Patent Application
20190285520
This application claims priority from prior Japanese Patent Application No. 2018-049010, filed on Mar. 16, 2018, entitled “Sample Processing Method, Sample Processing Chip, and Sample Processing Apparatus”, the entire contents of which are incorporated herein by reference.
The present invention relates to a sample processing method, sample processing chip and sample processing apparatus.
There is a demand for a technique for detecting a target component in a sample for each one molecule or for each one target component (digital detection). The target component is, for example, a nucleic acid, a protein, a cell, or the like. In digital detection, for example, a target component is included in one droplet for each molecule or each target component. This means that the target component is “segmented” into one molecule or one target component, because one molecule or one target component is arranged in a unit region composed of individual droplets. In order to segment the target component per molecule or every target component, it is required to dilute the target component at a high dilution ratio.
Japanese Patent Application Publication No. 2017-158491, as shown in FIG. 39, discloses a configuration for heating of a lower portion of a reservoir 903 of a sample processing chip 902 storing a mixture 901 of a target component 905 and a predetermined diluent by a heating unit 904, and the target component 905 is diluted to a high dilution ratio by agitation produced by thermal convection.
However, according to the agitation method of Japanese Patent Application Publication No. 2017-158491 described above, since the reservoir 903 is heated and the content is agitated by thermal convection, it takes time to completely agitate. Therefore, it is preferable to obtain a desired diluted mixture by agitating a short time.
The present invention is directed to obtaining a desired diluted mixture by agitating for a short time.
The sample processing method according to a first aspect of the present invention is a sample processing method comprising storing a processing liquid (11) containing a target component (10) and a diluent (12) for diluting the processing liquid (11) in a reservoir (110) of a sample processing chip (100), wherein the processing liquid (11) is diluted in order to prepare a droplet forming sample (13) for forming droplets individually encapsulating the target component (10), and agitating the processing liquid (11) and the diluent (12) in the reservoir (110) by introducing a gas into the reservoir (110).
In the sample processing method according to the first aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are mixed by the gas (bubbles) introduced into the reservoir (110), it is not necessary to heat the reservoir (110), and it is possible to reduce the time required for mixing as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. Since heating is not performed, it also is possible to suppress the target component (10) from changing due to heat.
In the sample processing method according to the first aspect, preferably, t the reservoir (110) is a storage tank which is tube-shaped and connected to a substrate (140) of the sample processing chip (100). In the agitating, the processing liquid (11) and the diluent (12) are agitated by introducing the gas from a bottom of the storage tank and rising the gas in the storage tank. According to this configuration, as compared with the case where the reservoir (110) is provided in the substrate (140) of the sample processing chip (100), the cross sectional area of the portion through which the gas passes can be increased so that the gas can easily pass through into the storage tank. In this way agitation can be performed in a shorter time.
In the sample processing method according to the first aspect, preferably, in the agitating, the gas is introduced into the reservoir (110) for a predetermined time of 0.1 seconds or more and 60 seconds or less to agitate the processing liquid (11) and the diluent (12). With such a configuration, it is possible to effectively shorten the agitation time as compared with thermal convection.
In this case, preferably, in the agitating, the processing liquid (11) and the diluent (12) are agitated by introducing the gas into the reservoir (110) at a pressure of 100 mbar or more and 1000 mbar or less. According to this configuration, it is possible to effectively stir the interior of the storage tank with a gas having a pressure of 100 mbar or more and 1000 mbar or less.
In the sample processing method according to the first aspect, preferably, in the storing, sending the processing liquid (11) to the reservoir (110) with the gas, after storing the diluent (12) in the reservoir. According to this configuration, the processing liquid (11) can be easily sent to the reservoir (110) by supplying the gas by the same method as the gas used for agitating.
In the sample processing method according to the first aspect, preferably, the sample processing chip (100) has an inlet (112) for introducing the gas. The method further comprises introducing the gas from the inlet (112) to deliver the processing liquid (11) to the reservoir (110), after storing the diluent (12) in the reservoir (110), and introducing the gas from a bottom portion of the reservoir (110) following the supply of the processing liquid (11) by the gas introduced from the inlet (112). According to this configuration, since it is possible to continuously perform the feeding of the processing liquid (11) to the reservoir (110) and the introduction of the gas into the reservoir (110) by the same operation, it is possible to shorten the processing time as compared with when feeding and agitating of the processing liquid (11) are performed by separate operations.
In the sample processing method according to the first aspect, preferably, the sample processing chip (100) has an inlet (112) for introducing the gas. The method further comprises introducing the gas from the inlet (112) in order to send the diluent (12) to the reservoir (110) from the inlet (112), after storing the processing liquid (11) in the reservoir (110), and introducing the gas from a bottom portion of the reservoir (110) following the sending of the diluent (12) by the gas introduced from the inlet (112). According to this configuration, since it is possible to continuously perform the feeding of the diluent (12) to the reservoir (110) and the introduction of the gas into the reservoir (110) by the same operation, it is possible to shorten the processing time as compared with when feeding and agitating of the diluent (12) are performed by separate operations.
In the sample processing method according to the first aspect, preferably, the sample processing chip (100) has a quantification unit (143). The method further comprises sending the processing liquid (11) quantified using the quantification unit (143) to the reservoir (110). According to this configuration, since the processing liquid (11) can be quantified by the quantification unit (143), a fixed amount of the processing liquid (11) can be delivered to the reservoir (110) to obtain a diluted mixture with a desired dilution ratio.
In this case, preferably, the quantification unit (143) comprises an inner cavity having a predetermined content amount formed in the sample processing chip (100). According to this configuration, it is possible to accurately quantify the treatment liquid (11) with an inner cavity having a predetermined internal capacity.
In the configuration in which the quantification unit (143) is formed by an inner cavity, preferably, the sample processing chip comprises a first flow path (141) and a second flow path (144) connected to the inner cavity of the quantification unit (143). Each of the first flow path (141) and the second flow path (144) has an on-off valve (147a, 147b). The first flow path (141) is connected to an inlet (141a) for the processing liquid (11). The second flow path (144) is connected to a disposal port (144a). The method further comprises quantifying the processing liquid (11) by bring the first flow path (141) and the second flow path (144) into an open state, delivering the processing liquid (11) from the first flow path (141) and filling the processing liquid (11) in the inner cavity of the quantification unit (143). According to this configuration, the processing liquid (11) can be accurately quantified when the processing liquid (11) is introduced into the inner cavity through the first flow path (141) and the second flow path (144) in an open state.
In this case, it is preferable that the sample processing chip further comprises a third flow path (145) and a fourth flow path (146) connected to the inner cavity of the quantification unit (143). Each of the third flow path (145) and the fourth flow path (146) has an on-off valve (147c, 147d). The third flow path (145) is connected to the reservoir (110). The fourth flow path (146) is connected to a gas supply unit (202) for feeding the gas. The method further comprises filling the processing liquid (11) in the inner cavity of the quantification unit (143) by bring the first flow path (141) and the second flow path (144) into an open state and the third flow path (145) and the fourth flow path (146) into closed state and delivering the processing liquid (11) to the reservoir (110) from the first flow path (141), and delivering the processing liquid (11) filled in the inner cavity of the quantification unit (143) with the gas from the gas supply unit (202) by bring the first flow path (141) and the second flow path (144) into the closed state and the third flow path (145) and the fourth flow path (146) into the open state. According to this configuration, the processing liquid (11) is accurately quantified by introducing the processing liquid (11) into the inner cavity when the first flow path (141) and the second flow path (144) are in an open state, and the quantified processing liquid (11) can be delivered to the reservoir (110) without residual when the third flow path (145) and the fourth flow path (146) are in the open state.
In this case, preferably, in the quantifying, the processing liquid is reciprocatingly moved between the first flow path (141), the second flow path (144), and the inner cavity. According to this configuration, it is possible to suppress the gas from remaining in the quantification unit (143) during quantification since the gas pre-existing in the first flow path (141), the second flow path (144) and the inner cavity can be discharged from the quantification unit (143) by the reciprocating movement of the processing liquid (11). In this way it is possible to quantify the processing liquid (11) more accurately.
In the configuration in which the sample processing chip (100) has the quantification unit (143), it is preferable that the sample processing chip (100) comprises a plurality of quantification units (143a, 143b) and reservoirs (110a, 110b) connected in series along the flow of the processing liquid (11). The method further comprises further diluting mixed solution containing the target component (10) diluted by one of the plurality of quantification units (143a, 143b) and one of the plurality of reservoirs (110a, 110b), by other of the plurality of quantification units (143a, 143b) and other of the plurality of reservoirs (110a, 110b) in a subsequent stage. According to this configuration, it is possible to effectively increase the dilution ratio by diluting in a plurality of stages.
In the sample processing method according to the first aspect, in the storing, a dilution ratio of the target component (10) is 10 times or more and 100,000 times or less. According to this configuration, the target component (10) can be diluted at a dilution ratio for dividing the target component (10) into one molecule or one component.
In the sample processing method according to the first aspect, preferably, the diluent (12) comprises a reagent (16) that reacts with the target component (10). According to this configuration, the target component (10) can be reacted and processed by the reagent (16) in a later process.
In this case, preferably, the method further comprises delivering the reagent (16) to the reservoir (110) that stores target component (10) and the diluent (12). According to this configuration, it is possible to mix the reagent (16) in addition to diluting the target component (10) via the reservoir (110).
In the configuration in which the reagent (16) reacting with the target component (10) is sent to the reservoir (110), the sample processing chip (100) preferably comprises a reagent quantification unit (143). The method further comprises delivering the reagent (16) quantified using the reagent quantification unit (148) to the reservoir (110).
In the sample processing method according to the first aspect, preferably, further comprises forming droplets (14) individually encapsulating the target component (10) contained in the prepared droplet forming sample (13) in a dispersion medium (15).
In the sample processing method according to the first aspect, preferably, the target component (10) is a component to be processed after pretreatment obtained by processing the sample. According to this configuration, the processing liquid (11) containing the target component (10) subjected to the pretreatment can be easily diluted by the reservoir (110).
In this case, preferably, the target component (10) is a nucleic acid, and the pretreatment of the target component (10) is a process of amplifying a nucleic acid in the sample. According to this configuration, the treatment liquid (11) containing the nucleic acid amplified as the target component (10) can be easily diluted by the reservoir (110).
In the configuration in which the target component (10) is a component to be processed after the pretreatment, preferably, the sample processing chip (100) has a processing flow path (150) for performing the pretreatment of the target component (10), and stores the target component (10) after the pretreatment in the reservoir (110). According to this configuration, after the pretreatment is performed by the processing flow path (150) of the sample processing chip (100), the processing liquid (11) can be sent to the reservoir (110) for dilution.
In the sample processing method according to the first aspect, preferably, a droplet (14) containing the prepared droplet forming sample (13) is formed in a dispersion medium (15). According to this configuration, the diluted processing liquid (11) can be made into droplets (14) in the dispersion medium (15).
In this case, preferably, the process of forming the droplet (14) containing the droplet forming sample (13) in the dispersing medium (15) is performed by the droplet forming flow path (180) provided with a first channel (181) through which the droplet forming sample (13) flows, a second channel (182) through which a dispersion medium (15) that is immiscible with the droplet forming sample (13) flows, and an intersection part (183) where the first channel (181) and the second channel (182) intersect each other. According to this configuration, the droplet forming sample (13) can be readily made into droplets (14) in the dispersion medium (15) by the droplet formation flow path (180).
In the configuration in which the droplet formation sample (13) is formed as a droplet (14) in the dispersion medium (15), the sample processing chip (100) preferably also includes a droplet formation flow path (180) and supplies a predetermined amount of the droplet forming sample (13) to the droplet formation flow path (180). According to this configuration, after diluting the processing liquid (11) by the reservoir (110), the droplet forming sample (13) is diluted by the droplet forming flow path (180) of the sample processing chip (100) to form droplets (14) in the dispersion medium (15).
In this case, preferably, the reservoir (110) and the droplet forming flow path (180) are provided separately in the sample processing chip (100). According to this configuration, dilution of the processing liquid (11) and formation of the droplet (14) can be performed by separate sample processing chips (100).
In the configuration in which the sample processing chip (100) has the droplet forming flow path (180), preferably, the reservoir (110) and the droplet forming flow path (180) are integrally connected to the sample processing chip (100). According to this configuration, the number of parts can be reduced as compared to when the reservoir (110) and the droplet forming channel (180) are provided in separate sample processing chips.
In the configuration in which the process of forming the droplet forming sample (13) as a droplet (14) in the dispersion medium (15) is performed by the droplet forming flow path (180), it is preferable that the sample includes a plurality of types of target components (10), and that the sample processing chip (100) has a plurality of droplet forming flow paths (180), and the amount of the droplet forming sample (13) to be supplied for each type of the target component (10) is calculated according to the abundance of the target component (10) type in the droplet forming sample (13), and the calculated amount of the droplet forming sample (13) of each type is supplied to a droplet forming flow path (180) provided for each type of target component (10). According to this configuration, it is possible to form droplets (14) of plural types of target components (10) in parallel using the sample processing chip (100).
In the sample processing method according to the first aspect, preferably, a reservoir (110c) is formed in a flat plate-like sample processing chip (100), and the sample processing chip (100) is arranged with the main plane of the sample processing chip (100) intersects the horizontal direction so that a gas is introduced from the bottom of the reservoir (110c) and agitates the processing liquid (11) and the diluent (12) by the rising gas in the reservoir (110c). According to this configuration, since the sample processing chip (100) can be formed in a flat plate shape, it is possible to reduce the size as compared with when providing a tubular storage tank.
In the sample processing method according to the first aspect, preferably, a predetermined amount of processing liquid (11) is stored in the reservoir (110) by controlling the flow rate and time of the processing liquid (11) that contains the target component (10) to be sent to the reservoir (110). According to this configuration, it is possible to quantify the processing liquid (11) without providing a space for quantification, so that it is possible to reduce the size of the sample processing chip (100).
A sample processing method according to a second aspect of the present invention is a sample processing method for processing a target component (10) in a sample, the method including storing a processing liquid (11) containing a target component (10) and a diluent (12) for diluting the processing liquid (11) in a reservoir (110) of the sample processing chip (100), preparing a droplet forming sample (13) by agitating the processing liquid (11) and a diluent (12) in the reservoir (110) by introducing a gas into the reservoir (110), and forming a droplet (14) containing one molecule or one target component (10) contained in the prepared droplet forming sample (13) in the dispersion medium (15).
According to the sample processing method of the second aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) can be mixed by the gas introduced into the reservoir (110) by configuring as described above (Bubbles), it is not necessary to heat the reservoir (110) and it is possible to reduce the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. The diluted processing liquid (11) also can be made into droplets (14) in the dispersion medium (15).
In the sample processing method according to the second aspect, preferably, the dilution ratio of the target component (10) is 10 times or more and 100,000 times or less. According to this configuration, the target component (10) can be diluted at a dilution ratio for dividing the target component (10) into one molecule or one component.
In the sample processing method according to the second aspect, preferably, the droplet (14) is formed in the dispersion medium (15) with a sample processing chip different from the sample processing chip (100) having the reservoir (110). According to this configuration, dilution of the processing liquid (11) and formation of the droplet (14) can be performed by separate sample processing chips (100).
A sample processing chip (100) according to a third aspect of the present invention is a sample processing chip (100) installed in a sample processing apparatus (200). The sample processing chip (100) comprises a reservoir (110) configured to store a processing liquid (11) containing a target component in a sample and a diluent (12) for diluting the processing liquid (11). The processing liquid (11) is diluted in order to prepare a droplet forming sample (13) for forming droplets (14) individually encapsulating the target component (10), and a gas supply unit (202) configured to supply a gas into the reservoir (110).
According to the sample processing chip (100) of the third aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are agitated by the gas (bubbles) introduced into the reservoir (110) by configuration as described above, it is not necessary to heat the reservoir (110), and it is possible to shorten the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. Since heating is not performed, it also is possible to suppress the target component (10) from changing due to heat.
In the sample processing chip (100) according to the third aspect, preferably, the reservoir (110) is formed by a tubular storage tank. According to this configuration, as compared with the case where the reservoir (110) is provided in the substrate (140) of the sample processing chip (100), the cross sectional area of the portion through which the gas passes can be increased so that the gas can easily pass through into the storage tank. In this way agitation can be performed in a shorter time.
In this case, preferably, the storage tank has an inlet (112) at the bottom portion, and the inlet (112) is arranged at a position where the central axis deviates from the central axis of the storage tank. According to this configuration, since the gas bubbles can be supplied from a position deviated from the center axis of the storage tank, it is possible to suppress the bubbles from contacting the entire circumference of the inner surface of the storage tank. In this way it is possible to suppress the liquid in the storage tank from rising from the liquid surface together with the bubbles, so that the liquid can be prevented from flowing out from the storage tank. As a result, contamination can be effectively suppressed.
In the configuration in which the reservoir (110) is formed by a tubular storage tank, it is preferable that a quantification unit (143) also is provided to quantify the processing liquid (11) sent to the reservoir (110). According to this configuration, it is possible to easily quantify a certain amount of processing liquid (11) for obtaining a diluted mixture with a desired dilution ratio by the quantification unit (143).
In this case, it is preferable that the substrate (140) on which the quantification unit (143) is provided, the quantification unit (143), and the reservoir (110) are connected and a first flow path is provided to move the processing liquid (11) from the quantification unit (143) to the reservoir (110), and that a storage tank of the reservoir (110) is connected on the substrate (140). According to this configuration, a predetermined amount of processing liquid (11) can be supplied from the quantification unit (143) provided on the substrate (140) to the storage tank connected on the substrate (140).
In the configuration including the substrate (140), preferably, the quantification unit (143) includes an inner cavity having a predetermined capacity formed on the substrate (140), and also includes a first flow path (141) connected to an inlet (141a) of the processing liquid (11), a second flow path (144) connected to a disposal port (144a), a third flow path (145) as a first connection flow path connected to the reservoir (110), and a fourth flow path (146) connected to a gas supply unit (202) for feeding a gas, wherein an on/off valve (147a, 147b, 147c, 147d) is respectively provided in each of the first flow passage (141), the second flow passage (144), the third flow passage (145) and the fourth flow passage (146). According to this configuration, the processing liquid (11) is quantified by the quantification unit (143) and the quantified processing liquid (11) is stored in the reservoir (110) by opening and closing the on/off valves (147a, 147b, 147c, 147d).
In the configuration in which the reservoir (110) is formed by a tubular storage tank, the storage tank preferably is formed so that its inner side surface is hydrophilic. According to this configuration, since enlargement of the bubbles in a state where the bubbles are attached to the inner side surface can be suppressed, it is possible to prevent the bubbles from contacting the entire circumference of the inner surface of the storage tank. In this way it is possible to suppress the liquid in the storage tank from rising from the liquid surface together with the bubbles, so that the liquid can be prevented from flowing out from the storage tank. As a result, contamination can be effectively suppressed.
In the configuration in which the reservoir (110) is formed by a tubular storage tank, the storage tank preferably is formed so that the cross sectional area in the horizontal direction becomes larger toward the upper part of the storage tank. According to this configuration, it is difficult for the rising bubble to come into contact with the inner side surface of the storage tank, so that it is possible to suppress the liquid in the storage tank from rising together with the bubbles rising from the liquid surface.
In the configuration in which the reservoir (110) is formed by a tubular storage tank, preferably, the storage tank has an inner cylinder (160) for allowing the introduced gas to ascend through the inside. According to this configuration, it is possible to suppress the liquid in the storage tank from rising above the liquid surface together with the bubbles since a pathway for bubbles is formed.
In the configuration including the quantification unit (143), it is preferable that a processing flow path (150) for pretreating the target component (10) in the sample, and a second flow path for the pretreated target component (10) from the processing flow path (150) to the quantification unit (143) are provided. According to this configuration, it is possible to quantify a desired amount of processing liquid (11) by transferring the processing liquid (11) containing the target component (10) subjected to the pretreatment to the quantification unit (143).
In the sample processing chip (100) according to the third aspect, it is preferable that a droplet forming flow path (180) for forming droplets (14) encapsulating the droplet forming sample (13) in the dispersion medium (15), and a third connection flow path for transferring the droplet forming sample (13) from the reservoir (110) to the droplet forming flow path (180) are provided; and a droplet forming quantification unit (185a, 185b, 185c, 185d) is provided in the third connection flow path. According to this configuration, it is possible to quantify the diluted processing liquid (11) and supply a desired amount to the droplet forming flow path (180), so that the desired droplet (14) can be readily formed.
A sample processing chip (100) according to a fourth aspect of the present invention is a sample processing chip (100) installed in a sample processing apparatus (200) and configured to prepare a droplet forming sample (13) containing a target component (10) in a sample supplied from the sample processing apparatus (200), and includes a reservoir (110) for storing a processing liquid (11) containing a target component (10), and a diluent (12) for diluting the processing liquid (11) for encapsulating one molecule or one component in a droplet (14), an inlet (112) for introducing a gas to the reservoir (110) disposed below the storage tank, and a filter (113) permeable to the gas disposed above the storage tank.
According to the sample processing chip (100) of the fourth aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are agitated by the gas (bubbles) introduced into the reservoir (110) by the configuration as described above, it is not necessary to heat the reservoir (110), and it is possible to shorten the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. Since heating is not performed, it also is possible to suppress the target component (10) from changing due to heat. When a gas is introduced into the storage tank, the liquid in the storage tank also can be prevented from leaking to the outside by the filter (113), so contamination can be effectively suppressed.
In the sample processing chip (100) according to the fourth aspect, the filter (113) preferably is formed of a polymer containing fluorine. According to this configuration, it is possible to effectively prevent the liquid from passing through the filter (113).
In the sample processing chip (100) according to the fourth aspect, the inlet (112) preferably is disposed at a position at which the central axis deviates from the central axis of the storage tank. According to this configuration, since the gas bubbles can be supplied from a position deviated from the center axis of the storage tank, it is possible to suppress the bubbles from contacting the entire circumference of the inner surface of the storage tank. In this way it is possible to suppress the liquid in the storage tank from rising from the liquid surface together with the bubbles, so that the liquid can be prevented from flowing out from the storage tank. As a result, contamination can be effectively suppressed.
A sample processing chip (100) according to a fifth aspect of the present invention is a sample processing chip (100) installed in a sample processing apparatus (200) for processing a target component (10) in a sample supplied by the sample processing apparatus (200), and includes a reservoir (110) for storing a processing liquid (11) containing a target component (10) and a diluent (12) for diluting the processing liquid (11), a gas supply unit (111) for supplying a gas into the reservoir (110), and a droplet forming flow path (180) for forming a droplet (14) encapsulating, in a dispersion medium (15), one molecule or one component of the target component (10) contained in the droplet forming sample (13) prepared by dilution in the reservoir (110).
According to the sample processing chip (100) of the fifth aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are agitated by the gas (bubbles) introduced into the reservoir (110) by the configuration as described above, it is not necessary to heat the reservoir (110), and it is possible to shorten the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. The diluted processing liquid (11) also can be made into droplets (14) in the dispersion medium (15).
A sample processing apparatus (200) according to a sixth aspect of the present invention comprises an installation unit (201) configured to be installed the sample processing chip (100) according to the third, fourth, or fifth aspect, and a supply unit (203) configured to supply the processing liquid (11) and the gas to the reservoir (110) of the sample processing chip (100).
In a sample processing apparatus (200) of the sixth aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are agitated by the gas (bubbles) introduced into the reservoir (110) by the configuration as described above, it is not necessary to heat the reservoir (110), and it is possible to shorten the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. Since heating is not performed, it also is possible to suppress the target component (10) from changing due to heat.
The sample processing apparatus (200) according to the sixth aspect preferably also includes a heating unit (207) for adjusting the temperature of the processing flow path (150) for pretreatment in the sample processing chip (100). According to this configuration, it is possible to dilute the processing liquid (11) by the reservoir (110) after performing pretreatment by heating.
In the sample processing apparatus (200) according to the sixth aspect, preferably, the sample processing chip (100) held by the chip holder (170) is installed as a cartridge (300) in the installation unit (201). According to this configuration, a plurality of samples can be processed in parallel by holding a plurality of sample processing chips (100) in the chip holder (170).
In this case, preferably, the chip holder (170) is formed in a frame shape provided with a hole (171) penetrating in the vertical direction, and holds the sample processing chip (100) by the frame. According to this configuration, since it is possible to access the sample processing chip (100) from both the upper side and the lower side, the heating unit (207) can be brought into contact with the sample processing chip (100) from the lower side, for example.
In the sample processing apparatus (200) according to the sixth aspect, preferably, the sample processing chip (100) is provided with a quantification unit (143) formed by an inner cavity having a predetermined capacity, a first flow path (141), a second flow path (144), a third flow path (145), and a fourth flow path (146) connected to the inner cavity and having an on/off valve (147a, 147b, 147c, 147d), wherein the first flow path (141) is connected to the inlet (141a) of the processing liquid (11), the second flow path (144) is connected to the disposal port (144a), the third flow path is connected to the reservoir (110), the fourth flow path (146) is connected to the supply unit (203) for supplying a gas; and a pressing part (206) for opening and closing the on/off valve (147a) of the first flow path (141), the on/off valve (147b) of the second flow path (144), the on/off valve (147c) of the third flow path (145), and the on/off valve (147d) of the fourth flow path (146). According to this configuration, the processing liquid (11) is quantified by the quantification unit (143) and the quantified processing liquid (11) is stored in the reservoir (110) by opening and closing the on/off valves (147a, 147b, 147c, 147d) via the pressing part (206).
In this case, it is preferable that a predetermined amount of processing liquid (11) is delivered to the reservoir (110) by feeding the processing liquid (11) from the first flow path (141) to fill the inner cavity of the quantification unit (143) when the first flow path (141) and the second flow path (144) are open and the third flow path (145) and the fourth flow path (146) are closed by the pressing unit (206), and feeding the processing liquid (11) filling the inner cavity of the quantification unit (143) via the supply unit t(203) to the reservoir (110) when the first flow path (141) and the second flow path (144) are closed and the third flow path (145) and the fourth flow path (146) are open by the pressing part (206). According to this configuration, the processing liquid (11) is accurately quantified by introducing the processing liquid (11) into the inner cavity when the first flow path (141) and the second flow path (144) are in an open state, and the quantified processing liquid (11) can be delivered to the reservoir (110) without residual when the third flow path (145) and the fourth flow path (146) are in the open state.
The sample processing apparatus (200) according to a seventh aspect of the present invention includes an installation unit (201) where a sample processing chip (100) for preparing a droplet forming sample (13) containing a target component (10) in a sample is installed, and a supply unit (203) for supplying the processing liquid (11) containing the target component (10) and a gas to the reservoir (110) of the sample processing chip (100), wherein the supply unit (203) introduces a gas into the reservoir (110) for a predetermined time of 0.1 second or more and 60 seconds or less.
In a sample processing apparatus (200) of the seventh aspect, since the processing liquid (11) and the diluent (12) in the reservoir (110) are agitated by the gas (bubbles) introduced into the reservoir (110) by the configuration as described above, it is not necessary to heat the reservoir (110), and it is possible to shorten the time required for agitation as compared with when using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time.
The invention makes it possible to obtain a desired diluted mixture.
Hereinafter, embodiments will be described with reference to the drawings.
An outline of a sample processing method according to an embodiment will be described with reference to the drawings.
The sample processing method according to the present embodiment is a sample processing method for processing a target component 10 in a sample using a sample processing chip 100 having a reservoir 110.
The sample processing chip 100 is configured to be capable of receiving a processing liquid 11 containing the target component 10, and is set in the sample processing apparatus 200 to thereby allow the sample processing apparatus 200 to perform sample processing using the cartridge type sample processing chip. The sample processing chip 100 also is a microfluidic chip having fine flow paths for performing desired processing steps. The flow path is, for example, a microchannel having a sectional dimension (width, height, inner diameter) of 0.1 μm to 1000 μm.
A sample obtained by collecting a fluid such as a body fluid and blood (whole blood, serum or plasma) from a patient and applying predetermined pretreatment to the collected body fluid or blood is injected into a sample processing chip 100. The target component 10 may be, for example, nucleic acids such as DNA (deoxyribonucleic acid), cells and intracellular substances, antigens or antibodies, proteins, peptides and the like. For example, when the target component 10 is a nucleic acid, an extract liquid from which nucleic acid is extracted by a predetermined pretreatment from blood or the like is injected into the sample processing chip 100.
The sample containing the target component 10 injected into the sample processing chip 100 is delivered into the sample processing chip 100 by the sample processing apparatus 200. In the course of delivering the sample, the processing of the target component 10 by one or a plurality of steps is performed in a predetermined order. As a result of the processing of the target component 10, a measurement sample suitable for analyzing a sample or a liquid sample suitable for processing using another apparatus is generated in the sample processing chip 100.
In the sample processing method of the present embodiment, the processing liquid 11 containing the target component 10 is diluted so that a molecule or one component of the target component 10 is contained in a droplet 14. That is, the droplet forming sample 13 for forming the droplet 14 including the target component 10 is prepared by diluting the target component 10. The droplets 14 are formed dispersed in a dispersion medium 15 such as oil. The droplet 14 includes not only the droplet forming sample 13 containing the target component 10 but also the reagent 16 for reacting with the target component 10. The reagent 16 includes, for example, a primer 17, a carrier 18, and the like.
In the present embodiment, the processing liquid 11 containing the target component 10 and the diluent 12 are stored in a reservoir 110. Then, by introducing gas into the reservoir 110, the processing liquid 11 and the diluent 12 in the reservoir 110 are agitated to dilute the processing liquid 11. In this way the droplet forming sample 13 for forming the droplet 14 including the diluted target component 10 is prepared.
Accordingly, since the processing liquid 11 and the diluent 12 in the reservoir 110 can be agitated by the gas (bubbles) introduced into the reservoir 110, it is unnecessary to heat the reservoir 110 and possible to shorten the time required for agitation as compared to using thermal convection. As a result, it is possible to obtain a desired diluted mixture by agitating a short time.
For example, by introducing a gas into the reservoir 110 for a predetermined time of 0.1 second or more and 60 seconds or less, the processing liquid 11 and the diluent 12 are agitated. For example, by introducing a gas into the reservoir 110 with a pressure of 100 mbar or more and 1000 mbar or less, the processing liquid 11 and the diluent 12 are agitated.
The dilution ratio of the target component 10 is 10 times or more and 100,000 times or less. In this way the target component 10 can be diluted at a dilution ratio for dividing the target component 10 into one molecule or one component.
In addition, the diluent 12 may contain a reagent 16 that reacts with the target component 10. In this way the target component 10 can be reacted and processed by later processing.
Overview of Sample Processing Chip
An outline of the sample processing chip 100 according to the present embodiment will be described with reference to
The sample processing chip 100 according to the present embodiment is a sample processing chip installed in a sample processing apparatus 200 for preparing a droplet forming sample 13 containing a target component 10 in a sample supplied from a sample processing apparatus 200.
The sample processing chip 100 also includes a reservoir 110 for storing a processing liquid 11 containing a target component 10 and a diluent 12 for diluting the processing liquid 11 so that one molecule or one component of the target component 10 is contained in the droplet 14, and a gas supply unit 111 for supplying a gas into the reservoir 110. Accordingly, since the processing liquid 11 and the diluent 12 in the reservoir 110 can be agitated by the gas (bubbles) introduced into the reservoir 110, it is unnecessary to heat the reservoir 110 and possible to shorten the time required for agitation as compared to using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time.
The sample processing chip 100 also includes a reservoir 110 having a tubular storage tank for storing a processing liquid 11 containing a target component 10, and a diluent 12 for diluting the processing liquid 11 to encapsulate one molecule or one component of the target component 10 in a droplet 14, an inlet 112 disposed below the reservoir to introduce gas into the reservoir 110, and a filter 113 permeable to the gas and disposed above the storage tank. In this way, when the gas is introduced into the storage tank, the liquid in the storage tank can be prevented from leaking to the outside by the filter 113, so contamination can be effectively suppressed.
Overview of Sample Processing Apparatus
The outline of the sample processing apparatus 200 according to the present embodiment will be described with reference to
The sample processing apparatus 200 according to the present embodiment is a sample processing apparatus for processing a target component 10 in a sample by using a sample processing chip 100.
The sample processing apparatus 200 also is provided with an installation unit 201 for installing the sample processing chip 100, a supply unit 203 that supplies the processing liquid 11 containing the target component 10 and the gas to the reservoir 110 of the sample processing chip 100. The supply unit 203 includes a gas supply unit 202 that supplies gas to the reservoir 110 of the sample processing chip 100, and a liquid supply unit 203a that supplies the processing liquid 11 to the reservoir 110 of the sample processing chip 100. Accordingly, since the processing liquid 11 and the diluent 12 in the reservoir 110 can be agitated by the gas (bubbles) introduced into the reservoir 110, it is unnecessary to heat the reservoir 110 and possible to shorten the time required for agitation as compared to using thermal convection. In this way it is possible to obtain a desired diluted mixed solution by agitating for a short time. The gas supply unit 202 and the liquid supply unit 203a may be integrally provided and may function as the supply unit 203. The gas supply unit 202 and the liquid supply unit 203a also may be provided separately and may function as the supply unit 203.
The thickness d of the substrate 120 is, for example, 1 mm or more and 5 mm or less. In this way the substrate 120 can be formed to have a sufficiently large thickness as compared with the flow path height (on the order of 10 μm to 500 μm) of the flow path formed in the fluid module 130. As a result, sufficient pressure resistance performance readily can be ensured for the substrate 120.
The substrate flow path 121 is, for example, a through-hole that penetrates the substrate 120 in the thickness direction. In addition to being connected to the flow path of the fluid module 130, the substrate flow path 121 functions as a port for supplying a liquid or a reagent into the sample processing chip 100 or as a port for recovering the liquid from inside the sample processing chip 100.
In the example of
The substrate flow paths 121 are arranged at a predetermined pitch, for example. In the example of
Each fluid module 130a-130c may have a different flow path shape. Each fluid module 130 may be disposed not only on the first surface but also on the second surface or only on the second surface.
In the structural example of
Each fluid module 130 (including a connection module) is connected to, for example, the substrate 120 by solid phase bonding. For the solid phase bonding, for example, a method in which the bonding surface is subjected to plasma treatment to form OH groups, and bonding surfaces are joined to each other by hydrogen bonding, or a method such as vacuum pressure welding or the like can be adopted. The fluid module 130 and the substrate 120 can be firmly bonded by solid phase bonding. The fluid module 130 also may be connected to the substrate 120 by an adhesive or the like.
In the example of
In the structural example of
The reservoir 110 is a tubular reservoir connected to the substrate 140 of the sample processing chip 100. The processing liquid 11 and the diluent 12 are agitated by introducing gas from the bottom of the storage tank of the reservoir 110 and rising gas in the reservoir. In this way the cross sectional area of the portion through which the gas passes can be increased so that the gas can easily pass through into the storage tank as compared with when the reservoir (110) is provided in the substrate (140) of the sample processing chip (100). As a result, agitation can be performed in a shorter time.
The diluent 12 is placed In the reservoir 110 in advance. The processing liquid 11 containing the target component 10 is supplied to the reservoir 110 via the first flow path 141 by the liquid supply unit 203a. For example, a predetermined amount of the treatment liquid 11 is stored in the reservoir 110 by controlling the flow velocity and time of the processing liquid 11 containing the target component 10 to be sent to the reservoir 110. In this way it is possible to quantify the processing liquid 11 even without providing a space for quantification, so that it is possible to reduce the size of the sample processing chip 100.
In the state in which the processing liquid 11 and the diluent 12 are contained in the reservoir 110, gas is supplied from the gas supply unit 111. At this time, the on/off valve 111a is in the open state. Specifically, the gas is supplied into the reservoir 110 via the inlet 112 disposed at the bottom of the reservoir 110.
The filter 113 is permeable to gas. On the other hand, the filter 113 transmits liquid with difficulty. That is, the filter 113 allows gas to escape from above the reservoir 110 and does not to allow liquid to pass therethrough. The filter 113 is arranged so as to cover the upper part of the reservoir 110. That is, when the gas is introduced into the reservoir 110, it is possible to suppress the liquid from ascending the reservoir 110 and flowing out from the reservoir 110 as the gas rises. The filter 113 may be formed in a cap shape and arranged above the reservoir 110. The filter 113 is made of, for example, a fluorine-containing polymer or a water-absorbing polymer. In this way it is possible to effectively suppress the liquid from passing through the filter 113. The filter 113 may be formed of a porous member. The filter 113 also may be formed of a sponge-like material.
The filter 113 also may be in the form of a film.
The droplet forming sample 13 prepared by the reservoir 110 is sent to the next step via the flow path 142. The liquid supply unit 203a includes, for example, a pump.
In the structural example of
The processing liquid 11 quantified using the quantification unit 143 is sent to the reservoir 110. Specifically, the quantification unit 143 is formed by an inner cavity having a predetermined capacity formed in the sample processing chip 100. The treatment liquid 11 also is supplied to the quantification unit 143 via the first flow path 141. At this time, more treatment liquid 11 is supplied than the amount quantified by the quantification unit 143. The excess treatment liquid 11 is sent to the disposal port 144a via the second flow path 144. In this way the quantification unit 143 is filled with a predetermined amount of treatment liquid 11.
One end of the first flow path 141 is connected to the inlet 141a of the treatment liquid 11, and the other is connected to the quantification unit 143. The on/off valve 147a is provided in the first flow path 141. One end of the second flow path 144 is connected to the disposal port 144a, and the other end thereof is connected to the quantification unit 143. The second flow path 144 is provided on/off valve 147b. One end of the third flow path 145 is connected to the reservoir 110, and the other end thereof is connected to the quantification unit 143. The on/off valve 147c is provided in the third flow path 145. One end of the fourth flow path 146 is connected to the gas supply unit 202 that supplies gas via the gas supply unit 111, and the other end thereof is connected to the quantification unit 143. The on/off valve 147d is provided in the fourth flow path 146.
After storing the diluent 12 in the reservoir 110, the treatment liquid 11 is delivered to the reservoir 110 by gas. In this way it is possible to carry out the feeding of the treatment liquid 11 to the reservoir 110 and the introduction of the gas into the reservoir 110 continuously and in the same operation, so that the feeding and agitating the treatment liquid 11 can be performed in a short time as compared with when it is carried out by the first embodiment.
Specifically, the on/off valves 147a and 147b are opened to bring the first flow path 141 and the second flow path 144 into an open state. The on/off valves 147c and 147d are closed, and the third flow path 145 and the fourth flow path 146 are closed. In this state, the treatment liquid 11 is fed from the first flow path 141 and fills the inner cavity of the quantification unit 143. Thereafter, the on/off valves 147a and 147b are closed, and the first flow path 141 and the second flow path 144 are closed. The on/off valves 147c and 147d are also opened, and the third flow path 145 and the fourth flow path 146 are opened. In this state, the treatment liquid 11 filling the inner cavity of the quantification unit 143 is sent by the gas from the gas supply unit 202. In this way a predetermined amount of the treatment liquid 11 is sent to the reservoir 110.
When the treatment liquid 11 is fed from the first flow path 141 and fills the inner cavity of the quantification unit 143, a fixed amount of the treatment liquid 11 also may be reciprocatingly moved between the first flow path 141, the second flow path 144, and the inner cavity of the quantification unit 143. In this way it is possible to suppress the gas from remaining in quantification unit 143 since the gas pre-existing in the inner cavity of the quantification unit 143 and the first flow path 141 is expelled from the quantification unit 143 by the reciprocating movement of the processing liquid 11. In this way it is possible to quantify the processing liquid (11) more accurately.
In the structural example of
In the structural example of
In the sample processing chip 100, a plurality of quantitative units 143 and reservoirs 110 are connected in series in this order along the flow of the treatment liquid 11. In the sample processing chip 100, the target component 10, which is diluted by the quantification part 143a and the reservoir 110 in an early stage, is further diluted by the quantitative part 143b and the reservoir part 110b in a later stage. In this way it is possible to effectively increase the dilution ratio by a plurality of stages of dilution.
In the structural example of
In a state where the target component 10 and the diluent 12 are stored in the reservoir 110, the reagent 16 for reacting with the target component 10 also is delivered to the reservoir 110. In this way mixing of the reagent 16 can also be performed in addition to diluting the target component 10 by the reservoir 110.
Specifically, the treatment liquid 11 containing the target component 10 quantified by the quantification unit 143 is delivered to the reservoir 110 containing the diluent 12. Thereafter, the reagent 16 quantified by the reagent quantification unit 148 is sent to the reservoir 110. The reagent quantification unit 148 is configured by, for example, an inner cavity formed in the sample processing chip 100. The inner cavity of the reagent quantification unit 148 has a predetermined capacity. On/off valves 149a and 149b are opened to open the flow path 148a and the flow path 148b. The on/off valves 149c and 149d are closed to close the flow path 148c and the flow path 148d. In this state, the reagent 16 is sent from the flow path 148a and is loaded in the reagent quantification unit 148. Thereafter, the on/off valves 149a and 149b are closed to close the flow path 148a and the flow path 148b. The on/off valves 149c and 149d are also opened to open the flow path 148c and the flow path 148d. In this state, the reagent 16 loaded in the reagent quantification unit 148 by a gas. In this way a predetermined amount of the reagent 16 is sent to the reservoir 110.
In the structural example of
Note that the main surface of the sample processing chip 100 may stand perpendicular to the horizontal direction or may be inclined.
In the structural example of
The diluent 12 is introduced via the diluent inlet 141c to the reservoir 110c. At this time, the on/off valve 141d is in an open state, and the on/off valves 111b, 141b, and 142b are in a closed state. Thereafter, the processing liquid 11 containing the target component 10 is introduced into the reservoir 110c via the inlet 141a. At this time, the on/off valve 141b is in an open state, and the on-off valves 111b, 141d, and 142b are in a closed state. Gas is introduced into the reservoir 110c from the inlet 112 via the gas supply unit 111. In this way the processing liquid 11 and the diluent 12 are agitated, and the droplet forming sample 13 is adjusted. Thereafter, the droplet forming sample 13 is sent from the reservoir 110c to the droplet forming sample supply unit 142a. At this time, the on/off valve 142b is in an open state, and the on/off valves 111b, 141b, and 141d are in a closed state.
An example of the sample processing chip 100 according to the present embodiment will be described with reference to
In the examples of
As shown in
The reservoir 110, the gas supply unit 111, the droplet forming sample supply unit 142a, the disposal port 144a, the sample supply tank 151, and the connection parts 153 and 154 are connected to each other via a tubular tank. The reservoir 110, the gas supply unit 111, the droplet forming sample supply unit 142a, the disposal port 144a, the sample supply tank 151, and the connection parts 153 and 154 are provided with connection holes at the lower side, and have a tubular shape extending upward.
The first flow path 141, the flow path 142, the quantification unit 143, the second flow path 144, the third flow path 145, the fourth flow path 146, the on/off valves 147a, 147b, 147c, 147d, 146i, the processing flow path 150, the flow path 152, the inner cavity 155, and the on/off valves 156a, 156b, 156c, 156d are provided within or on the main surface of the substrate 140.
The sample supply tank 151 is connected to the processing flow path 150 via the flow path 152. The processing flow path 150 is connected to the quantification unit 143 via the first flow path 141. The quantification unit 143 is connected to the disposal port 144a via the second flow path 144. The quantification unit 143 also is connected to the reservoir 110 via the third flow path 145. The quantification unit 143 also is connected to the gas supply unit 111 via the fourth flow path 146.
The reservoir 110 is connected to the inner cavity 155 via the on-off valve 156a. The lumen 155 is connected to the droplet forming sample supply unit 142a via the on/off valve 156b. The inner cavity 155 is connected to the connecting part 154 via the on/off valve 156c. The inner cavity 155 also is connected to the connecting part 153 via the on/off valve 156d.
The reservoir 110, the gas supply unit 111, the disposal port 144a, the sample supply tank 151, and the connection parts 153 and 154 are connected to the gas supply unit 202 of the sample processing apparatus 200. In this way a positive pressure and a negative pressure can be supplied to the reservoir 110, the gas supply part 111, the disposal port 144a, the sample supply tank 151, and the connection parts 153 and 154.
The on/off valve 147a is provided in the first flow path 141. The second flow path 144 is provided on/off valve 147b. The on/off valve 147c is provided in the third flow path 145. The on/off valve 147d is provided in the fourth flow path 146. The on/off valve 147i is provided In the flow path 152. The third flow path 145 is used as a first connection flow path for moving the processing liquid 11 from the quantification unit 143 to the reservoir 110. The first flow path 141 is used as a second connection flow path for transferring the pretreated target component 10 from the processing flow path 150 to the quantification unit 143.
The sample containing the target component 10 is supplied to the sample supply tank 151. For example, the user may measure and supply a predetermined amount of sample with a pipette or the like, or the sample may be dispensed and supplied by the sample processing apparatus 200. The sample in the sample supply tank 151 is sent to the processing flow path 150 via the flow path 152 when a positive pressure is applied from the gas supply unit 202. At this time, the on/off valve 147i is in the open state.
In the processing flow path 150, pretreatment of the target component 10 is performed. For example, the pretreatment of the target component 10 may be a process of amplifying a nucleic acid in the sample. That is, the target component 10 sent to the reservoir 110 is a component to be processed after the pretreatment obtained by processing the sample. The processing flow path 150 is subjected to pretreatment of the target component 10 by being heated. A positive pressure is applied from the gas supply unit 202 to the sample supply tank 151 after the processing in the processing flow path 150, whereby the processing liquid 11 is sent to the quantification unit 143 via the first flow path 141.
The quantification unit 143 quantifies the processing liquid 11 sent to the reservoir 110. The quantification unit 143 includes an inner cavity having a predetermined capacity formed on the substrate 140. For example, the quantification unit 143 quantifies about 1 μL to 100 μL of the processing liquid 11. For example, the quantification unit 143 quantifies about 10 μL of the processing liquid 11. A positive pressure is applied from the gas supply unit 202 to the gas supply unit 111, and a negative pressure is applied from the gas supply unit 202 to the reservoir 110, so that the processing liquid 11 is sent to the reservoir 110 via the flow path 145.
In the reservoir 110, the treatment liquid 11 is diluted with the diluent 12. The diluent 12 may be placed in the reservoir 110 in advance. A diluent 12 in an amount corresponding to a predetermined dilution ratio is placed in the reservoir 110. For example, several tens μL to several hundred μL of the diluent 12 are placed in the reservoir 110. For example, 190 μL of diluent 12 is placed in reservoir 110. For example, the dilution ratio of the treatment liquid 11 in the reservoir 110 is 10 times or more and 100,000 times or less.
The reservoir 110 agitates the treatment liquid 11 and the diluent 12 in the reservoir 110 by introducing gas. The gas is supplied via the gas supply unit 111. In this way a droplet forming sample 13 for forming a droplet 14 containing the diluted target component 10 is prepared in the reservoir 110. For example, the reservoir 110 is a tubular reservoir connected to the substrate 140 of the sample processing chip 100. Gas is introduced from the bottom of the reservoir 110 and the gas rises in the reservoir to agitate the treatment liquid 11 and the diluent 12. The droplet forming sample 13 is sent to the droplet forming sample supply unit 142a via the flow path 142 when the positive pressure is applied from the gas supply unit 202 to the reservoir 110 after adjustment of the reservoir 110. The flow path 142 is used as a third connection flow path for transferring the droplet forming sample 13 from the reservoir 110 to the droplet forming flow path 180.
In the example shown in
In the example shown in
In the example shown in
In the example shown in
The on/off valves 147a, 147b, 147c, 147d, 147i, 156a, 156b, 156c and 156d are opened and closed similar to the on/off valve 147 shown in
In the example shown in
As in the example shown in
As in the example shown in
The processing flow of the sample processing chip 100 of the example of
In
In
In
Then, a positive pressure is applied to the gas supply unit 111. In this way gas is introduced into the reservoir 110. The introduction time of the gas is, for example, about 0.1 second to 60 seconds together with the liquid transfer. Preferably, the introduction time of the gas is 0.4 seconds or more and 50 seconds or less together with the liquid transfer. More preferably, the introduction time of the gas is not less than 0.4 seconds and not more than 10 seconds together with the liquid transfer. In this way it is possible to agitate the interior of the reservoir 110 reliably in a short time. For example, the introduction time of the gas is about 0.4 seconds. In this way the processing liquid 11 and the diluent 12 are agitated in the reservoir 110, and the droplet forming sample 13 is adjusted.
In
As in the example shown in
In the sample processing chip 100 of the example of
The droplet forming flow path 180 includes a first channel 181 through which the droplet forming sample 13 flows, a second channel 182 through which flows the dispersion medium 15 that is immiscible with the droplet forming sample 13, and an intersection part 183 where the first channel 181 and the second channel 182 intersect each other. In this way the droplet forming flow path 180 makes it possible to easily convert the droplet forming sample 13 into the droplet 14 in the dispersion medium 15.
A predetermined amount of the droplet forming sample 13 also is supplied to the droplet forming flow path 180. The dispersion medium 15 also is supplied in accordance with the flow rate of the droplet forming sample 13. In this way the number of droplets 14 and the average particle diameter are controlled.
The sample processing chip 100 is provided with a dispersion medium supply unit 182a to which the dispersion medium 15 is supplied, a flow path 184, and an emulsion supply unit 184a for supplying the emulsion of formed droplets 14 for additional post-processing.
In the example of
When the sample includes a plurality of types of target components 10, the sample processing chip 100 may have a plurality of droplet forming flow paths 180 as shown in
In the example shown in
For example, the amount of the droplet forming sample 13 to be supplied for each type of the target component 10 is calculated according to the abundance of the plural kinds of target components 10 in the droplet forming sample 13. Then, the amount to be quantified by each of the droplet forming quantitative units 185a, 185b, 185c, 185d is set so as to supply the calculated amount of the droplet forming sample 13.
The droplet forming quantification unit 185 (185b, 185c, 185d) feeds the droplet forming sample 13 by controlling the opening and closing of the on/off valves 186a, 186b, 186c, 186d. Specifically, the on/off valves 186a and 186b are opened, and the on/off valves 186c and 186d are closed. In this state, the droplet forming sample 13 is fed from the reservoir 110 and fills the inner cavity of the droplet forming quantification unit 185a (185b, 185c, 185d). Thereafter, the on/off valves 186a and 186b are closed, and the on/off valves 186c and 186d are opened. In this state, the droplet forming sample 13 that loaded in the inner cavity of the droplet forming quantification unit 185a (185b, 185c, 185d) is transferred by the gas from the gas supply unit 202. In this way a predetermined amount of the droplet forming sample 13 is sent to the droplet forming flow path 180.
In the example of
In the example of
In the example of
In the example of
In the example of
The sample processing apparatus 200 is a sample processing apparatus for processing the target component 10 in the sample using the sample processing chip 100. The contents of sample processing are determined by the sample processing chip 100 to be used. The sample processing apparatus 200 can perform different types of sample processing depending on the type of the sample processing chip 100 to be used.
The sample processing apparatus 200 includes an installation unit 201, a gas supply unit 202, a solenoid valve 204, a solenoid valve 205, a pressing part 206, and a heating unit 207. The sample processing apparatus 200 also includes a control unit 210.
The control unit 210 controls each unit so that the sample processing chip 100 performs the processing of the sample. The control unit 210 includes a CPU and a memory.
When a processing unit used for various processing steps is installed in the sample processing apparatus 200, the control unit 210 may control these processing units. Units used for various processing steps include, for example, a heating unit or a cooling unit for controlling the temperature of the liquid, a magnet unit for exerting a magnetic force on the liquid, a camera unit for imaging the liquid, a detection unit for detecting a sample or a label in the liquid and the like. These processing units are provided corresponding to at least one of the plurality of fluid modules 130 and are configured to operate when executing the processing steps by the corresponding fluid modules 130.
The sample processing apparatus 200 can include a monitor 211, an input unit 212, a reading unit 213, and the like. On the monitor 211, the control unit 210 displays a predetermined display screen according to the operation of the sample processing apparatus 200. The sample processing apparatus 200 also may be connected to an external computer (not shown) and displayed on the monitor of the computer. The input unit 212 is composed of, for example, a keyboard, a mouse, and the like, and has a function of receiving information input. The reading unit 213 includes a code reader such as a bar code and a two-dimensional code, a tag reader such as an RFID tag, and has a function of reading information given to the sample processing chip 100. The reading unit 213 can also read information such as a sample container (not shown) for containing the sample.
The gas supply unit 202 can supply positive pressure and negative pressure to each section of the sample processing apparatus 200. The gas supply unit 202 includes a negative pressure generation unit 202a and a positive pressure generation unit 202b. The negative pressure generating unit 202a includes a negative pressure pump, for example. The positive pressure generating unit 202b includes, for example, a compressor. The gas supply unit 202 supplies positive pressure or negative pressure to the sample processing chip 100 via the solenoid valve 204. The liquid is sent in the sample processing chip 100 by the positive pressure and the negative pressure supplied by the gas supply unit 202. The positive pressure generating unit 202b of the gas supply unit 202 supplies positive pressure to the pressing part 206 via the solenoid valve 205. When a positive pressure is supplied, the pressing part 206 is exerted downward to press the on/off valve 147 of the sample processing chip 100 to bring it into a closed state. When the supply of the positive pressure is stopped, the pressing part 206 is exerted upward by an elastic member such as a spring, releases the pressure of the on/off valve 147 of the sample processing chip 100, and enters the open state. The gas supply unit 202 functions as a liquid supply unit that supplies a liquid and a gas supply unit that supplies a gas.
A plurality of solenoid valves 204 are provided. The solenoid valve 204 is individually controlled by the control unit 210, and the open/close state is switched. A plurality of solenoid valves 205 are provided. The solenoid valve 205 is individually controlled by the control unit 210, and the open/close state is switched. The heating unit 207 heats the sample processing chip 100.
Connectors 220 and 230 corresponding to the installation part 201 may be provided in the installation part 201.
A processing flow of installing the sample processing chip 100 of the example of
The sample processing is started in a state where the sample processing chip 100 is installed in the installation unit 201 of the sample processing apparatus 200. Note that in this case the sample containing the target component 10 is supplied to the sample supply tank 151, and the diluent 12 is supplied to the reservoir 110.
In step S1, the sample containing the target component 10 is sent to the processing flow path 150 (see
In step S2, pre-PCR is performed (see
In step S3, the pre-PCR processed processing liquid 11 is sent to the quantification unit 143 (see
In step S4, the quantitatively processed processing liquid 11 is sent to the reservoir 110 (
In step S5, the interior of the reservoir 110 is agitated by gas (bubbles). Specifically, after sending the processing liquid 11 to the reservoir 110, a positive pressure is continuously applied to the gas supply unit 111. In this way gas is introduced into the reservoir 110. The introduction time of the gas is, for example, about 0.4 second in conjunction with the liquid transfer. In this way the processing liquid 11 and the diluent 12 are agitated in the reservoir 110, and the droplet forming sample 13 is adjusted.
In step S6, the droplet forming sample 13 is fed (see
Next, an example of a specific assay using the sample processing chip 100 will be described.
In step S11, DNA is extracted from a sample such as blood by pretreatment (see
In step S12, the extracted DNA is amplified by pre-PCR processing (see
In step S13, the DNA is diluted with the diluent 12 (see
In step S14, an emulsion containing magnetic particles and the reagent 16 for amplification reaction and DNA is formed (see
In step S15, under the temperature control by the thermal cycler, the DNA binds to the primer 17 on the magnetic particle within each droplet 14 of the emulsion, and is amplified (emulsion PCR) (see
After amplifying the DNA on the magnetic particles, in step S16 the emulsion is destroyed and the magnetic particles containing the amplified DNA are taken out from the droplet 14 (emulsion break) (see
In step S17, the magnetic particles removed from the droplet 14 are washed in a BF separation step (primary cleaning). The BF separation step is a process step of removing unnecessary substances adhered to magnetic particles by allowing magnetic particles containing amplified DNA to pass through a washing liquid in a state of being magnetized by magnetic force. In the primary cleaning step, for example, a cleaning liquid containing alcohol is used. Alcohol removes the oil film on the magnetic particles and modifies the amplified double-stranded DNA into a single strand (see
After washing, in step S18 the DNA denatured to single strands on the magnetic particles is bound to a labeling substance 19 for detection (hybridization) (see
In step S19, the magnetic particles bonded to the labeling substance 19 are washed in a BF separation step (secondary washing). The secondary BF separation step is performed by the same process as the primary BF separation step. In the secondary washing step, for example, PBS (phosphate buffered saline) is used as a washing solution. PBS removes unreacted labeling substance (including labeling substance nonspecifically affixed to magnetic particles) not bound to DNA.
In step S20, DNA is detected via a hybridized labeling substance 19. DNA is detected, for example, by a flow cytometer. In the flow cytometer, magnetic particles containing DNA bound to the labeling substance 19 flow through a flow cell, and the magnetic particles are irradiated with laser light. The fluorescence of the labeling substance 19 emitted due to the irradiating laser light is detected.
The DNA may be detected by image processing. For example, magnetic particles containing DNA bound to the labeling substance 19 are dispersed on a flat slide or on a flow path, and the dispersed magnetic particles are imaged by a camera unit. The number of magnetic particles emitting fluorescence is counted based on the captured image.
Next, examples conducted to confirm the effect of the sample processing method of the present embodiment will be described. In this example, an experiment was conducted in which the processing liquid 11 containing the target component 10 and the diluent 12 were agitated by the reservoir 110. Experiments also were conducted using DNA as target component 10.
In the example, as shown in
In a comparative example shown in
In the comparative example, the DNA concentration of Sample A was about 600 pg/mL. The DNA concentration of Sample B was about 1400 pg/mL. That is, the concentration of DNA in the lower mixture was greater. That is, it was confirmed that, in the comparative example in which the agitation process by introducing the gas was not performed, and agitation of the processing liquid 11 and the diluent 12 was not carried out satisfactorily.
Next, a case where agitation is carried out by heat convection (Comparative Example) and a case where agitation is performed by bubbles (Example) will be described. In the example, the processing liquid 11 and the diluent 12 are supplied to the reservoir 110, the gas is introduced into the reservoir 110, and the inside of the reservoir 110 is agitated by bubbles. In the example, the processing liquid 11 also was diluted by 10 times or 50 times with the diluent 12. In the comparative example, the processing liquid 11 and the diluent 12 were supplied to the reservoir, the reservoir was heated, and the inside of the reservoir was agitated by heat convection. In the comparative example, the processing liquid 11 was diluted by 30 times or 50 times with the diluent 12.
As shown in
Note that the embodiments disclosed this time are examples in all respects and are not restrictive. The scope of the present invention is indicated not by the description of the above embodiments but by the scope of the claims, and includes meanings equivalent to the claims and all changes (modifications) within the scope thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-049010 | Mar 2018 | JP | national |
