Droplet processing device, droplet processing system, and droplet processing method

Abstract

To achieve technology that can more appropriately execute the process from collecting objects to processing samples.SOLUTION: A droplet processing device 1 includes a droplet levitation device 10, a sprayer 40, and a control unit 70. The droplet levitation device 10 generates ultrasonic or sound standing waves to hold droplets to be processed. The sprayer 40 sprays liquid or solid microparticles containing an object to be acted on the droplets held at nodes of the ultrasonic or sound standing waves. The control unit 70 controls the spraying of the microparticles by the sprayer 40.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a droplet processing device, a droplet processing system, and a droplet processing method. [Background technology]

[0002] 2. Description of the Related Art Conventionally, there are known techniques for collecting targets such as pathogens consisting of microorganisms such as viruses, chemical substances, or biomolecules, and performing various processes such as inspection or chemical reactions on samples containing the collected targets. For example, a test method is carried out in which a nasal swab is taken from a patient suspected of having a viral infection, the nasal swab is cultured in a culture medium, and then the virus contained in the culture medium sample is detected. Patent Document 1 describes a technique related to a specimen testing device that aims to rapidly detect nucleic acids in a sample. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2021-122197 Summary of the Invention [Problem to be solved by the invention]

[0004] However, when carrying out various processes on a sample containing a target object, for example, in each step of collecting the target object, amplifying and detecting the target object's genes, etc., it is required to appropriately collect the target object and handle the sample. For example, when taking a specimen such as a nasal swab from a patient suspected of having a viral infection, it is necessary to prevent the medical staff who take the specimen from becoming infected with the viral infection.In addition, it is necessary to prevent contamination of the sample to be tested with an unintended virus. In light of this situation, conventional techniques for processing samples have not been able to adequately resolve the above-mentioned concerns that arise during the process from collection of the subject to processing the sample.

[0005] An object of the present invention is to realize a technology that can more appropriately execute the process from collection of an object to processing of the sample. [Means for solving the problem]

[0006] In order to achieve the above object, a droplet processing device according to one aspect of the present invention comprises: a standing wave generating means for generating a standing wave of ultrasonic waves or sound waves for holding the droplets to be treated; A spraying means for spraying microparticles containing an object to be acted on the droplets held at the nodes of the ultrasonic or acoustic standing waves; A control means for controlling the spraying of the microparticles by the spraying means; The present invention is characterized by comprising: Effect of the Invention

[0007] According to the present invention, it is possible to realize a technology that can more appropriately carry out the process from collection of an object to processing of the sample. [Brief description of the drawings]

[0008] [Figure 1] 1 is a flowchart showing the flow of a droplet processing method to which the present invention is applied. [Diagram 2] 1 is a schematic diagram showing the overall configuration of a droplet processing apparatus 1 according to an embodiment of the present invention. [Diagram 3] 2 is a schematic diagram showing the functional configuration of a control unit 70. FIG. [Figure 4] 1 is a flowchart showing the flow of a microparticle spray process executed by the droplet processing apparatus 1. [Diagram 5] FIG. 13 shows the fluorescence spectrum of the collected capture droplets. [Figure 6]FIG. 13 is a graph showing the relationship between the distance between the capture droplet and the nebulizer and the measured fluorescence intensity. [Figure 7] FIG. 13 is a diagram showing the correspondence between Texas Red captured by the capture droplets and the number of aerosol particles. [Figure 8] FIG. 13 is an enlarged view showing a main part of the device configuration used in Verification 3. [Figure 9] FIG. 2 is a schematic diagram showing the results of measuring the fluorescence spectrum of a FITC solution. [Figure 10] FIG. 1 is a schematic diagram showing the principle of FRET detection of target DNA amplification using an HCR reaction. [Figure 11] FIG. 2 is a schematic diagram showing the predicted secondary structure of a primer. [Figure 12] FIG. 1 shows primer candidates and the values ​​of each parameter. [Figure 13] FIG. 2 is a schematic diagram showing the sequences of a primer set. [Figure 14] FIG. 1 shows the results of agarose gel electrophoresis after 24 hours of reaction. [Figure 15] FIG. 1 is a schematic diagram showing the time course of fluorescence of donor F using a fluorescently labeled primer set (531) (HCR reaction over 0 to 10 minutes). [Figure 16] FIG. 13 shows the time course of fluorescence of donor F (HCR reaction from 0 to 10 min) at different Itarget concentrations in levitated droplets. [Figure 17] This figure shows the time-dependent change in maximum fluorescence intensity in levitated droplets from immediately after the start of Itarget spraying to 10 minutes later. [Figure 18] Schematic diagram showing the sequence of a primer set for fluorescent detection of the new coronavirus (SARS-CoV-2). [Figure 19] FIG. 13 shows the time course of fluorescence of donor F2 at different Itarget concentrations in levitated droplets (HCR reaction from 0 to 10 min). [Figure 20] FIG. 1 is a schematic diagram showing the state of selective HCR reaction in suspended droplets of target DNA contained in a nebulized aerosol. [Figure 21]1A to 1C are schematic diagrams showing examples of configurations of a droplet processing apparatus 1 equipped with peripheral devices in various forms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Basic concept of the present invention] The present invention generates standing waves using sound waves or ultrasonic waves, levitates the droplets to be treated at the nodes of the standing waves, and then acts on the levitated droplets with microparticles such as mist-like microdroplets (aerosol), droplets, or droplet nuclei. The microparticles contain targets (pathogens consisting of microorganisms such as viruses, biomolecules (proteins, nucleic acids, etc.), fine particles (PM2.5, etc.), chemical substances, etc.) that are to be acted upon by the droplets, and as the microparticles act on (mix, dissolve, react, etc.) the levitated droplets, the targets contained in the microparticles are captured by the levitated droplets. In the following description, the levitated droplets will be referred to as "capture droplets" as appropriate.

[0010] In the present invention, the state in which the target object is captured by the capturing droplet is controlled by adjusting the conditions (spray conditions) under which the fine particles are sprayed onto the capturing droplet to act thereon. For example, the time for which the microparticles are allowed to act on the trapping droplets, the distance from the microparticle nozzle to the trapping droplets, the spray concentration of the microparticles, the ejection speed of the microparticles, etc. can be set as spray conditions. After the microparticles are sprayed onto the capture droplets under a predetermined spraying condition, the capture droplets serving as samples can be subjected to appropriate post-processing before various targeted processes (inspection, measurement, or recovery) can be carried out. Examples of post-processing that can be applied include gene amplification, antigen-antibody reaction, cell infection, and interaction with nanoparticles modified with recognition molecules.

[0011] By collecting (capturing) the target object in this process and performing various desired processes, the target object can be reacted with the capturing droplet in tiny amounts, allowing the sample to be controlled with high precision and made suitable for processing. In addition, the capturing droplets are handled while maintaining a non-contact interface in all directions, with none of the interface parts touching the container, thereby reducing the risk of contamination. Therefore, according to the present invention, appropriate handling of the sample can be performed from the process of collecting the object to the process of processing the sample. Therefore, according to the present invention, it is possible to realize a technology that can more appropriately carry out the process from collecting an object to processing the sample. Specific embodiments of the present invention will now be described.

[0012] [Droplet processing method] FIG. 1 is a flow chart showing the flow of a droplet processing method to which the present invention is applied. In FIG. 1, an example will be described in which an aerosol containing viruses (target objects) is sprayed as the microparticles, and the viruses are captured by the capturing droplets.

[0013] In step S1, a droplet to be captured is held in a levitated state at a node of a standing wave of an acoustic wave or an ultrasonic wave (droplet levitation step). When the capture droplets are levitated at a node of a standing wave of a sound wave or an ultrasonic wave, for example, a predetermined amount of the capture droplets can be ejected from a nozzle that ejects droplets at the position of the node of the standing wave.

[0014] In step S2, an aerosol containing a virus is sprayed onto the capture droplets (aerosol spraying step). The aerosol containing the virus is, for example, a specimen itself such as a patient's exhaled breath or cough (droplets), saliva, or a specimen extract (sheath liquid) from which the specimen has been extracted, which has been made into microdroplets. In the droplet processing method of this embodiment, spraying can be performed by using a sprayer while setting spraying conditions such as the time for which the aerosol acts on the capture droplets, the distance from the aerosol nozzle to the capture droplets, the aerosol spray concentration, and the aerosol spray speed.

[0015] In step S3, a gene amplification process (HCR: Hybridization Chain Reaction) is performed on the capture droplets sprayed with the aerosol (gene amplification step). Gene amplification by the HCR method can be performed at around room temperature (about 25° C.), and fluorescently labeled genes can be amplified. It should be noted that a gene amplification method other than the HCR method may be used as long as it is possible to amplify genes. In addition, when gene amplification is not required, the target substance may be detected without amplifying genes.

[0016] In step S4, the fluorescence of the capturing droplet after gene amplification treatment is measured to detect the presence or absence and concentration of viruses in the capturing droplet (target detection step). Here, the levitating capture droplets are irradiated with visible or ultraviolet light from a light source such as an LED, and the fluorescence spectrum is measured with a fluorescence spectrophotometer. The more targets (viral genes) there are in the capture droplets, the stronger the fluorescence spectrum observed. At this time, information that affects the fluorescence measurement results, such as the aerosol spray time or the distance between the sprayer and the capture droplets, may be output together with the fluorescence measurement results. It should be noted that a measurement method other than fluorescence measurement may be used as long as it is possible to detect the presence and concentration of viruses in the capture droplet.

[0017] In step S5, the capture droplets after fluorescence measurement are collected for post-processing or the like. For example, when other processing (such as processing to verify the effectiveness of a pharmaceutical ingredient) is performed using droplets in which the concentration of an object has been determined by fluorescence measurement, the capture droplets are handed over to subsequent processing together with the fluorescence measurement results. After step S5, the droplet processing method ends.

[0018] [Droplet processing device] Next, the droplet processing device used in this embodiment will be described. [composition] FIG. 2 is a schematic diagram showing the overall configuration of a droplet processing apparatus 1 according to one embodiment of the present invention. As shown in FIG. 2, the droplet processing device 1 includes a droplet levitation device 10, a liquid delivery pump 20, an air pump 30, a sprayer 40, a gene amplification device 50, a fluorescence measurement device 60, and a control unit 70.

[0019] The droplet levitation device 10 generates a standing wave using a vibrating member that generates sound waves or ultrasonic waves through vibration and a reflecting plate that reflects the sound waves or ultrasonic waves, and levitates and holds a droplet to be captured at the node of the standing wave. The liquid delivery pump 20 is composed of a pump that delivers liquid, such as a peristaltic pump, and supplies the liquid to be sprayed as an aerosol (specimen extract as sheath liquid) to the sprayer 40 via a silicon tube (outer diameter 4 [mm], inner diameter 2 [mm]) and a PEEK tube (inner diameter 0.25 [mm]). As the liquid delivery pump 20, for example, a peristaltic pump SJ-1211H (trademark) (manufactured by ATTO) or the like can be used.

[0020] The air pump 30 is composed of a pump that delivers gas, such as a vacuum pump, and supplies air for spraying the liquid supplied to the sprayer 40 by the liquid delivery pump 20 as an aerosol. As the air pump 30, for example, a Linikon LV-125A (trademark) (manufactured by NITTOH KOHKI Corporation) or the like can be used. The sprayer 40 atomizes the liquid supplied from the liquid feed pump 20 with air supplied from the air pump 30, and sprays the aerosolized liquid (specimen extract) toward the capture droplets. As the sprayer 40, for example, a sprayer kit AN5609 (trademark) (manufactured by Agilent) or the like can be used. When gas containing minute particles of the specimen is prepared, such as from the patient's breath or cough (droplets), the gas containing the minute particles of the specimen may be sprayed directly from the sprayer 40.

[0021] The gene amplification device 50 performs gene amplification processing based on the HCR method, PCR (Polymerase Chain Reaction) method, or the like, on the capture droplets held in the droplet levitation device 10. In the HCR method, the gene amplification device 50 performs the processing at around room temperature (approximately 25° C.) and amplifies fluorescently labeled genes. Alternatively, the gene amplification device 50 irradiates the droplets with electromagnetic waves or the like to change the temperature and amplify the genes. The fluorescence measuring device 60 irradiates the capture droplets held in the droplet levitation device 10 with light of a specific wavelength (visible light or ultraviolet light, etc.) from a light source such as an LED, and measures the fluorescence spectrum of the reflected light with a fluorescence spectrophotometer. The more detection targets (amplified genes) there are in the capture droplets, the stronger the fluorescence spectrum observed. The measurement results of the fluorescence measuring device 60 become information that represents the detection results of the detection targets (objects) in the capture droplets. It is to be noted that, together with the measurement results of the fluorescence measuring device 60, information that affects the measurement results, such as the aerosol spray time or the distance between the sprayer 40 (nozzle) and the capture droplets, may be output.

[0022] The control unit 70 is configured by an information processing device such as a PC (Personal Computer) or a PLC (Programmable Logic Controller), and controls the operation of the entire droplet processing device 1. For example, the control unit 70 switches on / off the operation of the droplet levitation device 10, the liquid feed pump 20, the air pump 30, the sprayer 40, the gene amplification device 50, and the fluorescence measurement device 60. The control unit 70 also accepts settings of spray conditions such as the time for spraying the aerosol onto the capture droplet by operating the liquid feed pump 20, the air pump 30, and the sprayer 40, the spray concentration of the aerosol, and the ejection speed of the aerosol, and can apply the aerosol to the capture droplet according to the set spray conditions. Furthermore, the control unit 70 controls the process of amplifying the gene in the capture droplet by the gene amplification device 50, and the process of detecting the detection target in the capture droplet by the fluorescence measurement device 60.

[0023] [Functional configuration] FIG. 3 is a schematic diagram showing the functional configuration of the control unit 70. As shown in FIG. As shown in Figure 3, in the control unit 70, a program for the microparticle spray processing described later is executed, and the functional configuration of the processor is formed to include a parameter setting unit 151, a standing wave generation control unit 152, a spray control unit 153, a gene amplification control unit 154, a detection processing unit 155, and a recording control unit 156.

[0024] The parameter setting unit 151 accepts settings of various parameters for operating the droplet processing device 1, such as parameters representing spray conditions for spraying an aerosol onto the capture droplets from the sprayer 40. The parameters representing the spray conditions can be manually input by an operator or automatically input by reading a setting file prepared in advance.

[0025] The standing wave generation control unit 152 controls the droplet levitation device 10 to generate a standing wave in the space between the vibration member and the reflector. The spray control unit 153 controls the liquid pump 20 and the air pump 30 to spray the aerosol from the sprayer 40. In this embodiment, the time for spraying the aerosol from the sprayer 40, the spray concentration of the aerosol, the ejection speed of the aerosol, and the like are determined according to the spray conditions set by the parameter setting unit 151.

[0026] The gene amplification control unit 154 controls the gene amplification device 50 to perform gene amplification processing based on the HCR method, PCR method, or the like, on the capture droplets held in the standing wave of the droplet levitation device 10. At this time, in the HCR method, the gene amplification control unit 154 performs the processing at about room temperature (about 25° C.) to amplify the fluorescently labeled genes. Alternatively, the gene amplification control unit 154 irradiates the droplets with electromagnetic waves or the like to change the temperature of the droplets and amplify the genes. When the HCR method is used, the gene amplification processing can be performed at about room temperature (about 25° C.), so that the gene amplification processing can be easily performed while the capture droplets are held in the droplet levitation device 10 without using a chamber or the like.

[0027] The detection processing unit 155 controls the fluorescence measuring device 60 to irradiate the capture droplets held by the droplet levitation device 10 with light of a predetermined wavelength (visible light, ultraviolet light, etc.) and measure the fluorescence spectrum of the reflected light. The record control unit 156 stores the measurement results of the fluorescence measuring device 60 in a non-volatile storage device as the detection results of the detection target in the capturing droplet.

[0028] [Operation] Next, the operation of the droplet processing apparatus 1 will be described. [Microparticle spray treatment] FIG. 4 is a flow chart showing the flow of the fine particle spray process executed by the droplet processing apparatus 1. The fine particle spraying process is started in response to an operation of the control unit 70 instructing the execution of the fine particle spraying process.

[0029] When the microparticle spraying process is started, in step S11, the parameter setting unit 151 receives settings of various parameters (parameters representing spraying conditions, etc.) for the operation of the droplet processing device 1. In step S12, the standing wave generation control unit 152 controls the droplet levitation device 10 to generate a standing wave in the space between the vibration member and the reflector. In step S13, the standing wave generation control unit 152 holds the capture droplet at the node of the generated standing wave. The capture droplet can be manually input into the droplet levitation device 10 by an operator, or can be automatically input into the droplet levitation device 10 by a droplet supplying device that supplies droplets from a nozzle.

[0030] In step S14, the spray control unit 153 controls the liquid pump 20 and the air pump 30 to cause the sprayer 40 to spray the aerosol under the set spray conditions. In step S15, the gene amplification control unit 154 controls the gene amplification device 50 to perform gene amplification processing based on the HCR method on the droplets to be captured held in the standing wave of the droplet levitation device 10. In step S16, the detection processing unit 155 controls the fluorescence measuring device 60 to irradiate the capture droplet held by the droplet levitation device 10 with light of a predetermined wavelength (visible light, ultraviolet light, etc.) and measure the fluorescence spectrum of the reflected light. This allows the detection target in the capture droplet to be detected (fluorescence measurement).

[0031] In step S17, the record control unit 156 stores the measurement result of the fluorescence measuring device 60 in the storage device as the detection result of the detection target in the capturing droplet. After step S17, the microparticle spray process ends.

[0032] [Specific verification] Next, specific verification results will be described for the steps performed in the droplet processing method according to the present invention. [Aerosol spraying process] [Verification 1] It was confirmed that in the aerosol spraying step of the droplet processing method, the amount of target object captured by the capturing droplet changes depending on the distance between the capturing droplet and the sprayer (nozzle) and the aerosol spraying time. Therefore, the dependence of the distance between the capture droplet and the sprayer and the spray time was examined. In this verification, in the droplet processing device 1 configured as shown in Figure 2, a PEEK tube with an inner diameter of 0.25 mm was used, a fluorescent substance (Texas Red) solution was sprayed from the sprayer 40, and the aerosol captured in the capture droplets was measured by measuring the fluorescence spectrum contained in the capture droplets.

[0033] Specifically, 10 μL of ultrapure water (Milli-Q water was used here) was levitated as a capture droplet, and Texas Red solution (50 μM) was sprayed for 5 or 10 minutes at distances of 5 cm, 20 cm, and 35 cm. After spraying, the capture droplet was collected, and the fluorescence spectrum (excitation wavelength 586 nm) was measured with a fluorescence spectrophotometer using a quartz cell with an optical path length of 1.5 mm. The Texas Red concentration of the collected capture droplet was calculated from the fluorescence intensity near 605 nm, which is the peak top of the spectrum of the capture droplet.

[0034] FIG. 5 shows the fluorescence spectrum of the collected capturing droplets. FIG. 5 shows the fluorescence spectrum when the spraying time was 5 minutes and the excitation wavelength was 489 nm. In FIG. 5, the spectrum of the undiluted solution is the spectrum of nebulized 50 μM Texas Red. FIG. 6 is a graph showing the relationship between the distance between the capture droplet and the spray and the measured fluorescence intensity. FIG. 6 shows the fluorescence intensity at about 605 [nm], which is the peak top of the spectrum, when the spraying time was 5 minutes and 10 minutes and the excitation wavelength was 586 [nm].

[0035] As shown in Figures 5 and 6, it was found that the longer the spray time and the closer the distance between the capture droplets and the sprayer, the higher the fluorescence intensity, i.e., the more Texas Red was captured in the capture droplets. Therefore, the amount of the target object captured by the capturing droplet depends on the distance between the capturing droplet and the sprayer and the spray time, and it is found that it is effective to appropriately control these.

[0036] [Verification 2] In the aerosol spraying step of the droplet processing method, verification was carried out as to whether or not it is possible to quantitatively determine the amount of aerosol captured by the capturing droplets. FIG. 7 is a diagram showing the correspondence between the amount of Texas Red captured by the capturing droplets and the number of aerosol particles. In FIG. 7, the Texas Red concentration can be estimated from the data in FIG. 6 described above. Then, from the volume of the collected trapping droplets and the Texas Red concentration, the number of particles of the Texas Red-containing aerosol trapped in the trapping droplets is calculated using the following formula (1). The diameter of one aerosol particle sprayed from the nebulizer was assumed to be 10 μm.

[0037]

number

[0038] As shown in Fig. 7, according to formula (1), the number of aerosol particles captured by the capture droplets is 5.1 × 10 6 At distances of 20 cm and 35 cm, the number of aerosol particles was reduced to 1 / 3.9 and 1 / 26 compared to a distance of 5 cm. In addition, when the spray time was 10 minutes, the number of particles did not simply double, but increased by 1.5 to 1.7 times. As described above, it is understood that the present invention makes it possible to quantitatively calculate the number of particles captured in the capturing droplet.

[0039] Generally, each sneeze produces about 40,000 particles with a diameter of 0.5 to 12 μm (= 4 × 10 4 It is known that aerosol particles are generated at a rate of about 10 to 100 times the amount of the airborne particles (J. Atkinson Ed., Natural Ventilation for Infection Control in Health-Care Settings, WHO Publication / Guidelines, 2009; ISBN-13: 978-92-4-154785-7). In this verification, the experimental conditions for the spraying time of 5 and 10 minutes were such that it was possible to collect about 10 to 100 times the amount of aerosol particles. Therefore, by shortening the aerosol spray time, it is possible to set conditions for capturing within the capturing droplets a number of aerosol particles equivalent to the amount of droplets expelled when a person sneezes.

[0040] [Verification 3] In the object detection step of the droplet processing method, it was verified whether the object could be appropriately detected in a state in which the capture droplet was levitated at a node of a standing wave of a sound wave or an ultrasonic wave. In this verification, we aimed to measure the fluorescence spectrum, and constructed a system that uses a reflection probe to measure the fluorescence spectrum of a levitating capture droplet. The device consisted of an LED light source controller (BAS, product code 013667), an LSM-470A LED light source module (470 [nm]) (BAS, product code 013674), a FLAME-S-VIS-NIR optical fiber spectrometer (BAS, product code 013467), and an R600-7-VIS / 125F reflected light measurement probe (BAS, product code 011281). The device was assembled so that the tip of the measurement fiber was located approximately 2 [mm] away from the capture droplet. The PC software used was "OceanView" (BAS, product code 013360).

[0041] FIG. 8 is an enlarged view showing a main part of the device configuration used in Verification 3. In this device configuration, an excitation wavelength of 470 [nm] was irradiated from an LED light source, and the fluorescence spectrum of a FITC (Fluorescein IsoThioCyanate) solution was measured behind a dark curtain. FIG. 9 is a schematic diagram showing the results of measuring the fluorescence spectrum of a FITC solution. In Figure 9, the measurements were performed under the following conditions: distance between the measurement fiber and the capture droplet: 2 mm, excitation wavelength: 470 nm, light source: 14 mA, percent flicker: 4%.

[0042] As shown in FIG. 9, it was confirmed that the intensity of the fluorescence spectrum increased in a FITC concentration-dependent manner and could be measured. That is, it was verified that the droplet processing method of the present invention can be carried out in a state in which the droplet to be captured is levitated at a node of a standing wave of a sound wave or an ultrasonic wave.

[0043] [Verification 4] We verified the HCR method as a feasible gene amplification step in the droplet processing method. When the target of detection is an infectious virus, its genes can be amplified and detected, but it is effective to select the HCR method, which allows amplification at around room temperature (25°C). The PCR method, which is often used for gene amplification, requires temperature rise and fall (55°C to 98°C), and there are problems with lack of temperature control technology and evaporation. In addition, the LAMP (Loop-Mediated Isothermal Amplification) method allows amplification at a constant temperature, but requires a high temperature of 60°C (approximately 35°C higher than room temperature). In these respects, the HCR method can be said to be a method suitable for levitating droplets.

[0044] When specifically performing fluorescence measurement using the HCR method, a hairpin-structured oligo DNA (Hp1) is modified with fluorescent molecules F (a donor fluorescent molecule) and Q (an acceptor, also called a quencher) near the terminal double strand so that fluorescence resonance energy transfer (FRET) occurs.

[0045] FIG. 10 is a schematic diagram showing the principle of FRET detection of target DNA amplification using the HCR reaction. As shown in Figure 10, when Hp1 forms a hairpin structure, FRET occurs between F and Q, and the fluorescence of F is quenched. If a target DNA (Itarget) is allowed to interact with this as an initiator, the hairpin Hp1 and Itarget form a double strand, which eliminates FRET and allows the fluorescence of F to be observed. Furthermore, the hairpin Hp2 interacts to form a double strand, and Hp1 further forms a double strand, further increasing the fluorescence intensity of F. Since the length of the double strand and the fluorescence intensity increase over time, it is possible to amplify and detect the target gene, even if it is present in a small amount.

[0046] [Verification 5] In the HCR method, which can be implemented as a gene amplification step in the droplet processing method, it is necessary to design primers modified with a fluorescent dye and a quencher in order to fluorescently detect viruses. We verified the design of the primers. First, a primer is designed to target the M1 gene of influenza A virus (IAV), and the progress of the HCR reaction is confirmed by agarose gel electrophoresis and fluorescence.

[0047] In a previous study1 (RM Dirks, NA Pierce, PNAS, 101(43), 15275-78 (2004).) in which the progress of the HCR reaction was measured by fluorescence, the length of the double-stranded part of HP1 was 18 bases and the length of the loop part was 6 bases. Therefore, the secondary structure of the primer set (531, 878) was predicted using the mFold webserver (http: / / www.unafold.org / mfold / software / download-mfold.php) and a previous study2 (J.-M. Rouillard, M. Zuker, E. Gulari., OligoArray 2.0: Thermodynamicaly improved oligonucleotide design for microarrays, Nucleic Acids Res. 31(12), 3057-3062 (2003).), and parameters (GC%, Tm value, ΔG) were calculated.

[0048] FIG. 11 is a schematic diagram showing the predicted secondary structure of the primer. FIG. 12 is a diagram showing candidate primers and the values ​​of each parameter, and FIG. 13 is a schematic diagram showing the sequences of the primer set. In Fig. 13, the underlined parts indicate the cohesive ends and their complementary sequences, and the double underlined parts indicate the loop structures and their complementary sequences. In addition, when fluorescently labeling, T in the box has Q, and A in the box has F.

[0049] As shown in FIG. 11, in this verification, a primer was designed in which a fluorescent dye (F) was attached to one end and a quencher (Q) was attached to the opposite end. In addition, the designed primer set was reacted in a test tube to confirm gene amplification as expected, and the reaction was confirmed by gel electrophoresis. The Hp1, Hp2 primer set (531) was heated in a heat block at 95°C for 2 minutes, and then cooled at room temperature (approximately 25°C) for 1 hour to form a hairpin structure. The Hp1 and Hp2 primers were diluted to a final concentration of 1 μM, and the target sequence (Itarget) was diluted to a final concentration of 10, 3.2, 1, 0.32, and 0.1 μM. After incubation at room temperature for 24 hours at a scale of 10 μL, the reaction was subjected to agarose gel electrophoresis and stained with ethidium bromide solution for 15 minutes.

[0050] FIG. 14 shows the results of agarose gel electrophoresis after 24 hours of reaction. As shown in Figure 14, it was confirmed that the HCR reaction proceeded at an Itarget concentration of 0.32 to 10 [μM]. As expected, the higher the Itarget concentration, the shorter the product length, and the lower the concentration, the longer the product length. It was also found that an Itarget concentration of 0.32 [μM] allowed the longest gene amplification. This reaction was also carried out using fluorescently labeled Hp1 and Hp2 primer set (531). Specifically, 4.5 μL of each primer was mixed in a measurement cell to make a total of 13.5 μL, and the time course of the fluorescence intensity of donor F was measured every 10 seconds for 10 minutes at an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm.

[0051] FIG. 15 is a schematic diagram showing the time course of fluorescence from donor F using a fluorescently labeled primer set (531) (HCR reaction for 0 to 10 minutes). FIG. 15 shows the results of measurement using a quartz cell and a fluorescence spectrophotometer. As shown in FIG. 15, it was confirmed that saturation was reached in about 3 minutes at Itarget concentrations of 0.32 [μM] to 3.2 [μM], enabling rapid detection.

[0052] [Verification 6] In the gene amplification step of the droplet processing method, it was confirmed whether the designed primer set amplifies genes in the floating capturing droplets. First, a hairpin structure was formed from the fluorescently labeled Hp1 and Hp2 primer set (531), and then 4.5 μL each of Hp1, Hp2, and Itarget (synthetic DNA) were acoustically levitated to a total of 13.5 μL (primer concentration 1 μM, target sequence (Itarget) concentrations 3.2, 1, 0.32, and 0.1 μM). The fluorescence spectrum was measured every 10 seconds for 10 minutes while irradiating the sample with light of an excitation wavelength of 470 nm using an optical fiber probe in a dark curtain, and the maximum fluorescence intensity (518 to 523 nm) was plotted.

[0053] FIG. 16 shows the time course of fluorescence of donor F at different Itarget concentrations in levitated droplets (HCR reaction from 0 to 10 min). As shown in Figure 16, the fluorescence of gene amplified by the optical fiber probe could be tracked at an Hp concentration of 1 [μM]. It can be seen that the fluorescence intensity reached saturation approximately 1 to 3 minutes after the start of the reaction at any Itarget concentration. That is, it is found that in the gene amplification step of the droplet processing method, the designed primer set is gene-amplified within the floating capturing droplet.

[0054] [Verification 7] In the target detection step of the droplet processing method, we verified the fluorescent observation of the HCR reaction of aerosol-containing DNA captured in levitating capture droplets. In this verification, an aerosol containing target DNA is sprayed onto the capture droplets, and the genes captured within the levitating capture droplets are amplified and detected. First, a hairpin structure was formed from the fluorescently labeled Hp1 and Hp2 primer set (531), and then the capture droplets were acoustically levitated to a total of 14 μL, with Hp1 and Hp2 at 7 μL each (primer concentration 1 μM). Itarget was diluted to a final concentration of 10 μM and sprayed at a spray distance of 20 cm for spray times of 60, 10, and 0 seconds. The fluorescence spectrum at an excitation wavelength of 470 nm was measured every 10 seconds for 10 minutes using an optical fiber probe in a dark curtain, and the maximum fluorescence intensity (518-523 nm) was plotted.

[0055] FIG. 17 shows the time course of the maximum fluorescence intensity in levitated droplets, from immediately after the start of Itarget spraying until 10 minutes later. FIG. 17 shows the change over time (0 to 10 minutes) in the fluorescence of donor F at different spray times (60 seconds, 10 seconds, 0 seconds) of Itarget (10 μM). As shown in FIG. 17, when the spraying time was 60 seconds, the fluorescence intensity reached saturation approximately 3 minutes after the start of the reaction, indicating that gene amplification was taking place within the capture droplets.

[0056] [Verification 8] In the above verification 5, an example of primer design was described in which the M1 gene of influenza A virus (IAV) was used as the target gene. However, primers can be designed for viruses other than influenza A viruses, and the progress of the HCR reaction can be confirmed by agarose gel electrophoresis or fluorescence. For example, primers can be designed targeting the novel coronavirus (SARS-CoV-2).

[0057] Figure 18 is a schematic diagram showing the sequences of primer sets for fluorescent detection of the new coronavirus (SARS-CoV-2). In Figure 18, the underlined parts indicate the cohesive ends and their complementary sequences, and the double underlined parts indicate the loop structures and their complementary sequences. In addition, when fluorescently labeled, the T in the box has Q2, and the A in the box has F2. By using the primer set shown in Figure 18, the progress of the HCR reaction for the new coronavirus (SARS-CoV-2) can be confirmed by agarose gel electrophoresis and fluorescence using the same procedure as in Verification 5 above.

[0058] [Verification 9] In the above verification 6, we confirmed whether the designed primer set would amplify genes of influenza A virus (IAV) within the floating capture droplets. Similarly, we confirmed whether the novel coronavirus (SARS-CoV-2) would amplify genes within the capture droplets using the primer set for the novel coronavirus (SARS-CoV-2) shown in verification 8 above. First, the fluorescently labeled Hp1 and Hp2 primer set (SARS-CoV-2) was allowed to form a hairpin structure, and then Hp1, Hp2, and Itarget (synthetic DNA) were acoustically levitated in the order of 4.5 μL each, for a total of 13.5 μL (primer concentration 1 μM, target sequence (Itarget) concentrations 10, 3.2, 1, and 0.32 μM). The fluorescence spectrum was measured every 10 seconds for 10 minutes while irradiating light with an excitation wavelength of 470 nm using an optical fiber probe in a dark curtain, and the maximum fluorescence intensity (518 to 523 nm) was plotted.

[0059] FIG. 19 shows the time course of fluorescence of donor F2 at different Itarget concentrations in levitated droplets (HCR reaction for 0 to 10 minutes). As shown in Figure 19, the fluorescence of gene amplified by the optical fiber probe could be tracked under the condition of Hp concentration of 1 [μM]. Fluorescence was observed about 10 minutes after the start of the reaction at Itarget concentrations of 3 [μM] and 10 [μM]. In other words, in the gene amplification step of the droplet processing method, the primer set for the new coronavirus (SARS-CoV-2) is gene amplified within the floating capture droplets.

[0060] [Verification 10] In the above verification 6, it was verified that by spraying an aerosol containing influenza A virus (IAV) onto capture droplets containing a primer set for detecting influenza A virus (IAV), gene amplification occurs within the floating capture droplets, making it possible to detect influenza A virus (IAV). Similarly, in the above verification 9, it was verified that by spraying an aerosol containing the novel coronavirus (SARS-CoV-2) onto capture droplets containing a primer set for detecting the novel coronavirus (SARS-CoV-2), gene amplification occurs within the floating capture droplets, making it possible to detect the novel coronavirus (SARS-CoV-2).

[0061] Here, we spray an aerosol containing influenza A virus (IAV) or the new coronavirus (SARS-CoV-2) onto capture droplets that contain a mixture of primer sets for detecting two types of genes, i.e., a primer set for influenza A virus (IAV) and a primer set for the new coronavirus (SARS-CoV-2), and verify whether it is possible to selectively detect either influenza A virus (IAV) or the new coronavirus (SARS-CoV-2) (the specific detection target).

[0062] First, for each primer set used in the above verifications 5 and 7, donor F and donor F2 were fluorescently labeled with Hp1 and Hp2 primer sets (531 for influenza A virus (IAV) and SARS-CoV-2 novel coronavirus) to form hairpin structures, and then the capture droplets were acoustically levitated to a total of 14 μL (primer concentration 1 μM). Itarget was diluted to 0.3 to 10 μM and sprayed at a spray distance of 20 cm for a spray time of 60 seconds. The fluorescence spectrum at an excitation wavelength of 470 nm (when detecting influenza A virus (IAV)) and the fluorescence spectrum at an excitation wavelength of 533 nm (when detecting SARS-CoV-2 novel coronavirus) were measured every 10 seconds for 10 minutes using an optical fiber probe in a dark curtain, and the maximum fluorescence intensity (520 nm or 580 nm) was plotted.

[0063] FIG. 20 is a schematic diagram showing the state of selective HCR reaction in suspended droplets of target DNA contained in a nebulized aerosol. In the example shown in Figure 20, a primer set of 531 for influenza A virus (IAV) and the novel coronavirus (SARS-CoV-2) was used, and verification was performed under the conditions of [Hp1] = [Hp2] = 1 [μM] and room temperature of 25 [℃].

[0064] As shown in Figure 20, in the time course of fluorescence of donor F and donor F2 at different Itarget concentrations (HCR reaction for 0 to 10 minutes), fluorescence was observed in the levitated droplets within approximately 10 minutes of the start of the reaction at all Itarget concentrations. In other words, by using capture droplets that contain a mixture of a primer set for influenza A virus (IAV) and a primer set for the novel coronavirus (SARS-CoV-2), the primer set corresponding to the genes contained in the sprayed aerosol is genetically amplified within the levitating capture droplets in the gene amplification step of the droplet processing method, making it possible to selectively detect either influenza A virus (IAV) or the novel coronavirus (SARS-CoV-2).

[0065] [Variation 1] In the above embodiment, the droplet processing apparatus 1 is provided with one peripheral device such as the sprayer 40, but the present invention is not limited to this. That is, the droplet processing apparatus 1 can include peripheral devices such as the sprayer 40 (the sprayer 40, a fluorescence measuring device, a droplet amount measuring device, a robot arm, etc.) in various forms as necessary. FIG. 21 is a schematic diagram showing an example of the configuration of a droplet processing device 1 equipped with peripheral devices in various forms. FIG. 21(A) shows a configuration in which the droplet processing apparatus 1 is equipped with three peripheral devices, and FIG. 21(B) shows a configuration in which the droplet processing apparatus 1 is equipped with four peripheral devices. In the configuration shown in FIG. 21, when a plurality of sprayers 40 are provided, microparticles (aerosols) containing the same or different target substances can be sprayed from the plurality of sprayers 40. Examples of applications of the droplet processing device 1 having a plurality of sprayers 40 include capturing, droplet manipulation, detection, and monitoring of two or more types of aerosols.

[0066] In addition, peripheral devices (such as a sprayer 40, a fluorescence measuring device, a droplet volume measuring device, a robot arm, etc.) can be installed on each side (e.g., 3 to 5 sides) of the droplet processing device 1 other than the support pillar supporting the droplet processing device 1. In this case, a robotic arm is set up to levitate and retrieve capture droplets with a motorized pipettor. The droplet volume measuring device makes it possible to quantitatively measure the amount of aerosol captured in droplets. The fluorescence measuring device is installed to detect the fluorescence of the contents of the captured aerosol. Alternatively, a camera may be provided instead of the fluorescence measuring device to observe the color.

[0067] These peripheral devices can be combined appropriately and installed in the droplet processing device 1. For example, when combining three peripheral devices, Combination (1): Robot arm / sprayer / fluorescence measuring device Combination (2): Robot arm / sprayer / droplet volume measuring device Combination (3): Robot arm / Sprayer A / Sprayer B Combination (4): Sprayer / Fluorescence measuring device / Droplet volume measuring device Combination (5): Sprayer A / Sprayer B / Fluorescence Measuring Device The droplet processing device 1 can be configured in various forms such as the above.

[0068] Incidentally, by providing a switching valve, a configuration may be adopted in which microparticles (aerosol) containing a plurality of targets are sprayed from one sprayer 40. With this configuration, it is possible to simultaneously spray microparticles containing multiple types of targets onto the capturing droplets, or to spray microparticles containing multiple types of targets in a time-division manner.

[0069] [Variation 2] In the above embodiment, an example has been described in which one droplet to be captured is held at a node of a standing wave generated by the droplet levitation device 10, but the present invention is not limited to this. For example, droplet levitation device 10 may generate a plurality of nodes of a standing wave and hold droplets to be captured at these nodes, thereby allowing droplet levitation device 10 to hold a plurality of droplets. At this time, the plurality of droplets held by the droplet levitation device 10 can be of the same type or different types. With this configuration, it is possible to spray microparticles onto a plurality of trapping droplets, making it possible to carry out a plurality of processes simultaneously.

[0070] [Variation 3] In the above embodiment, a case has been described in which fluorescence measurement is performed to detect the target object captured in the capturing droplet, but the present invention is not limited to this. That is, as long as it is possible to detect that a target object has been captured by a capturing droplet, various detection methods can be used. For example, when a virus is captured in a capturing droplet containing red blood cells, the target object can be detected by utilizing the agglutination action of the red blood cells. In this case, microparticles (aerosol) containing viruses are sprayed onto the capturing droplets containing red blood cells, and it is possible to detect that the target object has been captured by the capturing droplets by detecting the agglutination of the red blood cells in the capturing droplets.

[0071] The present invention can be modified and improved as appropriate within the scope of the effects of the present invention, and is not limited to the above-described embodiment. For example, the present invention can be applied to cases where the target objects are microorganisms such as viruses, dust, fine particles such as PM2.5, chemical compounds, etc., and can be used for monitoring or sampling these objects. As an example, the present invention can be used to detect microorganisms (such as infectious diseases viruses), and processes such as gene amplification, binding of antibodies to pathogens, emission of fluorescence or other light, and aggregation of microparticles (target objects) can be performed in the levitating capture droplets. Such a configuration can be used, for example, for diagnosing and monitoring infectious diseases. Furthermore, the present invention can be used to recover microorganisms (such as infectious diseases viruses), and in the levitating capture droplets, treatments such as infection of cells with viruses or cell culture can be carried out. Such a form can be used, for example, for surveillance of microorganisms.

[0072] As another example, the present invention can be used to detect fine particles (pollen, dust, soot, PM2.5, etc.), and processes such as binding of antibodies to the fine particles, emission of fluorescence, etc., and agglutination of the fine particles (target objects) can be performed in the levitating capture droplets. Such a configuration can be used, for example, for monitoring air pollution. The present invention can also be used to collect fine particles (pollen, dust, soot, PM2.5, etc.), and processes such as trapping the fine particles can be carried out in the floating trapping droplets. Such a configuration can be used, for example, for sampling particulate matter in the air.

[0073] As another example, the present invention can be used to detect compounds (such as VOCs (volatile organic compounds) and active oxygen), and processes such as antibody binding to the compounds, light emission such as fluorescence, chemical reactions, and enzyme reactions can be performed in the levitating capture droplets. Such a configuration can be used, for example, for compound monitoring. In addition, the present invention can be used to recover compounds (VOCs, active oxygen, etc.), and the levitating capture droplets can be used to capture chemical substances, etc. In the case of compounds that are unstable and susceptible to structural changes, a protective agent may be included in the capture droplets. Such a configuration can be used, for example, for compound sampling.

[0074] Furthermore, the present invention can be implemented by appropriately combining the above-described embodiments and specific application examples. The control processes in the above-described embodiments can be executed by either hardware or software. In other words, it is sufficient that the droplet processing apparatus 1 is provided with a function capable of executing the above-mentioned processes, and the functional and hardware configurations for realizing these functions are not limited to the above-mentioned examples.

[0075] The above embodiment shows an example of application of the present invention, and does not limit the technical scope of the present invention. In other words, the present invention can be modified in various ways, such as omissions and substitutions, without departing from the gist of the present invention, and various embodiments other than the above embodiment can be adopted. The various embodiments and modifications that the present invention can adopt are included in the scope of the invention described in the claims and their equivalents.

[0076] The droplet processing device 1 configured as above includes the droplet levitation device 10, the sprayer 40, and the control unit . The droplet levitation device 10 generates ultrasonic or sonic standing waves to hold droplets for processing. The atomizer 40 atomizes tiny liquid or solid particles, including the target substance, onto droplets held at nodes in a standing ultrasonic or sonic wave. The control unit 70 controls the spraying of the microparticles by the sprayer 40 . This allows the target object to be applied to the capture droplet in minute amounts, enabling the sample to be controlled with high precision and made suitable for processing. In addition, the capture droplet is handled while maintaining a non-contact interface in all directions, with none of the interface parts touching the container, reducing the risk of contamination. In other words, appropriate handling of the sample can be performed from the process of collecting the object to processing the sample. Therefore, it is possible to realize a technology that can more appropriately carry out the process from collecting an object to processing the sample.

[0077] The droplet processing device 1 includes a fluorescence measuring device 60 . The fluorescence measuring device 60 detects the target object contained in the atomized droplets of microparticles. This makes it possible to detect an object that has acted on the droplets by spraying the microparticles.

[0078] The fluorescence measurement device 60 detects objects in the droplets held at nodes of ultrasonic or acoustic waves. This allows detection of objects contained within the levitated droplet.

[0079] The droplet processing device 1 includes a gene amplification device 50 . The gene amplification device 50 amplifies the genes contained in the droplets. This allows the genes that have acted on the droplets to be detected in a state that makes them easier to detect.

[0080] The gene amplification device 50 amplifies genes in droplets held at nodes of ultrasonic or sonic waves. This allows a gene amplification process to be carried out in the levitated droplets.

[0081] The atomizer 40 sprays minute particles containing the target object for capture into droplets. The fluorescence measurement device 60 detects objects trapped in droplets from the microparticle spray. This makes it possible to capture targets among microparticles in droplets and detect the amount of the captured targets, etc.

[0082] The fluorescence measuring device 60 outputs the detection result of the target object using at least one of the spray time of the microparticles relative to the droplets or the distance between the sprayer 40 and the microparticles as an index. This makes it possible to grasp the detection result of the captured target object by correlating it with the spray time or spray distance of the microparticles.

[0083] The atomizer 40 atomizes small particles containing the target for mixing or reacting into droplets. The fluorescence measuring device 60 detects the components of the droplets with which the target substance is mixed or reacted due to the spray of microparticles. This makes it possible to carry out mixing of substances or chemical reactions in the levitated droplets and detect the results.

[0084] The fluorescence measuring device 60 outputs the detection result of the target object using at least one of the spray time of the microparticles relative to the droplets or the distance between the spray means and the microparticles as an index. This makes it possible to grasp the detection result of the target substance after mixing or reaction by correlating it with the spray time or spray distance of the microparticles. The droplet processing device 1 includes a plurality of sprayers 40. Each of the multiple atomizers 40 atomizes microparticles containing different targets into droplets held at nodes in a standing ultrasonic or sonic wave. This makes it possible to simultaneously spray microparticles containing multiple types of targets onto the levitated droplets, or to spray microparticles containing multiple types of targets in a time-division manner.

[0085] The fluorescence measuring device 60 is capable of detecting different types of target substances contained in the droplets, and detects a specific target substance contained in the sprayed microparticles among the different types of target substances. This makes it possible to selectively detect a specific target substance contained in a microparticle. [Explanation of symbols]

[0086] 1 Droplet processing device, 10 Droplet levitation device, 20 Liquid delivery pump, 30 Air pump, 40 Sprayer, 50 Gene amplification device, 60 Fluorescence measurement device, 70 Control unit, 151 Parameter setting unit, 152 Standing wave generation control unit, 153 Spray control unit, 154 Gene amplification control unit, 155 Detection processing unit, 156 Recording control unit

Claims

1. a standing wave generating means for generating a standing wave of ultrasonic waves or sound waves for holding the droplets to be treated; A spraying means for spraying liquid or solid microparticles including a target to be acted on the droplets held at the nodes of the ultrasonic or sound standing waves; A control means for controlling the spraying of the microparticles by the spraying means; A droplet processing device comprising:

2. The droplet processing apparatus according to claim 1 , further comprising a detection unit for detecting the target object contained in the droplets into which the fine particles are sprayed.

3. 3. The droplet processing apparatus according to claim 2, wherein the detection means detects the object in the droplet held at a node of the ultrasonic wave or sound wave.

4. 3. The droplet processing apparatus according to claim 1, further comprising a gene amplifying means for amplifying a gene contained in the droplet.

5. 5. The droplet processing apparatus according to claim 4, wherein the gene amplifying means amplifies the gene in the droplet held at a node of the ultrasonic wave or sound wave.

6. The spraying means sprays the microparticles including the object to be captured in the droplets; 4. The droplet processing apparatus according to claim 2, wherein the detection means detects the object captured in the droplets by spraying the fine particles.

7. The droplet processing device according to claim 6, wherein the detection means outputs a detection result of the target object using at least one of a spray time of the microparticles onto the droplets or a distance between the spray means and the microparticles as an index.

8. The spraying means sprays the microparticles containing the object to be mixed or reacted with the droplets, 4. The droplet processing apparatus according to claim 2, wherein the detection means detects components of the droplets mixed with or reacted with the target object by spraying the fine particles.

9. The droplet processing device according to claim 8 , wherein the detection means outputs a detection result of the target object using at least one of a spray time of the microparticles onto the droplets or a distance between the spray means and the microparticles as an index.

10. The spray means is provided in plurality, 3. The droplet processing device according to claim 1, wherein each of the plurality of spraying means sprays microparticles containing different objects onto the droplets held at nodes of ultrasonic or acoustic standing waves.

11. The droplet processing device according to claim 2 or 3, characterized in that the detection means is capable of detecting different types of the target object contained in the droplet, and detects a specific target object contained in the sprayed microparticles among the different types of the target object.

12. A standing wave generator that generates ultrasonic or sonic standing waves for holding the droplets to be treated; A spraying device that sprays liquid or solid microparticles including an object to be acted on the droplets held at the nodes of the ultrasonic or sound standing waves; A control device for controlling the spraying of the microparticles by the spraying device; A droplet processing system comprising:

13. a standing wave generating step of generating a standing wave of ultrasonic waves or sound waves for holding the droplets to be treated; a droplet holding step of holding the droplet at a node in the standing wave of the ultrasonic wave or sound wave generated in the standing wave generating step; a spraying step of spraying liquid or solid microparticles containing an object to be acted on the droplets held at the nodes of the ultrasonic or sonic standing waves; A method for processing droplets, comprising:

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