JP2019208395A - cartridge - Google Patents

Abstract

To generate multiple reaction fields in a short time.SOLUTION: This invention relates to a cartridge, wherein, a three-dimensional space is formed by a first axis and a second axis, and a third axis perpendicular to a bottom defined by the two axes, and the cartridge is installed on the bottom; and the cartridge is used to analyze an object contained in droplets. The cartridge has: a porous film that produces the plurality of droplets; and a holding part that holds the droplets.SELECTED DRAWING: Figure 1

Description

  The disclosure herein relates to cartridges.

  Conventionally, a nucleic acid sample digital PCR (dPCR) method using a water-in-oil emulsion as a reaction field has been proposed as a method for analyzing an analyte such as a compound or particles contained in a sample. As a method of dividing a reaction solution containing a sample into a number of physically independent reaction fields in digital PCR, a method of forming reaction solution droplets in oil, that is, a water-in-oil emulsion (W / O emulsion) There is a method of forming. In this method, each droplet in a water-in-oil emulsion is used as a reaction field (Patent Document 1).

Special table 2012-503773 gazette

  In the digital PCR method, generating a large amount of reaction field is effective from the viewpoint of improving the reliability of analysis results.

  However, there is a problem that it takes time to generate a large amount of reaction field.

  In view of the above problems, an object of the present invention is to generate a large amount of reaction field in a short time.

  It is to be noted that the present invention is not limited to the above-described object, and is an operational effect derived from each configuration shown in an embodiment for carrying out the invention described later, and also has an operational effect that cannot be obtained by conventional techniques. It can be positioned as one of other purposes.

  The cartridge according to the disclosure of the present specification is provided on the bottom surface in a three-dimensional space formed by a first axis, a second axis, and a third axis perpendicular to the bottom surface defined by the two axes. A cartridge that is installed and used for analyzing an object contained in a droplet, and has a porous film that generates a plurality of the droplets, and a holding unit that holds the droplets. To do.

  According to the disclosure of the present specification, a large amount of reaction field can be generated in a short time.

FIG. 3 is a diagram illustrating an appearance of a cartridge according to the first embodiment. The flowchart which shows an example of the whole process sequence in 1st Embodiment. The figure which shows a series of analysis systems which concern on 1st Embodiment. FIG. 10 is a diagram illustrating an appearance of a cartridge according to a second embodiment.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. However, the scope of the invention is not limited to the illustrated examples. Further, in this specification, the bottom surface of the cartridge is indicated by the X axis and the Y axis, and the Z axis indicates the height direction of the cartridge. That is, the cartridge is a cartridge installed on the bottom surface in a three-dimensional space formed by a first axis, a second axis, and a third axis perpendicular to the bottom surface defined by the two axes. is there.

<Embodiment 1>
The cartridge according to this embodiment is a cartridge that can be used for analysis of a sample. The sample here may be the specimen itself, or the specimen that has been subjected to pretreatment and adjustment for analysis, such as purification and concentration, chemical modification and fragmentation of the analyte. Also good. Examples of the analysis object include nucleic acids, peptides, proteins, enzymes, and the like, and these may coexist. The analysis target may be a molecule, a microparticle, a nanoparticle, or a virus, a bacterium, a cell, or the like to which at least one of a nucleic acid, a peptide, a protein, and an enzyme is bound or attached by a covalent bond. As an example of an analysis method, a digital PCR method is known. In the digital PCR method, a sample containing a nucleic acid to be analyzed is mixed with an amplification reagent for amplifying the nucleic acid, a fluorescent reagent for detecting the nucleic acid, etc., and diluted to obtain a number of physically independent reaction fields. Divide into Each droplet contained in the emulsion may be used as a reaction field in the digital PCR method. Then, PCR is performed independently in each of a large number of reaction fields (droplets) to amplify the nucleic acid. Based on the number of reaction fields in which nucleic acid was detected after amplification and the signal of the fluorescence reagent was detected (number of positive reaction fields) and the number of reaction fields in which no signal was detected after amplification (number of negative reaction fields) Thus, the concentration of the nucleic acid in the sample can be estimated.

  The cartridge according to this embodiment is formed of a transparent resin such as polycarbonate or glass. Specifically, the cartridge is formed by laminating a plurality of resin films or glasses having a predetermined thickness.

  For example, a cartridge is formed by laminating a plurality of resin films each having a predetermined thickness and having a punched channel shape. As a method for bonding the films to be laminated, surface activated bonding or molecular adhesion capable of bonding at a low processing temperature lower than the glass transition point of the resin can be used.

  In addition, since a temperature of about 100 ° C. is applied to the cartridge in the PCR processing step, the glass transition point of these materials is preferably higher than 100 ° C.

  The above-described cartridge material, flow path shape, formation method, and the like are merely examples, and are not limited thereto.

  Hereinafter, the configuration of the cartridge according to the present embodiment and a series of processes of digital PCR using the concentration analysis system will be described with reference to FIGS. 1 to 3.

  FIG. 1 is a view showing an appearance of a cartridge 100 according to the present embodiment.

  FIG. 1A is a plan view of the cartridge 100. FIG. 1B is a cross-sectional view showing the AA cross section of the cartridge 100. FIG. 1C is a cross-sectional view showing a BB cross section of the cartridge 100. In FIG. 1, the black portion of the cartridge 100 indicates a portion formed of resin or the like. In FIG. 1A, the upper portion (the holding portion in FIG. 1B) is shown to show the internal structure. The black portion of the upper surface 103 is shown for convenience.

  Hereinafter, the configuration of the cartridge 100 will be described with reference to FIGS.

  The first member 101 is a member having an opening into which a first liquid (water phase) for generating droplets is injected. As for the dimensions of the first member 101, it is desirable that the height in the Z-axis direction is about 12 [mm], the outer diameter is about 8 [mm], and the inner diameter is about 2 [mm]. The first liquid injected into the first member 101 is sent to the generation unit 102. The generation unit 102 has a plurality of holes, and a plurality of reaction fields that are physically independent from each other, that is, droplets, are generated when the injected first liquid passes through the plurality of holes. For example, the generation unit 102 is a member in which a plurality of holes are two-dimensionally arranged in a film-like member, and an open-cell porous body, a mesh in which fibers are arranged vertically and horizontally, and a through-hole in a single member Microchannels and emulsified membranes are used. The pore diameter of the substance used as the generation unit 102 is preferably about 10 to 30 [μm]. Moreover, it is preferable that the droplet diameter produced | generated from the production | generation part 102 is 30-95 [micrometer] in droplet diameter. Further, the CV (Coefficient of Variation) value of the generated droplets is preferably ± 5 to 20%, and more preferably ± 8 to 18%. In addition, it is desirable that the generation unit 102 is provided at the end of the first member 101. Furthermore, it is desirable that the first member 101 is provided substantially parallel to the opening direction of the opening of the first member 101. Note that the position where the generation unit 102 is provided is not limited to the above as long as it is a position where droplets can be generated. For example, the generation unit 102 may be provided in the holding unit 103. The generation unit 102 is bonded to the first member with, for example, an adhesive. Note that the bonding method is not limited to the above. The holding unit 103 holds a droplet generated by passing through the generation unit 102. The height of the holding portion 103 in the Z-axis direction is preferably about 20 to 100 [μm]. The second member 104 is a member into which the second liquid (oil phase) is injected and discharged. It is desirable that the second member 104 has the same size as the first member 101. In the cartridge 100 according to the present embodiment, the first member 101 and the second member 104 are each provided with an opening in the Z-axis direction, but may be provided on a side surface or a lower surface. In addition, the dimension of each said part is an example, and is not limited to this.

  Next, the overall processing in this embodiment will be described using the flowchart of FIG.

(S2000) (injecting the second liquid (oil phase))
In step S2000, the second liquid (oil phase) is injected from the second member 104 by the oil phase injection means 306 shown in FIG. The second liquid (oil phase) contains oil and a surfactant. The oil phase consists of a solvent that is incompatible with the aqueous phase and separates, and typically consists of oils such as aliphatic hydrocarbons and silicone oils.

  The oil phase injection unit 306 may be a unit that discharges a predetermined volume of liquid such as a syringe, a pump that pressurizes or depressurizes air in the flow path, and a combination of a pump and a valve. In addition, what is used for the oil phase injection | pouring means 306 is not limited above.

  In this step, the second liquid is injected from the opening of the second member 104, so that the cartridge 100 can be filled without passing through the generation unit 102, so that the filling time is shortened. Note that the second liquid may be injected from the opening of the first member 101. In addition, it is desirable that the second liquid injected by the oil phase injection unit 306 is injected so as to fill the entire cartridge 100, and at least an amount that allows the generation unit 102 to be immersed in the second liquid is injected.

(S2010) (Injecting the first liquid (aqueous phase))
In step S2010, the first liquid (aqueous phase) is injected by the aqueous phase injection means 305 and filled in the first member 101. The first liquid is composed of a reaction liquid, and the reaction liquid contains water, a sample, an amplification reagent, and a fluorescent reagent. The configuration of the reaction solution is not limited to this. The water phase injection unit 305 may be a unit that discharges a predetermined volume of liquid such as a syringe, a pump that pressurizes or depressurizes air in the flow path, and a combination of a pump and a valve. In addition, what is used for the water phase injection | pouring means 305 is not limited above.

(S2020) (Droplets are generated from the first liquid (aqueous phase))
In step S2020, the driving means pressurizes the inside of the cartridge 100 from the first member 101 or depressurizes the inside of the cartridge 100 from the second member 104, whereby the second liquid (oil phase) filled in the cartridge 100 is filled. ). Alternatively, the second liquid (oil phase) filled in the cartridge 100 is driven by applying pressure from both.

  In addition, pressure leakage can be prevented by attaching a pressure stopper to the member using the driving means of the first member 101 and the second member 104. Note that a pressure stopper is not always necessary. Further, the driving means may use the water phase injection means 305 and the oil phase injection means 306, or may newly use a different means. That is, various means can be used as the driving means as long as the liquid can be driven by applying pressure. As the second liquid (oil phase) is driven, the first liquid (water phase) filled in the first member 101 passes through the generation unit 102 and a plurality of droplets are generated simultaneously. That is, an emulsion is formed in which the second liquid (oil phase) is a continuous phase and the droplets containing the second liquid are dispersed phases.

  The method for forming the emulsion is not limited to the above.

  For example, a membrane emulsification method can be used. The membrane emulsification method is a method in which an emulsion is formed in a continuous phase slowly flowing on the side to be extruded by extruding a dispersed phase at a constant pressure through the emulsion membrane. The pumping emulsification method is a method of preparing an emulsion by sandwiching an emulsion film between a syringe that has collected a continuous phase and a syringe that has collected a dispersed phase, and by alternately extruding liquid from the two syringes and passing through the emulsion film. is there.

  In the pumping emulsification method, a mixture of a continuous phase and a dispersed phase may be collected in one of two syringes, and the other syringe may be empty. In the pumping emulsification method, a pumping type emulsification device in which an emulsification film is sandwiched between a pair of connectors each connectable to a syringe can be used.

  The membrane emulsification method is preferable because it can simplify the apparatus configuration of an apparatus for forming an emulsion and can form an emulsion with relatively small variation in droplet size.

  You may use the mechanical emulsification method which forms an emulsion by providing mechanical energy with the stirring apparatus and ultrasonic crushing apparatus etc. which are conventionally well-known emulsification methods.

  In the case where the cartridge 100 is formed of a material having low mechanical strength such as a thin layer of resin, the continuous phase is reduced by reducing the pressure from the second member 104 by the oil phase injection means 306 so that the cartridge 100 is not broken or peeled off. It is desirable to drive. If the cartridge 100 is a material having high mechanical strength, the continuous phase may be driven by applying pressure from the first member 101 by the water phase injection means 305.

(S2030) (add thermal cycle to cartridge)
In step S2030, PCR processing is performed on the nucleic acid sample contained in the droplet generated in step S2020 by the temperature adjusting unit 301 shown in FIG. Specifically, the temperature adjustment unit 301 includes a Peltier element and a controller, and generates a nucleic acid amplification reaction in the droplet held in the holding unit 103 of the cartridge 100. For example, a thermal cycler can be used as the temperature adjusting unit 301. As a nucleic acid amplification reaction, a PCR method or a LCR (Ligase Chain Reaction) method in which the reaction is allowed to proceed by subjecting a droplet (reaction field) to a thermal cycle can be used. In addition, the SDA (Strand Displacement Amplification) method, the ICAN (Isothermal and Chimerized Primer-Nuclide Amplified Nucleic acid) method, and the SDA (Strand Displacement Amplification) method in which the reaction proceeds by adjusting the temperature of the droplet (reaction field) without subjecting it to a thermal cycle. The Mediated Isometric Amplification) method or the like can also be used. Note that the apparatus used as the temperature adjusting means 301 and the nucleic acid amplification method are not limited to the above.

(S2040) (observing presence or absence of nucleic acid amplification product in droplet)
In step S2040, the cartridge 100 is transferred to the observation stage 308 and light such as fluorescence is measured. That is, the analysis object in the sample amplified in S2030 is analyzed. The fluorescence is measured by the illumination unit 309 and the observation unit 310, and the illumination unit 309 irradiates a plurality of droplets held by the holding unit 103 with light having a predetermined wavelength. As the illumination unit 309, an LED light, a halogen lamp, a fluorescent lamp, or the like can be used. The observation means 310 detects signals emitted from each of the plurality of droplets irradiated with light, and observes the presence or absence of the amplification product in the droplets. In addition, the droplet diameter is measured. As the observation means 310, a photodiode, a line sensor, an image sensor (imaging device), or the like can be used. Among them, an image sensor is preferably used because signals can be detected for a large number of droplets at once. . As the image sensor, a CCD (Charge Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor) image sensor can be used. The observation unit 310 may be a digital camera provided with an image sensor.

(S2050) (Estimate nucleic acid concentration of sample before nucleic acid amplification)
In step S2050, the processor (not shown) estimates the concentration in the sample from the ratio of the volume of the droplet without amplification product to the total volume of the sample from the result of the observation performed in S2040.

(Calculation of analyte concentration)
The calculation of the concentration of the analysis object can be performed by employing a concentration calculation method in digital analysis that is conventionally performed. A case will be described in which it is assumed that the number of analytes included in each reaction field is one or zero before the reaction in the temperature adjusting unit 301. In this case, the number x of reaction fields (positive reaction fields) in which the analysis object is detected is the number of analysis objects contained in the reaction solution of the volume Vs that is the object of detection of the analysis object by the observation unit 310. Can be considered. Therefore, the concentration λr of the analyte in the reaction solution can be calculated by the following formula (1). It should be noted that the volume Vs that is the target of detection of the analysis object by the observation unit 310 can be calculated based on information on the size of the reaction field acquired from the observation unit 310.
λr = x / Vs (1)

Further, for example, when it can be considered that a plurality of analytes can enter one reaction field before the reaction in the temperature adjusting unit 301, the concentration of the analyte is calculated by performing correction using the Poisson model. be able to. In this case, the concentration of the analysis object is calculated by estimating the average number C of the analysis objects included in each reaction field before the reaction in the temperature adjusting means 301. Specifically, with respect to the reaction field that is the object of detection of the analysis object by the observation unit 310, if the average number of analysis objects included in one reaction field is C, n analysis objects are included in one reaction field. The probability that an object is included is expressed by the following equation (2) from the Poisson model equation.
P (n, C) = (C ⁿ −e ^−C ) / n! ... Formula (2)

Here, the probability that one reaction field does not include any analysis object is represented by the following formula (3), where n = 0 in formula (2).
P (0, C) = e−C (3)

  If at least one analyte is included in one reaction field before the reaction in the temperature adjusting means 301, a signal can be detected from the reaction field, but before the reaction, We do not know the information on the number of analytes included. Therefore, the expression (3) is used based on the ratio of reaction fields in which the analyte is not detected (the ratio of reaction fields in which no signal is detected) to the total number of reaction fields to be detected by the observation unit 310. Thus, the number of analytes contained in the reaction solution to be detected is estimated.

Specifically, from the number of reaction fields in which a signal is detected or the number of reaction fields in which no signal is detected and the total number of reaction fields to be detected, the ratio F0 of reaction fields in which no signal is detected is calculated. calculate. Then, from the following equation (4), the average number C of the analytes included in one reaction field before the reaction in the temperature adjusting means 301 is estimated in the reaction field to be detected.
C = −ln (F0) (4)

Here, assuming that the average volume of the reaction field that the observation means 310 has detected as the analysis target is v, the concentration λr of the analysis target in the reaction solution can be calculated by the following equation (5). It should be noted that the average volume v that is an object of detection of the analysis object by the observation unit 310 can be calculated based on information about the size of the reaction field acquired from the observation unit 310.
λr = C / v (5)

  Note that the concentration λr of the analyte in the reaction solution is obtained by multiplying the average number C of the analytes by the number of reaction fields, the average volume v of the reaction fields, and the number of reaction fields. You may calculate based on the total volume of the reaction field obtained by multiplying.

  The concentration of the analyte in the reaction solution thus obtained is converted to the concentration of the analyte in the sample or sample by using the dilution factor when the reaction solution is prepared from the sample or sample. be able to.

  According to the above-described processing, it is possible to improve the quantitative accuracy and the reliability of the analysis result by performing analysis of the liquid droplets after filling the liquid droplets to be analyzed for each droplet diameter.

<Embodiment 2>
The cartridge 400 according to the present embodiment is a cartridge 400 that can be used for analyzing a sample in the same manner as the cartridge 100 according to the first embodiment. The cartridge 400 according to the present embodiment includes a member having a plurality of openings in addition to the first member 101 and the second member 104 as compared with the first embodiment. Thereby, a large amount of droplets can be generated at a higher speed. Hereinafter, the configuration and processing of this embodiment will be described with reference to FIG.

  The components of the cartridge 400 according to the present embodiment are the same as those of the cartridge 100 according to the first embodiment. However, since the third member 405 and the fourth member 406 are different in configuration from the first embodiment, the configuration will be described below. Other components are the same as those in the first embodiment, and thus the description thereof is omitted.

  FIG. 4 shows the configuration of the cartridge 400 according to this embodiment. FIG. 4A is a plan view of the cartridge 400. FIG. 4B is a cross-sectional view of the cartridge 400 taken along the line AA. FIG. 4C is a cross-sectional view of the cartridge 400 taken along the line BB. Hereinafter, the configuration of the cartridge 400 will be described with reference to FIG.

  The first member 401, the second member 404, the third member 405, and the fourth member 406 are provided on the holding portion 403 so as to have an opening in the third axial direction, respectively. Each of the member 401 and the third member 405 has a generation unit 402. That is, the cartridge 400 includes three or more members having an opening including the first member 401 and the second member 404, and at least one of the three or more members includes the generation unit 402. ing. In addition, the number of the plurality of members including the first member 401 and the second member 404 and the generation unit 402 in this embodiment is an example, and is not limited to the above. For example, only the third member 405 may be provided in addition to the first member 401 and the second member 404.

  In the present embodiment, the cartridge 400 includes a first member 401 and a third member 405 having a generation unit 402, and a second member 404 and a fourth member 406 not having the generation unit 402. Thereby, when a negative pressure is applied from the second member 404 or the fourth member 406 to drive the continuous phase, the first liquid (water phase) filled in the first member 401 and the third member 405 is discharged. A plurality of droplets are generated through the generation unit 402. Therefore, a larger amount of droplets can be generated in a shorter time than in the first embodiment. In addition, when the means for driving the second liquid (oil phase) that is a continuous phase is a means such as a syringe that has a predetermined stroke, until the pressure returns to a state where pressure can be applied again after the pressure is applied. take time. However, by providing the fourth member 406 in addition to the second member 404, pressure can be applied in order from each member, so that droplets can be continuously generated without loss of time. Furthermore, negative pressure may be applied from both the second member 404 and the fourth member 406. According to the above, since the applied pressure increases even when the same driving means as in the first embodiment is used, the driving speed of the continuous phase increases, and the speed at which the first liquid (water phase) permeates the generation unit 402 accordingly. Also goes up. That is, droplets can be generated at a higher speed.

  According to the present embodiment, by providing a member having a plurality of openings in addition to the first member 101 and the second member 104 in the first embodiment, a large amount of droplets can be generated at a higher speed. .

101 First member 102 Generation unit 103 Holding unit 104 Second member

Claims (8)

An object included in a droplet placed on the bottom surface in a three-dimensional space formed by a first axis, a second axis, and a third axis perpendicular to the bottom surface defined by the two axes. A cartridge used for the analysis of objects,
A porous membrane that produces a plurality of said droplets;
A holding unit for holding the droplet;
A cartridge characterized by comprising: A first member having an opening and a second member;
The cartridge according to claim 1, wherein the porous film is provided at an end of the first member.   The cartridge according to claim 2, wherein the porous film is provided substantially parallel to an opening direction of the opening of the first member.   The cartridge according to claim 2 or 3, wherein the first member and the second member have openings in the third axial direction. The cartridge further includes three or more members having openings including the first member and the second member;
The cartridge according to any one of claims 1 to 4, wherein at least one of the three or more members is provided with the porous film.   The cartridge according to any one of claims 1 to 5, wherein the porous film has a pore diameter of 10 to 30 [µm].   The cartridge according to any one of claims 1 to 6, wherein the holding portion has a height in the third axial direction of 20 to 100 [µm].   The cartridge according to any one of claims 1 to 7, wherein the analysis of the object contained in the droplet is a digital PCR method.

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