probe (GAPDH gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATCACTGAATCTCCCCTCCTCACA (SEQ ID NO: 4)
Second DNA probe (GUSB gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATCGTTTCTGGCCTGGGTTTTG (SEQ ID NO: 5)
Second DNA probe (HMBS gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATGATGACTGCCTTGCCTCCTC (SEQ ID NO: 6)
Second DNA probe (HPRT1 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATTAGTAGTGTTTCAGTAATGTTGACT (SEQ ID NO: 7)
Second DNA probe (RPL4 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATAAGAAGCCTGCTGCATAAAC (SEQ ID NO: 8)
Second DNA probe (RPLP1 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATAAGTGGAAGCAAAGAAAGAAGAATCC (SEQ ID NO: 9)
Second DNA probe (RPS18 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATGTGTCCGAGGCCAGCACA (SEQ ID NO: 10)
Second DNA probe (RPL13A gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATTCTAGAAGCAGAAATAGACTGGGAA (SEQ ID NO: 11)
Second DNA probe (RPS20 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATGAGATTGTTAAGCAGATTACTTCCA (SEQ ID NO: 12)
Second DNA probe (ALDOA gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATTTGCCCGCGCTCTTTCTTC (SEQ ID NO: 13)
Second DNA probe (B2M gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATATTCATATTTACTTCTTATACATTTGA (SEQ ID NO: 14)
Second DNA probe (EEF1G gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATAAAGCCTTCAATCAGGGCAA (SEQ ID NO: 15)
Second DNA probe (SDHA gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATCCAGGGAGCGTGGCACTT (SEQ ID NO: 16)
Second DNA probe (TBP gene, 2nd cDNA strand primer for specific analysis)
CCTCTCTATGGGCAGTCGGTGATCTCCAGTATTGCAGGACAGA (SEQ ID NO: 17)
Second DNA probe (VIM gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATAATCTTGTGCTAGAATACTT (SEQ ID NO: 18)
Second DNA probe (RPLP0 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATTCGGACGAGGATATGGGATT (SEQ ID NO: 19)
Second DNA probe (RPLP2 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATATGAGAAGAAGGAGGAGTCTG (SEQ ID NO: 20)
Second DNA probe (RPLP27 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATGGGAGGCCAAGGTCAAGT (SEQ ID NO: 21)
Second DNA probe (OAZ1 gene, primer for 2nd cDNA strand synthesis for specific analysis)
CCTCTCTATGGGCAGTCGGTGATAGAAGTTTCTTATTTGGAGTCT (SEQ ID NO: 22)

Subsequently, PCR amplification was performed using amplification primers (36 and 37) that bind to the amplification common sequence (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)), and PCR products derived from multiple types of genes ( 38) is prepared (FIG. 4 (steps 4 and 5)). Even if an amplification bias occurs between genes or molecules in this process, it is possible to correct the amplification bias using the molecular recognition tag sequence after acquiring next-generation sequencer data. Can be obtained.

Next, individual processing after RNAase treatment of the first bead will be described.
The reaction process is shown in FIGS. The DNA probe (50) fixed to the carrier comprises a T7 promoter sequence (SEQ ID NO: 24) from the 5 ′ end direction, a common sequence for PCR amplification (Forward direction, SEQ ID NO: 23), a cell recognition tag sequence, and a molecule recognition tag. It is composed of an array and a poly-T array. By introducing the T7 promoter sequence (SEQ ID NO: 24) into the DNA probe, the target sequence can be amplified by the subsequent cRNA (63) amplification step by IVT (In Vitro Transcription). That is, the T7 promoter sequence (SEQ ID NO: 24) is recognized by T7 RNA polymerase, and transcription (cRNA (63) amplification) reaction is started from the downstream sequence. Similarly, by introducing a common sequence for PCR amplification, it can be used as a common primer in the subsequent PCR amplification step. Further, by introducing a cell recognition tag sequence (for example, 5 bases) into a DNA probe, 4 ⁵ = 1024 single cells or regions can be recognized as described above. Furthermore, by introducing a molecular recognition tag sequence (for example, 15 bases) into a DNA probe, 4 ¹⁵ = 1.1 × 10 ⁹ molecules can be recognized. As described above, it is possible to recognize whether it is derived from a molecule. That is, since the amplification bias between genes generated in an amplification process such as IVT / PCR can be corrected, the amount of mRNA present in the sample can be quantified with high accuracy. The poly T sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA.

Next, each step of the reaction will be explained in order. As shown in FIG. 5 (step 1), mRNA (52) is captured by an 18-base poly-T sequence (51) which is a sequence complementary to the poly-A sequence (53) at the 3 'end of mRNA as described above. Next, the first cDNA strand (54) is synthesized to construct a cDNA library (FIG. 5 (step 1)). Next, a plurality of (up to several hundred genes) target gene-specific sequence primers (60) corresponding to the gene to be analyzed are annealed to the first cDNA strand (54) (FIG. 5 (step 2)), and 2nd by complementary strand extension reaction. A cDNA strand (61) is synthesized (FIG. 5 (step 2)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions. Thus, for a plurality of target genes, a common sequence for amplification (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) is provided at both ends, a cell recognition tag sequence, a molecular recognition tag sequence, and a gene-specific sequence Is synthesized in a double-stranded cDNA. In this embodiment, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ± 5 bases of 109 ± 8 bases upstream from the poly A tail of the target gene of OAZ1) were used as gene-specific sequences (SEQ ID NO: 3-22). This is to unify the amplification product size to about 200 bases. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from a single molecule (emulsion PCR, etc.) There is an effect that can be used directly.

Subsequently, T7 RNA polymerase is introduced into the pore to synthesize cRNA (63) (FIG. 5 (step 4)). Through this process, about 1000 copies of cRNA are synthesized. Furthermore, in order to synthesize double-stranded DNA for PCR, a plurality of (about several hundreds at the maximum) added with a common sequence for PCR amplification (Reverse (SEQ ID NO: 2)) using the amplified cRNA as a template A target gene-specific sequence primer (64) is hybridized (FIG. 6 (step 5)) to synthesize cDNA (65) (FIG. 6 (step 6)). Further, after cRNA is decomposed using an enzyme in the same manner as described above, double-stranded DNA for PCR (66) is synthesized by synthesizing 2nd strand using Forward common primer (SEQ ID NO: 23) (FIG. 6 (step 7)). This amplification product has the same length and can be directly applied to PCR and next-generation sequencers. Even if an amplification bias occurs between genes or molecules in this process, it is possible to correct the amplification bias using the molecular recognition tag sequence after acquiring next-generation sequencer data. It is the same as the above that can be obtained.

The common reaction part is described in detail.
A cell solution (phosphate buffer pH 7.5) adjusted to a concentration of about 100 cells / μL is dispensed to each chip, and the cells are captured by applying a negative pressure, and then the cell disruption solution is allowed to flow. Next, for cDNA synthesis, 585 μL of 10 mM Tris buffer (pH = 8.0) containing 0.1% Tween 20 and 40 μL of 10 mM dNTP and 5 × RT buffer (SuperScript III, Invitrogen) 225 μL, 0.1 M DTT 40 μL and RNaseOUT (Invitrogen) 40 μL and Superscript III (reverse transcriptase, Invitrogen) 40 μL were mixed, the solution filling the sheet was discharged from the outlet, and the solution containing reverse transcriptase was immediately injected from the inlet. Thereafter, the solution and the reaction vessel were raised to 50 ° C. and kept for 50 minutes to complete the reverse transcription reaction, and 1st cDNA having a sequence complementary to mRNA was synthesized (FIGS. 3 and 5 (step 1)).

Next, details of sample preparation for the second bead will be described.
After synthesizing the 1st cDNA strand, the reverse transcriptase was inactivated by maintaining at 85 ° C. for 1.5 minutes, cooled to 4 ° C., and then 10 mM Tris buffer (pH = 8.0) containing RNase and 0.1% Tween 20 After 10 mL is dispensed into a tube and reacted, the beads are allowed to settle, and the supernatant is discarded. In this step, RNA is degraded. Similarly, the same amount of alkali denaturant was dispensed and washed by removing the supernatant. Subsequently, 690 μL of sterilized water, 100 μL of 10 × Ex Taq buffer (TaKaRa Bio), 100 μL of 2.5 mM dNTP Mix, and 10 μM of each common sequence for PCR amplification (Reverse, SEQ ID NO: 2) were added to 20 gene-specific sequences. After preparing a reagent mixture by mixing 100 μL of primer Mix (SEQ ID NO: 3-22) and 10 μL of Ex Taq Hot start version (TaKaRa Bio), precipitate the beads by centrifuging the beads-mixed tube, and removing the supernatant. After removal, dispense the prepared reagent into the tube and mix with the beads. Thereafter, a cycle of 98 ° C. for 10 seconds, 43 ° C. for 60 seconds and 68 ° C. for 3 minutes was repeated 10 times, and then cooled to 10 ° C. to complete the reaction.

Subsequently, 495 μL of sterilized water, 100 μL of 10 × High Fidelity PCR buffer (Invitrogen), 100 μL of 2.5 mM dNTP mix, 100 μL of 50 mM MgSO _4, 40 μL of 10 μM PCR amplification common sequence primer (Forward, SEQ ID NO: 23) 100 μL and 10 μM PCR 100 μL of amplification common sequence primer (Reverse, SEQ ID NO: 2) and 15 μL of Platinum Taq Polymerase High Fidelity (Invitrogen) were mixed and dispensed into tubes. After that, the solution and the sheet are kept at 94 ° C. for 30 seconds, and the three-stage process of 94 ° C. for 30 seconds → 55 ° C. for 30 seconds → 68 ° C. for 30 seconds is repeated 40 cycles, finally kept at 68 ° C. for 3 minutes and then cooled to 4 ° C. Then, a PCR amplification step was performed (FIG. 3 (step 4)). Thereafter, PCR Purification Kit (QIAGEN) was used to remove residual reagents such as free PCR-amplified common sequence primers (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) and enzymes contained in this solution. Purify using etc. After applying the PCR amplification or the bridge amplification, this solution is applied to a next-generation sequencer of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) and analyzed.

Finally, details of the first bead sample preparation method will be described.
After synthesizing the first cDNA strand, the reverse transcriptase was inactivated by keeping at 85 ° C. for 1.5 minutes, cooled to 4 ° C., and then 10 mM Tris buffer containing RNase and 0.1% Tween 20 (pH = 8.0). 10 mL was dispensed into a tube to degrade the RNA. Similarly, the same amount of alkali denaturant was dispensed to settle the beads, and then the supernatant was removed to wash the residue and degradation products. Subsequently, 690 μL of sterilized water, 100 μL of 10 × Ex Taq buffer (TaKaRa Bio), 100 μL of 2.5 mM dNTP Mix, and 10 μM of each common sequence for PCR amplification (Reverse, SEQ ID NO: 2) were added to 20 gene-specific sequences. 100 μL of primer Mix (SEQ ID NO: 3-22) and 10 μL of Ex Taq Hot start version (TaKaRa Bio) were mixed and injected into a tube. Thereafter, the solution and the sheet are reacted at 95 ° C. for 3 minutes → 44 ° C. for 2 minutes → 72 ° C. for 6 minutes to anneal the primer-specific sequence using the 1st cDNA strand as a template (FIG. 5 (step 2)). A complementary strand extension reaction was performed to synthesize a 2nd cDNA strand.

Subsequently, 10 mL of 10 mM Tris buffer (pH = 8.0) containing 0.1% Tween 20 was dispensed into the tube to settle the beads, and then the supernatant was removed to wash the residue and degradation product. . Furthermore, 340 μL of sterilized water and AmpliScribe 10X Reaction buffer (EPICENTRE) 100 μL, 100 mM dATP 90 μL, 100 mM dCTP 90 μL, 100 mM dGTP 90 μL, 100 mM dUTP 90 μL, 100 mM DTT, and AmplTrE Prepared. Next, after removing the supernatant, the prepared reagent was dispensed into a tube, the temperature was raised to 37 ° C. and maintained for 180 minutes to complete the transcription reaction, and cRNA amplification was performed. As a result, the target portions of the 20 target genes are amplified, but the cRNA amplification product size is almost uniform at 200 ± 8 bases. Since the cRNA amplification product is contained in the supernatant of the bead solution, this solution is recovered. For the purpose of removing residual reagents such as enzymes contained in this solution, it is purified using PCR Purification Kit (QIAGEN) and suspended in 50 μL of sterile water. To this solution, 10 μL of 10 mM dNTP mix and 30 μL of 50 ng / μL random primer were mixed, heated at 94 ° C. for 10 seconds, lowered to 30 ° C. at 0.2 ° C./second, heated at 30 ° C. for 5 minutes, and further 4 Reduce to ° C. Thereafter, 20 μL of 5 × RT buffer (Invitrogen), 5 μL of 0.1 M DTT, 5 μL of RNase OUT, and 5 μL of SuperScript III are mixed, heated at 30 ° C. for 5 minutes, and the temperature is increased to 40 ° C. at 0.2 ° C./second. For the purpose of removing residual reagents such as enzymes contained in this solution, it was purified using PCR Purification Kit (QIAGEN), applied to PCR amplification, and then the next generation sequencer (NGS) of each company (Life Technologies, Illumina, Roche). ) To analyze.

As a result of sequence analysis, a large number of data including sequences other than the common sequence from the mRNA captured on the first bead and the mRNA captured on the second bead are obtained. The cell recognition tag is the same by rearranging the obtained data for each cell recognition tag sequence (or for each sequence including those sequences if other tag sequences for identifying the sample are present). The sequence data can be analyzed as data indicating gene expression in the same cell (if the additional tag is included, also by the sequence of the tag). That is, since the mRNA corresponding to the same cell has the same cell recognition tag sequence, it can be identified that the mRNA is derived from the same cell even if samples are prepared from different bead types (Fig. 2).

In this example, a flow cell device in which a plurality of chips as described in Example 1 are combined to increase the number of cells to be processed simultaneously will be described. FIG. 7A shows a top view and FIG. 7B shows a cross-sectional view. The flow cell device 701 includes a plurality of reaction chambers (702), and one chip (715) is disposed in the reaction chamber (702). A plurality of cell trapping holes are provided on the chip, and two types of beads to which DNA probes having different cell recognition tag sequences are fixed are filled immediately below. On this bead, mRNA for sequence analysis and mRNA for gene expression analysis are simultaneously captured to synthesize cDNA. Nucleic acid capture holes and cell recognition tag sequences are arranged in a one-to-one correspondence.

Also, the cells (706) flow from the common inlet (707) toward the common outlet (708) in the common flow path (705) on the flow cell device. At this time, the nucleic acid in the cell is individually captured for each hole in the two types of beads (first Sepharose beads and second magnetic beads) filled immediately below the cell capture holes on the chip 715. Due to the negative pressure applied through the common suction outlet (710), the solution in the common flow path (705) flows through the reaction area filled with cell trapping holes and beads toward the common suction flow path (709). . By this flow, mRNA in the cell is captured on the beads, and further, a cDNA synthesis reaction occurs on the beads.

For reaction after cDNA synthesis, the chip is taken out from the flow cell device, and two kinds of beads are collected in a tube (container). In order to take out the chip, the flow cell device has a combination of upper and lower parts (upper and lower in FIG. 7B) with the device separation boundary (720) as a boundary, and unscrew the upper and lower parts with this boundary as a boundary. The chip is recovered by separating. After taking out the chip, two kinds of beads are separated in a tube as in Example 1, and two kinds of samples are prepared by executing different reaction processes. To implement.

The data analysis method is the same as in Example 1. However, by inserting a different sequence for each chip into the PCR primer, gene analysis data for each chip and further for each cell trapping hole is obtained from the sequence analysis data. Is possible.

This example shows an example of simultaneous analysis of gene expression analysis in a single cell and protein analysis by mass spectrometry.

FIG. 8 shows a configuration diagram of the sample preparation chip in this example. The substrate (801) is made of a silicon substrate (other inorganic materials such as glass, silicon nitride, aluminum and copper may be used), and a through-hole (803) for capturing cells is formed on the two-dimensional lattice. It is arranged in a shape. As in Example 1, by applying a negative pressure to the membrane (807) side as a bead holding filter, the cells (804) are trapped in the cell trapping holes (803). Similar to the first embodiment, a reaction tank (802) is provided immediately below the captured cells, and a plurality of types of biomolecules to be analyzed are captured in this region. In this embodiment, the antibody (805) that specifically captures the protein that is the first measurement target biomolecule is directly fixed to the reaction vessel inner wall of the substrate (801). Furthermore, the reaction vessel is filled with beads (806) to which a DNA probe for capturing mRNA that is the second measurement target biomolecule is immobilized.

As in the case of Example 1, the second bead is used as the second bead for capturing mRNA that is the second biomolecule. The fixed probe on the bead is the same as the description corresponding to FIG. The beads are fixed with a poly-T probe to which a cell recognition tag sequence is added using magnetic beads. In order to analyze other than gene expression analysis, in order to analyze specific regions or microRNAs in the genome, specific sequences for these sequences may be used instead of poly-T sequences. Further, a random probe of about 6 bases may be used in place of the poly-T sequence in order to perform all nucleic acid sequence analysis.

On the other hand, the first biomolecule (protein or the like) is captured by the antibody immobilized on the substrate surface in the reaction vessel (802).

As in Example 1, after dropping the cell suspension station on the substrate (801), a negative pressure is applied to the side of the bead-holding porous membrane (807), so that the solution flow penetrating the cell trapping hole is generated. As a result, the cells (804) are trapped in the cell trapping holes (803).

After capture, a cleaning solution with an appropriate salt concentration (about 0.1 to 1M) is dropped onto the chip surface, and negative pressure is applied to cause the cleaning solution to flow into the reaction tank (802). Wash away the molecules. Thereafter, the membrane (807) is removed, and the beads and the substrate are separated (FIGS. 8B and 8C). The beads are collected in a tube (808), sample processing is performed in the same manner as the second bead in Example 1, and gene sequence analysis is performed with a next-generation sequencer (NGS). Since the cell recognition tag sequence is different for each reaction tank, gene expression analysis can be performed separately for each cell.

On the other hand, the first biomolecule captured by the antibody on the substrate (801) is individually ionized by laser for each reaction tank and analyzed by a mass spectrometer. That is, the substrate (801) on which the protein is captured is subjected to mass spectrometry (MALDI-TOF-MS analysis) retaining position information. Here, the antibody is immobilized on the inner wall of the reaction tank to capture the protein, but it goes without saying that the molecule to be immobilized may be changed depending on the molecule to be measured. Although the selectivity of the molecule to be measured is lowered, it is also possible to simply perform a surface treatment suitable for the object to be measured, such as making the surface hydrophobic or hydrophilic.

This includes immobilizing peptides on the inner wall, immobilizing sugar chain oligomers, immobilizing polyethylene glycol, phospholipid molecules, and similar molecules.

The sample processing flow of this example is summarized in FIG.
Next, a specific method of MALDI-TOF-MS analysis at the single cell level will be described. Since the measurement target molecule is a polymer, a 5 mg / mL sinapinic acid ethanol solution was used as a matrix agent. The substrate (801) is placed with the surface where the membrane (807) is in close contact, and the sinapinic acid solution is dropped. The chip with the matrix agent dripped is individually irradiated with a nitrogen laser (343 nm) in the reaction vessel, and the sample suction port of the TOF-MS apparatus is brought close to the chip to perform mass spectrometry. Since an apparatus (mass imaging) that performs different mass spectrometry for each laser excitation position is also commercially available, such an apparatus may be used.

Using the obtained mass spectrometry data for each position on the chip and the cell recognition tag, the gene expression data from the same position is correlated to analyze two types of biomolecules, protein and mRNA from the same cell. Can be executed in parallel.

In this example, an embodiment of the device of the present invention will be described. In the analysis of two or more kinds of biomolecules from a single cell, when one biomolecule is much smaller than the other, the trace amount of living body is different from the solid support for capturing the trace amount of biomolecule. The problem is that molecules are trapped. In order to alleviate this problem, it is effective to change the packing form of the solid support (beads in this embodiment).

FIG. 10 shows a diagram of a form of filling beads into the reaction vessel on the chip corresponding to this example. In this example, the first beads (5) for capturing a trace amount of mRNA are packed in the reaction tank (2) closer to the through hole (3), and the second beads for capturing a very small amount of mRNA. The beads (6) were filled at positions apart from the through holes in the reaction vessel (2). Thereby, the first biomolecule is preferentially captured by the first bead, and a trace amount of mRNA can be efficiently captured.

In this example, beads in which a DNA probe is immobilized on polystyrene beads having a diameter of 1 μm (through streptavidin and biotin) are used (the sequence of the immobilized DNA probe is the case of Example 1). Is the same). Therefore, since the separation according to the size of the beads cannot be performed, the second beads are collected at the bottom of the tube by the magnet 1001 and separated into a supernatant and a precipitate. At this time, since the second bead is recovered toward the precipitate, by resuspending this solution, a second bead solution is obtained, and the supernatant is allowed to settle the second bead with a magnet. By carrying out again and separating, the purity of the first beads in the supernatant is gradually increased. The precipitated second bead solution is mixed and the volume of the solution is controlled by precipitating the beads using a magnet. By repeating this process, the first and second beads are separated. The steps after this step are the same as those in the first embodiment.

In the above embodiment, only an example of capturing two types of biomolecules at the same time has been described. However, three types of biomolecules are collected from a single cell using beads having different presence and absence of magnetism and size, and separated. It is also possible to do. That is, the first bead captures mRNA for sequence analysis as sepharose beads (diameter 34 μm), the second bead captures mRNA for gene expression analysis as magnetic beads (diameter 1 μm), The third bead is a polystyrene bead having a diameter of 1 μm (non-magnetic and small in size) and captures microRNA for gene expression analysis (a specific probe for microRNA capture is immobilized on the bead). As the separation method, for example, the same method as in Example 1 can be used.

In this embodiment, an example of a method for analyzing two or more types of biomolecules from the same cell by confining the cell in a droplet in oil, instead of capturing the cell in the cell trapping hole on the substrate. Show.

FIG. 11 shows a structural diagram of a device for trapping cells and beads as a solid carrier in a droplet in a chip and capturing two or more types of biomolecules derived from cells on the beads. The chip (1101) is formed of glass, resin, or the like, and FIG. 11 (a) is a top view and FIG. 11 (b) is a cross-sectional view. The size of the cross section of the flow path (1102) is suitably several μm to several hundred μm in both vertical and horizontal directions (this flow path size roughly determines the droplet size), but here the length is 30 μm and the horizontal is 60 μm. Mineral oil (or oil) is allowed to flow through the flow path (1102) in the direction of the arrow (1103) at an appropriate flow rate (several μm / second to several cm / second). At the same time, the first beads (the same Sepharose beads as in the example) are suspended in a cell lysate (in this example, 100 mM Tris: HCl, pH 7.5 buffer containing 1% SDS, 500 mM NaCl, and 10 mM EDTA). Thus, it is introduced toward the flow path 1102 in the direction of the arrow (1104) at a speed at which a desired droplet interval can be obtained. Further, the cell lysate in which the second beads are suspended is caused to flow in the direction of arrow 1105 at the same speed as the first beads. At the same time, phosphate buffered saline with suspended cells is introduced in the direction of arrow 1106 at the same rate as the bead suspension. As a result, it is possible to generate a droplet (1107) in a state where each of the cell and the first and second beads is included in the droplet containing the cell lysate. The obtained droplets (1107) are generated in the number necessary for the analysis, and the cell recognition tag array is different for each droplet, and is the same array in the same droplet. Alternatively, the cell recognition tag sequence uses the same sequence in the same drop so that it differs from drop to drop. Such placement of the tag sequence in the droplet is realized by controlling the order of introduction of beads having different cell recognition tag sequences.

Incubate many of the resulting droplets for several minutes to tens of minutes to ensure that the mRNA is captured on the beads, and then emulsify the salt or surfactant to break up the emulsion and separate the aqueous solution from the oil. Add to. The recovered bead solution is separated into two types of beads in the same manner as in Example 1, and the subsequent reaction step is performed. This is also the same as in the first embodiment.

The sequences of the two types of mRNA for each cell are matched by the sequence of the cell recognition tag sequence or its order in gene expression analysis.

DESCRIPTION OF SYMBOLS 1 Substrate 2 Reaction tank 3 Cell capture hole 4 Cell 5 First solid support (bead)
6 Second solid support (beads)
7 Membrane 8 Separation tube 9 Separation filter 31 DNA probe 32 mRNA
33 1st cDNA strand 34 Primer 35 2nd cDNA strand 36 and 37 Primer for amplification 38 PCR product 301 Poly T sequence 302 Tag sequence for cell recognition 303 Common sequence for PCR amplification (Forward)
309 Common sequence for PCR amplification (Reverse)
311 Target gene specific sequence 50 DNA probe 51 Poly T sequence 52 mRNA
53 Poly A sequence 54 1st cDNA strand 60 Target gene specific sequence primer 61 2nd cDNA strand 63 cRNA
64 Target gene specific sequence primer 65 cDNA
66 Double-stranded DNA for PCR
701 Flow cell device 702 Reaction chamber 705 Common channel 706 Cell 707 Common inlet 708 Common outlet 709 Common suction channel 710 Common suction outlet 715 Chip 720 Device separation boundary 801 Substrate 802 Reaction tank 803 Cell capture hole 804 Cell 805 Antibody 806 Bead 807 Membrane 808 Tube 1001 Magnet 1101 Chip 1102 Flow path 1103 Direction in which mineral oil flows 1104 Direction in which the first bead suspension is introduced 1105 Direction in which the second bead suspension is introduced 1106 Direction in which the cell suspension is introduced 1107 droplet

Claims (10)

1. Means for separating cells;
   A single cell analysis device comprising a reaction field disposed in the vicinity of or within the separation means,
   The reaction field includes a first carrier that captures a first biomolecule extracted from the captured cells, and a second carrier that captures a second biomolecule different from the first biomolecule. Have an area to
   One or both of the first carrier and the second carrier comprise a tag sequence for identifying captured cells,
   The single cell analysis device, wherein the first carrier and the second carrier have different physical properties that can be separated from each other.
2. 2. The single cell analysis according to claim 1, wherein the separation means and the reaction field are a substrate provided with a plurality of cell-capturing holes arranged in a plane and a reaction tank arranged immediately below the holes. device.
3. The single cell analysis device according to claim 1, wherein the separation means and the reaction field are a flow path through which a nonpolar solvent flows and a droplet including a single cell formed in the nonpolar solvent.
4. The single cell analysis device according to claim 1, wherein the reaction field includes a carrier for capturing three or more kinds of biomolecules.
5. The single cell analysis device according to claim 1, wherein the first carrier and the second carrier differ in at least one physical property selected from the group consisting of size, weight, specific gravity, magnetism, and shape.
6. The single cell analysis device according to claim 1, wherein at least one of the first carrier and the second carrier is a magnetic bead.
7. The single-cell analysis device according to claim 1, wherein both the first biomolecule and the second biomolecule are mRNA.
8. The single-cell analysis device according to claim 1, wherein one of the first biomolecule and the second biomolecule is mRNA and the other is a protein.
9. The single cell analysis device according to any one of claims 1 to 8, wherein the cell is captured by the separating means, and the first biomolecule and the second biomolecule extracted from the captured cell are reacted with each other. In the first carrier and the second carrier, respectively, and the first carrier and the second carrier are separated by separating the first carrier and the second carrier based on the difference in physical properties. A single cell analysis method comprising processing a sample.
10. The single-cell analysis method according to claim 9, comprising analyzing which cell or position the processed sample is derived from using a cell identification tag sequence.

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