JP7247607B2 - Discharge device and discharge method - Google Patents

Description

The present invention relates to an ejection device and an ejection method.

It is known that when cells are used to evaluate the efficacy and toxicity of drugs, the resistance to reagents varies depending on the environment surrounding the cells. Therefore, when preparing multiple cell samples to be used in evaluation tests, it is very important to match the number of cells contained in each sample.

As a method for dispensing a specified number of cells, there is a method using a cell sorter to visually count and measure cells one by one from a dilute cell suspension, and a method for measuring droplets ejected from a cell suspension. A method of counting the number of cells in a droplet based on a photographed image is known (for example, Patent Document 1).

However, the dispensing method using a dilute cell suspension requires an enormous amount of time to prepare a large amount of samples when attempting to dispense more than 1,000 cells for efficacy and toxicity evaluation. In addition, there is a problem that the sample to be prepared has an excessive amount of liquid with respect to the cells. In addition, the method using the photographed image has the problem that the apparatus becomes large-scaled and complicated image analysis is required.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and can reduce the amount of liquid required to suspend particles, and can reduce the amount of particles contained in ejected droplets without performing complicated image processing. It is an object of the present invention to provide a discharge device capable of calculating the number.

The ejection device of the present invention as a means for solving the above problems is
droplet ejection means for ejecting droplets containing luminescent particles that emit light when irradiated with light;
a light irradiation means for irradiating the droplets discharged by the droplet discharge means with light;
a light detection means for detecting light emitted by the luminescent particles contained in the droplets upon receiving the light irradiated by the light irradiation means;
The presence or absence of the luminescent particles contained in the droplet is determined according to whether or not the light detection means detects the luminescence generated in the luminescent particles, and the luminescent particles in all the ejected droplets are obtained based on the presence or absence of the luminescent particles. and an average particle number calculation means for calculating an average number of particles, which is the average number of luminescent particles contained in one droplet in all droplets, from the ratio of droplets that do not contain The number is 1 or more and 2 or less .

As described above, according to the present invention, the amount of liquid required to suspend particles can be reduced, and the number of particles contained in an ejected droplet can be calculated without performing complicated image processing. A dispensing device is provided that can.

FIG. 1 is a schematic diagram showing an example of the ejection device of the present invention. FIG. 2 is a functional block diagram showing an example of a control device in the ejection device of the present invention. FIG. 3A is a conceptual diagram for explaining an example of a method for determining the presence or absence of luminescent particles. FIG. 3B is a conceptual diagram for explaining another example of a method for determining the presence or absence of luminescent particles. FIG. 4 is a conceptual diagram for explaining another example of the method for determining the presence or absence of luminescent particles. FIG. 5 is a diagram showing Poisson distributions when the average number of particles λ=1, 2, and 5. FIG. FIG. 6 is a diagram showing an example of simulation results of the average number of particles λ. FIG. 7 is a flowchart showing an example of average particle number calculation processing. FIG. 8 is a flowchart showing an example of dispensing processing. FIG. 9 is a flow chart showing another example of the dispensing process. FIG. 10 is a flow chart showing another example of the dispensing process. FIG. 11 is a diagram showing an example of design change of the discharge device of the present invention. FIG. 12 is a schematic diagram showing an example of a discharge device according to an embodiment of the invention.

The ejection apparatus of the present invention includes droplet ejection means for ejecting droplets containing luminescent particles that emit light upon being irradiated with light, and light irradiation means for irradiating the droplets ejected by the droplet ejection means with light. a light detection means for detecting light emission generated by the light emitting particles contained in the droplet upon receiving the light irradiated by the light irradiation means; The presence or absence of luminescent particles contained in the droplets is determined, and from the ratio of droplets not containing luminescent particles to all ejected droplets obtained based on the presence or absence of luminescent particles, one droplet in all droplets is determined. average particle number calculation means for calculating an average number of luminescent particles contained in the light emitting particles, and further includes other means as necessary.

Further, the ejection method of the present invention comprises a droplet ejection step of ejecting droplets containing luminescent particles that emit light upon being irradiated with light; a step of irradiating, a step of detecting light emitted from the luminescent particles contained in the droplet upon receiving the light emitted by the light irradiating means, and determining whether or not the light emitted from the luminescent particles is detected in the step of detecting light. The presence or absence of luminescent particles in the droplets is determined according to the presence of luminescent particles. an average particle number calculation step of calculating an average particle number, which is the average number of luminescent particles contained in the droplet, and further includes other steps as necessary.

The ejection method of the present invention can be suitably performed by the ejection device of the present invention. Further, the droplet discharging step can be suitably performed by droplet discharging means, the light irradiation step can be suitably performed by light irradiation means, and the light detection step can be suitably performed by light detection means. The particle number calculation step can be suitably performed by an average particle number calculation means.

The present invention is based on the knowledge that the conventional ejection device takes a long time to prepare a sample and count particles, and sometimes requires a large amount of liquid to prepare the sample.
In a conventional ejection device using image analysis, the number of particles in each droplet is counted based on an image obtained by a camera that captures an image of the droplet. Therefore, in the conventional ejection device, it may take time to acquire and analyze the data of the photographed image. Furthermore, with conventional ejection devices, it is sometimes difficult to determine the number of particles when there are multiple particles in a droplet, so it is necessary to use a dilute particle suspension, and a large amount of particles must be used. It takes a long time to prepare the required samples. In addition, in the conventional ejection device, a large amount of liquid is required for sample preparation due to the need to use a dilute particle suspension, which may lead to the use of excessive liquid.

On the other hand, the ejection apparatus of the present invention includes droplet ejection means for ejecting droplets containing luminescent particles that emit light when irradiated with light, and light for irradiating the droplets ejected by the droplet ejection means. irradiating means, light detecting means for detecting luminescence generated by the luminous particles contained in the droplet upon receiving the light irradiated by the light irradiating means, and determining whether or not the luminescence generated in the luminous particles is detected by the light detecting means. The presence or absence of luminescent particles in the droplets is determined according to the presence of luminescent particles. and average particle number calculation means for calculating an average particle number, which is the average number of luminescent particles contained in the droplet.
As a result, the ejection apparatus of the present invention does not require preparation of a dilute sample, so that the time required for sample preparation can be shortened. Use can be suppressed. Further, unlike the conventional ejection apparatus using image analysis, the ejection apparatus of the present invention does not necessarily have a camera for capturing images of droplets or a computer for performing complicated image processing. It can be made compact without a complicated configuration. That is, the ejecting apparatus of the present invention can reduce the amount of liquid required to suspend particles, and can calculate the number of particles contained in ejected droplets without performing complicated image processing.

Here, an example of the difference between an ejection apparatus according to an embodiment of the present invention and a conventional ejection apparatus using image analysis such as a technique for counting the number of cells in a droplet based on an image of the droplet is shown. It is shown in Table 1 below.

As described above, the ejection device of the present invention can solve various problems in conventional ejection devices and achieve the object of the present invention.

The present invention will be described below with reference to embodiments, but the present invention is not limited to the embodiments described later. In addition, in each figure referred to below, the same reference numerals are used for common elements, and the description thereof will be omitted as appropriate.

FIG. 1 is a schematic diagram showing the configuration of an ejection device 100 that is an embodiment of the present invention. The discharge device 100 of this embodiment is a device for dispensing a specified number of particles from a suspension of luminescent particles into a container 60, and includes a control device 10, droplet discharge means 20, and light irradiation means 30. , and a light receiving means 40 . The vessel 60 here corresponds to, for example, a well of a microwell plate.

FIG. 12 is a schematic diagram showing an example of the ejection device 100 according to the embodiment of the invention. The ejection device 100 will be described in detail with reference to FIG. 12 .
12, 10 is a control device, 20 is droplet ejection means, 21 is a liquid chamber of the droplet ejection means 20, 22 is a nozzle of the droplet ejection means, and 30 is light irradiation means. 40 is a light receiving means, and 50 is a droplet ejected from the nozzle 22 of the droplet ejecting means. In FIG. 12, 70 is a luminescent particle that emits light when irradiated with light, 81 is an arrow indicating laser light, 82 is an arrow indicating light to the light receiving means 40, and 83 is a droplet. is an arrow indicating the ejection direction of . Note that the light receiving means 40 is connected to the control device 10 .

As shown in FIG. 12, the liquid droplet ejection means 20 accommodates a particle suspension in which luminescent particles 70 are suspended in a liquid chamber 21, and the deformation of the piezoelectric element arranged in the liquid chamber 21 causes the luminescent particles 70 to can be ejected in the direction of an arrow 83 indicating the ejection direction.

The droplet ejection means 20 is means for ejecting droplets 50 containing the luminescent particles 70 .
The droplet ejection means 20 is not particularly limited and can be appropriately selected according to the purpose. Inkjet heads, cell sorters, and the like, such as electrostatic type ink jet heads, membrane vibration type ink jet heads using piezoelectric elements, and the like. Among these, the inkjet head of the membrane vibration type is preferable as the inkjet head. Membrane vibrating inkjet heads eject droplets using the inertial force of vibration. , damage such as pressure can be reduced.
The piezoelectric element used in the membrane vibration type inkjet head is not particularly limited and can be appropriately selected according to the purpose. For example, an element using lead zirconate titanate (PZT) is preferable.

The liquid chamber 21 is a liquid holding portion that holds a particle suspension in which the luminescent particles 70 are suspended, and a nozzle 22 that is a through hole is formed on the lower surface side. The liquid chamber 21 can be made of metal, silicon, ceramic, or the like, for example. The control device 10 is electrically connected to the piezoelectric element of the droplet ejection means 20, and applies a driving voltage to the piezoelectric element to deform the piezoelectric element, thereby ejecting the droplet 50 containing the luminescent particles 70 in the ejection direction. is discharged in the direction of an arrow 83 indicating .
Examples of the luminescent particles 70 include particles capable of receiving illumination light and emitting fluorescence (hereinafter also referred to as “fluorescent particles”). Examples of fluorescent particles include inorganic particles stained with a fluorescent dye, organic polymer particles stained with a fluorescent dye, cells stained with a fluorescent dye, and fluorescent proteins. Particles that emit light based on Raman scattering light instead of fluorescence can also be used.

The organic polymer particles dyed with a fluorescent dye are not particularly limited and can be appropriately selected according to the purpose. 10 μm to 14 μm in diameter).

Cells in cells stained with a fluorescent dye are not particularly limited and can be appropriately selected according to the purpose. cells. These may be used individually by 1 type, and may use 2 or more types together.

Eukaryotic cells are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, fungi, algae, and protozoa. These may be used individually by 1 type, and may use 2 or more types together. Among these, animal cells and fungi are preferred.

Adhesive cells may be primary cells directly collected from a tissue or organ, or primary cells directly collected from a tissue or organ that have been subcultured for several generations. Examples include differentiated cells and undifferentiated cells.

Differentiated cells are not particularly limited and can be appropriately selected according to the purpose. For example, hepatocytes, which are parenchymal cells of the liver; Endothelial cells such as cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; Epithelial cells such as epithelial cells; breast cells; pericytes; muscle cells such as smooth muscle cells and myocardial cells; renal cells; .

The undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. Unipotent stem cells such as vascular endothelial progenitor cells having unipotency; iPS cells and the like.

The fungus is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include molds, yeasts, and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, yeast is preferable because it can regulate the cell cycle and can use a haploid. The cell cycle means a process in which cell division occurs when cells multiply, and cells (daughter cells) produced by cell division become cells (mother cells) that undergo cell division again to produce new daughter cells.

The yeast is not particularly limited and can be appropriately selected according to the purpose. For example, Bar-1-deficient yeast with increased sensitivity to pheromones (sex hormones) that regulate the cell cycle to the G1 phase is preferred. When the yeast is Bar-1-deficient yeast, the abundance of yeast whose cell cycle cannot be controlled can be reduced, so an increase in the number of specific nucleic acids in the cells housed in the container can be prevented. be able to.

Prokaryotic cells are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include eubacteria and archaea. These may be used individually by 1 type, and may use 2 or more types together.

The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. For example, green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP : Yellow Fluorescent Protein) and the like. The fluorescent dye in the stained cells is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include Cell Tracker Orange and Cell Tracker Red.

When the particles aggregate, the concentration of the particles in the suspension and the concentration of the particles in the suspension may be adjusted by adjusting the concentration of the particles in the suspension containing the particles such as the luminescent particles 70 filled in the liquid chamber 21 of the droplet ejection means 20. , the number of particles in the suspension can be adjusted from the theory that the number of particles in the suspension follows a Poisson distribution. The liquid component of the suspension is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include ion-exchanged water.
The diameter of the droplet is not particularly limited and can be appropriately selected according to the purpose, but is preferably 25 μm or more and 150 μm or less. When the diameter of the droplet is 25 μm or more, the diameter of the droplet is not small, and it is difficult for the particles contained therein to have a small diameter. If the diameter is 150 μm or less, there is no problem as a droplet, and there is no need to increase the hole diameter of the inkjet head to eject the droplet, and the ejection of the droplet is less likely to be unstable. Further, it is preferable that R>3r, where R is the diameter of the droplet and r is the diameter of the particle. When R>3r, the diameter of the particles is not large relative to the diameter of the droplet, so that the edge of the droplet is less likely to affect the particle counting accuracy.

The light irradiation means 30 is means for irradiating the droplets 50 ejected from the droplet ejection means 20 with light. The light irradiation means 30 receives a synchronization signal from the control device 10 . The light irradiation means 30 to which the synchronizing signal is input irradiates the droplets 50 with laser light 81 as illumination light in accordance with the ejection timing of the droplets 50 by the droplet ejection means 20 .

As the light irradiating means 30 , it is preferable to irradiate light in synchronization with ejection of the droplets 50 by the droplet ejecting means 20 . This makes it possible to more reliably irradiate the droplets 50 ejected from the droplet ejection means 20 with light. Here, synchronizing means that the light irradiation means 30 irradiates the laser light 81 when the droplet 50 is discharged and reaches a predetermined position. That is, the light irradiation means 30 irradiates the droplets 50 with a delay of a predetermined time with respect to the discharge of the droplets 50 by the droplet discharge means 20 .

The light irradiation means 30 is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include solid lasers, semiconductor lasers, dye lasers, and the like. Examples of solid-state lasers include YAG lasers, ruby lasers, and glass lasers. Commercially available YAG lasers include, for example, Explorer ONE-532-200-KE (manufactured by Spectra Physics, Inc., output wavelength is 532 nm by SHG). Among these, it is preferable to be able to irradiate pulse light by pulse oscillation.

The pulse width of the pulsed light is not particularly limited and can be appropriately selected according to the purpose, but is preferably 10 μs or less, more preferably 1 μs or less.
The energy per unit pulse is not particularly limited and can be appropriately selected according to the purpose. Although it greatly depends on the optical system such as the presence or absence of light condensing, it is preferably 0.1 μJ or more, more preferably 1 μJ or more. . However, since photobleaching occurs depending on the fluorescent particles, it is preferable in this case to limit the energy per unit pulse or per unit time. Similarly, depending on the intended use of the fluorescent particles or other luminescent particles, light irradiation may have adverse effects, and in this case also it is preferable to limit the energy per unit pulse or unit time.

The light detecting means (light receiving means) 40 is a means for detecting (light receiving) the light emitted by the light emitting particles contained in the droplet upon receiving the light irradiated by the light irradiation means. The light receiving means 40 is electrically connected to the light irradiation means 30 and the control device 10 , and receives a synchronization signal from the control device 10 . The light receiving means 40 to which the synchronization signal is input receives the light 82 in synchronization with the timing at which the light emitting particles 70 emit light from the laser light 81 of the light irradiation means 30 .

The light receiving means 40 is an optical sensing device/module capable of measuring the presence or absence of the luminescent particles 70 contained in the droplet 50 . The light-receiving means 40 outputs light-receiving information to the control device 10 electrically connected thereto, and based on the information of these light-receiving elements, the control device 10 can measure the presence or absence of the luminescent particles 70 contained in the droplet 50. .

Since the light receiving means 40 only needs to be able to measure the presence or absence of the luminescent particles 70 in cooperation with the control device 10, a light sensing device/unit that acquires a small amount of information can be used. photomultiplier tube), avalanche photodiode (APD), pin photodiode (pin-PD), low resolution CMOS imaging device, etc. can be used. These are capable of sampling at high speed and high frequency, and at the same time, have high light utilization efficiency. An appropriate amount of information can be output to the controller ensuring that the

When an APD is used as the light receiving means 40 , the APD can output the amount of emitted light from the fluorescent particles 70 to the control device 10 as one-dimensional information. APD is an APD module, for example, APD410 (100 MHz band, 1 mm diameter, sensitivity 1 × 10 V / W, manufactured by Matsusada Precision Co., Ltd.), APD130A (50 MHz band, 1 mm diameter, sensitivity 2.5 × 10 V / W, Thorabo Japan) Co., Ltd.), C10508-01 (Band 100 MHz, Diameter 1 mm, Sensitivity 1.3 × 10 V / W, Hamamatsu Photonics Co., Ltd.), APD410A (Band 10 MHz, Diameter 1 mm, Sensitivity 2.7 × 10 V / W, Thorabo Japan (manufactured by Co., Ltd.), etc., can be selected in consideration of the appropriate band and sensitivity according to the purpose, the type of illumination light to be used, and the like.

It is preferable that the light receiving means 40 can receive the light 82 from the luminescent particles 70 in synchronization with ejection of the droplets 50 by the droplet ejecting means 20 . As a result, the droplets 50 ejected from the droplet ejection means 20 are irradiated with the laser light 81 from the light irradiation means 30, and the light 82 from the fluorescent particles 70 can be received more reliably. Here, synchronizing means, for example, when the droplet 50 is discharged and reaches a predetermined position, the droplet 50 is irradiated with the laser beam 81, and the light receiving means 40 emits light at the timing when the luminescent particle 70 emits light. 82 is meant to be received. That is, the light receiving means 40 detects the light 82 with a predetermined time delay with respect to the discharge of the droplets 50 by the droplet discharge means 20 and the irradiation of the laser light 81 by the light irradiation means 30 . Further, such delay adjustment can be realized by outputting a synchronizing signal considering the delay from each function generator by cooperating with a separate function generator. Furthermore, the function of this function generator may be integrated with the droplet discharge means 20 .

When the light from the luminescent particles 70 is weaker than the laser light 81 irradiated by the light irradiation means 30, the light receiving means 40 has a filter for attenuating the wavelength range of the laser light 81 on the light receiving surface side of the light receiving means 40. It is preferred to have By having this filter, the light receiving means 40 can receive the emitted light with less noise. As the filter, for example, a notch filter having an optical density of 6 or more that attenuates a specific wavelength range including the wavelength of light can be used.

The control device 10 can include, for example, a digitizer, a data logger, an oscilloscope, etc. as part thereof. A digitizer is a module (product) equipped with an ADC (analog-to-digital converter). For example, APX-510 (16bit/100MHz sampling, manufactured by Avaldata Co., Ltd.) provided as an expansion board for a personal computer (PC). , DIG-100M1002-PCI (10bit/100MHz sampling, manufactured by Contec Co., Ltd.). Also, the control device 10 may be realized by a plurality of PCs or the like, and in this case, the processing performed by the control device 10 may be distributed and executed. In addition, when the control device 10 has a plurality of PCs, each PC may be connected by a network. Furthermore, the processing performed by the control device 10 may be performed by a cloud computer.
By digitizing the analog information output from the light receiving means 40 using a digitizer or the like, the digitized information may be output to the information processing section which is a part of the control device 10 connected to the digitizer. Even though the acquired information of the digitized luminescent particles 70 is one-dimensional information and a small amount of acquired information, the information processing unit determines whether the fluorescent particles can The presence or absence of fluorescent particles can be determined and the presence or absence of fluorescent particles can be measured. The information processing unit, which is a part of the control device 10, can use a personal computer (PC) and image processing software installed in the PC.

Measuring the presence or absence of the luminescent particles 70 is a qualitative measurement that only needs to confirm the light emission based on the luminescent particles 70. Therefore, even if the amount of acquired information is small, the measurement is more accurate than the measurement of the number of the luminescent particles 70. In other words, it is possible to realize measurement with a high rate of correct answers. In addition, as an effect of the fact that the amount of acquired information may be small, the information acquisition operation and the output of acquired information regarding the light 82 by the light receiving means 40 and the information processing of the acquired information by the control device 10 are simplified. can be realized.

The configuration of the ejection device 100 of the present embodiment has been described above. Next, the functional configuration of the control device 10 will be described based on the functional block diagram shown in FIG. 2 .

As shown in FIG. 2, the control device 10 of the present embodiment has a liquid droplet ejection control section 12, a light emission detection section 13, a light irradiation control section 14, and an average particle number calculation means 19, and further includes necessary It has other means (parts) according to.
The average particle number calculation means 19 preferably has a determination unit 15 that performs determination processing, a calculation unit 17 that performs calculation processing, and preferably includes a counting unit 18 that performs counting processing. have In this embodiment, the average particle number calculation means 19 has a determination section 15 , a calculation section 17 and a counting section 18 . Note that the discharge device 100 can perform a dispensing process of dispensing a predetermined number of luminescent particles 70 into the container 60 through the cooperative operation of the respective means (units) described above.

The droplet ejection control unit 12 is means for controlling the droplet ejection means 20 to eject a predetermined amount of droplets.

The light irradiation control section 14 is means for controlling the light irradiation means 30 to irradiate the droplets discharged by the droplet discharge means 20 with light. Note that the light irradiation control unit 14 controls the irradiation timing of the light irradiation unit 30 so that the droplets are irradiated with light before the discharged droplets land on the container 60 . It is preferable to synchronize the timing.

The luminescence detection unit 13 is means for detecting luminescence of the luminescent particles contained in the droplets ejected by the droplet ejection unit 20 based on the detected intensity of the light received by the light receiving unit 40 .

The determining unit 15 in the average particle number calculating unit 19 determines whether or not the light receiving unit 40, which is an example of the light detecting unit, detects the light emission 82 generated in the light emitting particles 70. Judgment processing, which is processing for judging the presence/absence, is performed.

The calculation unit 17 in the average particle number calculation unit 19 calculates the proportion of the droplets 50 that do not contain the luminescent particles 70 in all the ejected droplets based on the presence or absence of the luminescent particles 70 (determination result of the determination unit 15). , calculation processing for calculating the average number of particles, which is the average number of luminescent particles 70 contained in one droplet 50 in all droplets.

More specifically, the calculation unit 17 divides the number of times the liquid droplets are judged to contain no light-emitting particles based on the determination result of the determination unit 15 by the total number of droplet ejection times (the total number of droplets). The obtained value is calculated as a ratio (probability) that the droplets ejected by the droplet ejection unit 20 do not contain the luminescent particles.
Subsequently, the calculator 17 calculates the average number of luminescent particles (hereinafter referred to as the average number of particles) contained in the droplets ejected by the droplet ejecting means 20 based on the calculated probability.
Further, in the present embodiment, the average number of particles calculated by the calculation unit 17 is an estimated value calculated based on the probability that the droplets ejected by the droplet ejection unit 20 do not contain the luminescent particles. Therefore, hereinafter, the expression that the calculation unit 17 estimates the average number of particles may be used.

The counting unit 18 in the average particle number calculation unit 19 multiplies the average number of particles estimated by the calculation unit 17 by the total number of droplets ejected by the droplet ejection unit 20 to obtain the total number of droplets ejected by the droplet ejection unit 20. Count the number of luminescent particles in the entire droplet. By doing so, the dispensing device 100 can dispense a prescribed number of particles into the container 60 .

The functional configuration of the control device 10 has been described above. Subsequently, the details of the processing executed by the determination unit 15 and the calculation unit 17 that constitute the control device 10 will be described.

First, the method by which the determining unit 15 determines the presence or absence of luminescent particles contained in a droplet will be described.

As shown in FIG. 3A, when the droplets ejected from the droplet ejecting means 20 contain one or more luminescent particles, the luminescent particles receive light emitted from the light irradiation means 30 and emit light, The emitted light is received by the light receiving means 40 . On the other hand, as shown in FIG. 3B, when the droplets ejected from the droplet ejection means 20 do not contain luminescent particles, the light receiving means 40 does not receive light.

Assuming this, the determination unit 15 compares the signal representing the ejection timing of the droplet ejection means 20 and the light detection intensity signal of the light received by the light receiving means 40 as shown in FIG. When a light detection intensity equal to or greater than a predetermined threshold value is detected in synchronization with the timing, it is determined that the droplet contains luminescent particles, and when a light detection intensity equal to or greater than the predetermined threshold value is not detected. , it is determined that the droplet does not contain the luminescent particles. The state in which the light detection intensity is detected in synchronization with the ejection timing of the droplet means the state in which the light detection intensity is detected without a delay of a predetermined threshold time or more from the ejection timing.

Next, a method for estimating the average number of particles by the calculation unit 17 will be described.

Assuming that the particle distribution in the luminescent particle suspension conforms to the Poisson distribution, it can be assumed that the particle distribution in the droplets ejected from the droplet ejection means 20 also conforms to the Poisson distribution. According to this assumption, the probability P(λ , x) can be represented by the following formula (1). In the following formula (1), e represents Napier's number, and x! represents the factorial of x.

As shown in the above formula (1), the probability P (λ, x) is a function of the average particle number λ and the particle number x of the luminescent particles contained in the droplet, so once the average particle number λ is determined, Poisson A distribution is determined. FIG. 5 shows the Poisson distribution for average particle number λ=1, 2, 5.

Since the probability P(x=0) that the ejected droplets do not contain any luminescent particles monotonously decreases with respect to λ, the average number of particles λ can be uniquely determined if P(x=0) is known. . Based on this, the calculation unit 17 obtains the probability P(x=0), and uses the obtained probability P(x=0) to estimate the average number of particles λ according to the above equation (2). When P(x=0) is large, that is, when there is a high probability that the ejected droplet does not contain the luminescent particles, λ becomes small. λ increases when the probability that the droplet containing no luminescent particles is small.

FIG. 6 shows simulation results of the average particle number λ estimated by the method described above. In this simulation, the average number of luminescent particles contained in a droplet is constant, random numbers are generated by a Poisson distribution as the number of luminescent particles contained in droplets ejected by the ejection device 100, and droplets are ejected a predetermined number of times ( 1,000 times, 3,000 times, 10,000 times, 30,000 times, 100,000 times, 300,000 times, 1,000,000 times). The average particle number λ was estimated based on the calculated probability P (x=0). In this simulation, the above trial was repeated 100 times, and the coefficient of variation (CV) was calculated from the average value and standard deviation of the average particle number λ obtained in the 100 trials.

FIG. 6 is a diagram plotting the results of 100 trials with the average number of particles λ on the horizontal axis and the coefficient of variation (CV) on the vertical axis. As shown in FIG. 6, the variation (CV) is the smallest when the average particle number λ is 1 to 2, and even when the average particle number λ is 5, the variation (CV) is within the allowable range. (Number of ejections: 100,000 → CV = 0.8%, Number of ejections: 1,000 → CV = 8.7%). That is, the average number of particles is preferably 1 or more and 2 or less. By setting the average number of particles to be 1 or more and 2 or less, the ejection device 100 can suppress variations in the number of luminescent particles contained in the liquid droplets and dispense a predetermined number of luminescent particles more accurately.
In addition, the CV is preferably 15% or less as the allowable range (allowable range of variation in the number of particles). When the range of variation in the number of particles is within the preferred range described above, it is possible to satisfy the standards of the Bioanalytical Method Validation Guidance for Industry by the US FDA (Food and Drug Administration).

The content of the processing executed by the determination unit 15 and the calculation unit 17 has been described above. Next, the dispensing processing executed by the ejection device 100 of this embodiment will be described. In addition, below, three types of execution modes provided in the ejection device 100 will be described in order.

(first execution mode)
In the first execution mode, the ejection device 100 presumes the average number of particles λ in cooperation with the droplet ejection control unit 12, the light emission detection unit 13, the light irradiation control unit 14, the determination unit 15, and the calculation unit 17. After that, the number of ejected luminescent particles is counted by the counting unit 18 using the estimated average number of particles λ, and the luminescent particles are dispensed. First, a calculation process for calculating (estimating) the average number of particles λ based on the flowchart shown in FIG. 7 will be described.

First, in step 101, the number of droplet ejections y (=the number of ejected droplets y) and the number of determinations k for determining that the droplets do not contain luminescent particles are set to initial values (0). .

In the subsequent step 102, one droplet of a predetermined liquid volume of the luminescent particle suspension is discharged from the droplet discharging means 20, and in the subsequent step 103, the number of times of discharge y is incremented by one. From the results of the simulation described in FIG. 6, the CV is low when the average number of particles is about 1 or more and 2 or less. It is difficult. Therefore, in order to set the average number of particles to 1 or more and 2 or less, it is preferable to control the particle concentration of the luminescent particle suspension (the same applies to other execution modes to be described later).

In subsequent step 104, the presence or absence of luminescent particles contained in the ejected droplet is determined by the method described above. If it is determined that the droplet contains luminescent particles (step 105, No), the process proceeds to step Proceeding to 107, it is determined whether or not the number of times of ejection y has reached the preset number of times N. If not (step 107, No), the process returns to step .

On the other hand, if it is determined that the droplet does not contain the light-emitting particles (step 105, Yes), the process proceeds to step 106, and the determination count k is incremented by one. After that, in subsequent step 107, it is determined whether or not the number of ejections y has reached the set number of times N. If not (step 107, No), the process returns to step .

Thereafter, the above-described series of processes (steps 102 to 107) are repeated until the number of ejections y reaches the set number of times N. When the number of ejections y reaches the set number of times N (step 107, Yes), the process proceeds to step 108 proceed to

In the subsequent step 108, the value obtained by dividing the determination number k at that time by the ejection number y at that time is calculated as the probability P (x=0) that the droplet does not contain the luminescent particles.

In subsequent step 109, the probability P(x=0) calculated in previous step 108 is used to estimate the average number of particles λ by the method described above.

Next, the dispensing process executed by the ejection device 100 will be described based on the flowchart shown in FIG.

First, in step 201, the expected value E of the number of droplet ejection times y and the number of dispensed luminescent particles (hereinafter referred to as the dispensed number) is set to an initial value (0).

In the following step 202, one droplet of a predetermined liquid volume of the luminescent particle suspension is discharged from the droplet discharging means 20, and in the subsequent step 203, the number of times of discharge y is incremented by one.

In the following step 204, based on the following equation (3), the average number of particles λ estimated in the previous calculation process is multiplied by the number of ejections y at that time to calculate the expected value E of the number of dispensing.

In the subsequent step 205, it is determined whether or not the expected value E has reached a preset target value. 204) are repeatedly executed. After that, when the expected value E reaches the target value (step 205, Yes), the process ends.

The first execution mode has been described above. In the conventional ejection device, sorting is generally performed using a low-concentration particle suspension under the condition that the average number of particles contained in the droplets is less than 1. For example, as the simulation results in FIG. 6 show, even when using a high-concentration particle suspension in which the average number of particles contained in a droplet is 1 to 5, the number of particles can be counted with high accuracy. Dispensing time can be shortened (the same applies to other execution modes to be described later).

(second execution mode)
In the first execution mode, the dispensing process is performed using the pre-estimated average particle number λ. 13, the light irradiation control unit 14, the determination unit 15, the calculation unit 17, and the counting unit 18 cooperate to simultaneously perform the estimation of the average number of particles λ and the dispensing process. The dispensing process in the second execution mode will be described below based on the flowchart shown in FIG.

First, in step 301, the number y of ejections of droplets, the number k of determinations that the droplets do not contain luminescent particles, and the expected value E of the number of dispensed are set to initial values (0).

In the subsequent step 302, one droplet of a predetermined liquid volume of the luminescent particle suspension is discharged from the droplet discharge means 20, and in the subsequent step 303, the number of times of discharge y is incremented by one.

In subsequent step 304, the presence or absence of luminescent particles contained in the ejected droplet is determined by the method described above. If it is determined that the droplet contains luminescent particles (step 305, No), the process proceeds to step Go to 307. On the other hand, if it is determined that the droplet does not contain any luminescent particles (step 305 , Yes), the process proceeds to step 306 , incrementing the number of determinations k by 1, and then proceeds to step 307 .

In the subsequent step 307, the value obtained by dividing the determination number k at that time by the ejection number y at that time is calculated as the probability P (x=0) that the droplet does not contain the luminescent particles.

In the subsequent step 308, the probability P(x=0) calculated in the previous step 307 is used to estimate the average number of particles λ by the method described above.

In the next step 309, the latest average number of particles λ estimated in the previous step 308 is multiplied by the number of ejections y at that time to calculate the expected value E of the number of dispensing.

In the subsequent step 310, it is determined whether or not the expected value E has reached a preset target value. 310) is repeatedly executed. During this time, every time a new droplet is ejected, the probability P (x=0) is recalculated, and the average particle number λ is reestimated based on the recalculated probability P (x=0). A value obtained by multiplying the latest average number of particles λ by the number of ejections y at that time is counted as the number of dispensing. Then, when the expected value E reaches the target value (step 310, Yes), the process ends.
That is, in the second execution mode, each time a new droplet is ejected, the average particle number calculating means obtains the ratio of droplets that do not contain luminescent particles in all droplets, and based on the obtained ratio, and multiplying the recalculated average particle number by the total number of droplets ejected by the droplet ejection means, the number of luminescent particles in all the droplets ejected by the droplet ejection means. to count.

The second execution mode has been described above. Since the second execution mode does not perform preprocessing for estimating the average number of particles λ, there is an advantage that the sample is not wasted and dispensing can be started immediately.

(Third execution mode)
In the second execution mode, the probability P(x=0) is calculated based on all the determination results determined by the determination unit 15. A probability P (x=0) is calculated based on the determination results obtained thereafter. The dispensing process in the third execution mode will be described below based on the flowchart shown in FIG.

First, in step 401, the number y of ejections of droplets, the number k of determinations that the droplets do not contain luminescent particles, and the expected value E of the number of dispensed are set to initial values (0).

In step 402, a timer for a predetermined time T is set.

At subsequent step 403, the same processing as steps 302 to 309 (see FIG. 9) in the second execution mode is executed.

In subsequent step 404, it is determined whether or not the expected value E has reached a preset target value.
As a result, if the target value has not been reached (step 404, No), it is determined whether or not the predetermined time T has elapsed in the following step 405, and if the predetermined time T has not elapsed (step 405, No), return to step 403 . On the other hand, if the predetermined time T has passed (step 405, Yes), in step 406, the ejection count y and determination count k are reset to the initial values (0), and the process returns to step 401.

After that, the above-described series of processes (steps 402 to 406) are repeatedly executed until the expected value E reaches the target value, and when the expected value E reaches the target value (step 404, Yes), the process ends. .
That is, in the third execution mode, the average particle number calculating means 19 calculates the number of luminescent particles in all droplets based on the information on the presence or absence of luminescent particles obtained after each elapse of a predetermined period of time. Determine the percentage of droplets that do not

The third execution mode has been described above. In the third execution mode, each time a preset predetermined time T elapses, the particle presence/absence determination results obtained before that time are discarded, and based on the determination results obtained after that, the probability P (x= 0) is recalculated, there is the advantage of minimizing the influence on the counting accuracy in the event of sedimentation of particles inside the droplet ejection means 20 or disturbance.

The dispensing process executed by the ejection device 100 of the present embodiment and the ejection method using the ejection device 100 have been described above. Explain how to count.

(Method for counting non-luminescent particles)
First, a suspension 1 containing arbitrary luminescent particles (e.g., fluorescent polymer particles) at a predetermined concentration, and arbitrary particles to be counted, which are non-luminescent particles that do not emit light when receiving light that causes the luminescent particles to emit light. A suspension 2 containing a predetermined concentration of sexual particles is prepared.

Next, the prepared suspension 1 having a known concentration and the suspension 2 having a known concentration are mixed at a predetermined mixing ratio (volume ratio) to prepare a mixed suspension.

Next, the prepared mixed suspension is set in the ejection device 100, and droplets of a predetermined liquid volume produced from the mixed suspension are continuously ejected into the container, and the same method as described above is applied to the container. Count the number of ejected luminescent particles.

Finally, the number of non-luminescent particles discharged into the container is calculated based on the counted number of luminescent particles and the mixing ratio (volume ratio) of the mixed suspension. Specifically, the number of the counted luminescent particles is multiplied by the mixture ratio of the mixed suspension (suspension 2 of non-luminescent particles/suspension 1 of luminescent particles). count as the number of
That is, when counting the number of non-luminescent particles in the present invention, a suspension with a known concentration of luminescent particles and a suspension with a known concentration of non-luminescent particles that do not emit light when irradiated with light are used. are mixed at a predetermined mixing ratio to prepare a mixed suspension, and when the droplets of the mixed suspension are discharged, the number of luminescent particles in all the discharged droplets is counted, and the counted luminescent particles It is preferable to count the number of non-luminescent particles in all ejected droplets based on the number of particles and the mixing ratio.

Using the method described above, even particles that do not themselves emit light can be counted using the ejection device 100 of the present embodiment. In the above-described method, the mixed suspension is prepared so that the average number of luminescent particles contained in the droplets ejected from the mixed suspension is 1 or more and 2 or less, based on the simulation results described in FIG. is preferable from the viewpoint of counting accuracy.

As described above, according to the present embodiment, it is possible to perform dispensing with high accuracy and in a short time with a simple configuration. It should be noted that the present invention is not limited to the above-described embodiments, and various design changes conceivable by those skilled in the art are included in the scope of the present invention.

For example, as shown in FIG. 11, the ejection device 100 includes light irradiation means for simultaneously irradiating n types of light (n is an integer of 2 or more; the same shall apply hereinafter) with different wavelengths, and n light sources corresponding to the n types of light. It is also possible to provide the light receiving means so that the number of n kinds of luminescent particles with different excitation wavelengths can be counted. In this case, for example, n types of cells emitting different fluorescences can be counted for each type to examine the ratio of cells, or to determine the expression ratio of a reporter gene (GFP reporter, etc.).

Each function of the above-described embodiment can be realized by a program written in C, C++, C#, Java (registered trademark), etc., and the program of the present embodiment can be implemented by hard disk devices, CD-ROMs, MOs, DVDs, etc. , a flexible disk, an EEPROM, an EPROM, or the like, and can be distributed, or can be transmitted over a network in a format that can be used by other devices.

DESCRIPTION OF SYMBOLS 10... Control apparatus 12... Droplet ejection control part 13... Light emission detection part 14... Light irradiation control part 15... Judgment part 17... Calculation part 18... Counting part 19... Average particle number calculation means 20... Droplet ejection means 30... Light Irradiation means 40... Photodetection means (light receiving means)
50... Droplet 60... Container 70... Luminescent particle 100... Ejection device

Japanese Patent Application Laid-Open No. 2018-17700

Claims (12)

droplet ejection means for ejecting droplets containing luminescent particles that emit light when irradiated with light;
light irradiation means for irradiating the light onto the droplets discharged by the droplet discharge means;
a light detection means for detecting the light emitted by the light-emitting particles contained in the droplets upon receiving the light emitted by the light irradiation means;
The presence or absence of the luminescent particles contained in the droplet is determined according to whether or not the light detection means detects the luminescence generated in the luminescent particles, and based on the presence or absence of the luminescent particles, the ejected liquid is obtained. The average number of particles for calculating the average number of the luminescent particles contained in one of the droplets in all the droplets from the ratio of the droplets that do not contain the luminescent particles in all the droplets. calculating means;
has
The ejection device , wherein the average number of particles is 1 or more and 2 or less . The average particle number calculating means multiplies the average particle number by the total number of droplets ejected by the droplet ejecting means to obtain the luminescent particles in all the droplets ejected by the droplet ejecting means. 2. The dispensing device of claim 1, wherein the number of . 3. The ejection device according to claim 1, wherein said average particle number calculating means calculates said average particle number based on the following formula.

(In the above formula, λ indicates the average number of particles, and P (x = 0) indicates the ratio.) The ejection device is
The light irradiation means for simultaneously irradiating n types of light with different wavelengths (n is an integer of 2 or more; the same shall apply hereinafter), and n light detection means corresponding to the n types of light,
The average particle number calculation means is
4. The ejection device according to claim 2, wherein the number of n types of luminescent particles having different excitation wavelengths is counted. The ejection device according to any one of claims 1 to 4, wherein the droplet ejection means is an inkjet head or a cell sorter. The ejection device according to any one of claims 1 to 5, wherein the luminescent particles are cells that are fluorescently stained or cells that retain fluorescent proteins. The average particle number calculation means is
obtaining the ratio each time a new droplet is ejected, and recalculating the average number of particles based on the obtained ratio;
By multiplying the recalculated average number of particles by the total number of droplets ejected by the droplet ejection means, the number of the luminescent particles in the total droplets ejected by the droplet ejection means is counted. The discharge device according to any one of claims 1 to 6 . droplet ejection means for ejecting droplets containing luminescent particles that emit light when irradiated with light;
light irradiation means for irradiating the light onto the droplets discharged by the droplet discharge means;
a light detection means for detecting the light emitted by the light-emitting particles contained in the droplets upon receiving the light emitted by the light irradiation means;
The presence or absence of the luminescent particles contained in the droplet is determined according to whether or not the light detection means detects the luminescence generated in the luminescent particles, and based on the presence or absence of the luminescent particles, the ejected liquid is obtained. The average number of particles for calculating the average number of the luminescent particles contained in one of the droplets in all the droplets from the ratio of the droplets that do not contain the luminescent particles in all the droplets. calculating means;
has
The average particle number calculation means is
obtaining the ratio each time a new droplet is ejected, and recalculating the average number of particles based on the obtained ratio;
By multiplying the recalculated average number of particles by the total number of droplets ejected by the droplet ejection means, the number of the luminescent particles in the total droplets ejected by the droplet ejection means is counted. An ejection device characterized by : The average particle number calculation means is
9. The ejection device according to claim 7 , wherein each time a predetermined period of time elapses, the ratio is obtained based on the information on the presence or absence of the luminescent particles obtained thereafter. a droplet ejection step of ejecting droplets containing luminescent particles that emit light when irradiated with light;
a light irradiation step of irradiating the light onto the droplets ejected in the droplet ejection step;
a light detection step of detecting the light emitted by the luminescent particles contained in the droplet receiving the light irradiated in the light irradiation step;
The presence or absence of the luminescent particles contained in the droplet is determined according to whether or not the luminescence generated in the luminescent particles is detected in the light detection step, and the ejection is obtained based on the presence or absence of the luminescent particles. The average number of particles for calculating the average number of the luminescent particles contained in one of the droplets in all the droplets from the ratio of the droplets that do not contain the luminescent particles in all the droplets. a calculation step;
including
In the average particle number calculation step,
obtaining the ratio each time a new droplet is ejected, and recalculating the average number of particles based on the obtained ratio;
By multiplying the recalculated average number of particles by the total number of droplets ejected in the droplet ejection step, the number of the luminescent particles in the total droplets ejected in the droplet ejection step is counted. A discharge method characterized by: When counting the number of non-luminescent particles,
A suspension having a known concentration of the luminescent particles and a suspension having a known concentration of the non-luminescent particles that do not emit light when irradiated with light are mixed at a predetermined mixing ratio to obtain a mixed suspension. further comprising a preparation step of preparing
In the average particle number calculation step,
counting the number of the luminescent particles in all the droplets ejected in the droplet ejection step when ejecting the droplets of the mixed suspension;
11. The ejection method according to claim 10, wherein the number of said non-luminescent particles in all said droplets ejected in said droplet ejection step is counted based on said counted number of said luminescent particles and said mixing ratio. In the preparation step,
12. The ejection method according to claim 11, wherein the mixed suspension is prepared so that the average number of particles is 1 or more and 2 or less.

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