JP4957893B2 - Isolation technology using integrated microwell array - Google Patents

Description

  The present invention relates to high-throughput screening in food inspection, clinical inspection, and water quality inspection, and more particularly to a technique for isolating a micromaterial in a sample using an integrated microwell array.

As a conventional cell isolation technique, a method using a cell sorter, a micropipette, dilution, and spin coating is known. In addition, a method of isolating beads bound to a peptide with a Poisson distribution using a periodically arranged microwell array is known (Non-patent Document 1).
Furthermore, Patent Document 1 discloses a technique for detecting lymphocytes using a microwell array chip.

However, these methods cannot efficiently separate and measure a large number of cells, microorganisms, and the like present in a specimen.
Patent No. 3372882 J. Biomol. Screen. (1998) vol.3, pp55-62

An object of the present invention is to rapidly detect biological materials such as food, biological samples such as blood, and bacteria, viruses, and cells contained in the environment.

The present invention provides a method for easily and rapidly isolating and measuring the following biological materials.
1． (1) supplying a sample solution to the supply unit of the microwell array in which a number of wells are formed along a flow path having a sample solution supply unit at one end;
(2) filling the well with the sample solution by flowing the sample solution from the supply unit to the flow path;
(3) A method for detecting a biological material present in a sample liquid, comprising the step of detecting a biological material present in the well.
2． Item 2. The method according to Item 1, wherein the biological material is a bacterium, a cell, a fungus, a spore, a virus, a protein, or a nucleic acid.
3． Item 3. The method according to Item 1 or 2, wherein the sample solution is caused to flow from the supply unit to the discharge unit by centrifugal force, capillary force, or gravity drop method.
Four. Item 4. The method according to any one of Items 1 to 3, wherein the number of biological materials in each well is 1 or 0.
Five. Item 5. The method according to any one of Items 1 to 4, wherein the biological material is a bacterium, a cell, a fungus, a spore, a virus, or a nucleic acid, and the biological material is detected using a PCR method.
6． A microwell array having a flow path formed from the center side to the peripheral side, a large number of wells formed along the flow path, and a sample liquid supply section formed at the center side end of the flow path. In the microwell array, the flow path and the well are formed inside the microarray body, and the flow direction of the flow path from the sample solution supply unit toward the peripheral side changes continuously or discontinuously.
7． Item 7. The microwell array according to Item 6, wherein a plurality of the supply section and the flow path are formed.
8． Item 8. The microwell array according to Item 6 or 7, wherein a sample solution discharge portion is formed at a peripheral side end of the flow path.
9． Item 9. The microwell array according to any one of Items 6 to 8, which has a disk shape.
Ten. Item 10. The microwell array according to any one of Items 6 to 9, wherein the flow path has a spiral shape.

  A large amount of biological material such as cells can be isolated at the same time by fractionating into a microwell array, which eliminates the need for expensive equipment, is easy to operate, and performs quantitative or qualitative evaluation very quickly. I can do it.

  In addition, the method that allows cells to be isolated by simply flowing in a microchannel using centrifugal force, etc., does not require a lid at the end as in a periodic microwell array, and is free from leaks, contamination, and air bubbles. There is no fear and practicality is high.

  According to the present invention, a solution containing cells, bacteria / virus-containing samples, DNA / proteins, etc. is partitioned into a large number using a microwell array and distributed, so that information for each cell and for each molecule is obtained. Can be obtained individually for classification or quantitative or qualitative evaluation.

  In this specification, examples of the biological material include cells (animals, plants), microorganisms (bacteria, fungi (including yeast), spores, etc.), viruses, proteins, nucleic acids (particularly DNA), and the like. Specifically, microorganisms (e.g., E. coli, food poisoning bacteria) contained in foods, clinical tests (antigens, antibodies, viruses, pathogenic bacteria and fungi, leukocytes, leukocytes, blood, serum, plasma, urine, saliva, etc.) It can be applied to red blood cells, proteins, markers, etc.) and water quality tests (ground water, rivers, lakes, waterworks, sewerage, industrial wastewater, domestic wastewater, etc.).

In the present invention, as a means for easily and quickly isolating biological material present in a sample,
(i) Utilizing a method in which bacteria are distributed in each fraction based on Poisson distribution by finely fractionating a solution or suspension of a small amount of biological material on a microwell array. To do. In particular, as the degree of subdivision, the fraction of biological materials such as bacteria, cells, viruses, proteins, and nucleic acids that are distributed redundantly decreases. Can be quantized.
(ii) After isolating the biological material based on (i), count the number of biological material present in the original sample by counting the number of microwells in which the biological material is present. And the like.

  If the biological material has DNA or RNA, a biological material suspension and PCR for identification of the target biological material as a means to conveniently and quickly isolate the biological material present in the sample By subdividing the solution or RT-PCR solution in a microwell array, a method is used in which biological material is distributed into each fraction based on the Poisson distribution. In particular, as fractionation decreases, the fraction of biological material that is distributed redundantly decreases, so that information on the presence or absence of biological material can be quantized. Count the number of biological materials (especially bacteria, cells, viruses) in the original sample solution by counting microwells containing biological material by detection of target DNA by real-time PCR.

  The number of wells formed in one channel is 10 or more, preferably 100 or more, more preferably 300 or more, and particularly 500 or more. The upper limit of wells formed in one channel is not particularly limited, but usually about 10,000 or less is sufficient.

  The flow path of the present invention is formed so that the direction in which the sample solution flows is different. In order to change the direction in which the sample liquid flows, the flow path may have a plurality of segments having different liquid flowing directions. In this case, the direction in which the sample solution flows changes discontinuously. Adjacent segments may continuously change the direction in which the sample liquid flows. Within each segment, the sample liquid flows in the same direction, and a connection is provided between the segments to change the direction in which the sample liquid flows. Also good. In order to cause the sample liquid to flow by centrifugal force, it is preferable that the adjacent channel segments have an angle θ. Alternatively, when the flow path is formed in a spiral shape, the flow direction of the liquid continuously changes (there is no segment). When forming in a spiral shape, a plurality of flow paths may be formed concentrically, or only one flow path may be formed.

  The flow path and well of the present invention are formed inside the microwell array main body so that the sample liquid is not dried (covered by the array main body). This is performed from the connected sample solution supply unit. The supply section is preferably located on the center side of the microwell array so that the sample solution can flow in the flow path by centrifugal force. It is preferable to provide a discharge part for discharging the sample solution to the outside at the peripheral side end of the flow path.

  Centrifugal force, capillary force, gravity, and the like can be used for flowing a solution or suspension of biological material in the flow path, and preferably centrifugal force is used. For example, if the centrifugal force is 500 to 7,000 rpm for 1 second to 3 minutes, the well can be filled with a solution or suspension of biological material.

  In the present invention, the sample solution supply section has 10% or more, preferably 30% or more, more preferably 50% or more, still more preferably 70% or more, particularly 90% or more of the wells formed in one channel. It is desirable to provide an amount that is filled with a solution or suspension of biological material. When the sample liquid is excessive than the total volume of the well, a sample liquid discharge portion is provided at the other end of the flow path, and excess sample liquid can be accumulated in the discharge portion. Or you may comprise so that an excess sample solution may flow out from a discharge part.

  One microwell array may have only one flow path, or a plurality of flow paths may be formed in one microwell array. In the case of having a plurality of independent flow paths, a large number of samples can be analyzed simultaneously, or a series of sample liquids having different dilution ratios can be prepared and analyzed by the array. A plurality of independent flow paths are preferably formed at symmetrical positions in order to form as many flow paths as possible in one array.

  The material of the microwell array main body may be any material such as polymer, ceramic, glass, metal, etc., but preferably a polymer, for example, siloxanes such as polydimethylsiloxane (PDMS). The material is preferably transparent in order to visually or optically observe the flow path and well conditions.

  The microwell array can take various shapes, and examples thereof include a disk shape, a triangle, a quadrangle (for example, a square), a regular hexagon, a regular octagon, and the like, and preferably a disk shape. The disc shape is particularly preferable for flowing the sample solution along the flow path by centrifugal force.

  The microwell array of the present invention can be manufactured, for example, by laminating a sheet or plate having a flow path and well pattern and a flat sheet or plate having a sample solution supply section. The sample liquid supply unit may be formed on a sheet or plate having a flow path and well pattern. Alternatively, a microwell array may be formed by bonding two sheets or plates having a flow path and a well pattern. A sheet having a flow path and well pattern may be formed by pressing with a mold, or by pouring a fluid material such as a polymer into the mold, or forming a convex pattern by thick film lithography. A fluid material may be poured onto the pattern, or may be formed directly on the sheet or plate by machining or etching using photolithography.

  Examples of a method for detecting a biological material include a method for immunological measurement using a labeled antibody, and a method for using PCR after disrupting cells, bacteria, viruses, and the like as necessary. The PCR method can be performed according to a conventional method.

  Hereinafter, one embodiment of the present invention is described based on a drawing. FIG. 1 shows a microwell array 1 having a large number of wells used in the present invention. A sample liquid supply section 5 for injecting a biological material solution / suspension is formed in the center of the compact disk (CD) type fluid device (microwell array), and the sample liquid discharge section is formed from the supply section 5 A large number of wells 4 are formed in the flow path 3 connecting the six. The well is formed such that when the biological material solution / suspension flows through the flow path, it is filled with the solution / suspension. One well is filled with a very small amount (1 pL to 10 nL) of solution / suspension, so if the biological material solution / suspension is sufficiently diluted, no more than one per well Of biological material can be included. Examples of the channel and well design include Type 1, Type 2, and Composite Type shown in FIG. 1B. After the biological material solution / suspension is flowed through the flow path, it is preferable to flow oil and cover each well with oil to prevent drying of the wells. Detection of biological material in a well is performed by introducing a PCR solution for target biological material identification into the well, performing PCR, and introducing fluorescently labeled antibodies (primary antibody, secondary antibody) into the well It can be done by things.

  In one preferred embodiment of the present invention, the CD-type fluid device has a plurality of material separation units consisting of biological material supply and discharge sections, flow paths and multiple wells as shown in FIG. For example, you may be comprised from one material separation unit which has a spiral flow path.

Hereinafter, the present invention will be described in more detail with reference to the drawings.
Example 1
[Isolation of beads]
First, it was examined whether each particle of the suspension could be isolated by using a microwell array type channel using beads.

Preparation of microwell array type flow path A reverse transfer pattern of a flow path was prepared as a flow path template. A thick film photoresist (SU-8 50) is spin-coated on a silicon wafer, heated at 65 ° C for 5 minutes, and then at 95 ° C for 15 minutes, and then irradiated with UV through a photomask on which a pattern equivalent to the flow path is drawn. Irradiation was performed and only the portion corresponding to the flow path was photocured. Again, after heating at 65 ° C. for 2 minutes and at 95 ° C. for 4 minutes, it was immersed in a developing solution to remove unpolymerized excess resist other than the reverse transfer pattern of the flow path. After air drying with an air gun, a poly (dimethylsiloxane) (PDMS) prepolymer (Dow Corning Sylgard 184) was poured into the mold structure and heat-cured to form a microwell array type flow path. In addition, a smooth PDMS sheet was produced by performing the same operation using a silicon wafer as a mold as a flow path lid. After through holes were formed only in the sample liquid supply section and discharge section, a PDMS sheet having a microwell array type flow path and a smooth PDMS sheet were bonded together by oxygen plasma treatment to form a closed flow path structure.

  As shown in Fig. 1, the microwell array channel is made using PDMS, and a plurality of channels with a width of 200μm and a depth of 32μm are placed on a disk with a diameter of 10cm and a thickness of 3mm. Zigzag in the circumferential direction. In each channel, microwells having a width of 400 μm and a length of 300 μm were continuously arranged at intervals of about 150 μm, arranged in a tuft on the circumferential side surface of the channel. Reservoirs with a diameter of 2 mm were provided at both ends of each flow path and used as solution injection and air vent. In particular, the junction between the reservoir and the flow path on the center side of the disk was made to have a reverse taper shape so that the solution could flow completely during injection by centrifugal force.

Preparation of bead suspension 3 μm diameter latex bead suspension PolybeadDyed Red (PolyScience, USA) is diluted with water and has a concentration of about 20,000 cells / mL on a hemocytometer (Elma Sales, Tokyo) It was confirmed.

Liquid feeding by centrifugal force 1 μL of the bead suspension was added to each reservoir on the center side of the disk of the microwell array channel. Depending on the wettability of the channel surface, some of the solution automatically flows into the channel due to the capillary force, but the solution is completely fed and the solution in the channel other than the microwells is discharged. Therefore, liquid feeding using centrifugal force was performed. Specifically, the disk after addition of the bead suspension was fixed to a spin coater (K-359 S-1, manufactured by Kyowa Riken Co., Ltd.) and rotated at 3,000 rpm for 30 seconds. As a result, no solution remains in the reservoir, the solution is fed into all the channels, and a small amount of solution is dispensed into each microwell arranged in a tuft. confirmed. At this time, since the volume of the solution held in each reservoir was a very small amount of about 3.4 to 3.6 nL, it was found that the solution gradually lost due to evaporation if it was exposed to air. Therefore, in order to prevent evaporation of the solution dispensed in each microwell, 3 μL of Fluorinert FC-40 (Sumitomo 3M Co., Ltd., Tokyo) was injected into one reservoir of each channel immediately after the disk rotation. . At this time, Fluorinert automatically flows into the flow path due to capillary force, but it was confirmed that the pre-injected bead suspension was not pushed out and remained in each microwell. .

Confirmation of bead isolation using microwell array-type channel After dispensing the bead suspension, the number of beads contained in each microwell was determined using a biological microscope BH-2 from Olympus Corporation (Tokyo). The measurement was performed using a biological microscope image high quality observation system ARGUS-20 of Hamamatsu Photonics Co., Ltd. (Hamamatsu City). The results are summarized in FIG. In the square microwell, the efficiency of bead isolation was different between upstream and downstream of the channel. This is considered to be because bacteria are likely to stay in the corners of the rectangular parallelepiped in the rectangular microwell while the bacterial suspension is flowing. From this, it became clear that a semicircular microwell having a curved surface with a smooth streamline is useful instead of a rectangular parallelepiped in which dead volume is likely to occur. It was also confirmed that the concentration of the original bead suspension calculated from the microwells to which the beads were distributed was equivalent to the result of the blood cell measurement plate. As a result, it was confirmed that it was possible to simultaneously isolate the beads and measure the concentration with an efficiency of 95% by a simple operation by simply flowing the microwell array channel.

Example 2
[Isolation of bacteria]
Based on the results from the bead suspension, bacterial isolation and weighing were studied.

Preparation of microwell array type channel A PDMS microwell array type channel having the semicircular microwells used above was prepared.

Preparation of Bacterial Suspension Two types of bacteria, Escherichia coli and Salmonella, were examined for isolation.
Escherichia coli strain DH5α is cultured at 37 ° C for about 12 hours using LB medium (1% Tryptone, 1% yeast extract, 0.5% NaCl dissolved in water, adjusted to pH 7.5, and sterilized). went.
Salmonella choleraesuis is cultured at 37 ° C for about 12 hours using NUTRIENT BROTH medium of 0.5% NaCl (0.5% Peptone, 0.3% Beef extract, 0.5% NaCl after preparation and sterilized). It was.
It was confirmed that the OD of these cultures was about 1, and diluted 10,000 times.

Liquid feeding by centrifugal force 1 μL of the diluted bacterial culture solution was added to each reservoir on the center side of the disk of the microwell array type flow path. Depending on the wettability of the channel surface, some of the solution automatically flows into the channel due to the capillary force, but the solution is completely fed and the solution in the channel other than the microwells is discharged. For this purpose, it was fixed on a spin coater, rotated at 3,000 rpm for 30 seconds, and sent by centrifugal force. In order to prevent evaporation of the solution dispensed in each microwell, 3 μL of Fluorinert FC-40 (Sumitomo 3M Co., Ltd., Tokyo) is injected into one reservoir of each flow channel immediately after the disk rotation. The air in the road was discharged.

Confirmation of Bacterial Isolation Using Microwell Array Type Channel After dispensing the bacteria, the number of bacteria contained in each microwell was observed and counted with a biological microscope. The results are summarized in FIG. The concentration of the original bead suspension calculated from the proportion of microwells into which bacteria were distributed was 1.1 × 10 ⁹ cells / ml for Escherichia coli and 1.0 × 10 ⁹ cells / ml for Salmonella. Since this was confirmed to be equivalent to the result of the blood cell measurement plate, it was confirmed that the concentration of the bacterial suspension could be measured quickly by the microwell array type flow path.

  The official method for testing bacteria in food requires several days, but according to the method of the present invention, the number of bacteria can be quickly determined. In addition to food, it can be applied to diagnosis of infectious diseases such as blood and environmental assessment including bioterrorism.

The design 1 and design 2 of the microwell array channel are shown. Effect of microwell shape and isolation of beads based on Poisson distribution using microwell array type channels. Isolation of Escherichia coli and Salmonella by microwell array channel

Explanation of symbols

1 Microwell array 2 Microwell array body 3 Flow path
3a segment
3b Connection section 4 Well 5 Sample solution supply section 6 Sample solution discharge section

Claims (10)

(1) supplying a sample solution to the supply unit of the microwell array in which a number of wells are formed along a flow path having a sample solution supply unit at one end;
(2) filling the well with the sample solution by flowing the sample solution from the supply unit to the flow path;
(3) including a step of detecting a biological material present in the well, wherein the flow path has a plurality of segments having different liquid flow directions, and the well is a semicircular microwell. A method for detecting biological material present in a sample liquid, characterized in that 2. The method of claim 1, wherein the biological material is a bacterium, cell, fungus, spore, virus, protein or nucleic acid. 3. The method according to claim 1, wherein the sample liquid is caused to flow from the supply unit to the discharge unit by centrifugal force, capillary force, or gravity drop method. The method according to claim 1, wherein the number of biological materials in each well is 1 or 0. The method according to any one of claims 1 to 4, wherein the biological material is a bacterium, a cell, a fungus, a spore, a virus, or a nucleic acid, and the biological material is detected using a PCR method. A microwell array having a flow path formed from the center side to the peripheral side, a large number of wells formed along the flow path, and a sample liquid supply section formed at the center side end of the flow path. The flow path and the well are formed inside the microarray body, and the flow path has a plurality of segments in which the flow direction from the sample solution supply part toward the peripheral side changes discontinuously, The microwell array, wherein the well is a semicircular microwell. The microwell array according to claim 6, wherein a plurality of the supply sections and flow paths are formed. The microwell array according to claim 6 or 7, wherein a discharge portion for the sample liquid is formed at a peripheral side end of the flow path. The microwell array according to any one of claims 6 to 8, which has a disc shape. The microwell array according to any one of claims 6 to 9, wherein the flow path has a spiral shape.

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