JP2022177832A - Droplet collection unit, droplet collection device, and method - Google Patents

Abstract

To allow for collecting droplets containing a controlled amount of microbes encapsulated therein.SOLUTION: A droplet collection unit according to an embodiment comprises a generation unit, detection unit, and a sorting unit. The generation unit generates droplets containing a microbe and a matrix that reacts with an enzyme originating from the microbe. The detection unit detects a reaction between the enzyme and the matrix in the droplets. The sorting unit sorts the droplets according to detection results of the reaction.SELECTED DRAWING: Figure 1

Description

Embodiments disclosed in the specification and drawings relate to droplet collection units, droplet collection apparatus and methods.

Conventionally, a method is known in which droplets are generated in a microchannel to enclose cells or bacteria. For example, there is known a technique of encapsulating cells or bacteria in droplets one by one using probability theory. In the case of stochastically encapsulating cells in this way, it is possible to separate only the droplets in which the cells are encapsulated by distinguishing and sorting the cells in the droplets with a microscope or the like.

JP 2018-38384 A

One of the problems addressed by the embodiments disclosed in the specification and drawings is collecting droplets with a controlled amount of microorganisms encapsulated in the droplets. However, the problems to be solved by the embodiments disclosed in this specification and drawings are not limited to the above problems. A problem corresponding to each effect of each configuration shown in the embodiments described later can be positioned as another problem.

A droplet collection unit according to an embodiment comprises a generator, a detector, and a sorter. The generator generates droplets containing a microorganism and a substrate that reacts with an enzyme derived from the microorganism. A detection unit detects a reaction between the enzyme and the substrate in the droplet. The sorting section sorts the droplets based on the detection result of the reaction.

FIG. 1 is a block diagram showing an example of the configuration of a droplet collection unit according to the first embodiment. FIG. 2 is a diagram showing an example of the microchannel chip according to the first embodiment. FIG. 3 is a diagram for explaining an example of processing by the droplet collecting unit according to the first embodiment; FIG. 4 is a diagram for explaining an example of a system according to the first embodiment; FIG. 5 is a diagram showing experimental results regarding the bacterial enzyme (β-galactosidase) according to the first embodiment. FIG. 6 is a diagram showing experimental results regarding a virus-derived enzyme (neuraminidase) according to the first embodiment.

Embodiments of the droplet collection unit, droplet collection apparatus and method are described in detail below with reference to the accompanying drawings. Also, the droplet collection unit, droplet collection apparatus and method according to the present application are not limited by the embodiments shown below. In addition, in the following description, common reference numerals are assigned to similar components, and duplicate descriptions are omitted.

(First embodiment)
First, an example of the configuration of the droplet collection unit according to the first embodiment will be described. FIG. 1 is a block diagram showing an example of the configuration of a droplet collecting unit 1 according to the first embodiment. The droplet collection unit 1 includes a processing circuit 11, a liquid sending section 12, a heating section 13, a light source section 14, a fluorescence detection section 15, a separation section 16, a recovery section 17, a liquid waste section 18, It has a microchannel chip 20 and collects droplets containing microorganisms such as viruses and bacteria.

Here, the droplet collection unit 1 can be configured to be connectable to a system that utilizes collected droplets. Specifically, the droplet collection unit 1 can be configured to be connectable to systems for various analyzes and tests using microorganisms encapsulated in droplets. For example, the droplet collection unit 1 can be configured to be conjugated to a system for introducing various factors into cells using viruses encapsulated in droplets as vectors.

A processing circuit 11 controls various processes in the droplet collecting unit 1 . For example, the processing circuit 11 is implemented by a processor. Here, the processing circuit 11 controls various processes in the droplet collecting unit 1, for example, according to various operations via an input interface provided in the droplet collecting unit 1 or the system described above.

The processing circuit 11 reads out and executes a program stored in a storage circuit (not shown) to perform a control function 11a, a detection function 11b, and a classification function 11c. Note that the control function 11a and the heating unit 13 are examples of a temperature control unit. Also, the detection function 11b, the light source section 14, and the fluorescence detection section 15 are examples of the detection section. Also, the sorting function 11c and the sorting unit 16 are examples of the sorting unit.

The control function 11 a controls delivery of liquid by the liquid delivery section 12 . Specifically, the control function 11a sends the liquid to be sent into the microchannel chip 20 by controlling the pump or the like included in the liquid sending section 12 . For example, the control function 11a controls the liquid sending unit 12 to send various sending liquids into the microchannel chip 20 for generating droplets containing microorganisms such as viruses and bacteria. The details of the delivery of the delivery liquid by the control function 11a will be described later.

Also, the control function 11 a adjusts the temperature of the fluid in the microchannel chip 20 by controlling the heating unit 13 . Specifically, the control function 11 a adjusts the temperature of droplets in the microchannel chip 20 . Details of temperature adjustment by the control function 11a will be described later.

The detection function 11b controls the light source unit 14 and the fluorescence detection unit 15 to detect the reaction inside the droplet. Specifically, the detection function 11b detects the reaction between the enzyme and the substrate in the droplet. More specifically, the detection function 11b emits light by controlling the light source unit 14, irradiates the droplets generated in the microchannel chip 20 with light, and emits fluorescence generated in the droplets. A reaction in the droplet is detected by detection by the fluorescence detection unit 15 . For example, the detection function 11b detects fluorescence based on a reaction between an enzyme derived from a microorganism and a substrate, and detects a reaction between the enzyme and the substrate within the droplet based on the detected fluorescence. Details of detection processing by the detection function 11b will be described later.

The sorting function 11 c sorts droplets generated in the microchannel chip 20 by controlling the sorting section 16 . Specifically, the sorting function 11c sorts droplets based on the detection result of the detection function 11b. More specifically, the sorting function 11c determines the amount of microorganisms contained in the droplet based on the detection result, and sorts the droplet based on the determination result. For example, the sorting function 11c determines the number of microorganisms contained in the droplet based on the detection result, and sorts the droplet based on the determination result. The details of the separation processing of droplets by the separation function 11c will be described later.

The liquid sending unit 12 sends the sending liquid into the microchannel chip 20 under the control of the control function 11a. Specifically, the liquid sending unit 12 sends various sending liquids into the microchannel chip 20 for generating droplets in which microorganisms are enclosed. For example, the liquid sending unit 12 includes a pump and the like, and sends various liquids into the microchannel chip 20 . The details of the liquid sent into the microchannel chip 20 by the liquid sending unit 12 will be described later.

The heating unit 13 adjusts the temperature of the fluid in the channels in the microchannel chip 20 under the control of the control function 11a. Specifically, the heating unit 13 adjusts the temperature of droplets in the microchannel chip 20 . For example, the heating unit 13 is arranged in contact or non-contact with the microchannel chip 20, and adjusts the temperature of droplets in the microchannel chip 20 by infrared heating, dielectric heating, or the like. The heating unit 13 may directly adjust the temperature of the fluid, or may adjust the temperature of the microchannel chip 20 to thereby adjust the temperature of the fluid. Adjusting the temperature of the container may be regarded as adjusting the temperature of the droplet.

Here, the heating unit 13 may be configured to have a temperature sensor and measure the temperature of the liquid droplets by the temperature sensor. In such a case, the control function 11 a adjusts the heating by the heating section 13 based on the temperature measured by the temperature sensor in the heating section 13 . That is, the control function 11a can also control the temperature of the droplets in the microchannel chip 20 so as to maintain the set temperature.

The light source unit 14 emits light under the control of the detection function 11b. Specifically, the light source unit 14 irradiates light onto droplets flowing through the channels of the microchannel chip 20 . Here, the light source unit 14 may be of any type as long as it can emit light capable of generating fluorescence from droplets. For example, a laser or an LED (light emitting diode) etc.

The fluorescence detection unit 15 is excited by the light emitted from the light source unit 14 and detects fluorescence generated from the droplet. Here, the fluorescence detection unit 15 may be of any type as long as it can detect the fluorescence emitted from the droplet. For example, an image sensor, a fluorescence microscope, a fluorometer, etc. realized by

For example, the fluorescence detection unit 15 is arranged at a position facing the light source unit 14 with the microchannel chip 20 interposed therebetween, detects the fluorescence generated by the excitation light emitted from the light source unit 14, and detects the detection result. Send to function 11b.

The sorting unit 16 sorts droplets flowing through the microchannel chip 20 under the control of the sorting function 11c. Specifically, the sorting unit 16 sorts the droplets according to the amount of microorganisms contained in the droplets. For example, the sorting unit 16 sorts droplets containing a predetermined amount or more of microorganisms. Also, for example, the sorting unit 16 sorts the droplets according to the number of microorganisms contained in the droplets. For example, the sorting unit 16 includes electrodes for applying an electric field to the channels of the microchannel chip 20, and applies an electric field to the channels of the microchannel chip 20 under the control of the sorting function 11c. The applied voltage separates the droplets by dielectrophoresis. For example, the sorting unit 16 separates droplets containing a desired number of microorganisms, other droplets (droplets not containing microorganisms, and droplets containing microorganisms other than the desired number). droplets) are separated by dielectrophoresis.

The collection unit 17 collects droplets separated by the separation unit 16 . Specifically, the collecting unit 17 collects droplets in which a desired number of microorganisms are encapsulated. The waste liquid unit 18 collects other droplets separated by the separating unit 16 .

The microchannel chip 20 includes microchannels and generates droplets. Specifically, the microfluidic chip 20 produces droplets encapsulating microorganisms and substrates that react with enzymes derived from the microorganisms. Details of the microchannel chip 20 will be described later. The microchannel chip 20 is an example of a generator.

The configuration of the droplet collection unit 1 according to the present embodiment has been described above. With such a configuration, the droplet collection unit 1 collects droplets having a known number of microbes encapsulated therein. As mentioned above, when encapsulating a predetermined number of cells or the like in a droplet, a stochastic approach is used. Here, for example, when generating droplets enclosing cells by a stochastic method, in addition to droplets enclosing a predetermined number of cells, a number larger than a predetermined number (or less than a predetermined number (number of cells) are generated, and droplets in which no cells are encapsulated (the number of encapsulated cells is 0) are generated. Therefore, when cells and the like are enclosed in droplets by a stochastic method, the cells in the droplets are identified and separated using a microscope or the like.

However, in the case of small-sized microorganisms such as viruses, it is difficult to observe with an optical microscope, and it is difficult to identify whether microorganisms are encapsulated in droplets and, if so, how many. It's not easy.

Therefore, the droplet collection unit 1 according to the present embodiment utilizes the reaction between the microorganism-derived enzyme and the substrate to determine whether or not the microorganism is enclosed in the droplet, and if so, whether or not the microorganism is enclosed. Collect droplets with a known number of microbes encapsulated in the droplet by identifying the number of droplets. Specifically, the droplet collection unit 1 stochastically generates droplets enclosing microorganisms in the microchannel chip 20, identifies each generated droplet based on an enzymatic reaction, and determines the identification result. The droplets are sorted accordingly.

Details of the droplet collection unit 1 according to the present embodiment will be described below. FIG. 2 is a diagram showing an example of the microchannel chip 20 according to the first embodiment. For example, the microchannel chip 20 includes a first injection port 21, a second injection port 22, a third injection port 23, a droplet generation region 24, an enzyme reaction region 25, and a detection region 26. , a separation area 27 , a recovery port 28 and a waste liquid port 29 . The droplet collection unit 1 can be configured such that the microchannel chip 20 can be replaced each time the droplet collection method described below is executed.

The first injection port 21 is connected to a first fluid supply unit for generating droplets, and the first fluid is injected by the liquid supply unit 12 sending out the first fluid. Here, the first fluid is a continuous phase fluid for generating droplets. For example, the continuous phase fluid is oil, and an oil suitable for the microorganisms to be encapsulated in the droplets is used as appropriate. A flow path 21 a is formed between the first injection port 21 and the droplet generation region 24 , and the continuous-phase fluid injected from the first injection port 21 is delivered to the droplet generation region by the liquid sending unit 12 . 24 direction.

The second injection port 22 is connected to a second fluid supply unit for generating droplets, and the second fluid is injected by the liquid supply unit 12 sending out the second fluid. Here, the second fluid is, for example, a suspension of microorganisms such as viruses, and its concentration is adjusted based on probability theory so that a predetermined number of microorganisms are enclosed in droplets.

The target microorganism may be any microorganism as long as it has an enzyme on its surface or inside and produces a product that can be optically detected by the reaction of the enzyme. For example, target microorganisms include Paramyxoviridae viruses having hemagglutinin-neuraminidase on the virus surface. Hemagglutin neuraminidase reacts with a substrate "MUNANA (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)" to produce a fluorescent substance "4-methylumbelliferone".

A flow path 22 a is formed between the second injection port 22 and the droplet generation region 24 , and the second fluid injected from the second injection port 22 is sent by the liquid feeding unit 12 to generate droplets. It flows in the direction of region 24 .

The third injection port 23 is connected to a third fluid supply unit for generating droplets, and the third fluid is injected by the liquid supply unit 12 sending out the third fluid. Here, the third fluid is, for example, a reagent containing a substrate that reacts with an enzyme contained on or inside the microorganism. For example, the third fluid is a reagent containing the substrate "MUNANA".

A flow path 23 a is formed between the third injection port 23 and the droplet generation region 24 , and the third fluid injected from the third injection port 23 is sent out by the liquid feeding unit 12 to generate droplets. It flows in the direction of region 24 .

Here, in the microchannel chip 20, the channel 22a and the channel 23a merge before the droplet generation region 24, so that the second fluid flowing through the channel 22a and the channel 23a pass. It mixes with the flowing third fluid before droplet generation region 24 to form a dispersed phase fluid for generating droplets. That is, in the microchannel chip 20, a dispersed phase fluid in which the microorganisms and the substrate are mixed is formed in the confluence region of the channel 22a and the channel 23a.

The concentration of the microorganisms in the second fluid and the concentration of the substrate in the third fluid are each adjusted so as to achieve optimum concentrations when mixed to form the dispersed phase fluid. In addition, conditions such as PH in the dispersed phase fluid are appropriately adjusted according to the target enzyme.

In the droplet generating region 24, the channel 21a through which the continuous-phase fluid flows and the channel through which the dispersed-phase fluid flows (the channel after the confluence region of the channels 22a and 23a) intersect. This region is connected to the flow path through which the dispersed phase fluid flows so that the flow path 21a faces the flow path. When the continuous-phase fluid and the dispersed-phase fluid are supplied to the droplet generation region 24 by the liquid sending unit 12, the continuous-phase fluid shears the dispersed-phase fluid to generate droplets. For example, the droplet generation region 24 generates droplets with volumes between femtoliters (fL) and picoliters (pL).

The droplet generation region 24 continuously generates a large number of droplets by sequentially delivering the continuous-phase fluid and the dispersed-phase fluid from the liquid delivery unit 12 . The droplets generated in the droplet generation region 24 are sequentially delivered to the enzyme reaction region 25 together with the continuous phase fluid in accordance with the delivery of the first to third fluids by the liquid delivery unit 12 .

The enzyme reaction area 25 has a channel 25 a for reacting the enzyme enclosed in the droplet with the substrate, and is connected to the droplet generation area 24 . The droplets generated in the droplet generation region 24 are sequentially flowed into the channel 25a together with the continuous phase fluid. Here, the enzyme reaction area 25 is an area whose temperature is adjusted by the heating section 13 . That is, the control function 11a controls the heating unit 13 to adjust the temperature of the droplet flowing through the flow path 25a of the enzyme reaction area 25 to the optimum temperature for the enzyme reaction. At this time, if a microorganism is enclosed in the droplet, the enzyme reacts with the substrate to generate a fluorescent substance. The channel 25a is formed with a length corresponding to the time required for the reaction between the enzyme and the substrate and the flow rate of the droplets.

The detection region 26 has a flow channel 26 a irradiated with light emitted from the light source unit 14 and is connected to the enzyme reaction region 25 . The droplets that have flowed through the channel 25a in the enzyme reaction region 25 are sequentially introduced into the channel 26a together with the continuous phase fluid. Here, the flow path 26 a is formed of a material that transmits the light emitted from the light source section 14 and fluorescence generated in the droplets, and is arranged between the light source section 14 and the fluorescence detection section 15 .

In the detection region 26 , light is emitted from the light source unit 14 to the channel 26 a, and fluorescence generated in the droplet is detected by the fluorescence detection unit 15 . That is, in the detection region 26, light is emitted from the light source unit 14 to droplets flowing through the flow path 26a, and fluorescence is generated when microorganisms are encapsulated in the flowing droplets and fluorescent substances are generated. detected. The detection function 11b controls the light source unit 14 to irradiate the flow path 26a with light, and controls the fluorescence detection unit 15 to acquire fluorescence detection information.

For example, the detection function 11b detects the number of microorganisms encapsulated in the droplet based on the fluorescence intensity detected by the fluorescence detection section 15. FIG. For example, the detection function 11b detects that the number of microorganisms enclosed in the droplet is "0" when the fluorescence detection unit 15 does not detect fluorescence. Further, the detection function 11b compares the reference information indicating the relationship between the number of microorganisms enclosed in the droplet and the fluorescence intensity and the fluorescence intensity detected by the fluorescence detection unit 15, thereby detecting the number of microorganisms enclosed in the droplet. detect the number of microorganisms Reference information indicating the relationship between the number of microorganisms enclosed in the droplet and the fluorescence intensity is obtained in advance and stored in a memory circuit (not shown).

The sorting area 27 has a branched flow path 27 a for sorting droplets and is connected to the detection area 26 . The flow path 27a is sequentially introduced with the continuous-phase fluid by droplets that have flowed through the flow path 26a in the detection region 26. As shown in FIG. Here, in the sorting area 27, sorting of droplets by the sorting unit 16 is performed. That is, in the sorting area 27, the operation of the sorting unit 16 under the control of the sorting function 11c separates droplets containing a predetermined number of microorganisms from other droplets.

Specifically, the sorting function 11c determines the number of microorganisms contained in the droplet based on the intensity of fluorescence resulting from the reaction between the enzyme and the substrate, and sorts the droplet based on the determination result. More specifically, the sorting function 11c controls the sorting unit 16 based on the detection result detected by the detection function 11b to separate droplets containing a predetermined number of microorganisms from other droplets. and separate. For example, the separation function 11c determines the number of microorganisms enclosed in the droplets based on the flow rate of the droplets (liquid feeding speed by the liquid feeding section 12) and the distance between the detection area 26 and the separation area 27. The passage timing in the sorting area 27 of droplets for which fluorescence corresponding to a predetermined number has been detected is specified. For example, the sorting function 11c identifies the passage timing in the sorting region 27 of the droplet whose fluorescence intensity indicates that the number of microorganisms enclosed in the droplet is "1."

Then, the sorting function 11c applies an electric field from the sorting unit 16 at the specified passage timing, thereby moving droplets containing a predetermined number of microorganisms enclosed in the droplets to the recovery port 28 side and recovering the droplets. Droplets are passed through a channel connected to port 28 . For example, the sorting function 11 c moves droplets containing “1” microorganisms to the recovery port 28 side and allows them to flow through the recovery port 28 . The separation region 27 is configured such that the liquid droplets flow through the channel connected to the waste liquid port 29 when the separation section 16 does not apply an electric field.

The recovery port 28 is connected to the recovery section 17 , and allows droplets containing a predetermined number of microorganisms enclosed in the droplets to flow through the recovery section 17 . The liquid waste port 29 is connected to the liquid waste section 18 , and allows droplets containing a number of microorganisms other than the predetermined number to flow through the liquid waste section 18 .

An example of processing by the droplet collecting unit 1 will be described below with reference to FIG. FIG. 3 is a diagram for explaining an example of processing by the droplet collecting unit 1 according to the first embodiment. Note that FIG. 3 shows droplet generation and droplet separation in the microchannel chip 20 . In addition, FIG. 3 shows a case where viruses are targeted as microorganisms.

For example, as shown in FIG. 3, a second fluid containing virus is supplied via channel 22a, and a third fluid containing substrate is supplied via channel 23a. Then, the second fluid and the third fluid are mixed in the confluence region of the channel 22a and the channel 23a to form a dispersed-phase fluid. When the dispersed-phase fluid reaches the droplet generation region 24 intersecting the channel 21a, it is sheared by the continuous-phase fluid supplied through the channel 21a into droplets.

Here, when virus-encapsulated droplets are stochastically generated, droplets not containing viruses or viruses other than the predetermined number are enclosed in addition to droplets containing a predetermined number of viruses. Droplets are generated.

In the droplet collecting unit 1 according to the present embodiment, a virus-derived enzyme is reacted with a substrate, and a predetermined number of virus-encapsulated droplets are detected based on fluorescence information obtained as a result of the reaction. , to separate droplets containing a predetermined number of micro-organisms from other droplets. That is, the droplet collection unit 1 collects droplets (with signal) in which a predetermined number of viruses are encapsulated and exhibits corresponding intensity of fluorescence, and other (no signal) droplets (no fluorescence and droplets exhibiting fluorescence other than the corresponding intensity). This allows the droplet collection unit 1 to collect droplets with a known number of microbes encapsulated therein.

As described above, the droplet collection unit 1 according to the present embodiment generates droplets containing microorganisms in the microchannel chip 20, and forms droplets based on the reaction between enzymes derived from microorganisms and substrates. The number of encapsulated microorganisms is identified and droplets with a predetermined number of encapsulated microorganisms are selectively collected.

Here, the shape of the microchannel chip 20 in the droplet collecting unit 1 is not limited to the shape shown in FIG. 2, and can be appropriately changed according to conditions such as droplet generation and enzyme reaction. In addition, the channel width and channel length of each channel in the microchannel chip 20 can be appropriately changed according to conditions such as droplet generation and enzyme reaction.

A droplet collection unit 1 having the above-described configuration can be configured to be connectable to a system that utilizes collected droplets. FIG. 4 is a diagram for explaining an example of a system according to the first embodiment; Referring now to FIG. 4, a system is shown comprising a droplet collection unit 1 collecting a known number of virus-encapsulated droplets and a droplet collection unit 2 collecting cell-encapsulated droplets.

In such a system, virus-encapsulated droplets and cell-encapsulated droplets are fused in order to introduce various factors into cells using the virus encapsulated in the droplets as a vector. For example, as shown in FIG. 4, the system includes a droplet collected by droplet collection unit 1 (one virus-encapsulated droplet) and a droplet collected by droplet collection unit 2 (one virus-encapsulated droplet). droplets encapsulating cells).

The droplet collection unit 1 according to the present embodiment uses a stochastic approach to generate virus-encapsulated droplets, but selectively generates a known number (for example, one) of virus-encapsulated droplets. can be separated into Therefore, by using the droplet collection unit 1, the above system can reliably fuse virus-encapsulated droplets with cell-encapsulated droplets, and the droplets can be fused efficiently. It can be carried out. In addition, since the droplet collection unit 1 can selectively separate droplets containing a known number of viruses, it is possible to easily control the amount of virus infection to cells.

In addition, when droplets are simply generated by a stochastic method, in addition to droplets in which a desired number (for example, one) of viruses is encapsulated, droplets in which no virus is encapsulated, and Droplets encapsulating a number of viruses other than the desired number are mixed, and the droplets cannot be fused efficiently in the above system.

In the above-described embodiment, a case was described in which the target microorganism was a Paramyxoviridae virus having hemagglutinin-neuraminidase. However, embodiments are not limited to this, as long as it has an enzyme on its surface or inside, and the reaction of the enzyme produces a product that can be optically detected. good. For example, target microorganisms include coronavirus family viruses having hemagglutinin esterase, viruses such as HIV (human immunodeficiency virus) having reverse transcriptase, and viruses such as Ebola virus having RNA-dependent RNA polymerase. Examples of target microorganisms include bacteria such as coliforms, Vibrio parahaemolyticus, Campylobacter, Enterobacter, and Bacillus.

Here, the result of the detection experiment of the enzymatic reaction in the droplet will be described with reference to FIGS. 5 and 6. FIG. In addition, FIG. 5 is a diagram showing experimental results regarding the bacterial-derived enzyme (β-galactosidase) according to the first embodiment. Moreover, FIG. 6 is a diagram showing experimental results regarding the virus-derived enzyme (neuraminidase) according to the first embodiment.

(Experimental Example 1: Experiment on enzymes derived from bacteria)
(Reagent used)
Phosphate Buffered Saline (PBS) (-): manufactured by Nacalai Tesque
Fluorescein Di-β-D-galactopyranoside (FDG) (fluorescein): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. β-galactosidase (β-galactosidase): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

(Experimental procedure)
FDG was prepared by adding it to PBS(-) so that the concentration was 100 μM. β-galactosidase was prepared by adding PBS(-) to a concentration of 10 pM. A 0.5% dSURF was prepared by diluting a fluorine-based surfactant dSURF (manufactured by Fluigent) with a fluorine oil HFE7500 (3M). An FDG solution and 10 pM β-galactosidase were fed at 2 μL/min from two dispersed phase inlets (injection ports) of a droplet generation microchannel (manufactured by Chipshop), and 0.5% dSURF was fed from a continuous phase inlet. Droplets were generated by feeding at 20 μL/min. As a control experiment, only the FDG solution and PBS(-) were fed at 2 μL/min from the two inlets of the dispersed phase, and 0.5% dSURF was fed at 20 μL/min from the inlet of the continuous phase. to generate droplets.

After droplets were generated by the above-described procedure, they were incubated at 37° C. for 1 hour and observed with a fluorescence microscope. The results are shown in FIG. In FIG. 5, the results of the control experiment are shown in the upper row, and the results of the experiment on β-galactosidase are shown in the lower row. In addition, in FIG. 5, the image obtained by observing the droplet after incubation in each experiment with a bright field is shown on the left side. The left side shows images obtained by observing the fluorescence of droplets after incubation in each experiment.

As shown in the bright field images on the left side of FIG. 5, it was confirmed that droplets were generated in each experiment. In addition, as shown in the lower image on the right side of FIG. 5, fluorescence inside the droplet was confirmed in the presence of β-galactosidase. On the other hand, in the control experiment, as shown in the upper image on the right side of FIG. 5, no fluorescence was observed inside the droplet. Thus, by utilizing the enzymatic reaction by β-galactosidase, it is possible to determine whether bacteria (eg, E. coli) are enclosed in the droplet.

(Experimental Example 2: Experiment on virus-derived enzyme)
(Reagent used)
1M Tris-HCl pH9.0: manufactured by Nippon gene 4-MUNANA: manufactured by sigma
Neuraminidase, Influenza A Virus, H1N1, Recombinant, Carrier-free: R&D systems

(Experimental procedure)
4-MUNANA was prepared by adding pH 9.0 1M Tris-HCl to a concentration of 1 mM. Neuraminidase was prepared by adding pH 9.0 1M Tris-HCl to a concentration of 21 μM. A 0.5% dSURF was prepared by diluting a fluorine-based surfactant dSURF (manufactured by Fluigent) with a fluorine oil HFE7500 (3M). A 4-MUNANA solution and 21 μM neuraminidase were each sent at 2 μL/min from the two dispersed phase inlets of the droplet generation microchannel (manufactured by Chipshop), and 0.5% dSURF was sent from the continuous phase inlet at 30 μL/min. Droplets were generated by sending the liquid. As a control experiment, only the 4-MUNANA solution and 1M Tris-HCl were fed from the two inlets of the dispersed phase at 2 μL/min, respectively, and 0.5% dSURF was fed from the inlet of the continuous phase at 30 μL/min. to generate droplets.

After droplets were generated by the procedure described above, they were incubated at 37° C. for 3 hours and observed with a fluorescence microscope. The results are shown in FIG. In FIG. 6, the results of the control experiment are shown in the upper row, and the results of the experiment on neuraminidase are shown in the lower row. In addition, in FIG. 6, the image obtained by observing the droplet after incubation in each experiment with a bright field is shown on the left side. The left side shows images obtained by observing the fluorescence of droplets after incubation in each experiment.

As shown in the bright field image on the left side of FIG. 6, it was confirmed that droplets were generated in each experiment. In addition, as shown in the lower image on the right side of FIG. 6, fluorescence inside the droplet was confirmed in the presence of neuraminidase. On the other hand, in the control experiment, as shown in the upper image on the right side of FIG. 6, no fluorescence was observed inside the droplet. Thus, by utilizing the enzymatic reaction by neuraminidase, it is possible to determine whether or not the virus is encapsulated in the droplet.

As described above, by detecting the fluorescence generated based on the enzymatic reaction, it is possible to determine whether or not the droplet contains microorganisms. That is, the droplet collection unit 1 can separate droplets in which microorganisms are enclosed and droplets in which no microorganisms are enclosed. The droplet collection unit 1 then sorts out the number of microorganisms enclosed in the droplets based on the intensity of the detected fluorescence.

Here, when separating droplets in which one microorganism is enclosed, fluorescence may be weak and detection may be difficult with a normal detection system. Therefore, the droplet collection unit 1 can also apply a high-sensitivity camera as the fluorescence detector 15 . Further, the droplet collecting unit 1 can increase the detection sensitivity by slowing down the flow rate of the liquid delivered from the injection port and lengthening the exposure time of the image sensor. In such a case, for example, the control function 11a controls the liquid sending unit 12 so as to send the liquid at the slowest speed, which is the speed of the liquid to be sent necessary for generating droplets. do. Further, the detection function 11b controls the shutter in the fluorescence detection section 15 so that the exposure time of the image sensor in the fluorescence detection section 15 is as long as possible within the permissible range.

As described above, according to the first embodiment, the microchannel chip 20 produces droplets containing microorganisms and substrates that react with enzymes derived from the microorganisms. The detection function 11b detects the reaction between the enzyme and the substrate in the droplet. The sorting function 11c sorts droplets based on the reaction detection result. Thus, the droplet collecting unit 1 according to the first embodiment allows collecting droplets with a controlled amount of micro-organisms encapsulated in the droplets.

For example, a virus direct fluorescence method using a fluorescently labeled antibody is known as a method for detecting a virus. However, since the virus direct fluorescence method uses an antibody to label the virus, the labeling substance affects various analyzes and tests using the virus. On the other hand, in the method according to the present embodiment, the number of viruses can be detected without labeling the viruses, and various analyzes and tests using viruses (e.g., single virus genome analysis, drug resistance test, etc.) have no effect on

Further, for example, a method using microwells is known as a method for detecting the presence or absence of a virus using a virus-derived enzyme. However, in the method using microwells, since the virus is encapsulated in oil, it is difficult to collect the virus, and after the virus is detected, it is difficult to use the detected virus for various analyzes and tests. On the other hand, in the method according to the present embodiment, viruses can be isolated by droplets using microchannels, and can be easily used for various analyzes and tests.

Further, according to the first embodiment, the sorting function 11c determines the amount of microorganisms contained in the droplet based on the reaction detection result, and sorts the droplet based on the determination result. Therefore, the droplet collection unit 1 according to the first embodiment enables sorting according to the amount of microorganisms encapsulated in the droplets.

Further, according to the first embodiment, the sorting function 11c determines the number of microorganisms contained in the droplet based on the reaction detection result, and sorts the droplet based on the determination result. The droplet collecting unit 1 according to the first embodiment thus makes it possible to quantify the number of micro-organisms encapsulated in droplets.

Moreover, according to the first embodiment, the detection function 11b detects fluorescence based on the reaction between the enzyme and the substrate. Therefore, the droplet collection unit 1 according to the first embodiment allows easy detection of the presence or absence of microorganisms in droplets.

Further, according to the first embodiment, the sorting function 11c discriminates the number of microorganisms contained in the droplet based on the fluorescence intensity based on the reaction between the enzyme and the substrate, and based on the determination result, the liquid Separate the drops. The droplet collecting unit 1 according to the first embodiment thus allows easy quantification of the number of microorganisms in the droplet.

Further, according to the first embodiment, the separation function 11c separates droplets generated in the microchannel chip 20 by dielectrophoresis. Therefore, the droplet collection unit 1 according to the first embodiment enables easy sorting of droplets in the microchannel.

Also according to the first embodiment, the control function 11a adjusts the temperature of the droplets. The droplet collection unit 1 according to the first embodiment thus allows the reaction of the enzyme and the substrate to be carried out at the optimum temperature.

(Second embodiment)
In the above-described first embodiment, a case has been described in which the presence or absence of a single type of microorganism is detected and droplets in which microorganisms are enclosed are separated. In the second embodiment, a case will be described in which a plurality of types of microorganisms are targeted and droplets in which microorganisms are enclosed are separated.

In the second embodiment, the second fluid injected from the second injection port 22 contains multiple types of microorganisms. Here, the plurality of microorganisms contained in the second fluid have different enzymes. In addition, each enzyme reacts with a different substrate. In addition, the concentration of microorganisms in the second fluid is stochastically a predetermined number (for example, one) regardless of the type of microorganisms when mixed with the third fluid to form a dispersed phase fluid. The concentration is adjusted so that the microorganisms are enclosed in the droplets.

Further, in the second embodiment, the third fluid injected from the third injection port 23 contains multiple types of substrates that react with the enzymes of the microorganisms contained in the second fluid. The concentrations of the respective substrates in the third fluid are adjusted so as to achieve optimum concentrations when mixed with the second fluid to form dispersed phase fluids.

The liquid sending unit 12 sends the above-described second fluid and third fluid to the microchannel chip 20 as in the first embodiment. The second fluid and the third fluid delivered to the microchannel chip 20 are mixed to form a dispersed phase fluid, and sheared by the continuous phase fluid in the droplet generation region 24 to form droplets.

Here, the droplets generated in the second embodiment include droplets containing only a plurality of substrates without microorganisms, droplets containing any microorganism and a plurality of substrates among a plurality of microorganisms, droplets containing a plurality of microorganisms and a plurality of substrates.

The detection function 11b according to the second embodiment detects whether fluorescence is detected by the fluorescence detection unit 15, and the wavelength (or spectrum) and intensity of the fluorescence detected by the fluorescence detection unit 15. FIG. That is, the detection function 11b detects the presence or absence of microorganisms in the droplet based on the presence or absence of fluorescence detection. Also, the detection function 11b detects the type and number of microorganisms encapsulated in the droplet based on the wavelength (or spectrum) and intensity of the fluorescence.

The sorting function 11c according to the second embodiment sorts droplets based on the detection result of the detection function 11b. Specifically, the sorting function 11c sorts droplets in which a predetermined number (for example, one) of microorganisms are encapsulated for each type of microorganism. For example, the sorting function 11c sorts for each wavelength (or spectrum) of fluorescence so as to collect droplets exhibiting a predetermined fluorescence intensity (for example, fluorescence intensity generated when one microorganism is encapsulated). control the unit 16;

Here, the microchannel chip 20 according to the second embodiment has a plurality of recovery ports 28, and a plurality of recovery units 17 are connected to each recovery port 28, respectively. In addition, the sorting unit 16 according to the second embodiment is formed so that the application of an electric field can be changed so that droplets for each type of microorganism can selectively move in the direction of the corresponding recovery port 28. be. Then, the sorting function 11c controls the sorting section 16 so that droplets for each type of microorganism are collected by different collecting sections 17, respectively.

As described above, according to the second embodiment, a plurality of droplets are separated separately according to the fluorescence detection result. Therefore, the droplet collection unit 1 according to the second embodiment makes it possible to detect and separate multiple types of microorganisms at the same time.

(Other embodiments)
Although the first and second embodiments have been described so far, various different forms other than the above-described first and second embodiments may be implemented.

In the above-described embodiment, the case of separating droplets in which one microorganism (virus) is encapsulated has been described. However, the embodiment is not limited to this, and may be a case of fractionating droplets in which a desired number of two or more microorganisms are encapsulated. In such cases, the concentration of microorganisms in the second fluid is adjusted. Based on the result of detection by the detection function 11b, the separation function 11c controls the separation unit 16 so that two or more droplets exhibiting fluorescence intensities corresponding to a desired number flow to the recovery port 28 side. do.

The droplet collecting unit 1 can also separate droplets according to the number of microorganisms enclosed in the droplets. That is, the sorting function 11c sorts the droplets according to the number of microorganisms contained in the droplets based on the determination result. In such a case, as in the second embodiment, the microchannel chip 20 has a plurality of recovery ports 28 and a plurality of recovery units 17 are connected to each recovery port 28 . The sorting unit 16 is formed so that the application of the electric field can be changed so that the droplets for each type of microorganism can selectively move in the direction of the corresponding recovery port 28 . Then, the sorting function 11c controls the sorting unit 16 so that droplets with different numbers of enclosed microorganisms are recovered by different recovering units 17, respectively, based on the difference in fluorescence intensity.

Moreover, in the above-described embodiments, the case of detecting fluorescence based on a fluorescent substance generated by an enzymatic reaction has been described. However, the embodiment is not limited to this, and may be a case of detecting luminescence based on a fluorescent substance produced by an enzymatic reaction.

Further, in the above-described embodiment, the case where droplets are separated by dielectrophoresis has been described. However, the embodiments are not limited to this, and any method may be used as long as it is possible to separate droplets.

Further, in the above-described embodiments, an example of a droplet collection unit configured to be connectable to a system that utilizes collected droplets has been described. However, embodiments are not limited to this, and the droplet collection method described above may be performed by a droplet collection device that is not configured to be connectable to a system that utilizes the collected droplets. Further, the droplet collection device may include, for example, a configuration for using collected droplets in addition to the configuration for executing the droplet collection method described above. For example, the droplet collection device may be configured to perform various analyzes and tests using microorganisms encapsulated in droplets. In such a case, the processing circuit included in the droplet collection device executes the above-described control function 11a, detection function 11b, and sorting function 11c, as well as a processing function for performing processing on the droplets sorted by the sorting function 11c. do. That is, the processing function controls analysis and testing using microorganisms encapsulated in fractionated droplets. Note that the processing function is an example of a processing unit.

Although FIG. 1 illustrates an example in which each processing function is implemented by a single processing circuit 11, the embodiment is not limited to this. For example, the processing circuit 11 may be configured by combining a plurality of independent processors, and each processor may implement each processing function by executing each program. Further, each processing function of the processing circuit 11 may be appropriately distributed or integrated in a single or a plurality of processing circuits and implemented.

Further, the term "processor" used in the description of each embodiment described above is, for example, a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), or an application specific integrated circuit (ASIC) , programmable logic devices (e.g., Simple Programmable Logic Devices (SPLDs), Complex Programmable Logic Devices (CPLDs), and Field Programmable Gate Arrays (FPGAs)), etc. means Here, instead of storing the program in the memory, the program may be configured to be directly embedded in the circuit of the processor. In this case, the processor realizes its function by reading and executing the program embedded in the circuit. Further, each processor of the present embodiment is not limited to being configured as a single circuit for each processor, but may be configured as one processor by combining a plurality of independent circuits to realize its function. good.

According to at least one embodiment described above, it is possible to collect droplets in which the amount of microorganisms encapsulated in the droplets is adjusted.

While several embodiments have been described, these embodiments are provided by way of example and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, changes, and combinations of embodiments can be made without departing from the scope of the invention. These embodiments and their modifications are included in the scope and spirit of the invention, as well as the scope of the invention described in the claims and equivalents thereof.

1 droplet collection unit 11a control function 11b detection function 11c separation function 13 heating section 14 light source section 15 fluorescence detection section 16 separation section 20 microchannel chip

Claims (17)

a generator that generates droplets containing a microorganism and a substrate that reacts with an enzyme derived from the microorganism;
a detection unit that detects a reaction between the enzyme and the substrate in the droplet;
a sorting unit that sorts the droplets based on the detection result of the reaction;
a droplet collection unit. 2. The droplet collector according to claim 1, wherein the sorting unit determines the amount of the microorganisms contained in the droplet based on the detection result of the reaction, and sorts the droplet based on the determination result. unit. 3. The droplet collector according to claim 2, wherein the sorting unit determines the number of the microorganisms contained in the droplet based on the detection result of the reaction, and sorts the droplet based on the determination result. unit. The droplet collection unit according to any one of claims 1 to 3, wherein the detection section detects fluorescence based on reaction between the enzyme and the substrate. The sorting unit determines the number of the microorganisms contained in the droplet based on the intensity of fluorescence resulting from the reaction between the enzyme and the substrate, and sorts the droplet based on the result of the determination. 4. A droplet collection unit according to clause 3. 6. The droplet collection unit according to claim 5, wherein the sorting section sorts the droplets according to the number of the microorganisms contained in the droplets based on the determination result. The droplet collection unit according to any one of claims 1 to 3, wherein the sorting section sorts the droplets generated in the microchannel by dielectrophoresis. The droplet collection unit according to any one of claims 1 to 3, further comprising a temperature adjustment section that adjusts the temperature of the droplets. a generator that generates droplets containing a microorganism and a substrate that reacts with an enzyme derived from the microorganism;
a detection unit that detects a reaction between the enzyme and the substrate in the droplet;
a sorting unit that sorts the droplets based on the detection result of the reaction;
a processing unit that performs processing on the separated droplets;
A droplet collector, comprising: producing a droplet containing a microorganism and a substrate that reacts with an enzyme derived from the microorganism;
detecting a reaction between the enzyme and the substrate in the droplet;
sorting the droplets based on the detection result of the reaction;
A droplet collection method comprising: 11. The droplet collection method according to claim 10, wherein the amount of the microorganisms contained in the droplet is determined based on the detection result of the reaction, and the droplet is sorted based on the determination result. 12. The droplet collecting method according to claim 11, wherein the number of said microorganisms contained in said droplet is determined based on the detection result of said reaction, and said droplet is sorted based on said determination result. The droplet collection method according to any one of claims 10 to 12, wherein fluorescence based on reaction between said enzyme and said substrate is detected. 13. The method according to claim 12, wherein the number of microorganisms contained in the droplet is determined based on the intensity of fluorescence resulting from the reaction between the enzyme and the substrate, and the droplet is sorted based on the determination result. Droplet collection method. 15. The droplet collection method according to claim 14, wherein the droplets are sorted according to the number of the microorganisms contained in the droplets based on the determination result. The droplet collection method according to any one of claims 10 to 12, wherein the droplets generated in microchannels are sorted by dielectrophoresis. The droplet collection method of any one of claims 10-12, further comprising adjusting the temperature of the droplets.

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