KR20150145162A - Micro-chip for diagnosis and integrated rotary diagnosis method using the same - Google Patents

Abstract

The present invention relates to a diagnosis technology through gene analysis. According to the present invention, provided is a microchip for diagnosis, which is characterized by comprising a unit process part separated from the center of rotation, wherein the unit process part comprises a detection part including ICS detecting pathogens; and a sample treatment part including a pretreatment part performing pretreatment on a sample and a diagnosis target material delivering part delivering a genetic material of a diagnosis target containing a genetic material amplified after the pretreatment to the detection part.

Description

TECHNICAL FIELD [0001] The present invention relates to a diagnostic microchip and an integrated rotary diagnostic method using the microchip. [0002] MICRO-CHIP FOR DIAGNOSIS AND INTEGRATED ROTARY DIAGNOSIS METHOD USING THE SAME [0003]

More particularly, the present invention relates to a diagnostic microchip for detecting pathogens through gene analysis and an integrated rotary diagnostic method using the same.

Generally, a diagnostic method through gene analysis involves a step of taking a patient sample, an RNA extraction step, a gene amplification step, an electrophoresis separation step, and a gene detection and discrimination step. However, conventionally, since each step is performed by a separate device and apparatus, an expensive analyzing apparatus and a large amount of sample are required, and it takes a lot of time for analysis, the sample is likely to be contaminated in the middle, There is a problem that diagnosis is difficult. In order to solve these problems, an integrated gene analyzing apparatus using a microchip based microfluidic has recently been developed. However, the conventional integrated gene analyzing apparatus is also required to have a complicated chip structure, a high production cost due to metal electrode patterning and a silicon / glass substrate infrastructure, operation complexity due to an external influent pump and a plurality of tube systems, There are problems such as low reproducibility of the apparatus, lack of automation of the system driving, and difficulty of on-site diagnosis due to limitations of miniaturization, so that improvement is required.

It is an object of the present invention to provide a diagnostic microchip in which pretreatment of a sample and pathogen detection are performed, and an integrated rotary diagnostic method for performing gene amplification and pathogen detection using the microchip.

It is another object of the present invention to provide a diagnostic microchip in which pretreatment of a sample is performed, and an integrated rotary diagnostic method for performing gene amplification and pathogen detection using the microchip.

According to an aspect of the present invention,

And a pretreatment unit for pretreatment the sample, the pretreatment unit including a trapping passage having an inlet and an outlet, and a trapping means filled in the trapping passage; A sample storage portion located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a sample is stored; A washing buffer liquid chamber located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a washing buffer solution is stored; And an elution buffer solution chamber located radially inward of the target material capturing portion and connected to an inlet of the trapping passage and providing a space in which an elution buffer solution is stored; A reaction solution chamber for providing a space in which a reaction solution necessary for the PCR process or the isothermal amplification process is stored; A discharge passage located radially outward of the target substance capturing portion and the reaction solution chamber and extending along the circumferential direction and connected to the outlet of the trapping passage and the reaction solution chamber at both ends; A waste liquid chamber located radially outwardly of the discharge passage and connected to the discharge passage; And a target material chamber disposed radially outwardly of the discharge passage and connected to the discharge passage, wherein a portion of the discharge passage, to which the reaction solution chamber is connected, and a portion to which the target material chamber is connected, Wherein a portion of the discharge passage, to which the outlet of the trapping passage is connected, and a portion to which the waste liquid chamber is connected, are opposed to each other, and a diagnostic method using the same.

According to the present invention, all of the objects of the present invention described above can be achieved. Specifically, a pre-treatment and a gene amplification process for a sample are performed in the unit cell provided in the diagnostic microchip by rotating the diagnostic microchip according to the present invention, and the genetic material amplified through the gene amplification process is provided in the unit cell Lt; RTI ID = 0.0 > ICS < / RTI >

1 is a perspective view of a diagnostic microchip according to a first embodiment of the present invention.
2 is a plan view of the diagnostic microchip shown in Fig.
3 is a plan view of the unit process unit shown in FIG.
4 is a perspective view showing the ICS shown in FIG.
FIG. 5 is a flowchart showing an integrated rotary diagnostic method according to the first embodiment of the present invention using the diagnostic microchip of FIG. 1;
FIG. 6 is a perspective view showing a diagnostic apparatus for performing the diagnostic method of FIG. 5; FIG.
FIG. 7 is a plan view schematically showing the configuration of the temperature control unit shown in FIG. 6;
FIG. 8 is a view illustrating a state in which a sample, a washing buffer solution, an eluting buffer solution, a reaction solution, and a running buffer solution are injected into a unit cavity through the diagnostic microchip preparation step shown in FIG.
FIG. 9 is a flowchart showing a detailed process of the preprocessing step shown in FIG.
FIGS. 10A to 10F are diagrams illustrating a process in which the preprocessing step shown in FIG. 9 is performed in a unit bank.
11 is a view showing a state in which the detection preparation step shown in FIG.
FIG. 12 is a view showing an example of the state of ICS in the unit bank after the detection step shown in FIG. 5 is performed.
13 is a perspective view showing a diagnostic microchip according to a second embodiment of the present invention.
14 is a plan view showing the unit processing unit shown in Fig.
15 is a flowchart showing an integrated rotary diagnostic method according to a second embodiment of the present invention using the diagnostic microchip shown in FIG.
FIG. 16 is a view showing a state in which a sample, a washing buffer solution, an eluting buffer solution and a running buffer solution are injected into the unit cavity through the diagnostic microchip preparation step shown in FIG.
FIG. 17 is a flowchart showing a detailed process of the preprocessing step shown in FIG.
FIGS. 18A to 18E are diagrams illustrating a process in which the preprocessing step shown in FIG. 17 is performed in a unit bank.
FIG. 19 is a diagram showing a state in which the detection preparation step shown in FIG. 15 is performed in the unit bank.
20 is a plan view of a diagnostic microchip according to the third embodiment of the present invention.
FIG. 21 is a plan view of the unit processing portion shown in FIG. 20; FIG.
FIG. 22 is a flowchart showing an integrated rotary body diagnostic method according to the third embodiment of the present invention using the diagnostic microchip of FIG. 20;
23 is a perspective view showing a diagnostic apparatus for performing the diagnostic method of Fig.
FIG. 24 is a plan view schematically showing the configuration of the temperature control unit shown in FIG. 23. FIG.
FIG. 25 is a view showing a state in which a sample, a washing buffer solution, an eluting buffer solution, and a reaction solution are injected into the unit cavity through the diagnostic microchip preparation step shown in FIG.
26 is a flowchart showing a detailed process of the preprocessing step shown in FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 and 2 show a diagnostic microchip according to a first embodiment of the present invention as a perspective view and a plan view. Referring to FIGS. 1 and 2, the diagnostic microchip 110 is generally disk-shaped, and the center of rotation O is provided at its center. The diagnostic microchip 110 includes a plurality of unit cells 120 which are located radially away from the rotation center O and are arranged in order along the circumferential direction. In the present embodiment, six unit copiers 120 are described. However, the present invention does not limit the number of unit copiers 120 to six. The present invention also includes the case where the diagnostic microchip 110 has five or less unit chambers 120 or seven or more unit chambers 120. Although the diagnosis microchip 110 is described as being in a disc shape in the present embodiment, the present invention does not limit the diagnosis microchip 110 to a disc shape. For example, the diagnostic microchip 110 may be formed by forming a groove pattern on a surface of a disk-shaped polycarbonate (PC) having a thickness of about 1 mm using a CNC milling machine, And ICS (Immunochromatographic Strip) (hereinafter referred to as ICS).

In FIG. 3, the unit unit 120 shown in FIGS. 1 and 2 is shown as a plan view together with the center of rotation O. FIG. 2 and 3, the unit control unit 120 includes a detection unit 120b, a sample processing unit 120a, and a running buffer supply unit 120c. The sample processing unit 120a and the running buffer liquid supply unit 120c are located on both sides in the circumferential direction of the rotation center O with the detection unit 120b interposed therebetween.

The detection unit 120b includes an ICS 180 that is housed in an ICS receiving groove 189 formed in the diagnostic microchip 110 and detects pathogens from a diagnostic target material.

3, the ICS 180 generally has a thin rod shape, extends generally along the radial direction with respect to the rotation center O, and has a sample processing unit 120a and a running buffer solution supply unit 120c along the circumferential direction, Respectively. ICS detects pathogens through immunochromatographic analysis. 3 and 4, the ICS 180 includes a support 181, a buffer solution loading pad 182, an absorption pad 183, a detection pad 184, a conjugate pad 182, 185).

The buffer 181 is attached to the upper surface of the support 181 by a buffer liquid loading pad 182, an absorbing pad 183 and a detecting pad 184 in the form of a bar elongated along the radial direction.

The buffer solution loading pad 182 is located at the radially outer end of both ends in the longitudinal direction of the support 181. The buffer solution loading pad 182 is adhered to the upper surface of the support 181. The buffer solution loading pad 182 absorbs the running buffer solution supplied from the running buffer solution supply part 120c. The running buffer solution absorbed into the buffer solution loading pad 182 flows uniformly in the radially inward direction.

The absorbing pad 183 is located apart from the buffer solution loading pad 182 at the radially inner end of both longitudinal ends of the support 181. The absorption pad 183 is bonded to the upper surface of the support 181. The absorption pad 183 absorbs the liquid so that the liquid flows smoothly from the buffer solution loading pad 182 toward the absorption pad 183.

The detection pad 184 extends between the buffer liquid loading pad 182 and the absorption pad 183. The detection pad 184 is bonded to the upper surface of the support 181 and both ends of the detection pad 184 are in contact with the buffer solution loading pad 182 and the absorption pad 183, respectively. Although not shown, the detection pad 184 is provided with a plurality of test lines and a control line. The detection is confirmed according to the color development state of the test line, and the passage of the sample is confirmed by color development of the control line.

The conjugate pad 185 is placed in contact with the buffer solution loading pad 182 at both ends in the longitudinal direction of the detection pad 184. The conjugate pad 185 is adhered to the upper surface of the detection pad 184 and the diagnostic substance is supplied to the conjugate pad 185 from the sample treatment section 120a. The conjugate pad 185 includes a flowable conjugate that specifically binds to the substance to be detected contained in the gene material to be diagnosed.

The sample processing unit 120a is located on one side of the ICS 180 in the circumferential direction. The sample processing unit 120a includes a preprocessing unit 120d for performing preprocessing on a sample and a diagnostic substance delivery unit 174 for delivering the genetic material amplified by the PCR process after the preprocessing to the detection unit 120b.

The pretreatment unit 120d includes a target substance capturing unit 125, a sample storage unit 130, a washing buffer chamber 140, a washing buffer solution introducing passage 145, elution buffer chamber 150, an eluting buffer solution flow control passage 156, a reaction solution chamber 150a, a reaction solution introduction passage 155a, a discharge passage 160, a waste solution chamber 165, And a target material chamber 170.

The target material capturing portion 125 has a trapping passage 126 in a zigzag shape and trapping means 128 such as a silica bead filled in the trapping passage 126. In the target substance capturing part 125, the substance containing the target substance in the sample introduced into the trapping passage 126 is captured by the capturing means 128. An inlet 126a and an outlet 126b are located at both ends of the trapping passage 126. [ The inlet 126a is positioned radially inward with respect to the center of rotation O and the outlet 126b is located radially outward. In the capturing means 128, a substance containing the target substance is adsorbed from the sample. In this embodiment, the capturing means 128 is described as being a silica bead. Although not shown in detail, a weir structure is formed at the downstream end of the catch passage 126, so that the catch means 128 is retained within the catch passage 126. [

The sample storage part 130 is in the form of a chamber and is located radially inward of the inlet 126a of the catching passage 126 and is connected to the inlet 126a of the catching passage 126. [ The sample storage unit 130 stores a sample. The extension passage 130a is connected to the inner end of the sample storage part 130 in the radial direction. The extension passage 130a extends generally along the radial direction and its radially outer end is connected to the sample storage part 130. [ A valve portion 130b formed of a capillary valve is provided at a position adjacent to the sample storage portion 130 in the extension passage 130a.

The cleaning buffer solution chamber 140 is in the form of a chamber and is positioned closer to the rotation center O than the sample storage part 130. The cleaning buffer solution chamber 140 is located on one side of the ICS 180 side of the extension passage 130a. The wash buffer solution chamber 140 stores the wash buffer solution. The wash buffer solution rinses and removes the remaining components from the trapped means 128, except for the target material, from the trapped means 128. The diagnostic microchip 110 is provided with a wash buffer solution injection hole for injecting the wash buffer solution into the wash buffer solution chamber 140. A valve 140a surrounding the cleaning buffer solution chamber 140 is provided around the cleaning buffer solution chamber 140. The valve portion 140a is a passive valve that uses a height difference to prevent the washing buffer solution stored in the washing buffer solution chamber 140 from easily passing. The washing buffer solution stored in the washing buffer solution chamber 140 flows through the valve portion 140a after the diagnostic microchip 110 rotates about the rotation center O and then flows into the washing buffer solution introducing passage 145).

The wash buffer solution introduction passageway 145 extends generally straight to connect the radially outer end of the wash buffer solution chamber 140 to the extension passageway 130a. The portion connected to the cleaning buffer solution chamber 140 in the cleaning buffer solution introducing passage 145 is located radially inward of the portion connected to the extending passage 130a. Further, a portion of the cleaning buffer solution introducing passage 145 connected to the extension passage 130a is located radially inward of the valve portion 130b.

The elution buffer solution chamber 150 is in the form of a chamber and is located closer to the rotation center O than the sample storage part 130. In addition, the eluting buffer solution chamber 150 is located across the cleaning buffer solution chamber 140 with the extension passage 130a therebetween. In the elution buffer solution chamber 150, an elution buffer solution is stored. The elution buffer solution separates the target material adsorbed by the trapping means 128 from the trapping means 128. In the diagnostic microchip 110, an elution buffer solution injection hole for injecting an elution buffer solution into the elution buffer solution chamber 150 is formed. A valve unit 151 surrounding the eluting buffer solution chamber 150 is provided around the elution buffer solution chamber 150. The valve portion 151 is a passive valve using a difference in height, so that the elution buffer solution stored in the elution buffer solution chamber 150 can not be easily passed. The elution buffer solution stored in the elution buffer solution chamber 150 flows over the valve portion 151 by the centrifugal force generated when the diagnostic microchip 110 rotates around the rotation center O, (156).

The elution buffer solution flow control passage 156 introduces the elution buffer solution stored in the elution buffer solution chamber 150 into the trapping passage 126 at an appropriate time. The elution buffer solution flow control passage 156 extends from the elution buffer solution chamber 150 and is connected to the extension passage 130b. The elution buffer solution flow control passage 156 extends substantially radially inwardly from the eluting buffer solution chamber 150 and then extends in a radially outward direction with a smooth curve. Accordingly, the eluting buffer solution flow control passage 156 has a switching curve portion 158 that converts the flow direction of the eluting buffer solution from inside to outside in the radial direction. The portion where the elution buffer solution flow control passage 156 is connected to the extension passage 130b is radially inward of the portion where the cleaning buffer solution introduction passage 145 is connected to the extension passage 130a.

The reaction solution chamber 150a is positioned between the sample storage part 130 and the ICS 180 in the circumferential direction. In the reaction solution chamber 150a, a reaction solution containing an enzyme, a primer, and other buffer solutions necessary for the PCR process or the isothermal amplification process is stored. The reaction solution enhances gene amplification efficiency. In the diagnostic microchip 110, a reaction liquid injection hole for injecting the reaction liquid into the reaction liquid chamber 150a is formed. A valve portion 151a surrounding the reaction liquid chamber 150a is provided around the reaction liquid chamber 150a. The valve portion 151a is a passive valve using a difference in height, so that the reacted liquid stored in the reaction liquid chamber 150a can not be easily passed. The reaction liquid stored in the reaction liquid chamber 150a flows over the valve portion 151a by the centrifugal force generated when the diagnostic microchip 110 rotates around the rotation center O and then flows into the reaction liquid introduction passage 155a ≪ / RTI >

The reaction liquid introduction passages 155a are provided with reaction liquid flow control passages 156a and connection passages 159a mutually connected to each other. The reaction liquid stored in the reaction liquid chamber 150a flows into the discharge passage 160 extending from the outlet 126b of the trapping passage 126 through the reaction liquid introduction passage 155a.

The reaction liquid flow control passage 156a extends from the reaction liquid chamber 150a and is connected to the connection passage 159a. The reaction liquid flow control passage 156a extends substantially radially inwardly from the reaction liquid chamber 150a and then smoothly curves and extends in a radially outward direction. Accordingly, the reaction liquid flow control passage 156a has the switching curve portion 158a for switching the flow of the reaction liquid from the radially inner side to the outer side.

The connection passage 159a extends radially outward from the downstream end of the reaction liquid flow control passage 156a and is connected to the discharge passage 160. [ A valve portion 159b formed by a height difference is formed on the connection passage 159a. The reaction liquid flowing through the connection passage 159a passes through the valve portion 159b when the rotational speed of the diagnostic microchip 110 increases.

The discharge passage 160 is located radially outward of the target substance catching portion 125 and the connecting passage 159a and extends substantially in the circumferential direction with respect to the rotation center O. [ A portion P1 connected to the connection passage 159a is positioned radially inside the end of the exhaust passage 160 near the ICS 180 in the circumferential direction, (P2) that is connected to the trapping passage (126) is located radially inward of the distal end (180).

The waste liquid chamber 165 is located radially outward of the discharge passage 160. The waste liquid chamber 165 is connected to the end of the connection passage 166 extending radially outward from the discharge passage 160. The portion P3 to which the connection passage 166 and the discharge passage 160 are connected is positioned to face the portion P2 that is connected to the trap passage 126 in the discharge passage 160. [ A plurality of (three in this embodiment) capillary valves 166a formed by the height difference are provided on the connection passage 166. The capillary valve 166a prevents the solution stored in the waste liquid chamber 165 from escaping to the outside. In the waste liquid chamber 165, unnecessary components other than the target substance are stored.

The target material chamber 170 is located radially outwardly of the discharge passage 160. The portion P4 where the target material chamber 170 and the discharge passage 160 are connected is located closer to the ICS 180 than the portion P3 where the connection passage 166 is connected to the discharge passage 160. [ In the target material chamber 170, the target material is stored. The target material stored in the target material chamber 170 is amplified by a PCR process or an isothermal amplification process. Hereinafter, a target substance amplified through an amplification process such as a PCR process or an isothermal amplification process is referred to as a 'gene substance to be diagnosed'.

The diagnosis target substance transfer unit 174 transfers the diagnosis target gene material contained in the target material chamber 170 to the detection unit 120b. The diagnosis target substance delivery portion 174 has a diagnosis target substance flow control passage 175. The diagnostic target material flow control passage 175 extends generally radially inwardly from the target material chamber 170 and then smoothly curves and is extended in a radially outward direction with the end turned to a conjugate pad (not shown) of the ICS 180 185). Accordingly, the diagnosis target substance flow control passage 175 is provided with the switching curve portion 175a for switching the flow of the diagnostic substance from the radially inner side to the outer side.

The running buffer solution supply unit 120c is located on the opposite side of the sample processing unit 220a with the detection unit 120b therebetween. The running buffer solution supply unit 120c supplies the running buffer solution to the buffer solution loading pad 182 of the ICS 180. [ The running buffer solution supply portion 120c includes a running buffer solution chamber 190, a running buffer solution receiving portion 190a, and a running buffer solution introducing passageway 191.

The running buffer solution chamber 190 is located opposite the sample processing unit 120a with the ICS 180 interposed therebetween. The running buffer solution chamber 190 stores the running buffer solution. The running buffer solution is composed of 25 mM sodium carbonate buffer (pH 9.6), 1% casein, 0.4 mM EDTA, 0.2% alpha-cyclodextrin, 0.2% triton-100, 0.4 M UREA and 0.1% sodium azide As one example, the present invention is not limited thereto. The running buffer solution assists the flow of the diagnostic gene material in the ICS 180.

The running buffer solution receiving portion 190a is located radially outward of the running buffer solution chamber 190 and a valve portion 190b is provided between the running buffer solution receiving portion 190a and the running buffer solution chamber 190 do. The valve portion 190b is a capillary-shaped manual valve formed by a height difference, so that the running buffer solution stored in the running buffer solution chamber 190 can not easily move to the running buffer solution receiving portion 190a. After the running buffer liquid stored in the running buffer liquid chamber 190 passes over the valve portion 190b by the centrifugal force generated when the diagnostic microchip 110 rotates around the rotation center O, 190a.

The running buffer solution introducing passage 191 connects the radially outer end of the running buffer solution receiving portion 190a and the buffer solution loading pad 182 of the ICS 180. [ The running buffer solution introduction passage 191 is provided with a running buffer solution flow control passage 191a. The running buffer solution flow control passage 191a has a smooth curve from the radial end portion of the running buffer solution receiving portion 190a and extends in a radially inward direction while being converted into a soft curve and is turned radially outward And again extends in a smooth curve, with the direction turning radially inward and then extending in a soft curve, with the direction turned radially outward. The running buffer liquid flow control passage 191a includes a first inside switching curve portion 192 and a second switching curve portion 193 which are arranged in order from the upstream side to the downstream side along the running direction of the running buffer liquid, And a second inside switching curve portion 194. The first inside switching curve portion 192 switches the flow of the running buffer solution from the inside to the outside of the radial direction to introduce the running buffer solution into the outside switching curve portion 193. The outer switching curve portion 193 switches the flow of the running buffer solution from the radially outer side to the inner side to flow the running buffer solution into the second inner switching curve portion 194. The second inside switching curve portion 194 switches the flow of the running buffer solution from the radially inner side to the outer side to flow the running buffer solution into the buffer solution loading pad 182 of the ICS 180. Valve portions 195 and 196 are provided between the first inside switching curve portion 192 and the outside switching curve portion 193 and between the second inside switching curve portion 194 and the buffer solution loading pad 182 do. The valve portions 195 and 196 are manual valves formed by the height difference. The running buffer solution flowing through the running buffer solution flow control passage 191a passes through the valve portions 195 and 196 when the rotational speed of the diagnostic microchip 110 increases.

An integrated rotary diagnostic method according to the first embodiment of the present invention using the diagnostic microchip 110 described with reference to FIG. 1 through FIG. 4 will now be described with reference to FIG. Before describing the diagnostic method shown in FIG. 5 in detail, the configuration of the diagnostic apparatus used therein will be described in detail. Fig. 6 shows a diagnostic apparatus for performing the diagnostic method shown in Fig. 6, the diagnostic apparatus 100 includes a temperature control unit 180a on which the diagnostic microchip 110 is mounted, a rotation drive unit 199a for rotating the diagnostic microchip 110 around the rotation axis X, .

The temperature regulating unit 180a includes a lower member 181a and an upper member 182a. The temperature regulating unit 180a regulates the temperature required for the PCR process. On the upper surface of the lower member 181a, the diagnostic microchip 110 is rotatably mounted on the temperature control unit 180a. The upper member 182a moves up and down with respect to the lower member 181a and receives the diagnostic microchip 110 therein. Referring to FIG. 7, a plurality of heating zones 185a, 185b, 185c, 185d, 185e and 185f are formed in the temperature regulating unit 180a in order along the circumferential direction. An insulator or cooling means 186 is provided between the heating zones 185a, 185b, 185c, 185d, 185e, 185f. The heating zones 185a, 185b, 185c, 185d, 185e, 185f may be formed by suitable heating means such as heating blocks. The plurality of heating zones 185a, 185b, 185c, 185d, 185e and 185f are arranged along the circumferential direction in the first heating zone 185a, the second heating zone 185b, the third heating zone 185c, A fourth heating zone 185d, a fifth heating zone 185e, and a sixth heating zone 185f. The first and fourth heating zones 185a and 185d provide the temperatures required for the denaturation step in the PCR process. The second and fifth heating zones 185b and 185e provide the temperatures required for the coupling step in the PCR process. The third and sixth heating zones 185c and 185f provide the temperatures required for the extension step in the PCR process. The three continuous heating zones 185a, 185b, and 185c (185d, 185e, and 185f) respectively form a unit temperature regulation period. That is, the temperature regulating unit 180a has three unit temperature regulating sections, each of which corresponds to each of the six unit cells 120 located along the circumferential direction in the microprocessing chip for diagnosis 110, And the required temperature is sequentially provided. When an isothermal amplification process is used instead of the PCR process, the temperature regulator 180a can be used instead of providing a temperature for the isothermal amplification process.

The rotation drive unit 199a includes a rotation drive motor that rotates the rotation center O of the diagnostic microchip 110 around the rotation axis X. [ The rotation driving unit 199a rotates the diagnostic microchip 110 to generate a centrifugal force required to move the liquid. In the PCR step, the target material chamber 170 of the diagnostic microchip 110 is rotated by the temperature control unit 180a, So as to be positioned in the heating region necessary for the heating operation.

5, the diagnosis method includes a diagnostic microchip preparation step S10, a diagnostic microchip mounting step S20, a preprocessing step S30, an amplification step S40, a detection preparation step S50 And a detecting step S60.

In the diagnostic microchip preparation step S10, the sample, the washing buffer solution, the elution buffer solution, the reaction solution, and the running buffer solution are injected into the diagnostic microchip 110 as shown in FIG. FIG. 8 shows a state in which the sample, the washing buffer solution, the elution buffer solution, the reaction solution, and the running buffer solution are injected in the unit well 120. 8, the sample S is stored in the sample storage part 130, the washing buffer solution W is stored in the washing buffer solution chamber 140, and the elution buffer solution E is stored in the elution buffer The reaction liquid M is stored in the reaction liquid chamber 150a and the running buffer liquid R is stored in the running buffer liquid chamber 190. The reaction liquid M is stored in the liquid chamber 150,

In the diagnostic microchip mounting step S20, the diagnostic microchip 110 into which the sample, the washing buffer solution, the elution buffer solution, the reaction solution, and the running buffer solution are injected is connected to the diagnostic apparatus 100 through the diagnostic microchip preparing step S10. And is accommodated in the temperature regulating portion 180a. At this time, the rotation center O of the diagnostic microchip 110 is positioned on the rotation axis X. The diagnostic microchip 110 accommodated in the temperature regulating part 180a is connected to the rotation driving part 199a and is rotatable with respect to the rotation axis X. [

In the preprocessing step S30, the target material contained in the sample stored in the sample storage unit 130 of the diagnostic microchip 110 is separated from other unnecessary components and stored in the target material chamber 170, And the running buffer solution is ready to be supplied to the ICS 180. A more detailed process of the preprocessing step S30 is shown as a flowchart in Fig. 9, the preprocessing step S30 includes a target substance capturing step S31, a washing buffer solution loading step S32, an elution buffer solution and a reaction solution introducing step S33, a reaction solution loading step S34 An elution buffer solution loading step S35, and a target material and running buffer solution supply preparation step S36. 10A to 10F show the state of the unit filter 120 in each step of the preprocessing step S30.

The target material trapping step S31 is a step in which the diagnostic microchip 110 rotates about the rotation center O at a first rotation speed (for example, 5000 RPM) for a predetermined time (for example, 10 seconds) (A). Here, the first rotational direction A is the direction in which the waste liquid chamber 165 rotates to move toward the target material chamber 170 side. The sample S stored in the sample storage part 130, the washing buffer solution W stored in the washing buffer solution chamber 140, the elution buffer solution chamber (S) stored in the washing buffer solution chamber 140 Each of the eluent buffer solution E stored in the reaction solution chamber 150, the reaction solution M stored in the reaction solution chamber 150a and the running buffer solution R stored in the running buffer solution chamber 190 are supplied to the valve portions 130a, 140a, 151 , 151a, and 190b. FIG. 10A shows the state of the unit filter 120 in the target substance capturing step S31. Referring to FIG. 10A, the sample S is adsorbed to the capturing means 128 through the target substance capturing part 125 by the centrifugal force, and the remaining unnecessary component S1 that is not adsorbed is adsorbed And then flows into the waste liquid chamber 165 through the discharge passage 160. The remaining unnecessary component S1 flows into the waste liquid chamber 165 without flowing toward the target material chamber 170 by the rotation direction A in the process of passing through the discharge passage 160. [ In the target material capturing step S31, the washing buffer solution W passes through the washing liquid introducing passage 145 and then flows into the target material capturing part 125 after the sample S. The eluent buffer solution E remains in the eluting buffer solution flow control passage 156 in the target substance capturing step S31 while moving to the previous position of the switching curve portion 158. [ In the target substance capturing step S31, the reaction liquid M is maintained in the state of moving to the previous position of the switching curve portion 158a on the reaction liquid flow control passage 156a. In the target material trapping step S31, the running buffer solution R is maintained in a state of being moved to the previous position of the first inside switching curve portion 192 on the running buffer liquid flow control passage 191a. After the target material capturing step S31, the washing buffer liquid loading step S32 is performed.

The washing buffer solution loading step S32 is a step in which the diagnostic microchip 110 is rotated at a second rotational speed (for example, 5000 RPM, which is the same as the first rotational speed) 4 minutes) in the first rotation direction (A). In the wash buffer liquid loading step S32, the wash buffer liquid W is transferred to the waste liquid chamber 165 across the discharge passage 160 after passing through the target substance capturing part 125 as shown in FIG. 10B by centrifugal force. Lt; / RTI > The washing buffer solution W does not flow toward the target material chamber 170 but flows into the waste liquid chamber 165 by the rotational direction A in the course of the washing buffer solution W passing through the discharge passage 160 . The SEPHOKE buffer solution W passes through the target material capturing portion 125 and cleans and removes the remaining unnecessary components except the target material among the substances trapped in the target material capturing portion 125. [ After the washing buffer solution loading step (S32), the elution buffer solution and the reaction solution introducing step (S33) are performed.

The eluting buffer solution and the reaction solution introducing step (S33) are performed by rapidly reducing the rotational speed of the diagnostic microchip 110 to zero. FIG. 10C shows the state of the unit cavity 120 in the elution buffer solution and the reaction solution introducing step (S33). 10C, in the elution buffer solution and the reaction solution introduction step S33, the elution buffer solution E passes through the switching curve portion 158 of the elution buffer solution flow control passage 156, and the reaction solution M The state passes the switching curve portion 158a of the reaction liquid flow control passage 156a. The running buffer solution R flows through the first inner switching curve portion 192 of the running buffer liquid flow control passage 191a and between the first inner switching curve portion 192 and the outer switching curve portion 193 The valve unit 195 is in a state of being moved. The passage of the elution buffer solution E through the switching curve portion 158, the passage of the reaction liquid M through the switching curve portion 158a and the passage of the running buffer solution R through the first inside switching curve portion 192 are abruptly decelerated Due to the centrifugal force reduction. After the elution buffer solution and the reaction solution introduction step (S33), the reaction solution loading step (S34) is performed.

The reaction liquid loading step S34 is a step in which the diagnostic microchip 110 rotates about the rotation center O at a third rotation speed (for example, 5000 RPM) for a predetermined time (for example, 30 seconds) And in the second rotation direction B, which is the opposite direction of the direction A, as shown in Fig. FIG. 10D shows the state of the unit cavity 120 in the reaction liquid loading step (S34). Referring to FIG. 10D, the reaction liquid M flows into the target material chamber 170 after passing through the discharge passage 160 through the connection passage 159a. The reaction liquid W flows into the target material chamber 170 without flowing toward the waste liquid chamber 165 by the rotation direction B in the course of the reaction liquid M passing through the discharge passage 160. The reaction liquid M that has flowed into the target material chamber 170 remains in a state of being moved before the switching curve portion 175a of the diagnosis target substance flow control passage 175. [ In addition, the elution buffer solution (E) separates the target material trapped in the target material capturing part (125) from the capturing means (128) while passing through the target material capturing part (125). The running buffer solution R passes through the valve portion 195 during the initial acceleration process of the reaction liquid loading step S34 and then passes through the outer switching curve portion 193 and the second inner switching curve portion 194 And remains in the moved state to the position.

The elution buffer solution loading step S35 is a step in which the diagnostic microchip 110 rotates about the rotation center O at a fourth rotation speed (for example, 5000 RPM, which is the same as the third rotation speed) 4 minutes) in the second rotation direction B. FIG. 10E shows the state of the unit cavity 120 in the elution buffer solution loading (S35). 10E, the elution buffer solution E flows into the target material chamber 170 through the discharge passage 160 together with the target material after passing through the target material capturing part 125 by the centrifugal force, Is mixed with the reaction solution (M). The elution buffer solution E does not flow toward the waste liquid chamber 165 as shown in the drawing and the target liquid in the target chamber 165 . The eluent buffer solution E flows into the target material chamber 170 through the elution buffer solution loading step S35. After the elution buffer solution loading step (S35), the target material and running buffer solution supply preparation step (S36) are performed.

The target material and running buffer solution supply preparation step (S36) is performed by rapidly reducing the rotational speed of the diagnostic microchip 110 to zero. FIG. 10F shows the state of the unit cavity 120 in the step S36 of preparing the target material and the running buffer solution. 10F, the reaction liquid M passes through the switching curve portion 175a of the diagnosis target substance flow control passage 175 and the running buffer solution R flows into the second buffer solution flow passage 191a Passes through the inner switching curve portion 194, and is moved to the valve portion 196. The passage of the reaction liquid M through the switching curve portion 175a and the passage of the running buffer solution R through the second inside switching curve portion 194 are caused by the centrifugal force reduction due to the sudden deceleration. After the preparation of the target material and the running buffer solution (S36), a PCR or isothermal amplification step (S40 in Fig. 7) is performed.

Referring again to FIG. 5, after the preprocessing step S30 is completed, the amplification step S40 by the PCR process is performed. The PCR process in the amplification step S40 is performed in the following manner. In the PCR process, the denaturation step, the combining step, and the elongating step are sequentially performed on the target material contained in each target material chamber 170 of the diagnostic microchip 110. This will be described in detail with reference to FIG. First, in a state where each of the target material chambers 170 is positioned correspondingly to the plurality of heating regions 185a, 185b, 185c, 185d, 185e, and 185f on a one-to-one basis, Regions 185a and 185d operate to perform a denaturation step for the target material contained in the target material chamber 170 located in the first and fourth heating regions 185a and 185d. Next, the diagnostic microchip 110 is rotated at a predetermined angle so that the target material chamber 170 containing the target material subjected to the denaturation step is positioned in the second and fifth heating zones 185b and 185e, . Hereinafter, 'constant angle' means an angle at which the target material chamber 170 moves to the adjacent heating region to perform the next step. Next, the second and fifth heating regions 185b and 185e are operated together with the first and fourth heating regions 185a and 185d. Accordingly, the denaturation step and the combining step are performed together. Next, the diagnostic microchip 110 is rotated by a predetermined angle to operate all the heating areas 185a, 185b, 185c, 185d, 185e, and 185f. Accordingly, the two target material chambers 170 of the plurality of target material chambers 170 are completed up to the elongation step, the coupling step is completed for the other two target material chambers 170, and the remaining two target materials The chamber 170 is completed up to the denaturation step. Next, after the diagnostic microchip 110 is rotated by a predetermined angle, the remaining heating regions 185b, 185c, 185e, and 185f except for the first and fourth heating regions 185a and 185d are operated. Thus, the two target material chambers 170 that have been completed from the previous step to the combining step are performed to the elongation step and to the joining step for the two target material chambers 170 that have been completed to the denaturation step in the previous step . Next, after the diagnostic microchip 110 is rotated by a predetermined angle, the third and sixth heating regions 185c, 185c, 185c, 185d, 185e, and 185f, which provide the temperature of the stretching step among the plurality of heating regions 185a, 185b, , 185f. Thus, the coupling to the two target material chambers 170 that have been completed from the previous step to the coupling step is performed up to the completion of the PCR for the target material contained in all the target material chambers 170. Here, only the case where the amplification step (S40) performs the PCR process has been described in detail, but the present invention is not limited thereto and includes the case of performing the isothermal amplification process. After the amplification step S40, the detection preparation step S50 is performed.

In the detection preparation step S60, the diagnosis microchip 110 is stopped for a predetermined time (for example, 4 minutes) at a fifth rotation speed (for example, 800 RPM) And rotating in the rotation direction A. FIG. 11 shows the state of the unit filter 120 in the detection preparation step (S60). 11, the diagnostic target gene material T 'stored in the target material chamber 170 is supplied to the conjugate pad 185 of the ICS 180 via the diagnostic substance transfer unit 174, The liquid R is supplied to the buffer liquid loading pad 182 of the ICS 180 through the running buffer liquid introduction passage 191. [ The passage of the running buffer solution R through the valve portion 196 is caused by an increase in the initial rotation speed in the detection preparation step S60. After the detection preparation step (S50), the detection step (S60) is performed.

In the detection step (S60), the ICS (180) detects pathogens from the target substance. 12 is a diagram showing an example of the state of the ICS 180 after the detection step is performed. Referring to Fig. 12, a control line 180b and a test line 180c indicating detection of a specific pathogen are colored. Although not shown, test lines at different positions are developed depending on the type of pathogens detected. In the detection step S60, it is preferable that the diagnostic microchip 110 does not rotate.

13 shows a diagnostic microchip according to the second embodiment of the present invention. Referring to FIG. 13, the diagnostic microchip 210 according to another embodiment of the present invention includes a unit spacer 220 different from the unit spacer 220 provided in the diagnostic microchip 110 shown in FIG. 2 Respectively. Fig. 14 shows the unit process section shown in Fig. Referring to FIG. 14, the unit control unit 220 includes a detection unit 120b, a sample processing unit 220a, and a running buffer liquid supply unit 220c. The sample processing unit 220a and the running buffer liquid supply unit 220c are located on both sides of the rotation center O in the circumferential direction with the detection unit 120b interposed therebetween. In the embodiment shown in Fig. 14 and the embodiment shown in Fig. 4, the same components have the same reference numerals. Therefore, a detailed description of the configuration of Fig. 14 denoted by the same reference numerals as those used in Fig. 4 will be omitted.

The detection unit 120b is configured in the same manner as the detection unit 120b shown in FIG. 4, and thus a detailed description thereof will be omitted.

The sample processing unit 220a is located on one side in the circumferential direction of the detection unit 120b. The sample processing unit 220a includes a preprocessing unit 220d for performing preprocessing on the sample, a diagnostic substance delivery unit 274 for delivering the genetic material amplified by the PCR process or the isothermal amplification process to the detection unit 120b, Respectively.

The pretreatment unit 220d includes a target substance capturing unit 125, a sample storage unit 230, a sample introduction channel 235, a washing buffer chamber 240, An elution buffer liquid introduction passage 255, an ejection passage 260, a waste liquid chamber 265, and a target material chamber 270 do.

The target material capturing unit 125 is configured in the same manner as the target material capturing unit 125 shown in FIG. 4, and thus a detailed description thereof will be omitted.

The sample storage portion 230 is located radially inward of the inlet 126a of the trapping passage 126. [ The sample storage unit 230 stores a sample. A sample injection hole (not shown) for injecting a sample into the sample storage part 230 is formed in the diagnostic microchip 210.

The sample introduction passage 235 connects the sample storage portion 230 and the inlet 126a of the trapping passage 126. [ The sample stored in the sample storage part 230 flows into the trapping passage 126 through the sample introduction passage 235 by the centrifugal force generated when the diagnostic microchip 210 rotates.

The cleaning buffer solution chamber 240 is located radially inward of the inlet 126a of the trapping passage 126 and is located closer to the rotation center O than the sample storage part 230. [ In the cleaning buffer solution chamber 240, a cleaning buffer solution is stored. The diagnostic microchip 210 is provided with a wash buffer solution injection hole (not shown) for injecting the wash buffer solution into the wash buffer solution chamber 240. The washing buffer solution stored in the washing buffer solution chamber 240 flows into the trapping passage 126 via the washing buffer solution introducing passage 245 by the centrifugal force generated when the diagnostic microchip 210 rotates.

The cleaning buffer solution introducing passage 245 has a capillary valve 245a and a connecting portion 245b mutually connected to each other. The washing buffer solution stored in the washing buffer solution chamber 240 flows into the trapping passage 126 through the washing buffer solution introducing passage 245. [

The capillary valve 245a is formed extending radially outward from the wash buffer solution chamber 245 to regulate the discharge of the wash buffer solution. The capillary valve 245a has a connection flow path 245a1 extending radially inwardly and outwardly and a plurality of branch portions 245a2 extending at both sides perpendicular to the connection flow path 245a1. The washing buffer solution flows into the connecting portion 245b through the connecting flow path 245a1, and the inflow speed is delayed by the plurality of branch portions 245a2.

The connecting portion 245b extends radially outward from the end of the capillary valve 245a and is connected to the inlet 126a of the trapping passage 126. [

The elution buffer solution chamber 250 is positioned radially inward of the inlet 126a of the trapping passage 126 and the distance from the rotation center O is similar to the wash buffer solution chamber 240. [ In the elution buffer solution chamber 250, an elution buffer solution is stored. In the diagnostic microchip 210, an elution buffer solution injection hole (not shown) for injecting an elution buffer solution into the elution buffer solution chamber 250 is formed.

The elution buffer solution introduction passages 255 have mutually connected elution buffer solution flow control passages 256 and connection passages 259. The elution buffer solution stored in the elution buffer solution chamber 250 through the elution buffer solution introducing passage 255 flows into the trapping passage 126.

The elution buffer solution flow control passage 256 extends from the elution buffer solution chamber 250 and is connected to the connection passage 259. The eluting buffer solution flow control passage 256 extends from the eluting buffer solution chamber 250 substantially radially outward and then forms a smooth curve and extends in a radially inward direction while being converted into a soft curve and a radially outward direction And is extended. Accordingly, the eluting buffer solution flow control passage 256 has an outer switching curve portion 257 provided on the upstream side and an inner switching curve portion 258 provided on the downstream side along the flow direction of the eluent. The outer conversion curve portion 257 converts the flow of the eluent liquid from the radially outer side to the inner side and flows into the inner conversion curve portion 258. The inner switching curve portion 258 turns the flow of the eluent liquid radially inward and flows into the connection passage 259.

The connection passage 259 extends radially outward from the flow control passage 256 and is connected to the inlet 126a of the catch passage 126. [

The discharge passage 260 extends from the outlet 126b of the catch passage 126. [ The discharge passage 260 is tilted with respect to the radial direction so as to move away from the rotational center O toward the downstream side so that the diagnostic microchip 210 rotates around the rotational center O Is moved along the discharge passage (260). The discharge passage 260 has a connection section 262 extending downstream so as to be close to the ICS 180. The waste liquid chamber 265 and the target material chamber 270 are connected to the connection section 262.

The waste liquid chamber 265 is located radially outward of the connection section 262 of the discharge passage 260. The waste liquid chamber 265 is connected to the connection section 262 of the discharge passage 260 through the connection passage 266. In the waste liquid chamber 265, unnecessary components other than the target substance are stored.

The target material chamber 270 is connected to the outside of the radial direction of the connection section 262 of the discharge passage 260. The target material chamber 270 is located downstream of the waste liquid chamber 265. In the target material chamber 270, the target material is stored. The target material stored in the target material chamber 270 is amplified by a PCR process or an isothermal amplification process.

The diagnosis target substance transfer unit 274 transfers the amplified target gene material to the detection unit 120b through the PCR process or the isothermal amplification process in the target material chamber 270. [ The diagnosis target substance transfer portion 274 has a receiving portion 274a and a supply capillary 274b.

The receiving portion 274a is located adjacent to the target material chamber 270 radially outward. The receiving portion 274a and the target material chamber 270 are connected by a passive valve installed therebetween. When the manual valve is above a certain rotational speed, a pressure due to the centrifugal force acts to open the valve. Alternatively, it may be in the form of being opened after PCR or by amplification at the same temperature using a material that is heated or opened by pressure. The receptacle 274a receives the diagnostic genetic material that has passed from the target material chamber 270.

The supply capillary tube 274b extends from the accommodating portion 274a and is connected to the detecting portion 120b. The diagnostic target gene material accommodated in the accommodating portion 274a is transferred to the conjugate pad 185 of the detection unit 120b via the supply capillary 274b.

The pretreatment unit 220a is connected to the discharge passage 260 and connected to the first auxiliary connection passage 275 leading to the detection unit 120b and the cleaning buffer solution chamber 240 and the elution buffer solution 250, And a second auxiliary connection passage 276 leading to the second auxiliary connection passage 120b. The first auxiliary connection channel 275 and the second auxiliary connection channel 276 are connected to each other through the detection unit 120b to facilitate movement of various liquids in the pre-processing unit 220a.

The running buffer solution supply unit 220c is located on the opposite side of the sample processing unit 220a with the detection unit 120b interposed therebetween. The running buffer solution supply unit 220c supplies the running buffer solution to the buffer solution loading pad 182 of the ICS 180. [ The running buffer liquid supply section 220c includes a running buffer liquid chamber 290, a running buffer liquid introduction passage 291, and a running buffer liquid supply passage 298. [

The running buffer solution chamber 290 is located opposite the sample processing unit 220a with the ICS 180 interposed therebetween. The running buffer solution chamber 290 stores the running buffer solution. Although not shown, the diagnostic microchip 210 is formed with a buffer solution injection hole for injecting the running buffer solution into the running buffer solution chamber 290. [

The running buffer solution introducing passage 291 has a running buffer liquid flow control passage 291a and a receiving space 291b mutually connected to each other. The running buffer solution stored in the running buffer solution chamber 290 flows into the supply passage 298 through the running buffer solution introducing passage 291. [

The running buffer solution flow control passage 291a extends smoothly from the radial end of the running buffer solution chamber 290 with a smooth curve and the direction changes radially inward and then forms a smooth curve and the direction is switched radially outward Extends again with a smooth curved line, with the direction turning radially inward, followed by a soft curve and the direction extending radially outwardly. Accordingly, the running buffer liquid flow control passage 291a includes a first outside switching curve portion 292 that is disposed in order from upstream to downstream along the flow direction of the running buffer liquid, 293, a second outside switching curve portion 294, and a second inside switching curve portion 295. [

 The first outside switching curve portion 292 turns the flow of the running buffer solution radially inward to introduce the running buffer solution into the first inside switching curve portion 293. The first inside switching curve portion 293 switches the flow of the running buffer solution from the inside to the outside in the radial direction to introduce the running buffer solution into the second outside switching curve portion 294. The second outside switching curve portion 294 switches the flow of the running buffer solution from the outside to the inside in the radial direction to flow the running buffer solution into the second inside switching curve portion 295. The second inside switching curve portion 295 switches the flow of the running buffer solution from the radially inner side to the outer side to introduce the running buffer solution into the accommodation space 291b.

 The running buffer liquid flow control passage 291a further includes a valve portion 297 positioned between the second inside switching curve portion 294 and the second inside switching curve portion 295. [ The configuration and operation of the valve portion 294 are the same as those of the valve portions 295 and 296 shown in Fig.

The accommodation space 291b is connected to the end of the running buffer liquid flow control passage 291a. In the accommodation space 291b, a running buffer solution that has passed through the running buffer solution flow control passage 291a is accommodated.

The running buffer solution supply passage 298 extends generally obliquely along the radial direction. The radially inner end of the running buffer solution supply passage 298 is located adjacent to the receiving space 291b and the radially outer end of the running buffer solution supply passage 298 is located adjacent to the buffer solution loading pad 182 of the ICS 180 ). The upstream end (i.e., the radially inner end) of the running buffer liquid feed passage 198 and the receiving space 291b are connected by a passive valve provided therebetween. Since the manual valve is the same as the manual valve located between the accommodating portion 274a and the target material chamber 270 described above, a detailed description thereof will be omitted. The running buffer solution of the accommodation space 291b is supplied to the buffer solution loading pad 182 of the ICS 180 via the running buffer solution supply passage 298. [

The running buffer solution supply unit 220c further includes a third auxiliary connection passage 299 extending from the running buffer solution chamber 290 and connected to the detection unit 120b. The movement of the running buffer solution in the running buffer liquid supply section 220c is facilitated by the third auxiliary connection passage 299. [

Now, an integrated rotary diagnostic method according to a second embodiment of the present invention using the diagnostic microchip shown in FIG. 13 will be described with reference to FIG. 15, the diagnostic method includes a microchip preparation step (S'10) for diagnosis, a microchip mounting step (S'20) for diagnosis, a preprocessing step (S'30), a running buffer solution moving step (S ' 40, an amplification step S'50, a detection preparation step S'60, and a detection step S'70.

In the diagnostic microchip preparation step (S'10), the sample, the washing buffer solution, the elution buffer solution and the running buffer solution are injected into the diagnostic microchip 210 as shown in Fig. FIG. 16 shows a state in which a sample, a washing buffer solution, an eluting buffer solution, and a running buffer solution are injected into the unit well 220. 16, the sample S is stored in the sample storage part 230, the washing buffer solution W is stored in the washing buffer solution chamber 240, and the elution buffer solution E is stored in the elution buffer And the running buffer solution R is stored in the running buffer solution chamber 290. [

In the diagnostic microchip mounting step (S'20), the diagnostic microchip 210 into which the sample, the washing buffer solution, the eluting buffer solution, and the running buffer solution are injected through the diagnostic microchip preparation step (S'10) And is accommodated in the thermostat 180a. The diagnostic microchip 210 accommodated in the temperature regulating part 180a is connected to the rotation driving part 199a and is rotatable.

In the preprocessing step S'30, the target material contained in the sample stored in the sample storage part 230 of the diagnostic microchip 210 is separated from other unnecessary components and stored in the target material chamber 270. A more detailed process of the preprocessing step S'30 is shown as a flowchart in Fig. Referring to FIG. 17, the pretreatment step S'30 includes a target substance capturing step S'31, a washing buffer solution loading step S'32, an eluting buffer solution introducing step S'33, A buffer solution loading step (S'34), and a target material collecting step (S'35). 18A to 18E show the state of the unit filter 220 in each step of the preprocessing step S'30.

The target material trapping step S'31 is performed in such a manner that the diagnostic microchip 210 is moved for a predetermined time (for example, 10 seconds) at a first rotational speed (for example, 5000 RPM) By rotating in the first rotation direction (A). Here, the first rotational direction A is the direction in which the target material chamber 270 rotates to move toward the waste liquid chamber 265 side. 18A shows the state of the unit filter 220 in the target substance capturing step (S'31). 18A, the sample (S in FIG. 16) stored in the sample storage part 230 passes through the target substance capturing part 125, and the substance including the target substance is adsorbed to the capturing means 128. FIG. The washing buffer solution W is not supplied to the target material capturing part 125 by the capillary valve 245a of the washing buffer solution introducing passageway 245 in the target material capturing step S'31. In the target material capturing step (S'31), the entire eluting buffer solution (E) remains in the eluting buffer solution chamber (150). In the target material capturing step (S'31), the entire running buffer solution R remains in the running buffer solution chamber 290.

The washing buffer solution loading step S'32 is a step in which the diagnostic microchip 210 rotates about the rotation center O at a second rotation speed (for example, 5000 RPM, which is the same as the first rotation speed) (For example, 4 minutes). The washing buffer solution W flows into the target material capturing part 125 by the action of the capillary valve 245a. FIG. 18B shows the state of the unit cavity 220 in the washing buffer solution loading step (S'32). Referring to FIG. 14 together with FIG. 18B, the washing buffer solution W passes through the target material capturing part 125 by the centrifugal force, and unnecessary components other than the target material in the target material capturing part 125, Is removed by washing. The sample S1 that has not been trapped in the trapping means 128 in the sample which has first passed through the target substance capturing portion 125 in the washing buffer liquid loading step S'32 is discharged through the discharge passage 260 into the waste liquid chamber 265 ). In the washing buffer solution loading step (S'32), the eluting buffer solution (E) is moved to a position between the outer switching curve portion (257) and the inner switching curve portion (258) on the eluting buffer solution flow control passage (256) It will remain in one state. In the washing buffer solution loading step (S'32), the running buffer solution (R) flows in the running buffer liquid flow control passage 291a between the first inside switching curve portion 292 and the first inside switching curve portion 293 And remains in a state of being moved to one position.

The eluting buffer solution introducing step S'33 is a step in which the diagnostic microchip 210 decelerates at a third rotational speed (for example, 100 RPM) significantly lower than the second rotational speed about the center of rotation O, (For example, 30 seconds) in the first rotation direction A. 18C shows the state of the unit filter 220 in the elution buffer solution introducing step (S'33). Referring to FIG. 14 together with FIG. 18C, the eluting buffer solution E is moved to the connection passage 259 through the inner switching curve portion 258. The passage of the inner switching curve portion 258 is caused by a decrease in the centrifugal force due to a sudden deceleration. The washing buffer solution W1 passing through the target material capturing part 125 in the elution buffer solution introducing step (S'33) is stored in the waste liquid chamber 165. [ At this time, the sample component S1 stored in the waste liquid chamber 265 and the washing buffer solution W1 completely fill the waste liquid chamber 265. [ In the eluting buffer solution introducing step (S'33), the running buffer solution R is moved to the valve portion 297 located between the second outside switching curve portion 294 and the second inside switching curve portion 295 State. The passage of the running buffer solution R through the first inside switching curve portion 293 is caused by a decrease in centrifugal force due to a sudden deceleration.

The elution buffer solution loading step S'34 is performed so that the diagnostic microchip 210 is accelerated to a fourth rotational speed (for example, 2000 RPM) which is significantly higher than the third rotational speed about the rotation center O, (For example, 30 seconds) in the first rotation direction A. FIG. 18D shows the state of unit unit 220 in the elution buffer solution loading step (S'34). Referring to Fig. 14 together with Fig. 18D, the elution buffer solution E is prepared so that the elution buffer solution E passes the target material capturing part 125 by the centrifugal force, and the target substance trapped in the target material capturing part 125 From the capture means 128. In the eluting buffer solution loading step S'34, the running buffer solution R passes through the valve portion 297 due to an increase in the rotational speed, and then passes through the second inside switching curve portion 294 and the second inside switching curve portion 295 in a state of being moved to one position.

The target material collecting step S'35 is a step in which the diagnostic microchip 210 is accelerated to a fifth rotational speed (for example, 5000 RPM) higher than the fourth rotational speed about the center of rotation O, (For example, 1 minute) in the first rotation direction A. 18E shows the state of the unit filter 220 in the target material collecting step (S'35). Referring to FIG. 14 together with FIG. 18E, the target material T separated from the trapping means 128 by the elution buffer solution E is stored in the target material chamber 270. In the target material collecting step (S'35), the running buffer solution R keeps moving to a position between the second outside switching curve portion 194 and the second inside switching curve portion 195.

Referring again to FIG. 15, after the preprocessing step S'30 is completed, the running buffer solution moving step S'40 is performed. In the running buffer solution liquid moving step S'40, the running buffer solution R moved to a position between the second outer curved portion 294 and the second inner switching curve portion 295 passes through the second inner transition curve portion 295, (295) to the accommodation space (291b). The running buffer solution moving step S40 is performed in such a manner that the diagnostic microchip 110 is rotated at a fifth rotational speed (for example, 100 RPM equivalent to the third rotational speed) significantly lower than the fifth rotational speed about the rotational center O And is rotated in the first rotation direction A for a predetermined time (for example, 30 seconds). After the running buffer solution moving step (S'40), the amplifying step performing step (S'50) is performed.

In the amplification step S'50, a PCR process or an isothermal amplification process is performed on the target material contained in each target material chamber 170 of the diagnostic microchip 210. [ Since the amplifying step S'50 is performed in the same manner as the amplifying step S40 shown in FIG. 5, a detailed description thereof will be omitted. After the amplification step S'50, the detection preparation step S'60 is performed.

In the detection preparation step (S'60), the diagnostic target gene material contained in the target material chamber 270 is passed through the passive valve to the receiving portion 274a and then passed through the supply capillary 274b to the conjugate pad 184 and the running buffer liquid contained in the accommodation space 291b is passed through the manual valve to the running buffer liquid supply passage 298 and then flows through the running buffer liquid supply passage 298 to the buffer liquid loading And is supplied to the pad 182. FIG. 19 shows the state of the unit filter 220 in the detection preparation step (S'60). 19, the gene material T 'to be diagnosed is supplied to the conjugate pad 184 of the ICS 180 via the receiving portion 274a and the supply capillary 274b, and the running buffer solution R And is supplied to the buffer liquid loading pad 182 of the ICS 180 via the running buffer liquid supply passage 298. After the detection preparation step (S'60), the detection step (S'70) is performed.

In the detection step S'70, the ICS 180 detects pathogens from the target substance. Since the detection step S'70 is performed in the same manner as the detection step S60 shown in FIG. 7, a detailed description thereof will be omitted.

20 shows a diagnostic microchip according to the third embodiment of the present invention. Referring to FIG. 20, the diagnostic microchip 310 includes a plurality of unit cells 320 which are located apart from the center of rotation O and are arranged in order along the circumferential direction. In the present embodiment, it is described that there are three unit copiers 320, but the present invention does not limit the number of the multiple coffer units 320 to three. In the present invention, the diagnostic microchip 310 may have two or less unit cells 320 or four or more unit cells 320. FIG. 21 is an enlarged view of the unit prism 320 shown in FIG. Referring to FIG. 21, the unit control unit 320 includes a preprocessing unit 320d having substantially the same configuration as the preprocessing unit 120d shown in FIG. In the embodiment shown in Fig. 21 and the embodiment shown in Fig. 3, the same components have the same reference numerals. Therefore, a detailed description of the configuration of FIG. 21 denoted by the same reference numerals as those used in FIG. 3 will be omitted.

The pretreatment section 320d includes a target substance capturing section 125, a sample storage section 130, a washing buffer chamber 140, a washing buffer solution introducing passage 145, elution buffer chamber 150, an eluting buffer solution flow control passage 156, a reaction solution chamber 150a, a reaction solution introduction passage 155a, a discharge passage 160, a waste solution chamber 165, And a target material chamber 170.

The integrated rotary diagnostic method according to the third embodiment of the present invention using the diagnostic microchip 310 described with reference to FIG. 20 and FIG. 22 will now be described with reference to FIG. Before describing the diagnostic method shown in FIG. 22 in detail, the configuration of the diagnostic apparatus used therein will be described in detail. Fig. 23 shows a diagnostic apparatus for performing the diagnostic method shown in Fig. 23, the diagnostic apparatus 300 includes a temperature control unit 380a on which the diagnostic microchip 310 is placed, a rotation drive unit 199a for rotating the diagnostic microchip 310 around the rotation axis X, . The diagnostic apparatus 300 performs an isothermal amplification process. In this embodiment, it is assumed that the RT-LAMP is used during the isothermal amplification process. In the present embodiment, the integrated rotation diagnostics device 300 is described as performing an isothermal amplification process. Alternatively, a PCR process may be performed instead of the isothermal amplification process, which is also included in the present invention. Referring to FIG. 23, the temperature regulating unit 380a includes a lower member 381a and an upper member 382a. The temperature regulator 380a regulates the temperature required for the isothermal amplification process. On the upper surface of the lower member 381a, the diagnostic microchip 310 is rotatably mounted on the temperature regulating portion 380a. The upper member 382a moves up and down with respect to the lower member 381a, and the diagnostic microchip 310 is received therein. Referring to FIG. 24, a plurality of heating regions 385 are formed in the temperature regulating portion 380a in order along the circumferential direction. The heating zone 385 may be formed by suitable heating means such as a heating block. In this embodiment, the number of the heating regions 385 is three, and each heating region 385 corresponds to the three unit flat portions 320. When a PCR process is used instead of the isothermal amplification process, the temperature regulator 380a may be used instead of providing a temperature for the PCR process. The detector 300a is a photodetector generally used in the fluorescence detection method and is movable relative to the temperature regulating portion 380a.

22, the diagnostic method includes a microchip preparation step S11 for diagnosis, a microchip mounting step S21 for diagnosis, a preprocessing step S31, an amplification step S41, a detection step S51, .

In the diagnostic microchip preparation step S11, the sample, the washing buffer solution, the elution buffer solution, and the reaction solution are injected into the diagnostic microchip 310 as shown in FIG. FIG. 25 shows a state in which a sample, a washing buffer solution, an eluting buffer solution, and a reaction solution are injected into the unit filter 320. 25, sample S is stored in sample storage 130, wash buffer solution W is stored in wash buffer solution chamber 140, elution buffer solution E is stored in elution buffer < RTI ID = 0.0 > And is stored in the liquid chamber 150, and the reaction liquid M is stored in the reaction liquid chamber 150a.

In the diagnostic microchip mounting step S21, the diagnostic microchip 310 into which the sample, the washing buffer solution, the eluting buffer solution, and the reaction liquid are injected is connected to the temperature regulating unit 300 of the diagnostic apparatus 300 through the diagnostic microchip preparing step S11. (380a). The diagnostic microchip 310 accommodated in the temperature regulating part 380a is connected to the rotation driving part 199a and is rotatable.

In the preprocessing step S31, the target material contained in the sample stored in the sample storage unit 130 of the diagnostic microchip 310 is separated from other unnecessary components and stored in the target material chamber 170. [ A more detailed process of the preprocessing step S31 is shown as a flowchart in Fig. 26, the preprocessing step S31 includes a step S311 of capturing a target substance, a washing buffer solution loading step S312, an eluting buffer solution and a reaction solution introducing step S313, a reaction liquid loading step S314 And an elution buffer solution loading step (S315). The target material capturing step S312 may be performed in the same manner as the target material capturing step S31 shown in FIG. The washing buffer solution loading step (S313) may be performed in the same manner as the washing buffer solution loading step (S32) shown in FIG. The elution buffer solution and the reaction solution introduction step (S313) can be performed in the same manner as the elution buffer solution and the reaction solution introduction step (S33) shown in Fig. The reaction solution loading step (S341) may be performed in the same manner as the reaction solution loading step (S34) shown in FIG. The elution buffer solution loading step S351 may be performed in the same manner as the eluting buffer solution loading step S35 shown in Fig.

Referring again to FIG. 22, after the preprocessing step S31 is completed, the amplifying step S41 is performed. In this embodiment, the gene amplification process is described as using an isothermal amplification process such as real-time RT-LAMP. The isothermal amplification process is performed by adjusting the target material contained in each target material chamber 170 of the diagnostic microchip 310 to a proper temperature by a corresponding heating zone 385. After the amplification step S41, the detection step S51 is performed.

The detection step S51 is performed by the fluorescence detection method for the diagnostic object contained in the target material chamber 170 using the detector 300a.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It is to be understood that the above-described embodiments may be modified or changed without departing from the spirit and scope of the present invention, and those skilled in the art will recognize that such modifications and changes are also within the scope of the present invention.

100: Integrated rotary diagnostics device 110: Diagnostic microchip
120: unit balancing unit 120a: sample processing unit
120b: Detection section 120c: Running buffer liquid supply section
120d: preprocessing unit 174:
180a: temperature control unit 199a: rotation driving unit

Claims (17)

And a unit processing unit located apart from the rotation center,
The unit processing unit includes:
A detection unit having an ICS for detecting a pathogen; And
And a sample processing unit including a pre-processing unit for pre-processing the sample, and a diagnostic substance delivery unit for delivering the genetic material to be diagnosed containing the amplified genetic material to the detection unit. The method according to claim 1,
The pre-
A target substance capturing portion having a capturing passage having an inlet and an outlet and a capturing means filled in the capturing passage;
A sample storage portion located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a sample is stored;
A washing buffer liquid chamber located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a washing buffer solution is stored; And
And an elution buffer solution chamber located radially inward of the target substance capturing part and connected to an inlet of the trapping passage and providing a space in which an elution buffer solution is stored. The method of claim 2,
Wherein the trapping passage connects the inlet and the outlet in a zigzag shape, and the inlet and the outlet are located radially inward and outward, respectively. The method of claim 3,
The pre-
A reaction solution chamber for providing a space in which a reaction solution required for the PCR process or the isothermal amplification process is stored,
A discharge passage which is located radially outward of the target substance capturing portion and the reaction solution chamber and extends along the circumferential direction and is connected to the outlet of the trapping passage and the reaction solution chamber at both ends,
A waste liquid chamber located radially outward of the discharge passage and connected to the discharge passage,
Further comprising a target material chamber radially outwardly connected to said discharge passage and connected to said discharge passage,
Wherein a portion of the discharge passage to which the reaction solution chamber is connected and a portion to which the target material chamber is connected are opposed to each other,
Wherein a portion of the discharge passage to which the outlet of the trapping passage is connected and a portion to which the waste liquid chamber is connected face each other. The method of claim 4,
The pretreatment unit may further include a valve unit surrounding the washing buffer solution chamber, the elution buffer solution chamber, and the reaction solution chamber,
Wherein the valve unit is a passive valve using a height difference. The method of claim 4,
Wherein the diagnostic substance delivery unit has a diagnostic substance flow control passage connecting the target substance chamber and the ICS,
Wherein the diagnosis target substance flow control passage has a switching curve portion for switching the flow of the diagnostic target from radially inward to outward. The method of claim 4,
Wherein the pre-processing unit further comprises an elution buffer liquid flow control passage for introducing the elution buffer solution stored in the elution buffer liquid chamber into the target substance capturing portion,
Wherein the elution buffer solution flow control passage has a switching curve portion for switching the flow of the eluting buffer solution from inside to outside in the radial direction. The method of claim 4,
Wherein the pre-processing unit further comprises a reaction liquid flow control passage extending from the reaction liquid chamber,
Wherein the reaction liquid flow control passage has a switching curve portion for switching the flow of the reaction liquid discharged from the reaction liquid chamber from the radially inner side to the outer side. The method of claim 4,
Wherein the pre-processing unit further comprises a capillary valve formed in a connection passage connecting the discharge passage and the waste liquid chamber. The method of claim 4,
The unit process unit may further include a running buffer liquid supply unit for supplying the running buffer liquid to the ICS,
Wherein the running buffer liquid supply portion includes a running buffer liquid chamber providing a space in which the running buffer liquid is stored, and a running buffer liquid flow control passage guiding the running buffer liquid to the ICS,
Wherein the running buffer fluid flow control passage includes a first inner changeover curve portion for changing the flow of the running buffer solution discharged from the running buffer solution chamber from the radially inner side to the outer side, An outer switching curve portion for switching the flow of the running buffer solution flowing outward in the radial direction and a second inner switching curve portion for turning the flow of the running buffer solution flowing radially inwardly by the outer switching curve portion into the radially outward direction, Wherein the microchip has a plurality of microchips. The method of claim 10,
The running buffer solution supply part further comprises a running buffer solution receiving part located radially outwardly of the running buffer solution chamber and connected to the running buffer solution flow control passage,
Wherein a passive valve formed by a height difference is provided between the running buffer solution chamber and the running buffer solution receiving portion. The method of claim 10,
Further comprising: a valve portion located between the first inside switching curve portion and the outside switching curve portion of the running buffer liquid flow control passage and beyond the second inside switching curve portion,
Wherein the valve unit is a passive valve formed by a height difference. The method of claim 2,
Wherein the pretreatment section further comprises a washing buffer solution introducing passage connecting the washing buffer solution chamber and an inlet of the trapping passage and an eluting buffer solution introducing passage connecting the inlet of the eluting buffer solution chamber and the trapping passage,
Wherein the wash buffer solution introduction passage has a capillary valve,
Wherein the elution buffer solution introduction passage has an elution buffer solution flow control passage having an inner transition curve portion for switching the flow of the elution buffer solution radially from the inside to the outside. And a unit processing unit disposed at a distance from the rotation center, wherein the unit processing unit comprises: a detection unit having an ICS for detecting pathogens; a preprocessing unit for performing preprocessing on the sample; Wherein the pretreatment unit comprises a target substance capturing unit having a trapping passage having an inlet and an outlet and a trapping means filled in the trapping passage, A sample storage portion located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a sample is stored, an elongated passage extending radially inward from the sample storage portion, , Located radially inward of the target material trapping portion and the wash buffer solution is stored A washing buffer solution introducing passage connecting the washing buffer solution chamber and the elongating passage, and a space located radially inward of the target material capturing portion and storing the eluting buffer solution. A reaction solution chamber for providing a space for storing a reaction solution required for a PCR process or an isothermal amplification process, an elution buffer solution flow chamber for connecting the elution buffer solution chamber and the extension passage, A reaction liquid flow control passage extending from the reaction liquid chamber; and a reaction liquid flow control passage extending radially outward of the target substance capturing portion and the reaction liquid chamber and extending along the circumferential direction, A discharge passage connected to the outlet of the discharge passage, And a target material chamber located radially outwardly of the discharge passage and connected to the discharge passage, wherein a portion of the discharge passage where the reaction solution chamber is connected and a portion where the target material chamber is connected Wherein the elution buffer solution flow control passage is opposed to each other and a portion where an outlet of the trapping passage is connected to the discharge passage and a portion to which the waste solution chamber is connected are opposed to each other, Wherein the reaction liquid flow control passage has a switching curve portion for switching the flow of the reaction liquid discharged from the reaction liquid chamber from the radially inner side to the outer side, Wherein the diagnosis target substance delivery unit is connected to the ICS, Wherein the diagnostic substance flow control passage comprises a diagnostic substance microchip having a conversion curve portion for converting the flow of the diagnostic target gene material stored in the target material chamber from the radially inner side to the outer side, As a method,
Preparing a diagnostic microchip for injecting a sample into the sample storage part, injecting a wash buffer solution into the wash buffer solution chamber, injecting an elution buffer solution into the elution buffer solution chamber, and injecting a reaction solution into the reaction solution chamber;
A preprocessing step of rotating the diagnostic microchip prepared by the diagnostic microchip preparation step to perform a pretreatment on the sample;
An amplification step of performing a PCR process or an isothermal amplification process on the target material pretreated in the pretreatment step; And
And a detection step of detecting a pathogen by transmitting the gene material to be diagnosed including the amplified gene material through the amplification step to the detection unit,
The pre-
A target material trapping step of rotating the diagnostic microchip in a first rotation direction so that the waste liquid chamber moves toward the target material at a first rotation speed to introduce the sample into the target material trapping portion;
A washing buffer liquid loading step of rotating the diagnostic microchip in the first rotation direction at a second rotation speed after the step of capturing the target material, to thereby introduce the washing buffer solution into the target material capturing part;
The rotation speed of the diagnostic microchip is reduced to 0 after the washing buffer solution loading step is performed so that the elution buffer solution and the reaction solution are mixed with the inside switching curve portion of the elution buffer solution flow control passage and the reaction solution flow control passage An eluting buffer solution and a reaction solution introducing step for allowing the solution to pass through the inner conversion curve portion;
The reaction liquid is loaded into the target material chamber by rotating the diagnostic microchip at a third rotation speed in a second rotation direction opposite to the first rotation direction after performing the elution buffer solution and the reaction liquid introduction step, step; And
And an elution buffer solution loading step of rotating the diagnostic microchip in the second rotation direction at a fourth rotation speed after the step of loading the reaction solution, so as to introduce the elution buffer solution into the target material chamber. 15. The method of claim 14,
Wherein the unit process section further comprises a run buffer liquid flow control passage through which the run buffer liquid is introduced into the ICS, the run buffer liquid flow control passage comprising a first inner switch for switching the flow of the running buffer liquid from radially inward to outward, An outer switching curve portion for switching the flow of the running buffer solution flowing radially outward by the first inner switching curve portion inward in the radial direction, and an outer switching curve portion for rotating the running buffer fluid flowing in the running buffer in the radial direction by the outer switching curve portion, And a second inner switching curve portion for switching the flow of the liquid radially outward,
The rotational speed of the diagnostic microchip is reduced to zero after the elution buffer solution loading step so that the diagnostic object substance stored in the target material chamber passes through the switching curve portion of the diagnostic substance flow control passage, Further comprising a step of supplying a target substance and a running buffer solution to pass through the second inside transition curve portion of the running buffer solution flow control passage. 18. The method of claim 17,
Wherein the running buffer liquid control flow passage further comprises a valve portion located past the second inner transition curve portion,
Further comprising a detecting preparation step in which the diagnosis target microorganism chip and the running buffer solution are supplied to the ICS by rotation of the diagnostic microchip that has been stopped after the amplification step. And a unit processing unit located apart from the rotation center,
The unit processing unit includes:
A trapping passage having an inlet and an outlet positioned radially outward of the inlet; and a trapping means having trapping means filled in the trapping passage;
A sample storage portion located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a sample is stored;
A washing buffer liquid chamber located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which a washing buffer solution is stored;
An elution buffer solution chamber located radially inward of the target substance capturing portion and connected to an inlet of the trapping passage and providing a space in which an elution buffer solution is stored;
A reaction solution chamber for providing a space in which a reaction solution necessary for the PCR process or the isothermal amplification process is stored;
A discharge passage located radially outward of the target substance capturing portion and the reaction solution chamber and extending along the circumferential direction and connected to the target substance capturing portion and the reaction solution chamber;
A waste liquid chamber located radially outwardly of the discharge passage and connected to the discharge passage;
And a target material chamber located radially outwardly of the discharge passage and connected to the discharge passage,
Wherein a portion of the discharge passage in which the waste liquid chamber is connected and a portion of the discharge passage in which the target material chamber is connected are spaced apart from each other in the circumferential direction,
Wherein a portion of the discharge passage which is connected to the reaction liquid chamber is positioned to face the portion of the discharge passage which is connected to the waste liquid chamber, And is positioned so as to face a portion to which the chamber is connected.

Images