Patent Application
20170016814
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Pursuant to 37 C.F.R. §1.52(e) (5), the Sequence Listing text file, identified as 072388_1261_Sequence_Listing_.txt, is 4,796 bytes and was created on Jun. 15, 2016. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
The present invention relates to a biomolecule analyzer.
Recently, the importance of a single-cell analysis in which an analysis is performed by paying attention to a difference in the genome, gene expression, or protein among individual cells when a genome analysis, a gene expression analysis, or a protein analysis is performed for a biological tissue composed of a large number of cells has started to be recognized. In a conventional analysis, DNA, RNA, or protein was extracted from a large number of cells (103 to 106 or more cells) sampled from a biological tissue as one type of sample, and the analysis was performed, and therefore, an average analysis in the sample was performed. Due to this, even if the existence amount in the individual cells of the DNA, RNA, or protein deviated from the average value, it was difficult to make an evaluation. The single-cell analysis is important as an analysis method for solving such a problem of averaging. In particular, in a study of cancer or iPSC (induced pluripotent stem cells), it is known that the behavior of a small number of cells is quite different from the average behavior of a biological tissue, and the importance of a single-cell analysis has been pointed out.
In a bulk analysis in which the average behavior of a large number of cells sampled from a biological tissue, the information regarding extremely various types of DNA, RNA, or protein can be obtained by one measurement. However, such a bulk analysis method cannot be simply applied to a single-cell analysis. In many bulk analysis methods, the minimum necessary sample size is 1,000 times to 1,000,000 times that at the single-cell level, and the method lacks necessary sensitivity in many cases. Then, in order to realize a single-cell analysis, it is necessary to isolate a single cell in the first place.
In the case of a tissue section or adherent culture cells, it is possible to isolate cells in principle by subjecting the cells to a chemical treatment such as a trypsin treatment thereby cleaving the bond between the cells.
Further, as described in NPL 1 and NPL 2, it is possible to dissect a specific cell group on a microscopic image by using a technique called laser microdissection or laser capture microdissection.
NPL 1: BioTechniques 27, 2: 362-367 (August 1999)
NPL 2: Cellular and Molecular Biology 44 (5), 735-746 (1998)
In the case where a bond between cells is cleaved by a chemical treatment such as a trypsin treatment as in the conventional method, the cells in a tissue fall apart and also due to the chemical treatment for isolation, a cell state, that is, the gene expression level, the protein level, etc. may be changed, which was also the matter of great concern.
Further, in laser microdissection or laser capture microdissection as described in NPL 1 and NPL 2, in order to dissect a tissue section with a thickness which is equal to or larger than at least the size of a cell using a laser, a dissection allowance of several micrometers or more is needed. This dissection allowance of several micrometers or more is comparable to the size of a cell. Therefore, in the case where the cells were in proximity to each other, it was difficult to isolate the cell without damaging the cells in proximity to each other. Further, there was also a problem that by disrupting neighboring cells, a biomolecule contained in the neighboring cells is mixed in a sample solution to decrease the measurement accuracy. In addition, the isolation of cells from a section composed of a plurality of layers could not be performed by a conventional technique because the conventional technique is a technique of performing dissection in the two-dimensional plane.
In view of this, an object of the present invention is to provide a biomolecule analyzer capable of collecting and analyzing a biomolecule in a single cell without damaging neighboring cells.
In order to achieve the above object, the biomolecule analyzer of the present invention is characterized by including a unit which obtains an optical image of a plurality of cells, a unit which disrupts a part or the whole of at least one cell of the plurality of cells, an array device in which regions for capturing a biomolecule in the cell released by the disrupting unit are arranged, and a unit which associates the region in which the biomolecule is captured in the array device with a portion corresponding to the cell disrupted by the disrupting unit in the optical image.
According to the present invention, a biomolecule such as DNA, RNA, or protein in adherent culture cells or cells in a tissue section is collected in the array device for each single cell, and a region in which the biomolecule is collected on the array device is associated with the cell on an optical image obtained by a microscope, whereby the biomolecule in the single cell can be analyzed without damaging neighboring cells while avoiding the contamination with the biomolecule from another cell. Other objects, configurations, and advantageous effects will become apparent from the following description of the embodiments.
Hereinafter, the present invention will be described in detail.
In the present invention, a part of an intracellular tissue (a cell membrane or the like) corresponding to an optical image of an adherent culture cell or a tissue section on a plate is disrupted by abrasion or the like using a laser, and a biomolecule to be measured among the biomolecules released from the disrupted cell is captured in a specific region of an array device. More generally, the biomolecule analyzer of the present invention includes a unit which obtains an optical image of a plurality of cells disposed on a plane, a unit which disrupts apart or the whole of at least one cell of the plurality of cells by convergent laser irradiation, convergent sound wave irradiation, needle perforation, hollow needle perforation, or the like, a unit (array device) which captures the biomolecule released to the outside of the cell by disruption for each cell in regions arranged in an array, and a unit which associates the region on the array device with the cell on the optical image.
That is, instead of performing isolation by dissection of cells, a part of an intracellular tissue such as a cell membrane is disrupted without isolating cells, and therefore, even if how a cell to be measured is adhered to neighboring cells, a part or the whole of the cell can be disrupted with high resolution obtained by a light collecting ability of a laser or the like without damaging the neighboring cells, and thus, a biomolecule in the cell can be collected.
Further, only a biomolecule of interest is captured using the array device without collecting the whole cells simultaneously with the neighboring cellular tissues, and therefore, in the subsequent sampling treatment, contamination with impurities other than the biomolecule of interest can be prevented, and thus, a highly accurate analysis can be achieved. In addition, the use of a reagent for an unnecessary sampling treatment for biomolecules other than the biomolecule of interest is no longer needed, and therefore, the cost for the analysis can be reduced.
According to the present invention, with respect to the optical image obtained by a fluorescence microscope, Raman microscope, or the like without disrupting cells, the quantitative information of a biomolecule such as a gene or a protein obtained by disrupting cells can be associated. This association enables the evaluation of detailed and dynamic characteristics of cells by associating data obtained by an imaging unit with which it is difficult to quantitatively evaluate a large number of types of biomolecules, but the cell can be measured alive with data obtained by a unit with which detailed quantitative data regarding the biomolecule can be obtained, but the dynamic characteristics of the cell cannot be evaluated because the cell is disrupted.
In the past, in order to analyze a large number of cells at a time at low cost, the present inventors developed a device for performing an analysis of gene expression in a large number of cells by constructing a cDNA library utilizing a porous membrane or the like and obtaining the two-dimensional distribution of gene expression (US Patent Application Publication No. 2012/0245053).
In a method using this conventional device, mRNA extracted from a cell observed by a microscope is captured on the device immediately below the cell, and then reverse transcription is performed, whereby an analysis of gene expression in the cell corresponding to the microscopic image of the cell can be performed without isolating the cell. Therefore, a problem that the association between the microscopic image of the cell at the time of isolation of the cell and the cell to be analyzed for gene expression is lost is avoided.
However, in the above conventional device, mRNA is captured on the device composed of a porous membrane immediately below the cell, and therefore, the device has a problem that if cells are overlapped in the axial direction perpendicular to the plane of the device, a cell in which the extracted mRNA is originally present cannot be specified. In addition, it was necessary to perform extraction and capture of a biomolecule such as mRNA from all the cells once on the device composed of a porous membrane, and thereafter perform a sample treatment for the subsequent analysis for all the cells even if there were a large number of cells for which it was not necessary to perform a detailed genome analysis, gene expression analysis, or proteome analysis at the time of optical imaging by a microscope. Therefore, the device also had a problem that a reagent for sample preparation is wasted. In the present invention, a single cell is disrupted by a laser or the like, and only a biomolecule in the cell can be analyzed, and therefore, the above problems can be solved.
Further, in the case where a cell sample placed on the above-mentioned conventional device composed of a porous membrane is subjected to optical imaging using a transmission microscope such as a differential interference contrast microscope or a nonlinear Raman microscope, there was a problem that light scattering occurs on the device to decrease the resolution of imaging. Also this problem can be solved by the present invention. That is, in the present invention, it is not necessary to perform microscopic observation in a state where cells are placed on the device, and therefore, in the case where a transmission microscope is used, the decrease in the resolution of an optical image due to light scattering can be avoided.
Further, in the case where an analysis of adherent culture cells is performed using the above-mentioned conventional device composed of a porous membrane, there was a problem that a material to which the culture cells are adhered is limited to a material composed of the porous membrane. The present invention is configured such that after an optical image is obtained by a microscope, the array device is brought close to a cell of interest, the cell is disrupted by a laser or the like, and a biomolecule in the cell is collected for each single cell, and therefore, a single-cell analysis can be performed by using a common plate to be used for adherent culture cells.
Hereinafter, the present invention will be described in further detail based on embodiments, however, the present invention is by no means limited thereto.
The biomolecule analyzer according to this embodiment includes a unit which obtains an optical image of a sample composed of adherent culture cells using a laser fluorescence microscope and a unit which disrupts the cell using a laser light source, and is a device capable of performing a gene expression analysis by capturing mRNA in the cell in an array device. In
The microscope system 1 includes a laser light source for fluorescence microscope 4. In this embodiment, as the laser light source for fluorescence microscope 4, a semiconductor laser with an output power of 50 mW and a continuous oscillation of 488 nm is used. Other than this, a laser with a wavelength of 405 nm or 633 nm may be used according to the variation of fluorescence desired to be observed, or it is also possible to output laser light with a plurality of wavelengths from the laser light source for fluorescence microscope 4 by using a dichroic mirror or an optical filter.
Further, the microscope system 1 includes a laser light source for cell disruption 5. In this embodiment, as the laser light source for cell disruption 5, a pulse laser with a 355 nm band (maximum average output power: 2 W, repetition frequency: 5 kHz) is used. The laser light source for fluorescence microscope 4 and the laser light source for cell disruption 5 perform multiplexing using a dichroic mirror 6 (edge wavelength: 409 nm). Incidentally, the laser light source for cell disruption 5 may be used as a light source for fluorescence microscope. In fact, in this embodiment, the laser light source for cell disruption 5 is used for observation of a nucleus.
As a fluorescent dye for dyeing cells, Annexin VFITC and Hoechst Dye are used in this embodiment. The former emits light at 535 nm by excitation at 488 nm, and the latter emits light at 465 nm by excitation at 355 nm. A cell membrane in the course of apoptosis is dyed with the former fluorescent dye, and a nucleus is dyed with the latter fluorescent dye.
As a dichroic mirror 7, for example, a dichroic mirror with an edge wavelength of 505 nm is used when performing FITC fluorescence imaging, and a dichroic mirror with an edge wavelength of 385 nm is used when performing Hoechst fluorescence imaging.
The microscope system 1 includes an objective lens 8. As the objective lens 8, for example, an objective lens with an NA of 0.8 and a magnification of 40 times is used.
The optical imaging of cells can be performed as follows. First, on a plate 20 having a bottom face with a cover glass thickness (0.18 mm) which is transparent and does not emit fluorescence, cultured adherent culture cells 21, 22, and 23 are placed, and the plate 20 is placed on a sample stage 10 provided with an open through-hole for light path in a central portion.
The sample stage 10 is driven at a pitch of 50 nm by a stage 9 movable in the X, Y, and Z directions. A fluorescence light receiving system is composed of a band-pass filter 11 which removes light with wavelengths other than fluorescence, an imaging lens 12, a PMT (photomultiplier tube) serving as an optical receiver 13, and a pinhole 14. By synchronously driving these members, an optical image of a plurality of cells is obtained. That is, a fluorescence signal from one point at which excitation light is collected is obtained by the optical receiver 13 while driving the XYZ stage 9, and an optical image is formed in a control system.
The biomolecule collection system 2 includes the array device in which regions for capturing a biomolecule such as mRNA released from the cell are arranged. For example, mRNA is captured in a plurality of regions of the array device for each single cell, and then, a reverse transcription reaction is performed in the array device, whereby a cDNA library can be constructed. In this embodiment, the array device is constructed from a porous membrane which is transparent and has a large number of through-holes formed perpendicularly to the plane, and is hereinafter referred to as “pore array sheet 30”. Further, a member in which a cDNA library is formed in the pore array sheet 30 is referred to as “cDNA library pore array sheet”.
In this embodiment, as the pore array sheet 30, a porous membrane made of aluminum oxide with a thickness of 80 μm and a size of 2 mm×2 mm and having a large number of through-holes with a diameter of 0.2 μm formed by anodization is used. In the pore array sheet 30, a separation wall 31 for separating the regions for capturing the biomolecule from each other can be formed. This separation wall 31 can be formed by a semiconductor process using polydimethylsiloxane (PDMS), and has a thickness of about 80 μm, and can be brought into close contact with the pore array sheet 30.
A top view of the pore array sheet 30 is shown in
As the array device, other than the pore array sheet 30 composed of a porous membrane formed by anodization of aluminum, a member having a large number of through-holes formed by anodization of a material such as silicon may be used. In addition, the array device may be constructed by providing a large number of through-holes in a silicon oxide thin film or a silicon nitride thin film using a semiconductor process. Further, as will be described later, a pore array sheet may be formed by packing beads with various sizes in box-shaped portions, or a film in which a monolithic column to be used as a column for liquid phase chromatography is formed thin can also be used as the array device. Further, membranes of various materials, that is, a cellulose membrane, a glass fiber membrane, a track-etched membrane, a nylon membrane, a polypropylene membrane, a PTFE membrane, and the like may be used. At this time, the separation wall 31 can be formed using other than the PDMS resin, a semiconductor material such as silicon or another resin material by combining known semiconductor processes. The present invention relates to the biomolecule analyzer incorporating the array device as described above therein, however, other than this, the above-mentioned array device, in which a plurality of regions capable of being associated with the optical image of a plurality of cells are arranged and formed, itself is provided as a kit for a biomolecule analysis. By using the kit of this array device in combination with the unit which disrupts a cell with a laser or the like by a user, an analysis of a biomolecule of a single cell can be efficiently performed.
As shown in
The structure of the electrode to be used for electrophoresis and the method for applying a voltage are not limited to the above-mentioned method. For example, in order to perform dielectrophoresis of a microgranule such as an exosome or a DNA fragment of 40 kb or more, and a protein of 10 kDa or more, an electrode structure as shown in
By capturing a peptide or a protein extracted from a single cell in the array device once, the measurement sensitivity when performing a mass analysis thereafter can be increased. That is, in a mass analysis of a tissue section using MALDI, it is necessary to mix a chemical substance called “matrix” with a sample in an appropriate ratio and perform irradiation with a laser. When the ratio is not appropriate, the efficiency of ionization of a molecule of interest is largely decreased. However, in MALDI for a tissue section, a matrix material is merely added from the surface, and therefore, a uniform material in an appropriate ratio cannot be obtained. Due to this, the ionization efficiency generally varies depending on the position and also decreases. However, by adsorbing and capturing a biomolecule of interest for each single cell, and thereafter performing MALDI for the array device having the molecule of interest adsorbed thereon, a mass analysis of a peptide or a protein in the cell in the tissue section can be performed with high efficiency. In addition, since only the biomolecule of interest is selectively captured, the ion suppression effect of impurities is also decreased, and thus, the ionization efficiency is further improved.
Next, an operation flow of the biomolecule analyzer according to this embodiment will be described. In
After the control system 3 confirms the completion of the movement, a voltage is applied to the platinum electrode 32 for electrophoresis, and at the same time, in order to disrupt the cell membrane of the cell to be measured, the cell is irradiated with laser light from the laser light source for cell disruption 5 (step 005). Here, the irradiation time can be set to, for example, 10 seconds, and the time for driving electrophoresis can be set to 60 seconds.
After completion of disruption of one cell and capture of the biomolecule in the cell, a specific region (for example, the region 301 at the first row and second column (1,2)) of the pore array sheet 30 is brought close to the vicinity (immediately above the cell in the configuration in
Here, the number of cells to be disrupted is set to one, however, in the case where coarser resolution data is desired to be obtained, with respect to one region 301 on the array device, mRNA released when a plurality of cells are disrupted and subjected to electrophoresis may be captured. As for the disruption at this time, a plurality of cells may be disrupted simultaneously, or a plurality of cells may be sequentially disrupted one by one without moving the array device.
Next, a sample preparation method for obtaining gene expression analysis data for each cell from the mRNA captured by the pore array sheet 30 will be described with reference to
In this embodiment, in order to capture mRNA, a poly T sequence is used as a part of the DNA probe 56, however, it goes without saying that in order to perform a microRNA analysis or a genome analysis, a part of a complementary sequence to the sequence desired to be analyzed may be used in place of the poly T sequence.
Next, a method for preparing the cDNA library pore array sheet will be described. A through-hole 55 formed in the pore array sheet 30 goes through the pore array sheet 30 in the thickness direction of the pore array sheet 30, and the through-holes 55 are completely mutually independent. The surface of the inner wall of the through-hole 55 is hydrophilic, and very few proteins are adsorbed on the surface, and thus, an enzymatic reaction efficiently proceeds. First, a silane coupling treatment or the like is performed for the surface of the pore array sheet 30, and the DNA probe 56 is immobilized on the surface of the through-hole 55. Since the DNA probe 56 is immobilized on the surface in a ratio of, for example, one per 30 to 100 nm2 in average, 4 to 10×106 DNA probes 56 are immobilized per through-hole 55. Subsequently, in order to prevent surface adsorption, the surface is coated with a surface coating agent. The surface coating may be performed simultaneously with immobilization of the DNA probe. The density of the DNA probe 56 is set to a density capable of capturing mRNA passing through this space with an efficiency of almost 100%.
Next, a method for preparing a cDNA library in the pore array sheet 30 by capturing mRNA 36 from a cell in the inside of the pore array sheet 30 will be described. As described above, the mRNA 36 which is released from a cell disrupted by irradiation with a laser and is negatively charged is subjected to electrophoresis by the platinum electrode 32, and guided to the inside of the through-hole 55 of the pore array sheet 30. As shown in
Here, a method for immobilizing the DNA probe 56 on the inside of the through-hole 55 of the pore array sheet 30 will be described. The surface of the through-hole 55 in the inside of the pore array sheet needs to be a surface on which the DNA probe 56 is immobilized at a high density and at the same time, nucleic acids such as mRNA and primers for PCR amplification, and proteins such as a reverse transcriptase and a polymerase are not adsorbed. Specifically, for example, a silane coupling agent for immobilizing the DNA probe 56 and a silanated MPC polymer for preventing adsorption are simultaneously immobilized in an appropriate ratio on the surface of the through-hole via a covalent bond, thereby realizing highly dense immobilization of the DNA probe 56 and stable prevention of adsorption of nucleic acids and proteins. Specifically, for example, first, a porous membrane made of alumina is immersed in an ethanol solution for 3 minutes. Then, a UVO3 treatment is performed for 5 minutes, followed by washing three times with ultrapure water. Subsequently, the membrane is immersed in an 80% ethanol solution containing 3 mg/ml of MPC0.8-MPTMSi0.2 (MPC: 2-methacryloyloxyethyl phosphorylcholine, MPTMSi: 3-methacryloxypropyl trimethoxysilane) (see, Biomaterials 2009, 30: 4930-4938, and Lab Chip 2007, 7, 199-206) serving as a silanated MPC polymer having an average molecular weight of 9700 (polymerization degree: 40), 0.3 mg/ml of a silane coupling agent GTMSi (GTMSi: 3-glycidoxypropyltrimethoxysilane, Shin-Etsu Chemical Co., Ltd.), and 0.02% acetic acid serving as an acid catalyst for 2 hours. After washing the membrane with ethanol, the membrane is dried in a nitrogen atmosphere and subjected to a heat treatment in an oven at 120° C. for 30 minutes. Subsequently, in order to immobilize the DNA probe, 0.05 M borate buffer (pH 8.5) containing 1 μM of the DNA probe (SEQ ID NO: 1) modified with an amino group at the 5′ end, 7.5% glycerol, and 0.15 M NaCl is ejected onto the pore array sheet by the same technique as an inkjet printer so that the DNA probe containing the tag sequence for cell discrimination (1024 types) which differs for each region (25 μm×25 μm) is ejected at 100 pL/region. Thereafter, a reaction is allowed to proceed at 25° C. for 2 hours in a humidified chamber. Finally, in order to block unreacted glycide groups and remove excess DNA probes, the sheet is washed with a sufficient amount of a borate buffer (pH 8.5) containing 10 mM Lys, 0.01% SDS, and 0.15 M NaCl for 5 minutes. After this washing liquid is removed, the sheet is washed with 30 mM sodium citrate buffer containing 0.01% SDS and 0.3 M NaCl (2×SSC, pH 7.0) at 60° C. to remove excess DNA probes. In this manner, immobilization of the DNA probe 56 and the surface treatment are completed.
Hereinafter, a method for preparing a cDNA library pore array sheet and obtaining a gene expression profile using a next generation (large-scale) sequencer will be described. As one example, a pore array sheet (a portion including 100 regions) after capturing mRNA is introduced into a 0.2-mL tube. Separately, 58.5 μL of 10 mM Tris buffer (pH 8.0) containing 0.1% Tween 20, 4 μL of 10 mM dNTP, 22.5 μK of 5× RT buffer (SuperScript III, Invitrogen Corporation), 4 μL of 0.1 M DTT, 4 μL of RNaseOUT (Invitrogen Corporation), and 4 μL of Superscript III (reverse transcriptase, Invitrogen Corporation) are mixed, and the resulting mixture is dispensed into the above-mentioned tube containing the pore array sheet. Thereafter, the temperature of the solution and the pore array sheet is increased to 50° C. and maintained for 50 minutes to complete the reverse transcription reaction, whereby the first strand cDNA strand 59 having a complementary sequence to the mRNA is synthesized (
After the first strand cDNA strand 59 is synthesized, the reverse transcriptase is inactivated by maintaining the temperature at 85° C. for 1.5 minutes, followed by cooling to 4° C. After the solution is discharged, 0.2 mL of 10 mM Tris buffer (pH 8.0) containing RNase and 0.1% Tween 20 is dispensed into the tube containing the pore array sheet, thereby degrading mRNA, and then, the same amount of an alkaline denaturant is made to flow therein in the same manner, thereby removing and washing off the residue and the degraded products in the through-hole. Subsequently, after the solution was discharged, 69 μL of sterile water, 10 μL of 10× Ex Taq Buffer (TaKaRa Bio, Inc.), 10 μL of 2.5 mM dNTP Mix, 10 μL of a primer mix of 20 types of gene-specific sequences (SEQ ID NOS: 3 to 22, 10 μM each) to which a common sequence for PCR amplification (Reverse, SEQ ID NO: 2) has been added, and 1 μL of Ex Taq Hot start version (TaKaRa Bio, Inc.) are mixed, and the resulting mixture is dispensed into the tube. Thereafter, a reaction is allowed to proceed while maintaining the solution and the pore array sheet under the following conditions: 95° C. for 3 minutes→44° C. for 2 minutes→72° C. for 6 minutes, whereby a target gene-specific sequence primer 60 is annealed by using the first strand cDNA strand 59 as a template (
Subsequently, 49.5 μL of sterile water, 10 μL of 10× High Fidelity PCR Buffer (Invitrogen), 10 μL of 2.5 mM dNTP mix, 4 μL of 50 mM MgSO4, 10 μL of 10 μM common sequence primer for PCR amplification (Forward, SEQ ID NO: 23), 10 μL of 10 μL common sequence primer for PCR amplification (Reverse, SEQ ID NO: 2), and 1.5 μL of Platinum Taq Polymerase High Fidelity (Invitrogen Corporation) are mixed. The solution present in the tube is removed, and immediately thereafter, the above-prepared solution is dispensed into the tube. Thereafter, the solution and the pore array sheet are maintained at 94° C. for 30 seconds, and then subjected to the following 3-step cycle: 94° C. for 30 seconds 55° C. for 30 seconds 68° C. for 30 seconds. The cycle is repeated 40 times. Finally, the solution and the pore array sheet are maintained at 68° C. for 3 minutes, and thereafter cooled to 4° C. In this manner, the PCR amplification step is performed (
Next, a method for reducing amplification bias using the tag sequence for molecule discrimination will be described. In
The pore array sheet prepared here can be used repeatedly, and with respect to a group of genes whose expression level is required to be known, a solution of a primer mix of target gene-specific sequences to which a common sequence primer for PCR amplification (Reverse, SEQ ID NO: 2) has been added is prepared, and in the same manner as described above, synthesis of the second strand cDNA strand, PCR amplification, and emPCR are performed, and then, an analysis may be performed using a sequencer. That is, by repeatedly using the cDNA library, highly accurate expression distribution measurement can be performed for necessary types of genes.
The size of the above-mentioned pore array sheet is 2×2 mm, and is a size capable of using a 0.2-mL tube as a reaction vessel, however, in general, the size of the pore array sheet may be made larger than 2×2 mm. However, in order to perform sample preparation shown in
Further, there are cases where after sample preparation is performed using a plurality of reaction vessels (in
Further, in the above description, a region with a unit size of 2×2 mm is cut from the pore array sheet 30 and introduced into the reaction vessel, however, each region 301 corresponding to a single cell may be cut and introduced into the reaction vessel. In this case, the tag sequence for cell discrimination for discriminating a cell is omitted, and in place of this, only the tag sequence for tube discrimination may be used. It is a matter of course that both tag sequences may be used.
In the microscope system 1 in
A differential interference contrast microscopic image is used only for observing the shape without using a fluorescent reagent, but is one of the measurement methods which have the smallest influence on cells when the cells should be returned in the body in regenerative medicine or the like. If a change in cell shape obtained from this image and a change in gene expression can be associated with each other, the system becomes a measurement system which can perform detailed cell classification with least damage to cells.
A biomolecule analyzer shown in
Further, as an example of the transmission microscope likewise, a CARS (Coherent anti-Stokes Raman scattering) microscope can also be used. In
A biomolecule analyzer in
According to the present invention, a cell is specified by an optical image obtained with a fluorescence microscope or the like, and the gene expression data can be obtained by associating the data with the cell image. By utilizing this function, the dynamic characteristics of the cell can be confirmed with high accuracy. A flowchart for performing such an analysis is shown in
Next, a method for performing cell classification by optical imaging will be shown. After an optical image is obtained with a microscope, for example, a gene expression analysis is performed for 20 cells among 180 cells, and a principal component analysis is performed, and a view obtained by plotting the top two principal components is shown in
Incidentally, in this example, a principal component analysis is used for clustering based on the gene expression in cells, however, it is also possible to apply various methods such as hierarchical clustering and k-means clustering. In addition, as the method for machine learning, various methods to be used for data mining, such as a support vector machine are known, and any of these may be used.
In this embodiment, an example in the case where a T7 promoter is used in place of PCR amplification will be described. A different point from the first embodiment is a method for preparing a sequencing sample. The procedure for sample preparation corresponding to
Next, the respective steps of the reaction will be sequentially described. As shown in
Next, a specific example of a series of steps will be described. After the first strand cDNA strand 59 is synthesized, the reverse transcriptase is inactivated by maintaining the temperature at 85° C. for 1.5 minutes, followed by cooling to 4° C. Thereafter, 10 mL of 10 mM Tris buffer (pH 8.0) containing RNase and 0.1% Tween 20 is injected from an inlet and discharged from an outlet, thereby degrading RNA, and then, the same amount of an alkaline denaturant is made to flow therein in the same manner, thereby removing and washing off the residue and the degraded products in the through-hole. Subsequently, 690 μL of sterile water, 100 μL of 10× Ex Taq Buffer (TaKaRa Bio, Inc.), 100 μL of 2.5 mM dNTP Mix, 100 μL of a primer mix of 20 types of gene-specific sequences (SEQ ID NOS: 3 to 22, 10 μM each) to which a common sequence for PCR amplification (Reverse, SEQ ID NO: 2) has been added, and 10 μL of Ex Taq Hot start version (TaKaRa Bio, Inc.) are mixed. The solution which fills the pore array sheet 51 is discharged from the outlet, and immediately thereafter, the above-prepared solution containing the reverse transcriptase is injected from the inlet. Thereafter, a reaction is allowed to proceed while maintaining the solution and the pore array sheet under the following conditions: 95° C. for 3 minutes→44° C. for 2 minutes→72° C. for 6 minutes, whereby the gene-specific sequence of the primer is annealed by using the first strand DNA strand 59 as a template (
Subsequently, 10 mL of 10 mM Tris buffer (pH 8.0) containing 0.1% Tween 20 is injected from the inlet and discharged from the outlet, thereby removing and washing off the residue and the degraded products in the through-hole. Further, 340 μL of sterile water, 100 μL of AmpliScribe 10× Reaction Buffer (EPICENTRE, Inc.), 90 μL of 100 mM dATP, 90 μL of 100 mM dCTP, 90 μL of 100 mM dGTP, 90 μL of 100 mM dUTP, 100 mM DTT, and 100 μL of AmpliScribe T7 Enzyme Solution (EPICENTRE, Inc.) are mixed. The solution which fills the pore array sheet is discharged from the outlet, and immediately thereafter, the above-prepared solution containing the reverse transcriptase is injected from the inlet. Thereafter, the temperature of the solution and the pore array sheet is increased to 37° C. and maintained for 180 minutes to complete a reverse transcription reaction, whereby cRNA amplification is performed. According to this, a region of interest of each of the 20 types of target genes is amplified, however, all cRNA amplification products have substantially the same size of 200±8 bases. The solution of the cRNA amplification product accumulated in the solution in the inside and outside of the through-hole is collected. Purification is performed using PCR Purification Kit (QIAGEN, Inc.) for the purpose of removing residual reagents such as enzymes contained in this solution, and the purified product is suspended in 50 μL of sterile water. In this solution, 10 μL of 10 mM dNTP mix and 30 μL of 50 ng/μL random primers are mixed. After the resulting mixture is heated to 94° C. for 10 seconds, the temperature of the mixture is decreased to 30° C. at 0.2° C./sec and the mixture is heated to 30° C. for 5 minutes, and then the temperature of the mixture is further decreased to 4° C. Thereafter, 20 μL of 5× RT buffer (Invitrogen Corporation), 5 μL of 0.1M DTT, 5 μL of RNaseOUT, and 5 μL of SuperScript III are mixed, and the resulting mixture is heated to 30° C. for 5 minutes, and the temperature of the mixture is increased to 40° C. at 0.2° C./sec. Purification is performed using PCR Purification Kit (QIAGEN, Inc.) for the purpose of removing residual reagents such as enzymes contained in this solution. After this purified product is subjected to emPCR amplification, an analysis is performed by subjecting the amplification product to a next generation sequencer.
In the first and second embodiments, laser light is used as the unit which disrupts a cell, however, a needle composed of any of various materials may be used. In
Further, as the method for disrupting the cell by perforating the cell membrane, other than the above-mentioned method, a method of concentrating ultrasound, a method of concentrating charged particles or electron beams, or the like can be used. Incidentally, the number of cells to be disrupted with the needle or the like may be one or a few. Further, the cells may be perforated one by one, or a plurality of cells may be disrupted at a time. The method is selected according to the target sampling resolution.
In this embodiment, a configuration for analyzing a peptide derived from a protein as a biomolecule is described. On the inside of a pore array sheet, an antibody which recognizes a protein or a peptide to be measured as the antigen is immobilized using a silane coupling agent instead of a DNA probe. The same immobilization conditions as described above may be used. The protein or peptide is not always negatively charged, and therefore, cannot be guided to the inside of the pore array sheet using electrophoresis. Accordingly, by using the pore array sheet as the plane of symmetry, a nozzle for sucking a solution is disposed on the opposite side to the cell to be measured. The solution sucked by the nozzle can be circulated by returning it to the inside of a plate. The inner diameter of the nozzle is, for example, 0.1 mm, and the suction rate can be set to 500 μL/sec. According to this, the flow of the solution is caused in the inside of the pore array sheet and in a region between the pore array sheet and the cell, and the biomolecule including a protein or a peptide released when the cell membrane is disrupted by a laser can be guided to the pore array sheet. Incidentally, the number of cells to be disrupted may be one or a few or so. The number of cells to be disrupted is selected according to the target sampling resolution.
After the biomolecule is captured in a specific region of the pore array sheet for each cell in this manner, the biomolecule is taken out from the plate, followed by drying. Then, 1 μL of sinapic acid at a saturated concentration is added thereto as a matrix agent for MALDI, and an analysis is performed in the same manner as common MALDI-TOF, whereby a single cell protein analysis with high sensitivity can be performed. Further, by performing the same treatment as described above without immobilizing an antibody specific to a specific biomolecule on the inside of the pore array sheet, it is also possible to analyze a protein or a peptide nonspecifically immobilized on the inner wall of the pore array sheet. In such a case, a single cell proteome analysis can be performed.
In this embodiment, a case where a bead array in which beads are packed on the surface is used as the array device in place of the pore array sheet composed of a porous membrane will be described. The configuration of a biomolecule analyzer is the same as that of the example in
The bead array 1701 can be produced as follows. First, a porous membrane made of alumina used in the pore array sheet of the first embodiment is cut into a size of 2 mm×2 mm. Onto this sheet, a PDMS resin film with a thickness of 100 μm in which 50-μm square through-holes are formed at a pitch of 100 μm is bonded. Thereafter, a solution of beads 1703 having a DNA probe containing a tag sequence for cell discrimination which is different for each region 1702 immobilized thereon is injected by a piezo injector to be used for an inkjet printer. The solution is sucked into the porous membrane side by a capillary effect, followed by drying, and therefore, only the beads 1703 are packed. In one region 1702, 104 to 105 beads 1703 can be packed. Here, as the beads 1703, for example, magnetic beads with a diameter of 1 μm coated with streptavidin can be used. As the DNA probe, a DNA probe terminally modified with biotin is used and immobilized on the surface of the bead through streptavidin. Such beads are commercially available from a lot of manufacturers. The array device produced in this manner is placed facing down on an XYZ stage 34 such that the opening portions of the regions 1702 face the cells.
Note that the present invention is not limited to the above-mentioned embodiments, but includes various modifications. For example, with respect to part of the constituent elements of the embodiments, it is possible to perform addition, deletion, or replacement using other constituent elements.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
This application is a U.S. National Phase application under 35 U.S.C. §371 of International Application No. PCT/JP2013/085093, filed on Dec. 27, 2013. The International Application was published in Japanese on Jul. 2, 2015 as WO 2015/097858 A1 under PCT Article 21(2). The contents of the above applications are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2013/085093 | 12/27/2013 | WO | 00 |
