KR101763510B1 - Microdevice integrated with ics and diagnosis method using the same - Google Patents

Microdevice integrated with ics and diagnosis method using the same Download PDF

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KR101763510B1
KR101763510B1 KR1020160001638A KR20160001638A KR101763510B1 KR 101763510 B1 KR101763510 B1 KR 101763510B1 KR 1020160001638 A KR1020160001638 A KR 1020160001638A KR 20160001638 A KR20160001638 A KR 20160001638A KR 101763510 B1 KR101763510 B1 KR 101763510B1
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lamp
solution
running buffer
channel
buffer solution
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KR20170017687A (en
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서태석
정재환
박현규
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한국과학기술원
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
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Abstract

According to the present invention, at least one unit prism (110) located apart from the center of rotation and disposed along the radial direction; And an RT-LAMP solution loading unit 120 for loading the RT-LAMP solution into each of the unit cavities. Each of the plurality of unit cavities includes a detection unit 111 having an ICS 180 for detecting pathogens, A RT-LAMP product supply part 115 disposed on one side in the circumferential direction with respect to the detection part and supplying an RT-LAMP product to the ICS, and a RT-LAMP product supply part 115 disposed on the opposite side of the RT- And a running liquid supply part (118) arranged to supply the running buffer solution to the ICS.

Description

TECHNICAL FIELD [0001] The present invention relates to a diagnostic microdevice having ICS and a rotary diagnostic method using the ICD.

The present invention relates to a microdevice for use in the detection of pathogens through gene analysis, and more particularly to a microdevice for diagnosing pathogens such as influenza viruses with high reliability by automating RT-LAMP reaction, Device and a diagnostic method using the same.

The pandemic influenza A virus has been reported to occur worldwide from about 250,000 to 500,000 deaths annually. The pandemic influenza A virus is spreading in more than 214 countries and causing a large number of deaths.

In particular, avian influenza (H5, H7, and H9 subtypes) are transmitted to humans and can have high mortality rates. Accordingly, it is necessary to develop a diagnostic tool for quickly carrying the influenza virus.

Reverse transcriptase polymerase chain reaction (RT-PCR) based gene analysis is considered to be an accurate diagnostic tool with the highest sensitivity for influenza A virus typing. However, RT-PCR methods require repeated thermal cycling processes and accurate temperature control measures, which leads to an increase in cost and time. To overcome these limitations, various isothermal amplification methods have been studied. Among these studies, LAMP is considered to be a fast, simple and highly sensitive isothermal amplification method. LAMP uses a combination of DNA polymerase and six LAMP primers to extend the target DNA template to 60-65 degrees Celsius within 1 hour.

RT-LAMP (reverse transcriptase loop-mediated isothermal amplification), in which a reverse transcriptase is added during the LAMP process, has been developed to apply LAMP for virus detection purposes. Various pathogens such as severe acute respiratory syndrome, influenza A virus and foot-and-mouth disease pathogens were analyzed by RT-LAMP method. The analysis of RT-LAMP products utilizes the turbidity analysis of fluorescence signals, colorimetric methods, etc. Particularly ICS-based colorimetric detection methods are widely used for low-cost and simple processes.

However, since the RT-LAMP reaction often causes contamination problems, a completely automated treatment process on the chip is required. Therefore, there is a need for an apparatus and method for automating RT-LAMP reactions and analyzing pathogens such as influenza virus with low cost and high reliability.

Accordingly, a problem to be solved by the present invention is to provide a diagnostic apparatus for analyzing a pathogen such as influenza virus with high reliability by automating the RT-LAMP reaction at low cost and a diagnostic method using the same.

According to an aspect of the present invention,

At least one unit prism (110) located apart from the center of rotation and disposed along the radial direction; And an RT-LAMP solution loading unit 120 for loading the RT-LAMP solution into each of the unit cavities. Each of the plurality of unit cavities includes a detection unit 111 having an ICS 180 for detecting pathogens, A RT-LAMP product supply part 115 disposed on one side in the circumferential direction with respect to the detection part and supplying an RT-LAMP product to the ICS, and a RT-LAMP product supply part 115 disposed on the opposite side of the RT- And a running liquid supply part (118) arranged to supply the running buffer solution to the ICS.

The RT-LAMP solution loading part includes a RT-LAMP solution dispensing channel 121 extending in a zigzag shape along the circumferential direction about a rotation center to form a capillary, And a plurality of RT-LAMP solution supply channels 125, which are connected to the RT-LAMP product supply unit at ends thereof.

The RT-LAMP solution supply channel 125 may extend radially outward from the outer protrusion 122 protruding radially outward from the RT-LAMP solution distribution channel.

The RT-LAMP solution loading unit may further include an annular air chamber 124 located radially inward of the RT-LAMP solution distribution channel and connected to the RT-LAMP solution distribution channel.

The air chamber may be connected to an inner protrusion 123 protruding radially inward from the RT-LAMP solution distribution channel.

The RT-LAMP solution supply channel is an even number, and two adjacent RT-LAMP solution supply channels along the radial direction of the plurality of RT-LAMP solution supply channels can be connected to the same RT-LAMP product supply channel.

The RT-LAMP product supply unit includes an RT-LAMP chamber 115a receiving a primer necessary for the RT-LAMP process and supplied with the RT-LAMP solution, and an RT- An RT-LAMP supply channel 117 may be provided.

The RT-LAMP product flow control channel further comprises an RT-LAMP product flow control channel connecting the RT-LAMP chamber and the RT-LAMP supply channel, And a switching curve portion 116a that converts the flow of the RT-LAMP product generated in the RT-LAMP chamber radially from the inside to the outside.

The RT-LAMP product supply unit may further include a capillary valve 116b located at the downstream end of the RT-LAMP product flow control channel.

The running buffer solution supply unit includes a running buffer solution chamber 118a for storing a running buffer solution and a running buffer solution introduction channel 119 for transferring the running buffer solution stored in the running buffer solution chamber to the ICS, The running buffer solution introduction channel includes a first inside switching curve portion 119c for switching the flow of the running buffer solution from inside to outside in the radial direction and a second inside switching curve portion 119c located downstream of the first inside switching curve portion, An outer switching curve portion 119d for switching from the outside to the inside and a second inside switching curve portion 119e located downstream of the outside switching curve portion and for changing the flow of the running buffer fluid from inside to outside in the radial direction Running buffer liquid flow control channel 119a.

The running buffer solution supply part may further include a capillary valve part 118c positioned radially outward of the running buffer solution chamber.

In order to achieve the object of the present invention, according to another aspect of the present invention,

A first pattern formation layer 140 on which the RT-LAMP solution loading part 120 is formed; An RT-LAMP chamber 115a connected to the first pattern formation layer and containing a primer necessary for the RT-LAMP process and receiving the RT-LAMP solution from the RT-LAMP solution loading section, a running buffer A second pattern formation layer 150 in which a liquid chamber 118a is formed; And an ICS (180) connected to the RT-LAMP chamber to receive the RT-LAMP product and to receive the running buffer solution from the running buffer solution chamber; And a third pattern formation layer in which an ICS receiving groove 171 in which the ICS is accommodated is formed and which is bonded to the second pattern formation layer on the opposite side of the first pattern formation layer.

The RT-LAMP solution loading unit includes an RT-LAMP solution distribution channel 121 formed in a zigzag shape extending in the circumferential direction about a rotation center on a surface facing the second pattern formation layer to form a capillary, LAMP solution supply channel 125 extending radially outward from the RT-LAMP solution distribution channel on a surface opposite to the second pattern formation layer, and an RT-LAMP solution supply channel 125 formed on the second pattern formation layer, And a connection hole 125a connecting the RT-LAMP chamber.

The RT-LAMP solution loading unit further includes an annular air chamber 124 formed on a surface facing the second pattern forming layer, and the air chamber 124 is located radially inward of the RT-LAMP solution distribution channel And can be connected to an inner protrusion 123 protruding radially inward of the RT-LAMP solution distribution channel.

The microdevice further comprises an RT-LAMP product supply channel (117) for supplying an RT-LAMP product to the ICS, wherein the RT-LAMP product supply channel is located between the second patterning layer and the third patterning layer A second channel portion 141 formed on a surface of the first pattern formation layer facing the second pattern formation layer, and a second channel portion 141 formed on the second pattern formation layer, A connection hole 153 for connecting the first channel part and the second channel part as holes, and a discharge hole 153 for discharging the RT-LAMP product by the ICS as a through hole formed in the second pattern formation layer can do.

The microdevice further includes a run buffer liquid supply channel (119b) for supplying a run buffer solution to the ICS, wherein the run buffer liquid supply channel is provided on a surface of the second pattern formation layer opposite to the third pattern formation layer A second channel section (143) formed on a surface of the first pattern formation layer opposite to the second pattern formation layer, and a second channel section (143) formed on the second channel formation section A connection hole 158 for connecting the first channel part to the second channel part and a discharge hole 159 for discharging the running buffer solution to the ICS as a through hole formed in the second pattern formation layer.

In order to achieve the object of the present invention, according to another aspect of the present invention,

A plurality of unit prongs (110) located apart from the rotation center and arranged in order along the radial direction; And an RT-LAMP solution loading unit (120) for uniformly loading the RT-LAMP solution into each of the unit cavities, wherein each of the plurality of unit cavities includes a detection unit (150) having an ICS A RT-LAMP product supply unit 115 disposed on one side in the circumferential direction with respect to the detection unit and supplying the RT-LAMP product to the ICS, and a RT-LAMP product supply unit 115 interposed between the detection unit and the RT- And a running liquid supply unit 118 disposed on the opposite side of the RT-LAMP chamber for supplying a running buffer solution to the ICS, wherein the RT-LAMP product supply unit includes an RT- Further comprising a LAMP product flow control channel (116), wherein the RT-LAMP product flow control channel comprises a switching curve portion (116a) for converting the flow of the RT-LAMP product produced in the RT-LAMP chamber from the radially inner side to the outer side ), And the run The buffer solution supply section includes a running buffer solution chamber 118a in which the running buffer solution is stored, a first inside switching curve section 119c for switching the flow of the running buffer solution from the inside to the outside in the radial direction, An outer switching curve portion 119d located downstream of the curved portion and switching the flow of the running buffer fluid from the outside to the inside in the radial direction and a downstream switching curve portion 119d located downstream of the outside switching curve portion, LAMP solution loading unit to load the RT-LAMP solution into the RT-LAMP solution loading unit, wherein the RT-LAMP solution loading unit includes a running buffer liquid flow control channel (119a) step; Rotating the diagnostic microdevice to supply the RT-LAMP solution to the RT-LAMP chamber; Stopping the rotation of the diagnostic microdevice to allow the passage of the outside switching curve portion 119d of the RT-LAMP solution and the passage of the first inside switching curve portion of the running buffer solution; Performing an RT-LAMP process in the RT-LAMP chamber to generate an RT-LAMP product; Rotating the diagnostic microdevice to supply an RT-LAMP product to the ICS; Stopping rotation of the diagnostic microdevice to allow passage of the second inner transition curve portion of the running buffer solution; And rotating the diagnostic micro device to supply the running buffer solution to the ICS.

According to the present invention, it is possible to achieve all of the above-mentioned problems to be solved by the present invention. Specifically, the present invention provides a rotatable multi-RT-LAMP-ICS microdevice to detect target viruses such as influenza A virus. Loading and dispensing of the RT-LAMP solution and the running buffer solution can be performed accurately on chip by only RPM control and microchannel design. In addition, the H1 and M genes amplified by isothermal reaction can be analyzed in the ICS immediately inside the chip, and as a result, the influenza A H1N1 subtype can be identified. Furthermore, it is possible to detect viruses even at a low concentration of 10 copies. By combining ICS and RT-LAMP in one device, highly reliable and portable virus detection devices can be realized.

1 is a perspective view showing a microdevice for diagnosis according to an embodiment of the present invention.
2A is a plan view showing the microdevice for diagnosis shown in FIG.
FIG. 2B is an enlarged view of one unit cavity and the RT-LAMP solution loading unit in FIG.
Fig. 3 is a perspective view showing the layers of the microdevice for diagnosis shown in Fig. 1 separated. Fig.
4 is a plan view of the cover layer shown in Fig.
5 is a plan view of the first pattern formation layer shown in FIG.
6 is a plan view of the second pattern formation layer shown in FIG.
7 is a plan view of the third pattern formation layer shown in Fig.
8 is a cross-sectional view taken along the line A-A 'in FIG. 2B.
9 is a cross-sectional view taken along the line B-B 'in FIG. 2B.
10 is a cross-sectional view taken along the line C-C 'in FIG. 2B.
11 is a cross-sectional view taken along the line D-D 'in FIG. 2B.
12A to 12H are diagrams illustrating a process of performing diagnosis in the microdevice shown in FIG.
Fig. 13 shows gene expression detection results according to the diagnostic method of the present invention.
FIG. 14 shows the results of ICS detection of gene expression over time by the diagnostic method of the present invention.
FIG. 15 shows LOD detection results according to the diagnostic method of the present invention.
FIG. 16 shows the results of specificity test for virus subtypes according to the diagnostic method of the present invention.

In order to solve the above problems, the present invention provides a direct-coupled centrifugal microdevice to perform a multiplex RT-LAMP reaction and detect ICS-based amplicons against influenza A virus type and subtype .

The RT-LAMP-ICS direct chip according to an embodiment of the present invention is patterned into three on a chip, and multistage gene expression is possible. Model of the influenza A H1N1 virus, in one embodiment of the invention, hemagglutinin (HA) and conserved matrix (M) gene expression was measured with a microdevice according to the invention for 1 hour , And subtype of influenza A H1N1 virus could be subtype.

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

FIG. 1 is a perspective view showing a microdevice for diagnosis according to an embodiment of the present invention, FIG. 2a is a plan view showing the microdevice for diagnosis shown in FIG. 1, FIG. 2b is an enlarged view to be. Referring to FIGS. 1 and 2B, the diagnostic microdevice 100 according to an embodiment of the present invention is generally disk-shaped, and its center of rotation O is provided at its center. The microdevice 100 for diagnostics includes a plurality of unit cells 110 arranged in a radial direction from the rotation center O and arranged in the circumferential direction in order and a plurality of unit cells 110 arranged in the order of RT- And an RT-LAMP solution loading unit 120 for uniformly loading the solution. In the present embodiment, it is described that there are three unit security units 110, but the present invention does not limit the number of unit security units 110 to three. The present invention also includes the case where the diagnostic microdevice has two or less unit processing parts or four or more unit processing parts. Although the diagnostic microdevice 100 is described as being in the form of a disk in the present embodiment, the present invention does not limit the diagnostic microdevice 100 to a disk shape.

The diagnostic microdevice 100 is a multi-layered structure, in which each layer of the diagnostic microdevice 100 is shown separately in Fig. 3, the diagnostic microdevice 100 includes a cover layer 130, a first pattern formation layer 140 located under the cover layer 130, and a second pattern formation layer 140 located under the first pattern formation layer 140 A first bonding film layer 160 which bonds the first pattern forming layer 140 and the second pattern forming layer 150 and a second bonding film layer 160 which is located below the second pattern forming layer 150, A plurality of ICS (Immunochromatographic Strip) (ICS) 180, and a second pattern formation layer 150, which are mounted on the third pattern formation layer 170, And a second bonding film layer 190 for bonding the third pattern forming layer 170 to each other. The cover layer 130, the first pattern forming layer 140, the second pattern forming layer 150, and the third pattern forming layer 170 are made of a synthetic resin material such as polycarbonate (PC). In the present invention, Are not limited thereto. In this embodiment, the cover layer 130 is 2 mm thick, each of the three patterning layers 140 is 1 mm thick, and each of the two bonding film layers 160 and 190 is 50 μm thick. Except for the cover layer 130, the remaining layers are precisely assembled using circular markers and strongly bonded by thermocompression bonding.

FIG. 4 is a plan view of the cover layer shown in FIG. 3, FIG. 5 is a plan view of the first pattern formation layer shown in FIG. 3, 3 is a plan view of the third pattern formation layer shown in Fig. 2A is a cross-sectional view taken along the line A-A 'in FIG. 2A, FIG. 9 is a cross-sectional view taken along the line B-B' in FIG. 2A, Is a cross-sectional view taken along the line D-D 'in FIG. 2A. Hereinafter, each configuration of the diagnostic microdevice 100 will be described in detail with reference to Figs. 1 to 11. Fig.

2A and 2B, the unit unit 110 includes a detection unit 111, an RT-LAMP product supply unit 115 disposed on one side in the circumferential direction with respect to the detection unit 111, And a running buffer liquid supply part 118 located on the opposite side of the RT-LAMP product supplying part 115. [

The detecting unit 111 includes an ICS 180 accommodated in an ICS receiving groove 171 formed in the third pattern forming layer 170 and detecting pathogens. The ICS receiving groove 171 is formed in the third pattern forming layer 170 on the surface facing the second pattern forming layer 150.

The ICS 180 has a generally thin rod shape and is accommodated in the ICS receiving groove 171 so as to extend generally along the radial direction with respect to the rotation center O and has an RT- And the buffer solution supply unit 118. ICS 180 includes a support 181, a buffer solution loading pad 182, an absorption pad 183, a detection pad 184, and a conjugate pad 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. When the ICS 180 is accommodated in the ICS receiving groove 171, the supporting member 181 contacts the bottom of the ICS receiving groove 171 and the buffer liquid loading pad 182, the absorption pad 183 and the detection pad 184 Is directed to the second pattern formation layer 150 side.

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 118. 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 attached to the upper surface of the detection pad 184 and the RT-LAMP product to be diagnosed is supplied from the RT-LAMP product supply part 115 to the conjugate pad 185. The conjugate pad 185 includes a flowable conjugate that specifically binds to the detection target substance contained in the RT-LAMP product.

The RT-LAMP product supply unit 115 includes an RT-LAMP chamber 115a, an RT-LAMP product flow control channel 116, and an RT-LAMP product supply channel 117.

The RT-LAMP chamber 115a is formed on the surface of the second pattern formation layer 150, which faces the third pattern formation layer 170. [ The RT-LAMP chamber 115a is filled with a freeze-dried primer combination (A) labeled with hapten. The surface of the second pattern formation layer 150 opposite to the third pattern formation layer 170 is provided with an RT-LAMP solution inflow space (not shown) located radially inward of the RT-LAMP chamber 115a, (Not shown). The heat shielding structures 101 and 102 are formed around the RT-LAMP chamber 115a. The heat shielding structures 101 and 102 are in the form of penetrating slots and are prevented from being thermally diffused into the ICS 180 during the RT-LAMP reaction by the heat shielding structures 101 and 102.

The RT-LAMP product flow control channel 116 is formed extending from the RT-LAMP chamber 115a on the surface of the second pattern formation layer 150, which faces the third pattern formation layer 170. [ The RT-LAMP product flow control channel 116 extends generally radially inwardly from the RT-LAMP chamber 115a and then smoothly curves and is extended in a radially outward direction with the end turned to the RT-LAMP product supply channel 117). Accordingly, the RT-LAMP product flow control channel 116 has a transition curve portion 116a that converts the flow of the RT-LAMP product from radially inward to outward. At the downstream end of the RT-LAMP product flow control channel 116, a circular capillary valve (e.g., 1 mm deep) 116b is patterned to prevent the solution from leaking to the ICS 180 during the RT-LAMP reaction .

The RT-LAMP product supply channel 117 extends from the end of the RT-RAMP product flow control channel 116 to the RT-LAMP product feed channel 117, which extends obliquely outward generally radially outward, To the conjugate pad 185 of the substrate 180. The RT-LAMP product supply channel 117 includes a first channel portion 152 formed in the second pattern formation layer 150, a second channel portion 141 formed in the first pattern formation layer 140, A connection hole 153 formed in the first pattern formation layer 150 and a discharge hole 155 formed in the second pattern formation layer 150.

The first channel portion 152 is formed on a surface of the second pattern formation layer 150 that faces the third pattern formation layer 170. The first channel portion 152 extends obliquely toward the ICS 180 from the end of the RT-RAMP product flow control channel 116 generally radially outward.

The second channel portion 141 is formed on a surface of the first pattern formation layer 140 facing the second pattern formation layer 150. The second channel portion 141 extends generally radially outward from the radial end of the first channel portion 152 and is positioned at the upper end of the conjugate pad 185 of the ICS 180.

The connection through hole 153 is a through hole formed in the second pattern formation layer 150 and connects the first channel part 152 and the second channel part 141.

The through hole 155 is a through hole formed in the second pattern formation layer 150 and connects the radially outer end of the second channel portion 141 and the upper portion of the conjugate pad 185 of the ICS 180. The RT-LAMP product exiting through the vent hole 155 is supplied to the conjugate pad 185 of the ICS 180.

The running buffer solution supply part 118 is located across the RT-LAM product supply part 115 with the detection part 111 therebetween. The running buffer solution supply unit 118 supplies the running buffer solution to the buffer solution loading pad 182 of the ICS 180. [ The running buffer solution supply portion 118 includes a running buffer solution chamber 118a, a running buffer solution receiving portion 118b, and a running buffer solution introduction channel 119. [

The running buffer solution chamber 118a is located opposite the RT-LAMP product supply portion 115 with the ICS 180 therebetween. A running buffer solution is stored in the running buffer solution chamber 118a. The running buffer solution is composed of 25 mM sodium carbonate buffer solution (pH 9.6), 1% casein, 0.4 mM EDTA, 0.2% alpha-cyclodextrin, 0.2% Triton-100, 0.4 M UREA, The present invention is not limited thereto as an example. The running buffer solution assists in the flow of the RT-LAMP product, which is the target gene material, in the ICS (180). The running buffer solution chamber 118a is formed on the surface of the second pattern formation layer 150 that faces the third pattern formation layer 170. [ The volume of the running buffer solution chamber 118a is 50 mu L.

The running buffer solution reservoir 118b is located radially outward of the running buffer solution chamber 118a and the running buffer solution chamber 118a is located in the second pattern formation layer 150 and the third pattern formation layer 170, And is formed on the opposite surface. A valve portion 118c is provided between the running buffer solution receiving portion 118b and the running buffer solution chamber 190. [ The valve portion 118c is a manual valve in the form of a capillary (depth 50 占 퐉) formed by a height difference so that the running buffer solution stored in the running buffer solution chamber 118a can not easily move to the running buffer solution receiving portion 118b . After the running buffer liquid stored in the running buffer liquid chamber 118a is passed through the valve portion 118c by centrifugal force generated when the diagnostic microdevice 100 rotates around the rotation center O, 118b.

The running buffer solution introduction channel 119 connects the radially outer end of the running buffer solution receiving portion 118b and the buffer solution loading pad 182 of the ICS 180. [ The running buffer solution introduction channel 119 has a running buffer solution flow control channel 119a and a running buffer solution supply channel 119b.

The running buffer solution flow control channel 119a is formed to extend from the radially outer end of the running buffer solution receiving portion 118b on the surface opposite to the third pattern forming layer 170 in the second pattern forming layer 150 . The running buffer liquid flow control channel 119a is formed by a smooth curve from the radial end portion of the running buffer liquid receiving portion 118b 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. Accordingly, the running buffer liquid flow control channel 119a includes a first inside switching curve portion 119c and a second switching curve portion 119d 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 119e. The first inside switching curve portion 119c switches the flow of the running buffer solution from the inside to the outside of the radial direction to introduce the running buffer solution to the outside switching curve portion 119d. The outer switching curve portion 119d switches the flow of the running buffer liquid from the radially outer side to the inward side to flow the running buffer solution into the second inner switching curve portion 119e. The second inside switching curve portion 119e turns the flow of the running buffer solution from the inside to the outside in the radial direction to flow the running buffer solution into the running buffer solution supply channel 119b. The running buffer solution flow control channel 119a is treated with a Vistex solution to become hydrophilic.

The running buffer solution supply channel 119b extends from the end of the running buffer solution flow control channel 119a to the ICS 180 by extending the running buffer solution slanting generally radially outwardly from the running buffer solution flow control channel 119a, To the buffer solution loading pad 182 of FIG. The running buffer solution supply channel 119b includes a first channel portion 157 formed in the second pattern formation layer 150, a second channel portion 143 formed in the first pattern formation layer 140, 150, and a discharge hole 159 formed in the second pattern formation layer 150. The connection hole 158 is formed in the second pattern formation layer 150,

The first channel part 157 is formed on the surface of the second pattern formation layer 150 facing the third pattern formation layer 170. The first channel portion 157 extends obliquely toward the ICS 180 from the end of the running buffer liquid flow control channel 119a generally radially outward.

The second channel portion 143 is formed on a surface of the first pattern formation layer 140 that faces the second pattern formation layer 150. The second channel portion 143 extends generally radially outward from the radial end of the first channel portion 157 and is positioned at the top of the buffer solution loading pad 182 of the ICS 180.

The connection through hole 158 is a through hole formed in the second pattern formation layer 150 and connects the first channel part 157 and the second channel part 143.

The through hole 159 is a through hole formed in the second pattern formation layer 150 and connects the radially outer end of the second channel portion 143 and the upper portion of the buffer solution loading pad 182 of the ICS 180 . The running buffer liquid discharged through the discharge opening 159 is supplied to the buffer liquid loading pad 182 of the ICS 180. [

The RT-LAMP solution loading unit 120 uniformly loads the RT-LAMP solution into each of the plurality of unit cells 110. In this example, an RT-LAMP cocktail (23 μL volume) was mixed with a 2 μL volume of viral RNA template solution as an RT-LAMP solution. The RT-LAMP solution loading section 120 includes an RT-LAMP solution distribution channel 121, an air chamber 124, a plurality of RT-LAMP solution supply channels 125, a sample injection port 126, A channel 127, a sample outlet 128, and an outlet connection channel 129.

The RT-LAMP solution distribution channel 121 is formed by extending in the zigzag shape along the circumferential direction about the rotation center O. The RT-LAMP distribution channel 121 is formed on the surface of the first pattern formation layer 140 opposite to the second pattern formation layer 150, for example, with a width of 580 mu m and a depth of 150 mu m to form a capillary. The RT-LAMP solution distribution channel 121 is positioned radially inward of the RT-LAMP solution inflow space 151. In this embodiment, six outward protrusions 122 protruding radially outward in the RT-LAMP solution distribution channel 121 are described, which is twice the number of the RT-LAMP chambers 115a, The number of the RT-LAMP chambers 122 may vary depending on the number of the RT-LAMP chambers 115a. In the RT-LAMP solution distribution channel 121, the RT-LAMP solution is automatically filled by the capillary phenomenon, and the solution filled in the RT-LAMP solution distribution channel 121 is stably stored and does not leak into the neighboring channel. This is due to the channel depth difference.

The air chamber 124 has an annular shape centered on the rotation center O and is formed at a depth of, for example, 500 탆 on the surface of the first pattern formation layer 140 facing the second pattern formation layer 150. The air chamber 124 is radially inward of the RT-LAMP solution distribution channel 121 and is connected to an inner protrusion 123 protruding radially inwardly of the RT-LAMP solution distribution channel 121, The air pressure is applied to the RT-LAMP solution distribution channel 121.

A plurality of RT-LAMP solution supply channels 125 are formed in the first patterning layer 140 on the surface facing the second patterning layer 150. Each of the RT-LAMP solution supply channels 125 extends radially outward from the outer protrusion 122 of the RT-LAMP solution distribution channel 121, -LAMP solution inflow space 151. In this case, The ends of the two connected RT-LAMP solution supply channels 125 are connected to the RT-LAMP solution inflow space 151 by the connection holes 125a formed in the second pattern formation layer 150.

The sample injection port 126 is for injecting the RT-LAMP solution and is formed in the first pattern formation layer 140 in the form of a through-hole.

The inlet connection channel 127 is formed in the first pattern formation layer 140 on the surface facing the second pattern formation layer 150 and connects the sample injection port 126 and one end of the RT-LAMP solution distribution channel.

The sample outlet 128 is for discharging the RT-LAMP solution and is formed in the first pattern forming layer 140 in the form of a through-hole.

The outlet connection channel 129 is formed on a surface of the first pattern formation layer 140 opposite to the second pattern formation layer 150 and connects the sample outlet 128 to the other end of the RT-LAMP solution distribution channel.

The diagnostic microdevice 100 described above is manufactured as follows. First, a mixture of RT-LAMP H1 and M gene primers (0.3 μL each) was dropped on the RT-LAMP chamber 115a of the second pattern formation layer 150 before bonding each layer, Lt; / RTI > The RT-LAMP primer mixture consisted of 10 pmol of each outer primer (F3 and B3), 100 pmol of each inner primer (FIP and BIP), and 50 pmol of each loop primer (LF and LB) at a volume of 3 μL . The overall thickness of the microdevice according to the present invention was 3.10 mm except for the cover layer 130 and the cover layer 130 was applied to the device before the isothermal amplification process of the RT-LAMP-ICS microdevice, For example.

A diagnostic method using the diagnostic microdevice 100 of the present invention will be described as follows.

First, the RT-RAMP solution and the running buffer solution are injected into the micro device 100 before the cover layer 130 is finally bonded. The RT-RAMP solution was prepared by mixing RT-LAMP cocktail (23 μL volume) with 2 μL volume of viral RNA template solution and injecting 7 μL of RT-LAMP solution through sample inlet 126 of microdevice 100 Respectively. The RT-LAMP solution (S) directly fills the RT-LAMP solution distribution channel 121 by capillary force within one minute, as shown in FIG. 12A. The running buffer solution (R) (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, 0.1% sodium azide) As shown in 12a, 50 占 를 was loaded into the running buffer solution chamber 118a. Simultaneously with the loading of the RT-LAMP solution (S) and the running buffer solution (R), the sample inlet 126, the sample outlet 128 and the running buffer solution chamber 118a are tightly sealed with an adhesive film, prevent.

Next, the micro device 100 is rotated for 1 minute at a rotation speed of 4000 RPM using a rotating device. The relative position of the micro device 100 at this time is shown in FIG. 12B. As shown in FIG. 12B, the RT-LAMP solution S is accurately divided into three parts and moves uniformly through the RT-LAMP solution supply channel 125 toward the three RT-LAMP chambers 115a, LAMP solution S is injected into the RT-LAMP chamber 115a by 2 L as shown in FIG. 12C and the RT-LAMP solution S is injected into the RT-LAMP chamber 115a by the switching curve 116a. As shown in FIG. The lyophilized freeze-dried primer combination (A) in the RT-LAMP chamber 115a is rehydrated by the loaded RT-LAMP solution (S). The remaining RT-LAMP solution that has not flowed into the RT-LAMP chamber 115a moves to the sample discharge port (128 in FIG. 12A) and the sample discharge port (128 in FIG. 12C, the running buffer solution R passes through the valve portion 118c and is received in the running buffer solution receiving portion 118b and is connected to the first inward switching of the running buffer tank flow control channel 119a So that it can not pass through the curved portion 119c.

Next, when the rotation is stopped, the RT-LAMP solution S passes through the switching curve portion 116a and the running buffer solution R passes through the first inner switching curve portion 119c and then stops This state is as shown in Fig. 12D. Next, the RT-LAMP reaction is performed at 66 degrees Celsius for 40 minutes using the microdevice 100 to which the cover layer 130 is bonded after bonding the cover layer 130 in FIG. 3, RT-LAMP product (T) is obtained. The RT-LAMP product (T) is labeled with biotin from biotin-dUTP and hapten from the loop primer.

After the RT-RAMP reaction, microdevice 100 is again rotated at 800 RMP for 1 minute to feed the amplified RT-LAMP product (T) to ICS 180, as shown in FIG. 12f. 12F, the RT-LAMP product T is supplied to the conjugate pad 185 of the ICS 180 via the RT-LAMP product supply channel 117, and the running buffer solution R is supplied to the outer switching Passes through the curve 119d, and then stops. In this state, when the rotation is stopped, the running buffer solution R passes through the second inside switching curve portion 119e, and is further rotated at 800 RPM for 30 seconds. As shown in FIG. 12G, 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 channel 119b. Thereafter, the rotation is stopped, and the RT-LAMP product is loaded into the conjugate pad 185 and bound by the biotin-streptavidin interaction with the gold nanoparticles.

The binding of the gold nanoparticles to the RT-LAMP product migrates to the test line area and is captured within 15 minutes through hapten and anti-hapten interaction (see FIG. 12H).

The expression of M gene and / or HA gene can be determined according to the colorimetric ICS detection result. Test line 1 (labeled with Digoxigenin antibody) indicates M gene expression and test line 2 (labeled with Texas Red) indicates HA gene expression.

The monophlase target of the H1 and M genes of the influenza A H1N1 virus was detected using the RT-LAMP-ICS microdevice according to the present invention. In one embodiment of the invention, the H1 and M gene target primer combinations were dried in different RT-LAMP chambers. As a negative control, no primer combination was injected into one RT-LAMP chamber. The RT-LAMP solution containing the influenza A H1N1 virus template was simultaneously loaded into the sample inlet and the three RT-LAMP reaction chambers. Multiple monoplex gene expression detection was performed in ICS (see Fig. 13). H1 gene expression was confirmed by the purple line of test line 2 (see Fig. 13 (b) (i)). M gene expression was also confirmed by positive signals in test line 1 and control line (see Fig. 13 (b) (ii)). However, in the negative control experiment, purple lines were confirmed only in the control line (see Fig. 13 (b) (iii)).

In order to demonstrate the speed-improving effect in detecting influenza A virus using the microsystem according to the present invention, the multiple RT-LAMP reaction time was adjusted to 60 minutes to 20 minutes.

Influenza A H1N1 viral RNA was used as template and H1 and conserved M genes were selected as targets. In one RT-LAMP chamber, the H1 and M gene primer combinations were loaded together and then lyophilized. The amount of viral RNA was fixed at 10 6 .

All tests were repeated at least 3 times. Influenza A H1N1 virus was successfully detected through the identification of three purple lines, the three purple lines representing H1 and M gene expression. As the reaction time increased, the band signal became stronger and sharper (see FIG. 14). Even at RT-LAMP reaction times of 30 min, multiplex H1 and M gene products were detected via ICS. At the 20 minute reaction time, the band signal in ICS appeared too blurry and the detection was not confirmed with high reliability.

Despite the small amount, influenza viruses are likely to spread to epidemics, so a highly sensitive diagnostic approach is needed. In order to confirm the sensitivity of the multiplex RT-LAMP-ICS microdevice according to the present invention, a detection limit (LOD) method is employed. Influenza A H1N1 viral RNA was serially diluted 10-fold and subjected to multiplex RT-LAMP reaction at 60 ° C for 40 minutes. 40 minutes is the reaction time chosen to increase the success even with a small number of RNAs. Multiplex RT-LAMP reactions were performed at least three times. As shown in Fig. 15, the multiplexed targets of the H1 and M genes were successfully amplified and detected in ICS (three band signals). A positive signal was also detected in test lines 1 and 2, even when the H1N1 viral RNA was 10 copies. The total process time was 55 minutes including the RT-LAMP reaction time of 40 minutes.

To determine the selectivity of the multiplex RT-LAMP reaction, three different reactions were performed. One target template (influenza A H1N1 viral RNA) was used for this in a single RT-LAMP-ICS microdevice. Three types of RT-LAMP primers targeting the H1 and M genes, the H3 and M genes, and the H5 and M genes were prepared, and subtypes of influenza A H1N1, influenza A H3N2 and influenza A H5N1, respectively.

Each primer combination is separately loaded into different RT-LAMP chambers and then dried. The RT-LAMP solution containing influenza A H1N1 viral RNA is separated into three RT-LAMP chambers and a multiplex isothermal reaction at 66 ° C for 40 minutes is performed simultaneously.

Figure 16 shows the result of a multiplex RT-LAMP reaction in the device.

16, only the RT-LAMP chamber containing the H1 & M gene target primer combination produced three bands on ICS. H3 and H5 target primers were not specific for influenza A H1N1 viral RNA, Showed only two positive bands (test line 1 and control line for the M gene). These results indicate that rapid and specific detection of influenza A virus is possible according to the combination of the specific primer design and the multiplex RT-LAMP-ICS device according to the invention.

As described above, the present invention provides a rotary type multi-RT-LAMP-ICS microdevice to detect target viruses such as influenza A virus. Loading and dispensing of the RT-LAMP solution and the running buffer solution can be performed accurately on chip by only RPM control and microchannel design. In addition, the H1 and M genes amplified by isothermal reaction can be analyzed in the ICS immediately inside the chip, and as a result, the influenza A H1N1 subtype can be identified. Furthermore, it is possible to detect viruses even at a low concentration of 10 copies. By combining ICS and RT-LAMP in one device, highly reliable and portable virus detection devices can be realized.

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: microdevice 110:
111: Detection unit 115: RT-LAMP product supply unit
115a: RT-LAMP chamber 116: RT-LAMP product flow control channel
117: RT-LAMP product supply channel 118: Run buffer liquid supply unit
118a: Running buffer solution chamber 118b: Run buffer solution reservoir
119: Running buffer solution introduction channel 119a: Runnig buffer solution flow control channel
119b: Run buffer liquid supply channel 120: RT-LAMP solution loading unit
130: Cover layer 140: First pattern forming layer
150: second pattern forming layer 160: first bonding film layer
170: Third pattern forming layer 180: ICS
190: second bonding film layer

Claims (17)

At least one unit prism (110) located apart from the center of rotation and disposed along the radial direction; And
And an RT-LAMP solution loading unit (120) for loading the RT-LAMP solution into each of the unit cavities,
Each of the plurality of unit cavities includes a detection unit 111 having an ICS 180 for detecting pathogens, an RT-LAMP product supply unit 110 disposed on one side in the circumferential direction with respect to the detection unit and supplying the RT- And a running liquid supply unit (118) disposed on the opposite side of the RT-LAMP product supply unit (115) with the detection unit interposed therebetween to supply the running buffer solution to the ICS, characterized in that the diagnostic microdevice . The method according to claim 1,
The RT-LAMP solution loading unit includes:
An RT-LAMP solution distribution channel 121 formed to extend in a zigzag shape along the circumferential direction about the rotation center to form a capillary,
And a plurality of RT-LAMP solution supply channels (125) extending radially outward from the RT-LAMP solution distribution channel and having ends connected to the RT-LAMP product supply channel. The method of claim 2,
Wherein the RT-LAMP solution supply channel (125) extends radially outward from an outer protrusion (122) protruding radially outwardly from the RT-LAMP solution distribution channel. The method of claim 2,
The RT-LAMP solution loading unit includes:
Further comprising an annular air chamber (124) connected to the RT-LAMP solution distribution channel,
Wherein the air chamber is located radially inward of the RT-LAMP solution distribution channel. The method of claim 4,
Wherein the air chamber is connected to an inner protrusion (123) protruding radially inward in the RT-LAMP solution distribution channel. The method of claim 2,
The two RT-LAMP solution supply channels 125 are even numbered, and two RT-LAMP solution supply channels 125 adjacent to each other in the radial direction of the plurality of RT-LAMP solution supply channels are connected to the same RT- Wherein the microdevice is connected to the microdevice. The method according to claim 1,
The RT-LAMP product supply unit includes an RT-LAMP chamber 115a receiving a primer necessary for the RT-LAMP process and supplied with the RT-LAMP solution, and an RT- And an RT-LAMP supply channel (117). The method of claim 7,
The RT-LAMP product supply unit further includes an RT-LAMP product flow control channel 116 connecting the RT-LAMP chamber and the RT-LAMP supply channel 117,
Wherein the RT-LAMP product flow control channel comprises a switching curve portion (116a) for converting the flow of the RT-LAMP product generated in the RT-LAMP chamber radially from the inside to the outside, The method of claim 8,
Wherein the RT-LAMP product supply further comprises a capillary valve (116b) located at the downstream end of the RT-LAMP product flow control channel. The method according to claim 1,
The running buffer solution supply part
A running buffer solution chamber 118a in which a running buffer solution is stored,
And a running buffer solution introduction channel (119) for transferring the running buffer solution stored in the running buffer solution chamber to the ICS,
The running buffer solution introducing channel includes a first inside switching curve portion 119c for switching the flow of the running buffer solution from the inside to the outside in the radial direction and a second inside switching curve portion 119c located downstream of the first inside switching curve portion, An outer switching curve portion 119d for switching the direction from the outside to the inside and a second inside switching curve portion 119e located downstream of the outside switching curve portion and for switching the flow of the running buffer solution from inside to outside in the radial direction , And a running buffer liquid flow control channel (119a). The method of claim 10,
Wherein the running buffer solution supply part further comprises a valve part (118c) of a capillary structure located radially outward of the running buffer solution chamber. A first pattern formation layer 140 on which the RT-LAMP solution loading part 120 is formed;
An RT-LAMP chamber 115a connected to the first pattern formation layer and containing a primer necessary for the RT-LAMP process and receiving the RT-LAMP solution from the RT-LAMP solution loading section, a running buffer A second pattern formation layer 150 in which a liquid chamber 118a is formed; And
An ICS (180) connected to the RT-LAMP chamber to receive the RT-LAMP product and to receive the running buffer solution from the running buffer solution chamber; And
And a third pattern formation layer in which an ICS receiving groove (171) in which the ICS is accommodated is formed and which is bonded to the second pattern formation layer on the opposite side of the first pattern formation layer. The method of claim 12,
The RT-LAMP solution loading unit includes:
An RT-LAMP solution distribution channel 121 formed in a zigzag shape extending in the circumferential direction around the rotation center on the surface facing the second pattern formation layer to form a capillary,
An RT-LAMP solution supply channel 125 extending radially outward from the RT-LAMP solution distribution channel on a surface facing the second pattern formation layer,
And a connection hole (125a) formed in the second pattern formation layer and connecting the RT-LAMP solution supply channel and the RT-LAMP chamber. 14. The method of claim 13,
The RT-LAMP solution loading unit further includes an annular air chamber 124 formed on a surface facing the second pattern formation layer,
The air chamber (124) is radially inward of the RT-LAMP solution distribution channel and connected to an inner protrusion (123) protruding radially inward of the RT-LAMP solution distribution channel. device. The method of claim 12,
Further comprising an RT-LAMP product supply channel (117) for feeding the RT-LAMP product to the ICS,
Wherein the RT-LAMP product supply channel comprises:
A first channel portion 152 formed on a surface of the second pattern formation layer opposite to the third pattern formation layer and a second channel portion 152 formed on a surface of the first pattern formation layer opposite to the second pattern formation layer 141), a connection hole (153) connecting the first channel part and the second channel part as a through hole formed in the second pattern formation layer, and a through hole formed in the second pattern formation layer And a discharge aperture (153) for discharging the LAMP product. The method of claim 12,
And a running buffer solution supply channel 119b for supplying a running buffer solution to the ICS,
Wherein the running buffer solution supply channel comprises:
A first channel portion 157 formed on a surface of the second pattern formation layer opposite to the third pattern formation layer and a second channel portion 157 formed on a surface of the first pattern formation layer facing the second pattern formation layer 143), a connection hole (158) connecting the first channel part and the second channel part as through holes formed in the second pattern formation layer, and a through hole (158) formed in the second pattern formation layer And a discharge hole (159) for discharging the buffer solution. A plurality of unit prongs (110) located apart from the rotation center and arranged in order along the radial direction; And an RT-LAMP solution loading unit (120) for uniformly loading the RT-LAMP solution into each of the unit cavities, wherein each of the plurality of unit cavities includes a detection unit (150) having an ICS A RT-LAMP product supply unit 115 disposed on one side in the circumferential direction with respect to the detection unit and supplying the RT-LAMP product to the ICS, and a RT-LAMP product supply unit 115 interposed between the detection unit and the RT- And a running liquid supply unit 118 disposed on the opposite side of the RT-LAMP chamber for supplying a running buffer solution to the ICS, wherein the RT-LAMP product supply unit includes an RT- Further comprising a LAMP product flow control channel (116), wherein the RT-LAMP product flow control channel comprises a switching curve portion (116a) for converting the flow of the RT-LAMP product produced in the RT-LAMP chamber from the radially inner side to the outer side ), And the run The buffer solution supply section includes a running buffer solution chamber 118a in which the running buffer solution is stored, a first inside switching curve section 119c for switching the flow of the running buffer solution from the inside to the outside in the radial direction, An outer switching curve portion 119d located downstream of the curved portion and switching the flow of the running buffer fluid from the outside to the inside in the radial direction and a downstream switching curve portion 119d located downstream of the outside switching curve portion, And a running buffer liquid flow control channel (119a) having a second inside switching curve section (119e)
Loading the RT-LAMP solution into the RT-LAMP solution loading section;
Rotating the diagnostic microdevice to supply the RT-LAMP solution to the RT-LAMP chamber;
Stopping the rotation of the diagnostic microdevice to allow the passage of the outside switching curve portion 119d of the RT-LAMP solution and the passage of the first inside switching curve portion of the running buffer solution;
Performing an RT-LAMP process in the RT-LAMP chamber to generate an RT-LAMP product;
Rotating the diagnostic microdevice to supply an RT-LAMP product to the ICS;
Stopping rotation of the diagnostic microdevice to allow passage of the second inner transition curve portion of the running buffer solution;
And rotating the diagnostic microdevice to supply the running buffer solution to the ICS.
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