Cartridge for genetic analysis and genetic analysis device comprising same

Abstract

The present invention relates to a cartridge for genetic analysis and a genetic analysis device comprising same and, in particular, to an analytical tool which can be manufactured by a 3D printer and can accommodate a gene amplification reaction and a solution therefor, and a device capable of analyzing a gene on-site and transmitting the results of analysis to the outside.

Description

Genetic analysis cartridge and genetic analysis device including the same

[0001] The present invention relates to a cartridge for genetic analysis and a genetic analysis device including the same, and more particularly, to an analysis tool capable of being manufactured using a 3D printer and capable of accommodating a genetic amplification reaction and a solution therefor, and a device capable of analyzing genes on site and transmitting the analysis results to an external source.

[0002] The World Health Organization (WHO) has reported 762.79 million confirmed cases of COVID-19 and more than 6.9 million deaths since the outbreak began in December 2019.

[0003] Despite the remarkable speed of mRNA vaccine development, a recent surge in infections caused by omicron variants in vaccinated populations has raised concerns about the efficacy of existing vaccines.

[0004] The protective efficacy of currently available vaccines against infections and symptomatic diseases caused by omicron variants appears to be inferior to that of previous variants, and this efficacy declines rapidly after vaccination.

[0005] Therefore, rapid diagnostic testing is still considered essential to protect against COVID-19.

[0006] While it is understandable that the unprecedented demand for testing led to global shortages of medical supplies early in the pandemic, local lockdowns and social distancing policies in some countries have disrupted global manufacturing and trade, leading to ongoing shortages of medical equipment and analytical tools.

[0007] Additionally, the current standard for COVID-19 diagnostic systems approved by the U.S. Centers for Disease Control and Prevention (CDC) requires sophisticated real-time fluorescence thermocyclers and biological assays based on quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

[0008] qRT-PCR is widely used in molecular diagnosis due to its high sensitivity and excellent specificity, but it requires various expensive equipment and trained analysts, and the analysis time usually takes 4-6 hours, and it takes more than 24 hours to provide the analysis results to the patient.

[0009] The purpose of the present invention is to provide an analysis tool that can be manufactured using a 3D printer and can accommodate a gene amplification reaction and a solution therefor, and a device that can analyze genes on site and transmit the analysis results to an external source.

[0010] To achieve the above object, the present invention may include a chamber block coupled to a microfluidic chip and having a plurality of chambers formed therein; a syringe tube installed in the chamber block and communicating with the chamber; and a rotary valve communicating with the chamber or the microfluidic chip.

[0011] In order to achieve the above other objects, the present invention may include a cartridge coupled with a microfluidic chip; a fluid flow unit for flowing a fluid between the microfluidic chip and the cartridge; and a magnetic body rotating unit for rotating a magnetic body and placing it on the side of the cartridge.

[0012] In the present invention, the microfluidic chip and cartridge can be manufactured using a 3D printer.

[0013] In the present invention, the cartridge may include a chamber block coupled to a microfluidic chip and having a plurality of chambers formed therein; a syringe tube installed in the chamber block and communicating with the chamber; and a rotary valve communicating with the chamber or the microfluidic chip.

[0014] In the present invention, a cartridge has a slide groove formed along the height direction of the chamber block on the side of the chamber block so that the side end of the microfluidic chip can be coupled.

[0015] In the present invention, a cartridge has a valve coupling groove formed at the bottom of the chamber block along the height direction of the chamber block, and a plurality of cartridge paths are formed inside the chamber block so as to be in communication with the valve coupling groove.

[0016] In the present invention, the cartridge euros can be arranged on the inner surface of the valve coupling groove at an angle of 60 degrees along the circumference of the valve coupling groove based on the center of the valve coupling groove.

[0017] In the present invention, the cartridge has a valve path formed inside the rotary valve so that it can communicate with the cartridge path.

[0018] In the present invention, the valve path may include a chamber connection path formed at the upper portion of the rotary valve; and an air discharge path formed at the lower portion of the rotary valve.

[0019] In the present invention, the chamber connecting passage may include a combined passage formed to extend a certain length from the upper surface of the rotary valve along the longitudinal direction of the rotary valve; and a pair of branch passages formed to branch from the combined passage and extend to the outer surface of the rotary valve along the diametric direction of the rotary valve.

[0020] In the present invention, the angle between the branch channels can be formed at 150 degrees.

[0021] In the present invention, the fluid flow unit may include a valve rotation unit that is coupled to a rotary valve and rotates the rotary valve; and a plunger transfer unit that is installed with a plunger inserted into a syringe tube and linearly moves the plunger along the length of the syringe tube.

[0022] The present invention may further include a heater disposed opposite the microfluidic chip; and a cooling fan disposed opposite the heater.

[0023] The present invention may further include an image sensor that is arranged opposite to a microfluidic chip and captures images of the microfluidic chip at regular time intervals to generate image information; and a control unit that analyzes a fluorescent signal generated from the microfluidic chip based on the image information and transmits the result to the outside.

[0024] The genetic analysis device according to the present invention uses magnetic beads to extract RNA from a solution in which a virus sample is dissolved in a cartridge, and a microfluidic chip coupled to the cartridge can perform a genetic amplification reaction on the solution in which RNA has been extracted.

[0025] In addition, the genetic analysis device automatically measures and processes the fluorescence signals generated in the reaction chamber of the microfluidic chip, and based on IoT technology, transmits the analysis results to a smartphone in real time via a wireless network such as the Internet or Wi-Fi, enabling accurate and rapid diagnosis in the field at the same time.

[0026] Additionally, microfluidic chips and cartridges can be manufactured using a 3D printer, ensuring a stable supply.

[0027] Fig. 1 (a) is a perspective view showing a state in which a cartridge is separated from a genetic analysis device according to one embodiment of the present invention.

[0028] Fig. 1 (b) is a perspective view showing a state in which a cartridge is coupled to the genetic analysis device illustrated in Fig. 1 (a).

[0029] Figure 2 is an exploded perspective view of the genetic analysis device illustrated in (b) of Figure 1.

[0030] Figure 3 (a) is an exploded perspective view showing a product manufactured using an actual 3D printer using the microfluidic chip and cartridge illustrated in Figure 2.

[0031] Figure 3 (b) is a perspective view showing a product manufactured using an actual 3D printer using the microfluidic chip and cartridge illustrated in Figure 2.

[0032] Figure 4 is a one-sided perspective view of the internal configuration accommodated in the housing illustrated in Figure 2.

[0033] Figure 5 is a perspective view of the other side of the internal configuration accommodated in the housing illustrated in Figure 2.

[0034] Fig. 6 is a perspective view showing a state in which a cartridge is separated from the fluid flow section illustrated in Figs. 4 and 5.

[0035] Figure 7 is a conceptual diagram showing the connection relationship between the genetic analysis device illustrated in Figure 1 (b) and a smartphone.

[0036] Figure 8 is a plan view of the microfluidic chip illustrated in Figure 6.

[0037] Figure 9 is a perspective view of the cartridge illustrated in Figure 6.

[0038] Figure 10 is a BB section diagram shown in Figure 9.

[0039] Fig. 11 (a) is a plan view of the cartridge illustrated in Fig. 9.

[0040] Figure 11 (b) is a bottom view of the cartridge illustrated in Figure 9.

[0041] Figure 12 is an exploded perspective view of the cartridge illustrated in Figure 9.

[0042] Fig. 13 is a schematic diagram of the valve coupling groove shown in Fig. 11 (b).

[0043] Figure 14 is a section AA diagram shown in Figure 9.

[0044] Fig. 15 is a perspective view of the rotary valve illustrated in Fig. 12.

[0045] Fig. 16 is a plan view of the rotary valve illustrated in Fig. 12.

[0046] Figure 17 is a CC section diagram shown in Figure 9.

[0047] Figure 18 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the dissolution chamber to the mixing chamber.

[0048] Figure 18 (b) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the mixing chamber to the dissolution chamber.

[0049] Figure 19 is a side view of the cartridge illustrated in Figure 9, showing a state in which the magnetic body rotates and is placed or separated from the side of the cartridge.

[0050] Figure 20 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the first washing chamber to the mixing chamber.

[0051] Figure 20 (b) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the mixing chamber to the first washing chamber.

[0052] Figure 21 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the second washing chamber to the mixing chamber.

[0053] Figure 21 (b) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the mixing chamber to the second washing chamber.

[0054] Fig. 22 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which fluid flows from the dissolution chamber to the mixing chamber.

[0055] Figure 23 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the mixing chamber to the reagent chamber.

[0056] Figure 23 (b) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the reagent chamber to the mixing chamber.

[0057] Figure 24 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove illustrated in Figure 13, and shows a state in which a fluid flows from a mixing chamber to a microfluidic tip through a solution outlet channel.

[0058] Figure 25A is a diagram showing the extracted RNA concentration according to sample concentration using the genetic analysis device illustrated in Figure 1 (a).

[0059] Figure 25B is a diagram showing a fluorescence image generated during an amplification reaction in the microfluidic chip illustrated in Figure 1 (a).

[0060] FIG. 25C is a diagram showing the display shown in FIG. 7.

[0061] Figure 25D is a diagram comparing the critical time for each sample concentration using the genetic analysis device shown in Figure 1 (a) with the critical time for each sample concentration using a standard thermocycler.

[0062] Figure 26A is a diagram showing the results of 19 clinical sample tests using the genetic analysis device illustrated in Figure 1.

[0063] Figure 26B is a diagram showing a decision tree of the genetic analysis device illustrated in Figure 1.

[0064] Figure C of Figure 26 is a diagram showing the identification status of a sample based on the decision tree illustrated in Figure B of Figure 26.

[0065] Figure 26D is a conceptual diagram showing the state in which data derived from the genetic analysis device illustrated in Figure 1 is transmitted by mail and output to a smartphone.

[0066] In order to explain in detail the technical idea of ​​the present invention to a degree that a person having ordinary skill in the art to which the present invention pertains can easily practice the present invention, the most preferred embodiment of the present invention will be described with reference to the attached drawings.

[0067] First, when adding reference signs to the components of each drawing, it should be noted that identical components are given the same signs whenever possible, even if they are shown on different drawings.

[0068] In addition, when describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description is omitted.

[0069] Hereinafter, a genetic analysis device according to an embodiment of the present invention will be described with reference to FIGS. 1 to 26.

[0070] FIG. 1 (a) is a perspective view showing a state in which a cartridge is separated from a genetic analysis device according to an embodiment of the present invention, FIG. 1 (b) is a perspective view showing a state in which a cartridge is coupled to the genetic analysis device shown in FIG. 1 (a), and FIG. 2 is an exploded perspective view of the genetic analysis device shown in FIG. 1 (b).

[0071] Referring to FIG. 1 (a) to FIG. 2, a genetic analysis device (100) may include a housing (110), a cover (111), a closing plate (112), a battery (113), a microfluidic chip (200), a cartridge (300), a fluid flow portion (120), and a magnetic rotating portion (140).

[0072] The housing (110) is in the form of a polyhedron with an empty interior, such as a hexahedron or a cylinder. An open hole (110a) is formed on the upper surface of the housing (110), a fluid flow part (120) is arranged inside, and a cover (111) can be hingedly connected to the upper end of the housing (110).

[0073] The closing plate (112) can be placed in the opening (110a) to close the upper part of the housing (110), and can be fixed to the inner surface of the housing (110) using a known fixing member such as a bolt, screw, piece, clamp, or a known adhesive member such as an adhesive.

[0074] Hereinafter, the fixation between the components of the genetic analysis device (100) described later can also be achieved through a fixing member and an adhesive member, so a detailed description will be omitted.

[0075] The battery (113) is a lithium ion battery, is placed inside the housing (110), and can supply power to the components forming the genetic analysis device (100).

[0076] The closing plate (112) has a cartridge insertion hole (112a) formed therein, and a cartridge (300) can be inserted through the cartridge insertion hole (112a) to be combined with the fluid flow portion (120).

[0077] Fig. 3 (a) is an exploded perspective view showing a product manufactured using an actual 3D printer based on the microfluidic chip and cartridge shown in Fig. 2, and Fig. 3 (b) is a perspective view showing a product manufactured using an actual 3D printer based on the microfluidic chip and cartridge shown in Fig. 2.

[0078] Referring to (a) and (b) of FIG. 3, the microfluidic chip (200), the cartridge (300), and the rotary valve (330) included in the cartridge (300) can be manufactured using a 3D printer, thereby enabling stable supply.

[0079] The dimensions of the completed cartridge are 30X25X40mm in width, length, and height, and the width of the cartridge (300) including the microfluidic chip (200) when the microfluidic chip (200) is combined with the cartridge (300) is 45mm, which can be made into a compact size.

[0080] The genetic analysis device (100) can be manufactured using a known material having a certain rigidity, such as steel or synthetic resin, except for the microfluidic chip (200) and cartridge (300).

[0081] FIG. 4 is a one-sided perspective view of the internal configuration accommodated in the housing illustrated in FIG. 2, FIG. 5 is a other-sided perspective view of the internal configuration accommodated in the housing illustrated in FIG. 2, and FIG. 6 is a perspective view showing a state in which a cartridge is separated from the fluid flow portion illustrated in FIGS. 4 and 5.

[0082] Referring to FIGS. 4 to 6, the fluid flow unit (120) is a configuration for flowing fluid between the microfluidic chip (200) and the cartridge (300), and can be positioned and fixed inside the housing (110) via a holder (114).

[0083] The stand (114) is a frame in which a component disposed inside the housing (110), including the fluid flow portion (120), is mounted and fixed, and can be fixed to the inner surface of the housing (110).

[0084] The fluid flow unit (120) includes a valve rotation unit (121) and a plunger transfer unit (123). The valve rotation unit (121) is a motor, and the rotation shaft (121a) of the motor can be coupled to the rotary valve (330) of the cartridge (300).

[0085] That is, the motor rotates the rotary valve (330) in the forward or reverse direction so that multiple chambers formed inside the cartridge (300) can be selectively connected.

[0086] The plunger transfer unit (123) may include a plunger (123a), a plunger, a rack bar (123b), and a motor (123c).

[0087] The plunger (123a) is fixed to the end of the plunger, the plunger and the rack bar (123b) are arranged in parallel and fixed to each other, and the rotational axis of the motor (123c) can be engaged with the rack bar (123b).

[0088] That is, the rotational force of the motor (123c) is transformed into linear motion by the rack bar (123b), and in response to this, the plunger (123a) is raised and lowered so that the plunger (123a) can move linearly inside the cartridge (300).

[0089] The magnetic rotating part (140) includes a magnetic body (130), a rotating rod (142), and a motor (141). One end of the rotating rod (142) is fixed to the rotational axis of the motor (141), and the magnetic body (130) can be fixed to the other end of the rotating rod (142).

[0090] That is, the motor (141) rotates the rotary rod (142) so that the magnetic body (130) can be placed on or separated from the outer surface of the cartridge (300).

[0091] When the magnetic body (130) is placed on the outer surface of the cartridge (300), the magnetic force of the magnetic body (130) reacts with the magnetic beads contained in the solution, so that the magnetic beads can be attached to the inner surface of the cartridge (300).

[0092] The genetic analysis device (100) may further include a heater (151), a cooling fan (152), an image sensor (160), and a control unit (170).

[0093] The heater (151) is a plate-shaped resistance heater, which is arranged to face one side of the microfluidic chip (200), applies heat to the microfluidic chip (200), and can control the heat temperature applied to the microfluidic chip (200) by PID control during genetic amplification reaction analysis using Arduino Nano and a thermistor.

[0094] The cooling fan (152) is positioned opposite to the heater (151) to cool the heater (151) and the microfluidic chip (200).

[0095] The image sensor (160) is positioned facing the other side of the microfluidic chip (200), and a CMOS sensor combined with a 520 nm band pass filter and a 488 nm LED can be used to measure the fluorescence signal coming from the reaction chamber (220) of the cartridge (300).

[0096] An incident hole (115) is formed at the top of the stand (114), and a light beam of a certain range output from an LED can pass through the incident hole (115) and be irradiated onto the reaction chamber of the microfluidic chip (200), and the fluorescence intensity generated in the reaction chamber during a gene amplification reaction is strengthened by the light beam, so that genetic analysis can be performed more easily.

[0097] The image sensor (160) can generate image information by taking pictures of the reaction chamber of the microfluidic chip (200) at regular intervals.

[0098] The control unit (170) can be mounted on a printed circuit board and can diagnose pathogens by analyzing fluorescent signals generated from a microfluidic chip (200) based on image information.

[0099] Figure 7 is a conceptual diagram showing the connection relationship between the genetic analysis device illustrated in Figure 1 (b) and a smartphone.

[0100] Referring to FIG. 7, the genetic analysis device (100) further includes a display (180), and the display (180) is a 3-inch touch screen and is installed on the upper surface of the cover (111) so that image information or analysis values ​​of fluorescent signals can be displayed.

[0101] The control unit can control the operation of the components of the genetic analysis device (100), including the valve rotation unit, based on preset values ​​input to the display (180).

[0102] The control unit automatically measures and processes the fluorescence signal generated from the microfluidic chip (200), and in addition to the display (180), can transmit the analysis results in real time to the smartphone (1) of the patient or analysis personnel via a wireless network such as the Internet or Wi-Fi based on IoT technology, thereby enabling accurate diagnosis and rapid on-site diagnosis at the same time.

[0103] Figure 8 is a plan view of the microfluidic chip illustrated in Figure 6.

[0104] Referring to FIG. 8, the microfluidic chip (200) may include a fluid plate (210), a reaction chamber (220), a waste chamber (230), and a passive valve (240).

[0105] A solution inlet tube (211) and an air outlet tube (212) are formed protrudingly on one side of the fluid plate (210), and the solution inlet tube (211) and the air outlet tube (212) are combined with the cartridge so that the microfluidic chip (200) and the cartridge can be mutually communicated.

[0106] The reaction chamber (220) is composed of a plurality of chambers, spaced apart from each other, and formed on the other side of the fluid plate (210), and the inner surface can be coated with a primer capable of reacting with target RNA.

[0107] A solution inlet channel (213) is formed in the fluid plate (210) to allow the solution inlet pipe (211) and the reaction chamber (220) to communicate with each other, and an air outlet channel (214) is formed to allow the reaction chamber (220) and the air outlet pipe (212) to communicate with each other.

[0108] The solution inflow channel (213) may include a solution flow channel (213a) and a solution separation channel (213b).

[0109] One end of the solution flow channel (213a) is connected to the solution inlet pipe (211), one end of the solution collection channel (213b) is connected to the other end of the solution flow channel (213a), and the other end of the solution collection channel (213b) can be connected to each reaction chamber (220).

[0110] The air outlet channel (214) includes an air flow channel (214a) and an air collection channel (214b), and one end of the air flow channel (214a) is connected to the air outlet pipe (212), one end of the air collection channel (214b) is connected to the other end of the air flow channel (214a), and the other end can be connected to the reaction chamber (220).

[0111] The waste chamber (230) is formed in the air flow channel (214a) and can receive and accommodate excess solution that the reaction chamber (220) was unable to accommodate.

[0112]

[0113] In a microfluidic chip (200), a solution flowing from a cartridge (300) flows through a solution inlet tube (211) along a solution inlet channel (213) and is divided into a plurality of reaction chambers (220) and received, and a gene amplification reaction can occur by heat applied from a heater (151).

[0114] The passive valve (240) is a channel section coated with a hydrophobic coating, which means a section in which a hydrophilic solution does not flow when suction force is not applied by the plunger transfer section (123, shown in FIG. 6).

[0115] That is, by forming a passive valve (240) in the air extraction channel (214b), a gene amplification reaction can be performed while the solution is contained in the reaction chamber (220).

[0116] Fig. 9 is a perspective view of the cartridge illustrated in Fig. 6, Fig. 10 is a BB section view illustrated in Fig. 9, Fig. 11 (a) is a plan view of the cartridge illustrated in Fig. 9, and Fig. 11 (b) is a bottom view of the cartridge illustrated in Fig. 9.

[0117] Referring to (b) of FIGS. 9 to 11, the cartridge (300) may include a chamber block (310), a syringe tube (320), and a rotary valve (330).

[0118] The chamber block (310) is formed as a polyhedron including a hexahedron, and can be formed by partitioning a dissolution chamber (312), a mixing chamber (311), a first washing chamber (313), a second washing chamber (314), a dissolution chamber (315), and a reagent chamber (316).

[0119] An injection hole (310a) is formed for each chamber on the upper surface of the chamber block (310), through which a solution, magnetic beads (10), etc. can be injected.

[0120] A slide groove (310b) is formed along the height direction of the chamber block (310) on the side of the chamber block (310) so that the side end of the microfluidic chip (200) can be slidably coupled.

[0121] An air inlet (317) and a solution outlet (318) can be formed at the bottom of the slide home (310b).

[0122] The air outlet tube (212, shown in FIG. 8) of the microfluidic chip (200) is inserted into the air inlet port (317), and the solution inlet tube (211, shown in FIG. 8) of the microfluidic chip (200) is inserted into the solution outlet port (318), so that the cartridge (300) and the microfluidic chip (200) can be connected to each other.

[0123] Fig. 12 is an exploded perspective view of the cartridge illustrated in Fig. 9, and Fig. 13 is a schematic diagram of the valve coupling groove illustrated in (b) of Fig. 11.

[0124] Referring to FIGS. 12 and 13, a valve coupling groove (319) is formed along the height direction of the chamber block (310) at the bottom of the chamber block (310) so that a rotary valve (330) can be inserted.

[0125] The rotary valve (330) and the valve coupling groove (319) are formed to correspond to each other in a cylindrical shape, and an axial coupling groove (331) is formed on the lower surface of the rotary valve (330) so that the rotary shaft (121a, shown in FIG. 6) of the valve rotation part (121) can be inserted.

[0126] In order to enable the rotational force of the rotary shaft (121a, shown in FIG. 6) to be transmitted to the rotary valve (330), the shaft coupling groove (331) and the end of the rotary shaft (121a) can be formed to correspond to each other in the shape of a polyhedron, including a triangular prism.

[0127] A cartridge path (340) is formed inside the chamber block (310) so that the valve coupling groove (319), the dissolution chamber (312), the mixing chamber (311), the first washing chamber (313), the second washing chamber (314), the elution chamber (315), the reagent chamber (316), and the microfluidic chip can be mutually connected.

[0128] The cartridge euro (340) may include a mixing chamber euro (341), a dissolution chamber euro (342), a first washing chamber euro (343), a second washing chamber euro (344), a dissolution chamber euro (345), a reagent chamber euro (346), and a solution outflow euro (347).

[0129] The mixing chamber passage (341) can communicate the upper surface of the valve coupling groove (319) and the mixing chamber (311), and the dissolution chamber passage (342) can communicate the inner surface of the valve coupling groove (319) and the dissolution chamber (312).

[0130] The first washing chamber passage (343) can connect the inner surface of the valve coupling groove (319) and the first washing chamber (313), and the second washing chamber passage (344) can connect the inner surface of the valve coupling groove (319) and the second washing chamber (314).

[0131] The elution chamber euro (345) can connect the inner surface of the valve coupling groove (319) and the elution chamber (315), and the reagent chamber euro (346) can connect the inner surface of the valve coupling groove (319) and the reagent chamber (316).

[0132] The solution outlet (347) can communicate with the inner surface of the valve coupling groove (319) and the solution outlet (318, shown in (a) of FIG. 11).

[0133] That is, the mixing chamber flow path (341), the dissolution chamber flow path (342), the first washing chamber flow path (343), the second washing chamber flow path (344), the dissolution chamber flow path (345), and the reagent chamber flow path (346) can be arranged on the inner surface of the valve coupling groove (319) at an angle of 60 degrees (θ2) along the circumference of the valve coupling groove (319) based on the center of the valve coupling groove (319).

[0134] Figure 14 is a section AA diagram shown in Figure 9.

[0135] Referring to FIG. 14, the syringe tube (320) may be formed integrally with the chamber block (310) or may be separately fixed to the chamber block (310).

[0136] One end of the syringe tube (320) is formed with a through hole (321), and the syringe tube (320) and the mixing chamber (311) can be mutually connected through a flow path (322) formed between the syringe tube (320) and the mixing chamber (311).

[0137] And, the plunger (123a, shown in FIG. 6) can be inserted and placed inside the syringe tube (320) through the other end of the syringe tube (320).

[0138] That is, as the plunger (123a, shown in FIG. 6) moves linearly, positive or negative pressure is formed inside the mixing chamber (311), so that the fluid contained inside the mixing chamber (311) can flow out of the mixing chamber (311) or flow into the mixing chamber (311).

[0139] Fig. 15 is a perspective view of the rotary valve illustrated in Fig. 12, Fig. 16 is a plan view of the rotary valve illustrated in Fig. 12, and Fig. 17 is a CC section view illustrated in Fig. 9.

[0140] Referring to FIGS. 15 to 17, the rotary valve (330) may be connected to at least one of the dissolution chamber (312), the mixing chamber (311), the first washing chamber (313), the second washing chamber (314), the elution chamber (315), the amplification reagent chamber (316), and the microfluidic chip (200) illustrated in FIG. 13.

[0141] For this purpose, a valve passage (350) is formed inside the rotary valve (330) so as to be connected to the cartridge passage (340, as shown in FIG. 13).

[0142] The valve path (350) includes a chamber connection path (351) and an air discharge path (352). The chamber connection path (351) may be formed at the upper portion of the rotary valve (330), and the air discharge path (352) may be formed at the lower portion of the rotary valve (330).

[0143] The chamber connection path (351) is 3-way and may include a combined path (351a) and a branch path (351b).

[0144] The mixing channel (351a) is connected to the mixing chamber channel (341, shown in FIG. 13) and can be formed to extend a certain length from the upper surface of the rotary valve (330) along the longitudinal direction of the rotary valve (330).

[0145] The branch channel (351b) is formed as a pair, branches off from the end of the combined channel (351a) and extends along the diameter of the rotary valve (330) to the outer surface of the rotary valve (330), and can be connected to at least one of the dissolution chamber channel (342), the first washing chamber channel (343), the second washing chamber channel (344), the dissolution chamber channel (345), the reagent chamber channel (346), and the solution discharge channel (347) illustrated in FIG. 13.

[0146] The angle (θ1) between the branch flow path (351b) can be formed as 150 degrees, and as described above, the mixing chamber flow path (341), the dissolution chamber flow path (342), the first washing chamber flow path (343), the second washing chamber flow path (344), the dissolution chamber flow path (345), and the reagent chamber flow path (346) illustrated in FIG. 13 can be arranged on the inner surface of the valve coupling groove (319) at a distance of 60 degrees (θ2) from each other along the circumference of the valve coupling groove (319).

[0147] That is, when the mixing chamber flow path (341) and the combined flow path (351a) are connected, the rotary valve (330) rotates so that one side of the branch flow path (351b) can be connected to any one of the mixing chamber flow path (341), the dissolution chamber flow path (342), the first washing chamber flow path (343), the second washing chamber flow path (344), the dissolution chamber flow path (345), the reagent chamber flow path, and the solution discharge flow path (347).

[0148] In addition, the other side of the branch channel (351b) is sealed by being in contact with the inner surface of the valve coupling groove (319), so that airtightness can be maintained.

[0149] Since the branch passages (351b) are formed in a pair, the rotational amount of the rotary valve (330) is reduced compared to when there is only one branch passage (351b), thereby reducing fatigue of the valve rotation part (121).

[0150] The cartridge euro (340) further includes an air inlet passage (348), and the air inlet passage (348) can be arranged on the inner surface of the valve coupling groove (319) apart from the solution outlet passage (347) along the height direction of the chamber block (310).

[0151] The air exhaust path (352) is a 1-way path, and is formed between the outer surface of the rotary valve (330) and the lower surface of the rotary valve (330), i.e., the shaft coupling groove (331), and can be connected to the air inlet path (348).

[0152] An air discharge groove (332, shown in FIG. 15) may be formed between the outer surface of the rotary valve (330) and the inner surface of the shaft coupling groove (331).

[0153] That is, the internal air of the cartridge (300) flows along the air discharge path (352) and the air inlet path (348) and is discharged to the shaft coupling groove (331), and then can be discharged from the rotary valve (330) through the air discharge groove (332).

[0154] Hereinafter, the flow process of the solution contained in the cartridge will be described with reference to (a) of FIG. 18 to FIG. 24.

[0155] Figure 18 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the dissolution chamber to the mixing chamber.

[0156] And, (b) of Fig. 18 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which a fluid flows from a mixing chamber to a dissolution chamber.

[0157] And, Fig. 19 is a side view of the cartridge illustrated in Fig. 9, showing a state in which the magnetic body rotates and is placed or separated on the side of the cartridge.

[0158] Referring to (a) to (19) of FIG. 18, first, the volumes of the dissolution chamber (312), the first washing chamber (313), the second washing chamber (314), the elution chamber (315), and the reagent chamber (316) are 1200 μL, 1200 μL, 1200 μL, 600 μL, 600 μL, and 1000 μL, respectively, and a designated solution is injected into each chamber.

[0159] Then, magnetic beads are injected into the dissolution chamber (312) to prepare, and a sample is collected using a cotton swab to create a virus sample solution.

[0160] After injecting 150 μL of virus sample solution into the dissolution chamber (312) using a pipette, as shown in FIG. 2, a cartridge (300) coupled with a microfluidic chip (200) is inserted into the cartridge insertion hole (112a) and coupled to the fluid flow portion (120).

[0161] Afterwards, the rotary valve (330) rotates so that the branch flow path (351b) is connected to the dissolution chamber flow path (342), and the plunger (123a, shown in FIG. 6) descends so that the solution contained in the dissolution chamber (312) flows into the mixing chamber (311).

[0162] While the solution is contained in the dissolution chamber (312), the magnetic body (130) rotates and is placed on the outer surface of the mixing chamber (311), and as the solution flows from the dissolution chamber (312) to the mixing chamber (311) and is contained in the mixing chamber (311), the magnetic beads (10) can be adhered to the inner surface of the mixing chamber (311) as shown in FIG. 19.

[0163] At this time, after waiting for 30 seconds so that the magnetic beads (10) can adhere to the inner surface of the mixing chamber (311), the solution contained in the mixing chamber (311) flows back into the dissolution chamber (312).

[0164] Afterwards, as the plunger (123a, shown in FIG. 6) rises, the solution contained in the mixing chamber (311) flows back into the dissolution chamber (312), thereby improving the dissolution efficiency of the virus and the RNA capture effect on the magnetic beads (10).

[0165] Hereinafter, redundant descriptions of the rotation of the rotary valve (330), the elevation of the plunger, and the rotation of the magnetic body (130) will be omitted.

[0166] Figure 20 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the first washing chamber to the mixing chamber.

[0167] And, (b) of Fig. 20 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which fluid flows from the mixing chamber to the first washing chamber.

[0168] Referring to (a) and (b) of FIG. 20, thereafter, the branch flow path (351b) is connected to the first washing chamber flow path (343) and the magnetic body (130, shown in FIG. 19) is separated from the outer surface of the mixing chamber (311).

[0169] Then, the solution received in the first washing chamber (313) flows to the mixing chamber (311) and then flows back to the first washing chamber (313). By performing this flow back and forth three times, washing of the magnetic beads (10) can be performed.

[0170] Afterwards, the magnetic body (130) is placed on the outer surface of the mixing chamber (311), and the magnetic beads (10) are in close contact with the inner surface of the mixing chamber (311) and remain in the mixing chamber (311), and the solution contained in the mixing chamber (311) flows to the first washing chamber (313).

[0171] Figure 21 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the second washing chamber to the mixing chamber.

[0172] And, (b) of Fig. 21 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which fluid flows from the mixing chamber to the second washing chamber.

[0173] Referring to (a) and (b) of Fig. 21, the secondary washing of the magnetic beads (10) is the same as the primary washing described above.

[0174] That is, at the same time that the branch channel (351b) is connected to the second washing chamber channel (344), the magnetic body (130) is separated from the outer surface of the mixing chamber (311).

[0175] Then, the solution received in the second washing chamber (314) flows to the mixing chamber (311) and then flows back to the second washing chamber (314), and this flow is repeated three times.

[0176] Afterwards, a magnetic body (130, shown in FIG. 19) is placed on the outer surface of the mixing chamber (311), and the magnetic beads (10) are in close contact with the inner surface of the mixing chamber (311) and remain in the mixing chamber (311), and the solution contained in the mixing chamber (311) flows to the second washing chamber (314).

[0177] Fig. 22 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which fluid flows from the dissolution chamber to the mixing chamber.

[0178] Referring to FIG. 22, thereafter, the branch flow path (351b) is connected to the elution chamber flow path (345), and the solution contained in the elution chamber (315) flows to the mixing chamber (311).

[0179] The solution contained in the elution chamber (315) is nuclease-free water, and RNA captured by the magnetic beads (10) can be dispersed in the solution to form an RNA solution. To enhance this dispersion effect, the flow of the solution between the elution chamber (315) and the mixing chamber (311) is repeated several times.

[0180] After repeated flow, the solution is finally received in the mixing chamber (311), and the magnetic body (130) is placed on the outer surface of the mixing chamber (311) so that the magnetic beads (10) are in close contact with the inner surface of the mixing chamber (311).

[0181] Figure 23 (a) is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Figure 13, and shows a state in which fluid flows from the mixing chamber to the reagent chamber.

[0182] And, (b) of Fig. 23 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove shown in Fig. 13, and shows a state in which a fluid flows from a reagent chamber to a mixing chamber.

[0183] Referring to (a) and (b) of FIG. 23, thereafter, the branch flow path (351b) is connected to the reagent chamber flow path (346), and the solution contained in the mixing chamber (311) flows to the reagent chamber (316) and is mixed with the reagent solution already contained in the reagent chamber (316) to form an amplification reaction mixture.

[0184] Afterwards, the solution contained in the reagent chamber (316) flows into the mixing chamber (311).

[0185] Figure 24 is a schematic diagram showing a state in which a rotary valve is coupled to the valve coupling groove illustrated in Figure 13, and shows a state in which a fluid flows from a mixing chamber to a microfluidic tip through a solution outlet channel.

[0186] Referring to FIG. 24, the branch channel (351b) may be connected to the solution outlet channel (347), and as shown in FIG. 17, the air exhaust channel (352) may be connected to the air inlet channel (348).

[0187] That is, the solution contained in the mixing chamber (311) can flow along the solution outlet path (347) and be introduced into the solution inlet channel (213) through the solution inlet tube (211) of the microfluidic chip (200) as shown in FIG. 8.

[0188] And, air can flow from the air outlet channel (214) of the microfluidic chip (200) and flow along the air inlet channel (348) and the air outlet channel (352) as shown in FIG. 17 and be discharged to the outside of the cartridge (300).

[0189] The solution introduced into the microfluidic chip is received in the reaction chamber as described above, and a gene amplification reaction is performed. The fluorescent signal generated in the reaction chamber is measured to diagnose the pathogen.

[0190] Figure 25A is a diagram showing the extracted RNA concentration according to sample concentration using the genetic analysis device illustrated in Figure 1 (a).

[0191] Figure 25B is a diagram showing a fluorescence image generated during an amplification reaction in the microfluidic chip illustrated in Figure 1 (a).

[0192] FIG. 25C is a diagram showing the display shown in FIG. 7.

[0193] Figure 25D is a diagram comparing the critical time for each sample concentration using the genetic analysis device shown in Figure 1 (a) with the critical time for each sample concentration using a standard thermocycler.

[0194] Referring to FIGS. 25A to 25D, the diagnostic performance of the genetic analysis device of the present invention was evaluated through sensitivity tests, specificity tests, clinical sample tests, and comparison tests with a standard thermocycler.

[0195] The limit of detection (LOD) is an important indicator in genetic diagnosis, and to determine the LOD of the genetic analysis device of the present invention, an LOD test was performed using a heat-inactivated SARS-CoV-2 sample purchased from ATCC.

[0196] Dilute the stock sample to 1Х10 -2 1Х10 in 3 A series of concentrations ranging from genome copies / μL were prepared, and 150 μL of sample was used for each test.

[0197] In genetic diagnostics, the sample preparation step is crucial for successful and sensitive detection of target pathogens. To confirm RNA extraction by sample preparation, an aliquot of extracted RNA was collected before transfer to the microfluidic chip and its concentration was measured using a Nanodrop One spectrophotometer.

[0198] The extraction results in the cartridge are 1Х10 -1 We demonstrated that purified RNA could be effectively recovered from viral samples at a concentration of 10 copies / μL, and the concentration of extracted RNA was measured to be 0.52±0.12 ng / μL (Figure 25A).

[0199] After successfully recovering purified RNA from the virus sample, four amplification reactions were performed using the reaction chamber of the microfluidic chip.

[0200] This facilitated the amplification and detection of target genes of viral RNA, further confirming the presence of viruses in the samples, and demonstrated real-time fluorescence monitoring of gene amplification in a microfluidic chip. 2 Amplification assays were performed using input sample concentrations of genome copies / μL, and fluorescence intensity was monitored throughout the process.

[0201] Fluorescence images of the reaction chamber captured by the image sensor during the amplification reaction are shown in Fig. 25B, and the results show that the fluorescence intensity of the reaction chamber containing the primer sets targeting 18S rRNA, As1e and N genes increased during the reaction process, whereas the negative control did not change.

[0202] As part of data processing, fluorescence images of four reaction chambers were captured every minute, fluorescence intensities were calculated, and displayed on a display. As shown in Fig. 25C, the analysis results of the gene amplification curve after the test was completed were displayed as a screenshot, and the 5-parameter log-logistic (5PLL) fitting fluorescence intensities of each reaction chamber were presented to show the progress of the amplification.

[0203] The bottom of the display shows the calculated critical time of the amplification curve and the final diagnostic results, providing a comprehensive analysis of the amplification response.

[0204] In a comparative test with a standard thermocycler, serially diluted samples of inactivated SARS-CoV-2 were utilized in both the genetic analysis device of the present invention and the standard thermocycler.

[0205] Testing of the standard thermocycler was performed as follows:

[0206] First, RNA was extracted from the sample using a cartridge-based method, and after the elution step, the extracted RNA was pipetted for use in the amplification reaction in a thermocycler.

[0207] Each reaction consisted of 5 μL of amplification reaction mixture (RM), 0.4 μL of enzyme mixture (EM), 0.4 μL of SYBR green, 2.4 μL of primer mixture, 0.8 μL of nuclease-free water, and 1 μL of RNA sample.

[0208] The amplification reaction was carried out at 65 degrees for 1 hour, and the fluorescence intensity of the amplification mixture was measured at 1-minute intervals.

[0209] In contrast, testing of the genetic analysis device of the present invention was performed on cartridges and microfluidic chips, from sample preparation to amplification analysis.

[0210] This allowed us to compare the accuracy, sensitivity, and specificity of the genetic analysis device with a standard thermocycler.

[0211] Fully integrating all steps into a single cartridge can significantly simplify the testing process, increasing the speed and user-friendliness of analysis and reducing the risk of contamination and human error by requiring fewer steps of manual pipetting and sample transfer.

[0212] Additionally, the cartridge's compact size and IoT-based genetic analysis device make it easier to use in various genetic analysis devices and field testing applications.

[0213] Figure 25D shows that the genetic analysis device of the present invention detects As1e and N genes of SARS-CoV-2 with LOD of 1Х10 -1 We demonstrate high sensitivity for COVID-19 identification by detecting genome copies / μL, which corresponds to 15 genome copies per sample (150 μL volume).

[0214] In addition, a comparison of the critical times between a standard thermocycler, BioRad CFX Connect, and the genetic analysis device of the present invention revealed that the molecular diagnostic performance of the genetic analysis device of the present invention was similar to that of the standard thermocycler, proving that it is a reliable and accurate diagnostic tool for detecting SARS-CoV-2.

[0215] FIG. 26A is a diagram showing the results of 19 clinical sample tests using the genetic analysis device shown in FIG. 1, FIG. 26B is a diagram showing a decision tree of the genetic analysis device shown in FIG. 1, FIG. 26C is a diagram showing the identification status of a sample based on the decision tree shown in FIG. 26B, and FIG. 26D is a conceptual diagram showing the state of data derived from the genetic analysis device shown in FIG. 1 being sent by e-mail and output on a smartphone.

[0216] Referring to FIGS. 26A to 26D, clinical samples were also tested to demonstrate the practical application of the genetic analysis device of the present invention.

[0217] Virus samples were obtained from five patients infected with COVID-19, 14 patients infected with other respiratory viruses, three positive control samples (commercial virus samples from ATCC), and five negative control samples.

[0218] As shown in Figure 26A, real-time fluorescence detection was performed in the reaction chamber, and the threshold time of the target gene in each sample was determined based on the threshold of fluorescence, and three genes (As1e and N genes of SARS-CoV-2 and human 18S rRNA gene) were successfully detected in both SARS-CoV-2 patient samples and positive control samples.

[0219] Samples from patients infected with other respiratory viruses and negative controls amplified only the 18S rRNA gene, whereas other negative controls containing only water showed no signs of amplification.

[0220] For SARS-CoV-2 samples, the critical time is shortest for the 18S rRNA gene and longest for the As1e gene due to its abundance in human cells.

[0221] The total diagnostic time was 70 minutes, including 10 minutes of sample preparation and 60 minutes of amplification reaction with real-time analysis of fluorescence signals.

[0222] As illustrated in B of FIG. 26, after obtaining diagnostic data from the genetic analysis device of the present invention, a final decision can be made using a decision tree based on possible situations.

[0223] Initially, the threshold of the positive control chamber containing 18S rRNA as a template was examined, a negative result indicates that the sample does not contain housekeeping RNA, suggesting that the sample was collected improperly, whereas a positive result indicates the threshold of the negative control chamber.

[0224] The negative control chamber is designed to identify cross-contamination issues or primer-dimer occurrence between reaction chambers, indicating a false positive result if the negative control gives a positive result.

[0225] If the negative control correctly shows a negative result, the presence of SARS-CoV-2 genes (As1e and N genes) is tested, and if both show a positive result, the sample is positive for COVID-19.

[0226] Conversely, a negative result from both tests suggests that the patient's sample is not infected with COVID-19.

[0227] The proposed genetic analysis system of the present invention, based on this decision-making process, was able to automatically and accurately identify contaminated samples regardless of the presence or absence of SARS-CoV-2, taking into account all issues related to proper sample collection or chip operation (Figure 26, C).

[0228] In addition, as described above, the genetic analysis device of the present invention is integrated with an IoT function that enables communication with a computer or smartphone via the Internet (see diagram D in FIG. 26).

[0229] That is, once the genetic diagnosis is complete, the genetic analysis device can compile the raw data, analyze the results, record other metadata such as time, location, and notes, and send them to a designated email address.

[0230] These measures will enable scientists and technicians at the Centers for Disease Control and Prevention to easily collect and audit data, which will be invaluable in real-time, continuous tracking of the early stages of outbreaks like COVID-19, enabling proactive containment.

[0231] As described above, the best embodiments have been disclosed in the drawings and specifications. While specific terminology has been used herein, it is solely for the purpose of describing the present invention and is not intended to limit the meaning or scope of the invention as defined in the claims. Therefore, those skilled in the art will understand that various modifications and equivalent embodiments are possible. Accordingly, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A chamber block combined with a microfluidic chip and having a plurality of chambers formed; A syringe tube installed in the chamber block and communicating with the chamber; and A rotary valve communicating with the chamber or the microfluidic chip; A cartridge for genetic analysis containing .

2. Cartridge combined with microfluidic chip; A fluid flow unit for flowing fluid between the microfluidic chip and the cartridge; and A magnetic rotating part that rotates a magnetic body and places it on the side of the cartridge; A genetic analysis device including:

3. In paragraph 2, The above microfluidic chip and cartridge are a genetic analysis device manufactured using a 3D printer.

4. In paragraph 2, The above cartridge, A chamber block combined with a microfluidic chip and having a plurality of chambers formed; A syringe tube installed in the chamber block and communicating with the chamber; and A rotary valve communicating with the chamber or the microfluidic chip; A genetic analysis device including:

5. In paragraph 4, The above cartridge, A genetic analysis device in which a slide groove is formed along the height direction of the chamber block on the side of the chamber block and the side end of the microfluidic chip is coupled thereto.

6. In paragraph 4, The above cartridge, A valve coupling groove is formed at the bottom of the chamber block along the height direction of the chamber block, A genetic analysis device in which a plurality of cartridge channels are formed inside the chamber block and communicate with the valve coupling groove.

7. In paragraph 6, The above cartridge euro is, A genetic analysis device arranged on the inner surface of the valve coupling groove at a distance of 60 degrees from each other along the circumference of the valve coupling groove based on the center of the valve coupling groove.

8. In paragraph 6, The above cartridge, A genetic analysis device in which a valve path is formed inside the rotary valve and is connected to the cartridge path.

9. In paragraph 8, The above valve oil is, A chamber connecting passage formed at the upper portion of the rotary valve; and An air exhaust path formed at the bottom of the above rotary valve; A genetic analysis device including:

10. In paragraph 9, The above chamber connection path is: A joint flow path formed to extend a certain length from the upper surface of the rotary valve along the longitudinal direction of the rotary valve; and A pair of branch channels branched from the above-mentioned joint channel and formed to extend along the diameter of the rotary valve to the outer surface of the rotary valve; A genetic analysis device including:

11. In paragraph 10, A genetic analysis device in which the angle between the above branch channels is formed at 150 degrees.

12. In paragraph 4, The above fluid flow section is, A valve rotation part that is combined with the above rotary valve and rotates the above rotary valve; and A plunger transport unit having a plunger inserted into the syringe tube and linearly moving the plunger along the length of the syringe tube; A genetic analysis device including:

13. In paragraph 2, A heater disposed opposite to the microfluidic chip; and A cooling fan positioned opposite the above heater; A genetic analysis device further comprising:

14. In paragraph 2, An image sensor that is positioned opposite to the microfluidic chip and captures images of the microfluidic chip at regular intervals to generate image information; and A control unit that analyzes a fluorescent signal generated from the microfluidic chip based on the image information and transmits it to the outside; A genetic analysis device further comprising:

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