KR102350660B1 - Centrifugal injection device with variable wing - Google Patents

Abstract

Disclosed is a variable-wing centrifugal injection device capable of preventing the occurrence of air pockets in a corner or edge region of a reaction space. Variable-wing centrifugal injection device, the storage container; a rotating body disposed inside the storage container; A first rotation-supporting part that is rotatably connected to one end of the rotating body, grips the first PCR cartridge at an angle and supports the first PCR cartridge at various inclination angles according to the rotation of the rotating body; and a second rotation-supporting part that is rotatably connected to the other end of the rotating body, grips the second PCR cartridge at an angle, and supports the second PCR cartridge at various inclination angles according to the rotation of the rotating body. include Accordingly, the liquid sample can be evenly distributed in the reaction space by centrifugal force by rotating the PCR cartridge vertically so that the plane of the PCR cartridge into which the liquid sample is injected is vertical.

Description

CENTRIFUGAL INJECTION DEVICE WITH VARIABLE WING

The present invention relates to a variable-wing centrifugal injection device, and more particularly, to a variable-wing centrifugal injection device capable of evenly distributing a liquid sample to a corner or an edge region of a reaction space of a PCR cartridge.

Gene amplification technology is an essential process in molecular diagnosis, and is a technology that repeatedly replicates and amplifies a specific nucleotide sequence of a trace amount of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample. Among them, polymerase chain reaction (PCR) is a representative gene amplification technology and consists of three steps: DNA denaturation, primer bonding, and DNA extension. Since the step is dependent on the temperature of the sample, DNA can be amplified by repeatedly changing the temperature of the sample.

Previously, PCR was generally performed in 96 or 384-well microplates. When higher throughput is required, conventional PCR methods in microplatelets are not cost effective or efficient. On the other hand, reducing the PCR reaction volume may reduce the consumption of reagents and shorten the amplification time at the reduced thermal mass of the reaction volume. This strategy can also be implemented in an array form (m×n), resulting in many smaller reaction volumes. The use of any array also allows for scalable and high-throughput analysis with increased quantitative sensitivity, dynamic range and specificity.

So far, digital polymerase chain reaction (digital PCR or dPCR) has been performed using array formats. Results from digital PCR can be used to detect and quantify concentrations of rare alleles, provide absolute quantification of nucleic acid samples, and also measure fold changes with low nucleic acid concentrations. In general, increasing the number of replicates increases the accuracy and reproducibility of digital PCR results.

The array format of most quantitative polymerase chain reaction (qPCR) platforms is designed for assay experiments on a sample-by-sample basis, where PCR results must be addressable for post-mortem analysis. However, for digital PCR, the specific location or well of each PCR result is not critical, only the number of positive and negative replicates per sample may be analyzed.

In the case of such digital PCR, since the size of the reaction space is very small and the number is very large, it is difficult to put a sample into all of the reaction spaces.

Also, since the sample is not injected to the corner or edge region of the reaction space, air pockets may be formed in the corner or edge region of the reaction space. These air pockets expand or contract due to temperature changes during the PCR process, resulting in errors in the test results.

Korea Patent Publication No. 2017-0024827 (2017. 03. 08.) (Title of the invention: qPCR cartridge equipped with micro-channel film reactor, nucleic acid extraction module and qPCR reaction composition module and high-speed qPCR system using the same) Korean Patent Application Laid-Open No. 2016-0086937 (2016. 07. 20.) (Title of the invention: system and method for loading liquid samples) Korean Patent Registration No. 10-1484408 (2015. 01. 13.) (Title of the invention: Rotational PCR method using microchip and microchip for the same) Korea Patent Registration No. 10-1513273 (April 13, 2015) (Title of the invention: Rotary PCR device and PCR chip)

Accordingly, it is an object of the present invention to provide a variable-wing centrifugal injection device capable of evenly distributing a liquid sample to a corner or an edge region of a reaction space of a PCR cartridge.

In order to achieve the above object of the present invention, a variable-wing centrifugal injection device according to an embodiment includes a receiving container; a rotating body disposed inside the storage container; A first rotation-supporting part that is rotatably connected to one end of the rotating body, grips the first PCR cartridge at an angle and supports the first PCR cartridge at various inclination angles according to the rotation of the rotating body; and a second rotation-supporting part that is rotatably connected to the other end of the rotating body, grips the second PCR cartridge at an angle, and supports the second PCR cartridge at various inclination angles according to the rotation of the rotating body. include

In one embodiment, the rotating body, the central portion; Extending obliquely upward from one side of the central portion, the first rotation - a first wing comprising one end portion rotatably connected to one side of the support portion, and the other end portion rotatably connected to the other side of the first rotation - support portion wealth; And extending obliquely upward from the other side of the central portion, the second rotation- a second end including one end portion rotatably connected to one side of the support portion, and the second end portion rotatably connected to the other side of the second rotation-support portion It may include a wing part.

In one embodiment, the variable-wing centrifugal injection device is connected to the lower portion of the first rotation-supporting part and the inner wall of the receiving container, and when the rotating body does not rotate, the first PCR cartridge is rotated at a first inclination angle. a first guide member for guiding to be disposed, and for guiding the first PCR cartridge to an inclination angle greater than or equal to 90 degrees greater than the first inclination angle by centrifugal force when the rotating body rotates; and the second rotation-connected to the lower part of the support part and the inner wall of the receiving container, and guides the second PCR cartridge to be disposed at a first inclination angle when the rotating body does not rotate, and when the rotating body rotates, the The second PCR cartridge may further include a second guide member for guiding at an inclination angle greater than or equal to 90 degrees greater than the first inclination angle by centrifugal force.

In one embodiment, each of the first guide member and the second guide member may include a spiral spring or a silicon piston bumper.

According to this variable-wing centrifugal injection device, the liquid sample can be evenly distributed in the reaction space by centrifugal force by rotating the PCR cartridge vertically so that the plane of the PCR cartridge into which the liquid sample is injected is vertical. That is, by configuring the variable-wing centrifugal injection device so that the liquid sample is distributed even in the corners or edge regions of the well array, which is the reaction space, by centrifugal force, it is possible to prevent air pockets from being generated in the reaction space. Accordingly, it is possible to prevent an error caused by an air pocket in the PCR test result.

1 is a perspective view schematically illustrating a variable-wing centrifugal injection device according to an embodiment of the present invention.
FIG. 2 is an exploded perspective view illustrating a cartridge for PCR employed in the variable-wing centrifugal injection device shown in FIG. 1 .
3A is a perspective view schematically illustrating a state before rotation of the variable-wing centrifugal injection device shown in FIG. 1 , and FIG. 3B is a perspective view schematically illustrating a state during rotation of the variable-wing centrifugal injection device shown in FIG. 1 . to be.

Hereinafter, a variable-wing centrifugal injection device according to the present invention will be described in more detail with reference to the accompanying drawings. Since the present invention can have various changes and can have various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to the specific disclosed form, it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

In describing each figure, like reference numerals have been used for like elements. In the accompanying drawings, the dimensions of the structures are enlarged than the actual size for clarity of the present invention.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In the present application, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It is to be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

In addition, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, preferred embodiments of the present invention will be further described with reference to the accompanying drawings. The same reference numerals are used for the same components in the drawings, and repeated descriptions of the same components are omitted.

The present invention relates to a structure for mounting samples, sample volumes or reaction volumes in a predetermined arrangement on a substrate, and more particularly, to mounting the samples in a predetermined arrangement of respective reaction positions in a substrate.

In various embodiments, apparatus, apparatus, systems, and methods for loading samples in an article used to detect targets in large, small volume samples are provided. Such targets may be any suitable biological target, but include DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (eg, circulating tumors). cells), or other suitable target biomolecules. In various embodiments, these biological components are used in applications such as fetal diagnosis, multiplex dPCR, virus detection, and quantitation standardization, genotyping, sequence validation, mutation detection, genetic biology, rare allele detection, and copy number change detection. It may also be used in conjunction with various PCR, qPCR, and/or dPCR methods and systems.

In general, although applicable to quantitative polymerase chain reaction (qPCR) in which large amounts of samples are processed, it should be appreciated that any suitable PCR method may be used in accordance with the various embodiments described herein. Suitable PCR methods include, for example, digital PCR, allele specific PCR, asymmetric PCR, ligationmediated PCR, multiplex PCR, nested PCR, qPCR, casting PCR, genome walking, bridging ( bridge) PCR, but are not limited thereto.

As described below in accordance with various embodiments described herein, reaction locations include, but are not limited to, for example, through holes, sample holding areas, wells, indentations, spots, cavities, and reaction chambers. doesn't happen

The various embodiments described herein are particularly suitable for digital PCR (dPCR). In digital PCR, a solution containing a relatively small number of target polynucleotides or nucleic acid sequences may further partition each sample into a large number of smaller samples, whereby each sample typically contains one molecule of the target nucleotide sequence. It may or may not contain the target nucleotide sequence. Subsequently, when samples are thermally cycled in a PCR protocol, procedure, or experiment, a sample comprising a target nucleotide sequence is amplified to produce a positive detection signal while samples containing no target nucleotide sequence are detected without amplification. It does not generate a signal. Poisson statistics may be used to correlate the number of target nucleotide sequences in the original solution with the number of samples that produce a positive detection signal.

For a typical dPCR protocol, procedure, or experiment, to tens or hundreds of thousands of samples in a simple and cost-effective way, i.e. samples having a volume of less than a few nanoliters, one or approximately one nanoliter, or less than one nanoliter, respectively There is an advantage in that the initial sample solution can be partitioned. Since the number of target nucleotide sequences can be very small, it may also be important in those environments where the total contents of the initial solution are incorporated to account for multiple reaction sites.

Embodiments described herein address these and other dPCR design constraints by dispensing the initial sample solution to a plurality of reaction sites in a manner that considers all, or all, essential, of the sample solution.

Because of the high-throughput PCR assays and dPCR methods, a strategy that uses an array format to reduce the reaction volume of a liquid sample while increasing the number of reactions performed at one time may be applied. The arrangement of reaction volumes of the liquid sample may be a substrate having a plurality of reaction locations. Reaction locations can be, but are not limited to, through holes, wells, recesses, points, cavities, reaction chambers, or any structures capable of holding a sample according to various embodiments described herein. doesn't happen In some embodiments, the through hole or wells may be tapered in diameter.

If the reaction volume of the liquid sample is reduced so that more reactions can be performed within a given region, the density of the reaction volumes may be increased. For example, an array of reaction sites configured with a diameter of 300 μm through through holes in the substrate may include a reaction volume of about 30 nL. For example, by reducing the size of each through hole in the array to 60 to 70 μm in diameter, for example, each reaction volume may be 100 pL of a liquid sample. Reaction volumes according to various embodiments described herein may be from about 1 pL to 30 nL of liquid sample. Moreover, the dynamic range may be increased by using one or more dilutions of the liquid sample.

1 is a perspective view schematically illustrating a variable-wing centrifugal injection device according to an embodiment of the present invention.

Referring to FIG. 1 , a variable-wing centrifugal injection device according to an embodiment of the present invention includes a storage container 110 , a rotating body 120 , a first rotation-supporting part 130 , a second rotation-supporting part 140 , It includes a first guide member 150 and a second guide member 160, and rotates the cartridge for PCR to be mounted. In this embodiment, the cartridge for PCR may be a cartridge for digital PCR, or a cartridge for real-time PCR.

The storage container 110 includes a storage body 112 and a storage cover 114, and includes a rotating body 120, a first rotation-supporting part 130, a second rotation-supporting part 140, a first guide member ( 150) and the second guide member 160 is accommodated.

The housing body 112 has a cylindrical shape and a motor for rotating the rotating body 120 and a controller for controlling various operations such as varying the number of rotations or executing a rotation operation may be disposed therein. Various operation buttons for receiving a user's operation are disposed on the outer surface of the housing body 112 .

The storage lid 114 has a dome shape and is fastened to the storage body 112 . The storage cover 114 may include a transparent material so that the user can check the rotation state of the cartridge for PCR.

The rotating body 120 includes a central part 122 , a first wing part 124 , and a second wing part 126 , and is disposed inside the storage container 110 .

The first wing portion 124 extends obliquely upward from one side of the central portion 122 , and the first rotation-supporting portion 130 has one end portion rotatably connected to one side, and the first rotation-supporting portion 130 of the first wing portion 124 . It includes the other end rotatably connected to the other side.

The second wing portion 126 extends obliquely upward from the other side of the central portion 122, and the second rotation-supporting portion 140 has one end portion rotatably connected to one side, and the second rotation-support portion 140 of the second wing portion 126. It includes the other end rotatably connected to the other side.

The first rotation-support unit 130 is rotatably connected to one end of the rotating body 120, and grips the first PCR cartridge at an angle and according to the rotation of the rotating body 120, the first PCR cartridge (not shown). ) is configured to support it at various inclination angles. For example, when the revolutions per minute (RPM) of the rotating body 120 is 100, the first rotation-supporting part 130 may be maintained at 50 degrees with respect to the ground, and when the RPM of the rotating body 120 is 200 The first rotation-supporting portion 130 may be maintained at 60 degrees with respect to the ground. As such, according to the RPM of the rotating body 120, the first rotation-supporting part 130 may be maintained at a maximum of 90 degrees with respect to the ground.

The second rotation-support unit 140 is rotatably connected to the other end side of the rotating body 120 , and obliquely grips the second PCR cartridge (not shown) and according to the rotation of the rotating body 120 for the second PCR It is configured to support the cartridge at various angles of inclination. For example, when the revolutions per minute (RPM) of the rotating body 120 is 100, the second rotation-supporting part 140 may be maintained at 50 degrees with respect to the ground, and when the RPM of the rotating body 120 is 200 The second rotation-supporting portion 140 may be maintained at 60 degrees with respect to the ground. As such, according to the RPM of the rotating body 120, the second rotation-supporting part 140 may be maintained at a maximum of 90 degrees with respect to the ground.

The first guide member 150 is connected to the lower part of the first rotation-supporting part 130 and the inner wall of the receiving container 110, and when the rotation body 120 is not rotated, the first PCR cartridge is placed against the ground. Guides to be disposed at an inclination angle of 1, and when the rotating body 120 rotates, the first PCR cartridge is guided at an inclination angle greater than or equal to 90 degrees greater than the first inclination angle by centrifugal force. The first guide member 150 may employ a spiral spring, a cylinder piston bumper, or the like for buffering the first rotation-supporting part 130 .

The second guide member 160 is connected to the lower portion of the second rotation-supporting part 140 and the inner wall of the receiving container 110, and when the rotation body 120 is non-rotated, the second PCR cartridge is placed against the ground. Guides to be disposed at an inclination angle of 1, and when the rotating body 120 rotates, the second PCR cartridge is guided at an inclination angle greater than or equal to 90 degrees greater than the first inclination angle by centrifugal force. The second guide member 160 may include a spiral spring. The second guide member 160 may employ a spiral spring, a cylinder piston bumper, or the like for cushioning the second rotation-supporting part 140 .

FIG. 2 is an exploded perspective view illustrating a cartridge for PCR employed in the variable-wing centrifugal injection device shown in FIG. 1 .

Referring to FIG. 2, the cartridge 200 for PCR includes a case for accommodating the PCR module 210 and the PCR module 210, and the first rotation shown in FIG. 1 - the support part 130 and/or the second It can be rotated by being mounted on the rotation-supporting part 140 . The case may include a body 220 disposed below the PCR module 210 and a cover 230 disposed above the PCR module 210 and coupled to the body 220 to accommodate the PCR module 210 . .

The PCR module may include a silicon chamber 212 , a well array 214 , a CMOS image sensor 216 and a PCB 216 .

The silicon chamber 212 includes a membrane switch (not shown) protruding upward, and an inlet (not shown) formed on one side of the membrane switch for injection of a liquid sample. The lower edge region of the silicon chamber 212 abuts the edge region of the PCB 216 . In the lower central region of the silicon chamber 212 , a space with a retracted shape is formed to accommodate the CMOS image sensor 216 and the well array 214 mounted on the PCB 216 . The silicon chamber 212 may be made of a material such as PDMS. The silicone chamber 212 has flexibility, transparency, PCR compatibility and low autofluorescence, and can be injection molded.

A well array 214 is attached to the bottom surface of the silicon chamber 212 and is disposed over the CMOS image sensor 216 . The well array 214 may be formed of an etched silicon material. Well array 214 includes a plurality of micro-wells (approximately 20,000) serving as PCR reaction sites. The shape, size and volume of the microwells can be controlled. The micro-wells have a shape through which upper and lower portions are penetrated. In this embodiment, the micro-well may have a thickness of less than 250 um and a pitch of less than 70 um. The micro-well may have various shapes, such as a hexagonal shape, a circular shape, and the like. Well array 214 may be treated with a hydrophilic coating. The well array 214 may be attached to the silicon chamber 212 by plasma or thermal or UV curing adhesive.

A CMOS image sensor 216 is disposed below the well array 214 and captures PCR reaction products in micro-wells of the well array 214 . Specifically, the CMOS image sensor 216 is disposed to correspond to the substrate hole formed in the PCB 216 and receives light emitted from the well array 214 to measure the PCR reaction product performed in the PCR device. The top surface of the CMOS image sensor 216 may be coated with a thin layer of a material such as PDMS to form the bottom of the micro-well after sealing.

The PCB 216 houses the CMOS image sensor 216 . A ventilation hole 152 for vacuum processing may be formed in the PCB 216 so that the liquid sample injected through the inlet is quickly provided to the well array 214 . The vent 152 communicates with the space between the CMOS image sensor 216 and the well array 214 .

In this embodiment, the cartridge 200 for PCR may further include a heater (not shown) for thermal circulation. As used herein, thermal cycling uses a thermal cycler, isothermal amplification, thermal convention, infrared mediated thermal cycling, or helicase dependent amplification. It may include the step of In some embodiments, a chip may be integrated with an embedded heating element. In various embodiments, the chip may be integrated with semiconductors. Detection of a target according to various embodiments may, for example, alone or in combination with fluorescence detection, positive or negative ion detection, pH (pH) detection, voltage detection, or current detection, but is not limited thereto.

The cartridge 200 for PCR may further include a ring member 250 and a button member 260 .

The ring member 250 surrounds the interface between the body 220 and the cover 230 coupled to each other. The ring member 250 may function to block the liquid sample from flowing out while the body 220 and the cover 230 are fastened to each other.

The button member 260 is disposed between the body 220 and the cover 230 and is exposed through a large hole formed in the center of the cover 230 . The button member 260 may be configured to provide a pressing function to the user so that the liquid sample injected into the PCR module 210 is evenly distributed inside the PCR module 210 .

3A is a perspective view schematically illustrating a state before rotation of the variable-wing centrifugal injection device shown in FIG. 1 , and FIG. 3B is a perspective view schematically illustrating a state during rotation of the variable-wing centrifugal injection device shown in FIG. 1 . to be.

As shown in Fig. 3a, after injecting the liquid sample into the PCR cartridge, the variable-wing centrifugal injection device is rotated to evenly distribute the liquid sample in the reaction space, the first PCR cartridge (CT1) into which the liquid sample is injected. is disposed on the first rotation-supporting part 130 , and the second PCR cartridge CT2 into which the liquid sample is injected is placed on the second rotation-supporting part 140 . In Figure 3a, the two rotation-supporting parts (130, 140), respectively, showing that the cartridge for PCR is shown, but one rotation-supporting part only, it is also possible to arrange the cartridge for PCR.

As shown in Figure 3b, when the rotation body 120 is rotated about the central axis, the plane of each of the first PCR cartridge (CT1) and the second PCR cartridge (CT2) is moved in a direction perpendicular to the ground while The liquid sample is moved away from the central axis by centrifugal force. The liquid sample moved by centrifugal force is evenly distributed in the reaction spaces of the first PCR cartridge (CT1) and the second PCR cartridge (CT2). According to the rotational speed of the rotating body 120, the movement angle of each cartridge may be changed, and the time to be completely distributed in the reaction space may be changed.

As described above, according to the present invention, the liquid sample can be evenly distributed in the reaction space by centrifugal force by rotating the PCR cartridge vertically so that the plane of the PCR cartridge into which the liquid sample is injected is vertical. That is, by configuring the variable-wing centrifugal injection device so that the liquid sample is distributed even in the corners or edge regions of the well array, which is the reaction space, by centrifugal force, it is possible to prevent air pockets from being generated in the reaction space. Accordingly, it is possible to prevent an error caused by an air pocket in the PCR test result.

Although the above has been described with reference to the embodiments, those skilled in the art can variously modify and change the present invention within the scope without departing from the spirit and scope of the present invention described in the claims below you will understand

The present invention has industrial applicability that can be used for research, medical, disaster prevention, livestock breeding, pet treatment, etc. as a device for testing biochemical substances, blood testing device, disease testing device, and the like.

110: storage container 112: storage body
114: storage cover 120: rotating body
122: central 124: first wing
126: second wing 130: first rotation-support part
140: second rotation-support 150: first guide member
160: second guide member CT1, CT2, 200: cartridge for PCR
210: PCR module 212: silicon chamber
214 well array 216 CMOS image sensor
216: PCB

Claims (4)

storage container;
a rotating body disposed inside the storage container;
a first rotation-supporting part connected to one end of the rotating body to be rotatably connected to an inclined grip for a first PCR cartridge and to support the first PCR cartridge at various inclination angles according to the rotation of the rotating body; and
A second rotation-supporting part configured to be rotatably connected to the other end of the rotating body, to grip the second PCR cartridge at an angle, and to support the second PCR cartridge at various inclination angles according to the rotation of the rotating body. but,
Each of the first and second rotation-supporting parts is variably maintained at 50 degrees to 90 degrees with respect to the ground according to the number of revolutions per minute of the rotating body to rotate the first and second PCR cartridges by centrifugal force to the liquid sample A variable-wing centrifugal injection device, characterized in that by evenly distributing in the reaction space to block the air pockets from being created in the reaction space. According to claim 1, wherein the rotating body,
center;
Extending obliquely upward from one side of the central portion, the first rotation - a first wing comprising one end rotatably connected to one side of the support portion, and the other end portion rotatably connected to the other side of the first rotation - support portion wealth; and
Extending obliquely upward from the other side of the central portion, the second rotation- a second wing comprising one end rotatably connected to one side of the support and the other end rotatably connected to the other side of the second rotation-support portion A variable-wing centrifugal injection device comprising a portion. The method of claim 1,
The first rotation-connected to the lower portion of the support portion and the inner wall of the receiving container, guides the first PCR cartridge to be disposed at a first inclination angle when the rotation body does not rotate, and when the rotation body rotates, the second 1 The first guide member for guiding the cartridge for PCR at an inclination angle greater than or equal to 90 degrees greater than the first inclination angle by centrifugal force; and
The second rotation-connected to the lower part of the support part and the inner wall of the receiving container, guides the second PCR cartridge to be disposed at a first inclination angle when the rotating body does not rotate, and when the rotating body rotates, the second 2 Variable-wing centrifugal injection device, characterized in that it further comprises a second guide member for guiding the cartridge for PCR at an inclination angle greater than the first inclination angle and less than or equal to 90 degrees by centrifugal force. 4. The variable-wing centrifugal injection device according to claim 3, wherein each of the first guide member and the second guide member includes a helical spring or a silicon piston bumper.

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