KR20220096143A - Immunoassay automation device - Google Patents

Abstract

The present invention relates to an automated immunoassay device which comprises: a lower plate in the form of a microplate and having a plurality of wells; an upper plate in the form of a microplate cover disposed on an upper part of the lower plate and having an automation function added and includes a reagent injection nozzle disposed on a lower part of the upper plate and injecting a reagent into each well of the lower plate, a reagent suction nozzle disposed on the lower part of the upper plate and sucking reagent from each well, a microfluidic passage disposed inside the upper plate and connected to each nozzle, and a tubing coupling unit disposed on a front surface of the upper plate and connected to the microfluidic passage; a pump connected to the tubing coupling unit of the upper plate through tubing; and a reagent vessel connected to the pump through the tubing.

Description

Immunoassay automation device

The present invention relates to an immunoassay automation apparatus for automating the sample processing process of various steps required in the conventional immunoassay experiment.

(1) Related fields

A related field is immunoassay, an experiment that requires repeated sample processing such as dispensing of reagents, incubation, and washing when performing an immunoassay experiment with a sample for analysis attached to the surface of a microplate. For example, Figure 1 shows the ELISA (Enzyme-Linked Immuno-Sorbent Assay) procedures.

(2) Applicable products and methods

Related products and methods can be applied to various antigen-binding material-based immunoassays, and can be used for diagnostic kits (virus and cancer diagnostic kits) and cell analysis using the same. For example, FIG. 2 shows (left) an immunoassay technique used for diagnosing a virus and (right) cancer.

(3) Areas expected to be applied in the future

Related products and methods are applicable to ELISA and cellular immunoassay, for example, immunoassay experiments that require repeated reagent treatment for analysis of samples attached to the slide glass surface and microplate well surface. is applicable to

(4) the most relevant prior art

In the conventional immunoassay, reagent dispensing and washing process were repeated manually in microplate wells.

In the case of a microplate washer for automating immunoassay experiments, when the microplate is mounted on the equipment, the position of the plate is adjusted with the nozzle fixed to the equipment, and the washing reagent is injected and suctioned into the well to remove it. Washing of the microplate wells was automated by repeating. For example, FIG. 3 shows a microplate washer having only a washing function.

The automated ELISA processing system enables dispensing and washing of reagents using a robot arm built into the equipment.

(5) Problems of the prior art

In the case of performing the conventional immunoassay, the sample to be analyzed is attached to the surface of the microplate, the reagent is dispensed, incubated for a reaction time, and then washed with a washing reagent was repeated several times. Since all existing methods are performed manually by the experimenter, a long time and labor are required, and there is a problem that a difference may occur in the reproducibility of the experiment result depending on the skill of the experimenter.

In order to solve the problems of conventional immunoassay experiments, automated equipment such as microplate washers and ELISA workstations have been proposed. However, since such equipment must be used by mounting the microplate on a bulky external equipment, space utilization is poor. In addition, there is a problem that contamination of the sample may occur in the process of opening and closing the microplate cover to dispense and wash the reagent in the well. For example, Figure 4 shows an ELISA workstation with wash and reagent handling functions.

It is an object of the present invention to provide an automated immunoassay apparatus capable of reducing the time and labor required for an experiment, reducing the difference in experimental results depending on the skill level of the experimenter, having a small volume, and reducing sample contamination.

In order to achieve the above object, the present invention is in the form of a microplate, the lower plate having a plurality of wells; An upper plate in the form of a microplate cover disposed on the upper part of the lower plate and with an added automation function, a reagent injection nozzle disposed on the lower part of the upper plate and injecting reagents into each well of the lower plate, and a reagent injection nozzle disposed on the lower part of the upper plate and sucking reagents from each well an upper plate having a reagent suction nozzle, a microfluidic passage disposed inside the upper plate and connected to each nozzle, and a tubing coupling part disposed in front of the upper plate and connected to the microfluidic passage; a pump connected to the tubing coupling part of the upper plate through tubing; And it provides an assay automation device comprising a reagent vessel connected through the pump and tubing.

In the present invention, the lower plate is disposed on the lower surface and has a groove into which the photocurable adhesive is injected, and can be coupled to the cover glass through the photocurable adhesive.

In the present invention, the lower plate is formed around the well and has a stepped region having an inclined surface, and the reagent injection nozzle may be disposed in the stepped region.

In the present invention, the lower plate has a hole formed on one side of the well, and the reagent suction nozzle can be inserted into the hole.

In the present invention, a plurality of reagent injection nozzles are arranged per well, and at least one reagent suction nozzle is arranged per well, and each nozzle is individually injected through a microfluidic passage for reagent injection and reagent suction. It may be connected to a tubing coupling for inhalation of solvents and reagents.

In the present invention, a set of tubing coupling portions having the same number as a set of nozzles disposed per well are formed as a single set in the center of the upper plate, and the microfluidic passages are divided by the number of wells from the single set of tubing coupling portions. can

In the present invention, the plurality of microfluidic passages branched from one tubing coupling part have the same length and structure from the tubing coupling part to the nozzle, so that the same amount of reagent can be injected or sucked into each well.

In the present invention, the upper plate is manufactured through 3D printing, and by increasing or decreasing the number of branches of the microfluidic passage starting from a single set of tubing couplings, it is applicable to the lower plate having a different number of wells.

In the present invention, the pump is a diaphragm type micro water pump, the number of pumps and containers is the same as the number of tubing coupling parts, and the pump is divided into a reagent injection pump and a reagent discharge pump each having an inlet and an outlet, and the container is a reagent It is divided into a storage vessel and a reagent discharging vessel, and the inlet of the reagent inlet pump, the outlet of the market storage vessel and the reagent infusion pump, and the reagent injection tubing coupling unit are respectively connected by tubing, the outlet of the reagent discharging pump and the market discharging vessel And the inlet of the reagent discharge pump and the reagent suction tubing coupling portion may be connected to each other by tubing.

In the present invention, the reagent is divided into a reaction reagent and a washing reagent, and the reaction completion reagent or washing reagent inside each well is removed through the suction nozzle of each well by the operation of the reagent discharge pump, and at the same time, the reagent injection pump is operated The washing reagent is dispensed through the nozzle for injection of the washing reagent by means of a continuous flow of the washing reagent, enabling washing of the inside of the well.

In the present invention, after attaching the analyte sample to the surface of the cover glass and bonding the cover glass to the lower surface of the lower plate, the upper plate is covered on the lower plate and the pump is operated. By setting the operation time of the pump according to the assay protocol to be performed, the reagent Dispensing, incubation, and washing can be handled automatically.

The immunoassay test automation apparatus according to the present invention adds a function for automating the immunoassay experiment to the existing microplate cover, which had no special function during the immunoassay experiment performed on the microplate. Specifically, microfluidic passages and nozzles for dispensing and inhaling reagents exist in the microplate cover itself, so that after closing the cover on the plate where the sample is prepared, dispensing and dispensing reagents into the microplate wells without opening the cover in all experimental procedures Incubation and washing processes can be automated. Therefore, it is possible to reduce the time and labor required for the experiment, and to reduce the difference in experimental results according to the skill level of the experimenter. In addition, since the microplate cover itself is used as an automated device for the experiment, the volume is small, and immunoassay can be performed without opening the cover throughout the sample processing process, thereby reducing sample contamination.

1 shows the ELISA procedures.
2 shows (left) an immunoassay technique used for diagnosing viruses and (right) cancer.
3 shows a microplate washer having only a washing function.
4 shows an ELISA workstation with washing and reagent handling functions.
5 is a plan view of the upper plate of the automated immunoassay method according to the present invention.
6 is a plan view of the lower plate of the automated immunoassay method according to the present invention, wherein the upper view shows the upper side of the lower plate, and the lower view shows the lower side of the lower plate.
7 is a front view of an automated immunoassay method according to the present invention.
8 is a side view of an automated immunoassay method according to the present invention.
9 is a state diagram of a reagent injection operation of the automated immunoassay method according to the present invention.
10 is a front view of the lower plate of the automated immunoassay method according to the present invention.

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

The device according to the present invention includes a micro-plate consisting of a bottom of a cover glass to which a sample can be attached and a micro-plate cover with an automation function added, and a reagent is dispensed inside the micro-plate cover. There is a channel for dispensing and incubating reagents without opening the cover by using the microplate cover itself as an automated device. It relates to a device capable of automating the immunoassay sample processing process, such as (incubation) and washing (washing).

5 to 9 , the apparatus for automating analysis experiments according to the present invention may include an upper plate 100 , a lower plate 200 , a pump 300 , a container 400 , and the like. The assay automation apparatus according to the present invention can be applied to immunoassays (ELISA, cellular immunoassay, etc.), PCR (Polymerase Chain Reaction), etc., and can be used for diagnostic kits and cell analysis using the same. In particular, the immunoassay automation device is a device for automating the immunoassay experiment process performed on the microplate, and includes a microplate-shaped lower plate 200 and an immunoassay sample manufactured by combining a cover glass for attaching an assay sample. and a top plate 100 that is a microplate cover including a dispensing channel.

Referring to FIG. 5 , the upper plate 100 is disposed on the lower plate 200 , which is a microplate, and is in the form of a microplate cover to which an automation function is added, a tubing coupling unit 110 , and a microfluidic passage 120 . , the nozzle 130 may be provided.

The tubing coupling unit 110 serves as a connector to which the tubing 500 is coupled. The tubing coupling part 110 is disposed in a slightly protruding shape on the front surface (front) of the upper plate 100, and is internally connected to the microfluidic passage 120, and externally through the tubing 500, the pump ( 300) can be connected.

Referring to FIG. 7 , the tubing coupling unit 110 may be configured as a set unit, and one set includes tubing coupling units 111a, 111b, 111c, 111d, 111e for injecting a plurality of reagents (dispensing or supplying) and It may be composed of at least one reagent suction (or discharge) tubing coupling portion (112). The number of tubing coupling portions 111a to 111e for reagent injection in one set may be, for example, 2 to 10, 3 to 8, or 4 to 6, respectively. The number of tubing coupling portions 112 for aspirating reagents in one set may be, for example, 1 to 3, 1 to 2, or 1 piece. A plurality of tubing coupling portions (111a to 111e, 112) in one set may be arranged to form a substantially circular shape. The number of tubing coupling portions 111a to 111e and 112 in one set may be the same as the number of nozzles in one set of nozzles 130 disposed per well 204 .

The tubing 500 and 510 mounted on the tubing coupling parts 111a to 111e for reagent injection are connected to the market storage containers 410 to 450, and the reagents in the reagent storage containers 410 to 450 are stored inside the upper plate 100. It is for injection into the microfluidic passage 120 . The tubing 500 and 510 mounted on the tubing coupling part 112 for discharging the reagent is for discharging the reagent or washing reagent after the reaction from the well 204 of the lower plate 200, and is connected to the reagent discharging container 460. do.

Referring to FIG. 7 and the like, the tubing coupling unit 110 is a set disposed in the center of the upper plate 100 in order to provide an efficient fluid flow while reducing the number of the pump 300 and the container 400, that is, It may consist of a single set. However, the number, position, arrangement, size, etc. of the tubing coupling portion 110 may be appropriately changed as necessary.

The microfluidic passage 120 is disposed inside the upper plate 100 and serves as a passage connecting each tubing coupling unit 110 and the nozzle 130 . As such, inside the upper plate 100 , there is a microfluidic passage 120 capable of dispensing and suctioning the same amount of reagents in the microplate well 204 of the lower plate 200 . The microfluidic passage 120 may be disposed in the vertical direction and the horizontal direction inside the upper plate 100, and specifically, may be composed of a combination of a plurality of vertical and horizontal passages that are connected or branched to each other. The diameter of the microfluidic passage 120 is not particularly limited, and may be appropriately selected in the range of several micrometers to several millimeters.

7 and 8 , the microfluidic passage 120 may be configured as a set unit, similar to the tubing coupling unit 110 , and one set includes a plurality of reagent injection microfluidic passages 121a, 121b, 121c. , 121d, 121e) and at least one microfluidic passage 122 for inhaling reagents. Each of the microfluidic passages 120 may be independently connected to a corresponding individual tubing coupling 110 .

The microfluidic passage 120 may branch as many as the number of wells 204 from a single set of tubing coupling portions 110 disposed in the center of the upper plate 100 . In this case, based on the central tubing coupling portion 110, it may be arranged approximately left and right symmetrical. In addition, the plurality of microfluidic passages 120 branched from one tubing coupling unit 110 have the same length and structure from the tubing coupling unit 110 to the nozzle 130 , so that the same amount of flow to each well 204 is The reagent may be injected or aspirated. In this way, the microfluidic passage 120 starting from one tubing coupling unit 110 is branched into a plurality of microfluidic passages 120 having the same length and structure and can be connected to the nozzle 130 under the upper plate 100, Accordingly, the reagent injected from one tubing coupling unit 110 may be dispensed in the same amount to each well 204 . However, the number of branches of the microfluidic passage 120 , branch structure, flow passage structure, arrangement, size, and the like may be appropriately set as needed.

The nozzle 130 is disposed under the upper plate 100 to inject a reagent into each well 204 of the lower plate 200 or to suck a reagent from each well 204 . As described above, a nozzle 130 for injecting and sucking a reagent into the well 204 is disposed under the upper plate 100 .

Referring to FIG. 5 , the nozzle 130 may be configured in a set unit like the microfluidic passage 120 and the tubing coupling unit 110 , and one set includes a plurality of reagent injection nozzles 131a, 131b, 131c. , 131d, 131e) and at least one microfluidic passage 132 for aspirating reagents. Each of the nozzles 130 may be independently connected to a corresponding individual microfluidic passage 120 and a tubing coupling unit 110 . That is, each of the nozzles 130 may be connected to the individual reagent injection and reagent suction tubing coupling units 110 through the microfluidic passage 120 for each reagent injection and reagent suction. Specifically, for example, the nozzle 131a - the microfluidic passage 121a - the tubing coupling portion 111a each other, the nozzle 131b - the microfluidic passage 121b - the tubing coupling portion 111b each other, and the nozzle 131c - Microfluidic passage (121c) - Tubing coupling portion (111c) each other, nozzle (131d) - Microfluidic passage (121d) - Tubing coupling portion (111d) to each other, nozzle (131e) - Microfluidic passage (121e) - Tubing coupling The parts 111e, the nozzle 132, the microfluid passage 122, and the tubing coupling part 112 may be connected to each other.

The reagent injection nozzles 131a to 131e are arranged in plurality per well 204 (one set), and the number of reagent injection nozzles 131a to 131e in one set is, for example, 2 to 10 pieces. , 3 to 8, or 4 to 6 may be present. The reagent aspiration nozzle 132 is arranged at least one per well 204 (one set), and the number of reagent aspiration nozzles 132 in one set is, for example, 1 to 3, 1 to 2 It may be a dog, or one. For one well 204 , a set of nozzles 131a to 131e and 132 may be arranged to form a substantially circular shape corresponding to the shape of the well 204 . The number of sets of nozzles 130 may be equal to the number of wells 204 . However, the number, position, arrangement, size, etc. of the nozzles 130 may be appropriately changed as needed.

As illustrated in FIG. 5 , five reagent injection nozzles 131a , 131b , 131c , 131d , 131e and one reagent suction nozzle 132 may be disposed per well 204 . When the upper plate 100, which is the microplate cover, is covered, five nozzles for reagent injection (131a to 131e) and one nozzle for aspirating reagent 132 are placed on the lower part of the upper plate 100 so that they are included within the diameter of each well 204. can be positioned. The nozzles 131a to 131e and 132 configured for one well 204 may all be branched from individual tubing coupling portions 110 .

The upper plate 100 may include a rim member extending downward from the lower surface where the microfluidic passage 120 ends, and accordingly, an empty space may be formed inside the rim member from the lower surface to the lower end of the rim member. The reagent injection nozzles 131a to 131e may protrude from the lower surface and be disposed in the empty space, and the reagent suction nozzle 132 may protrude longer from the lower surface to beyond the lower edge of the edge member for smooth suction, Accordingly, a height difference may be formed between the two types of nozzles.

The upper plate 100 may be manufactured through 3D printing using a polymer material, etc., and by increasing or decreasing the number of branches of the microfluidic passage 120 starting from the single set of tubing coupling portions 110, the well ( It is also applicable to the lower plate 200 with a different number of 204). As such, by increasing or decreasing the number of branches of a channel (microfluidic passage 120) starting from one reagent injection unit (tubing coupling unit 110), the number of wells 204 is also applicable to the lower plate 200 of the microplate. do.

Referring to FIG. 6 and the like, the lower plate 200 may be configured as one plate. In FIG. 6 , the upper view is a single lower plate 200 viewed from the upper surface 200a side, and the lower view is a lower plate 200 viewed from the lower surface 200b side. 7 and 8 , the upper portion of the lower plate 200 may have a slightly shorter length and width than the lower portion, and accordingly, the edge member of the upper plate 100 may be fitted with the upper portion of the lower plate 200 .

The lower plate 200 may be in the form of a microplate having a plurality of (preferably four or more) wells 204, and a cover glass is attached to the lower surface thereof to be used as a reagent reaction chamber for immunoassay experiments. can The microplate may refer to a microwell plate having a plurality of wells 204 used for analysis and diagnostic experiments, and the like. The lower plate 200 may be manufactured through injection molding using a polymer material or the like.

The lower plate 200 may include a plurality of wells 204 . The number of wells 204 may be, for example, 2 to 10,000, more specifically 6, 12, 24, 48, 96, 384, 1,536, 3,456, 9,600. Each well 204 can contain tens of nanoliters to several milliliters of liquid sample. Each well 204 is preferably circular when viewed from above, but other shapes such as oval and polygonal (square, etc.) are also possible.

The lower plate 200 may be partitioned for each well 204 . Each well 204 may be formed through the lower plate 200, and the lower part of the well 204 is sealed by a cover glass mounted on the lower part of the lower plate 200 to form a reagent accommodating space and a reagent reaction chamber. can

A groove 202 may be formed in the lower surface 200b of the lower plate 200 , and a photocurable adhesive may be injected into the groove 202 , and may be coupled to the cover glass through the photocurable adhesive. In this way, the photocurable adhesive is injected into the groove 202 on the lower surface of the lower plate 200 and the cover glass can be combined by exposure, so that the cover glass surface-treated suitable for the sample to be analyzed can be combined. do.

The shape of the groove 202 may vary depending on the shape of the well 204 , but one groove 202 may be formed along the perimeter of the well 204 . Specifically, the groove 202 includes one main groove formed along the perimeter of the well 204 adjacent to the well 204 ; and a plurality of (eg, 5 to 20) sub grooves arranged at regular intervals along the circumference of the main groove and extending outwardly from the main groove. All of the sub grooves are integrally connected to the main groove, and thus may be configured as one integral groove as a whole.

As can be seen in FIG. 6 , the groove 202 may have a sun shape when viewed as a whole. In this way, by integrally forming a plurality of sub-grooves in one main groove, the bonding force with the cover glass can be more effectively increased. In addition, a partition wall separating them is formed between the groove 202 and the well 204 to prevent the adhesive injected into the groove 202 from flowing into the well 204 . In addition to the examples in the drawings, the shape and size of the groove 202 may be appropriately set as necessary.

A hole 203 may be formed at one side of the well 204 , and a reagent suction nozzle 132 may be at least partially inserted into the hole 203 . As described above, there is a hole 203 through which the reagent suction nozzle 132 of the upper plate 100 can be inserted on one side of the well 204, so that the end of the nozzle 132 and the cover glass are in contact with the well 204 ) It can be washed by effectively sucking the reagent inside. The hole 203 may be formed to correspond to the position, number, shape, and size of the reagent suction nozzle 132 . The hole 203 may be formed through the lower plate 200 and may be directly connected to the well 204 .

On the upper surface 200a side of the lower plate 200, a step 201 may be formed around the well 204, and a plurality of reagent injection nozzles 131a to 131e may be disposed in the stepped area 201. have. In this way, the step 201 is provided at the edge of the well 204 to prevent damage to the sample attached to the cover glass when the reagent is injected from the top plate 100 . That is, the reagent injection nozzles 131a to 131e are positioned at the portion where the step 201 exists at the edge of the well 204 of the lower plate 200 to inject the reagent, thereby minimizing damage to the analysis sample. After the reagent injected from the reagent injection nozzles 131a to 131e falls on the stepped region 201 , it may flow into the well 204 inside the stepped region 201 . The reagent injection nozzles 131a to 131e may be spaced apart from the stepped region 201 in the height direction.

The step 201 is at a lower height than the upper surface 200a of the lower plate 200 , and may be directly connected to the well 204 at the same height. The step 201 may be formed over the entire perimeter of the well 204 except for the hole 203, and has a shape corresponding to the well 204 and an appropriate area corresponding to the reagent injection nozzles 131a to 131e. can be formed with A separate through hole may be formed in the stepped region 201 to allow the reagent to flow well.

Referring to FIG. 10 , the stepped area 201 may include an inclined surface. The inclined surface may be formed over the entire step area 201 or may be formed in a partial area of the step area 201 . The inclination angle of the inclined surface may be, for example, 10 to 50 degrees, or 20 to 40 degrees. In this way, a slope (slope) exists in the step region 201, and the reagent discharged from the reagent injection nozzles 131a to 131e of the upper plate 100 falls onto the step 201 having a slope (slope), By designing the positions of the reagent injection nozzles 131a to 131e, the reagent can be injected without damaging the sample attached to the well 204. In addition, since there is a slope in the step 201 , the reagent discharged from the reagent injection nozzles 131a to 131e may flow into the well 204 without pooling on the step 201 and be injected, thereby reacting with the sample This may occur normally, and even in the washing process of the reagent, the residue may be completely washed without accumulating in the step 201 portion.

9, the pump 300 is connected to the tubing coupling part 110 and the reagent container 400 of the upper plate 100 through tubing 500, 510 to inject a reagent into the upper plate 100 or to the upper plate ( 100) to inhale and discharge reagents. The pump 300 may be a diaphragm micro water pump. The number of pumps 300 may be the same as the number of tubing coupling units 110 . The pump 300 may have an inlet 301 and an outlet 302 , and may be divided into reagent injection pumps 310 , 320 , 330 , 340 , 350 and a reagent discharge pump 360 . The inlet 301 of the reagent infusion pumps 310 to 350 and the market storage containers 410 to 450 may be connected with tubing 510, and the outlet 302 of the reagent infusion pumps 310 to 350 and the reagent injection The tubing coupling portions 111a to 111e for use may be connected to the tubing 500 . In addition, the discharge part 302 of the reagent discharge pump 360 and the market discharge container 460 may be connected with tubing 510 , and the inlet 301 of the reagent discharge pump 360 and the reagent suction tubing coupling part 112 may be connected to tubing 500 .

Referring to FIG. 9 , the reagent container 400 serves to store the reagent for injection and to receive the discharged reagent. The reagent vessel 400 may be connected to the pump 300 through tubing 510 . The number of reagent vessels 400 may be the same as the number of tubing coupling units 110 and pumps 300 . The reagent container 400 may be divided into reagent storage containers 410 , 420 , 430 , 440 , 450 and a reagent discharge container 460 . Reagents can be divided into reaction reagents and washing reagents. By the operation of the reagent discharge pump 360, the reaction completion reagent or washing reagent inside each well 204 is removed through the suction nozzle 132 of each well 204, and at the same time, the reagent injection pumps 310 to 350 The washing reagent is dispensed through the washing reagent injection nozzles 131a to 131e by operation of

After attaching the analyte sample to the surface of the cover glass and bonding the cover glass to the lower surface of the lower plate 200, the upper plate 100 is covered on the lower plate 200 and the pump 300 is operated, but the pump according to the analysis protocol to be performed By setting the operating time of 300, it is possible to automatically process the dispensing of reagents, incubation and washing.

The operation of the immunoassay automation apparatus may include automation of reagent dispensing, incubation, and washing processes that are sample processing processes of an immunoassay experiment.

Dispensing of the reagent may be performed as follows.

The reagent storage container (410 to 450), the inlet 301 of the diaphragm-type micro water pump (310 to 350), and the tubing coupling parts (111a to 111e) of the front part of the upper plate 100 and the diaphragm-type micro water pump (310 to) An outlet 302 of 350 is connected to respective tubing 500 and 510 . When the water pumps 310 to 350 are operated, the reagents inside the reagent storage containers 410 to 450 pass through the pumps 310 to 350 and flow into the microfluidic passages 121a to 121e inside the upper plate 100 .

The microfluidic passages 121a to 121e starting from the reagent dispensing tubing coupling portions 111a to 111e are branched into a plurality (eg, four), and the nozzles 131a to 131e under the upper plate 100 are branched. is connected with Since the plurality of channels 121a to 121e branched from one tubing coupling part 111a to 111e have the same length and structure from the tubing coupling part 111a to 111e to the nozzles 131a to 131e, one tubing coupling The reagents introduced from the parts 111a to 111e are discharged in the same amount from the nozzles 131a to 131e to each well 204 . Since the reagent discharged from the nozzles 131a to 131e falls on the part where the step 201 at the edge of the well 204 exists, it is possible to inject the reagent without affecting the analysis sample attached to the cover glass surface. .

A process of washing the reagent in which the reaction is completed in the well 204 may be performed as follows.

At least one tubing coupling part 112 of a plurality of (eg, 6) tubing coupling parts 110 present in the front part of the upper plate 100 is the well 204 of the reaction completion reagent or washing reagent. It is for discharge.

The reagent suction tubing coupling 112, the fluid inlet 301 of the diaphragm-type micro water pump 360, and the reagent discharge container 460 and the fluid outlet 302 of the diaphragm-type micro water pump 360 are provided. Each of the tubing 500, 510 is connected.

When the water pump 360 is operated, the reaction completion reagent or washing reagent inside the well 204 flows into the microfluidic passage 122 inside the upper plate 100 through the suction nozzle 132 of each well 204 . After being discharged, it passes through the pump 360 and is discharged to the reagent discharge container 460 .

Then, by simultaneously performing the dispensing process of the washing reagent through the washing reagent injection nozzle 131a and the removal of the washing reagent through the suction nozzle 132 at the same time, the washing reagent continues to flow and the residual reagent inside the well 204 is removed. has a cleaning effect.

Therefore, by attaching a sample for analysis to the cover glass surface of the lower plate 200, covering the upper plate 100, and setting the operating time of the diaphragm-type micro water pump 300 according to the immunoassay protocol to be performed Reagent processing such as dispensing, incubation, and washing of reagents for analysis experiments can be automated.

In short, the device according to the present invention is a device for automating the immunoassay test process performed on the microplate, and the microplate-shaped lower plate 200 and the upper plate 100 which is a microplate cover to which the immunoassay test process automation function is added. Including, when the microplate cover 100 is closed and the pump 300 is operated, it is an immunoassay automation device that can automatically process the immunoassay sample processing of reagent dispensing, incubation, and washing process.

In addition, there is a microfluidic passage 120 capable of dispensing the same amount of reagent into each well 204 inside the microplate upper plate 100 , and a nozzle 130 is present in the lower part of the upper plate 100 to provide microfluidic It may be functionalized to inject reagents into the plate wells 204 or to aspirate reagents inside the wells 204 .

In addition, a step 201 exists at the edge of the well 204 of the lower plate 200 of the microplate, and the position of the reagent injection nozzles 131a to 131e under the upper plate 100 is positioned at the edge of the well 204 by a step 201. By placing it in the part where the reagent is present, it is possible to inject the reagent while minimizing damage to the analyte sample when injecting the reagent.

In addition, a plurality of reagent injection nozzles 131a to 131e and at least one reagent suction nozzle 132 are configured for one well 204 , and each nozzle 130 is an individual tubing coupling unit 110 . ) to inject and aspirate reagents into the well 204 through the internal microfluidic passage 120 .

In addition, the upper plate 100 is manufactured through 3D printing, but by increasing or decreasing the number of branches of the channel 120 started from one reagent injection unit 110, the number of wells 204 is different in the lower plate 200 of the microplate. Applicable.

In addition, the reagent storage containers 410 to 450 and the injection part 301 of the diaphragm type micro water pumps 310 to 350 are coupled to each other by tubing 510, and the discharge part of the diaphragm type micro water pumps 310 to 350 302 may be connected to the tubing coupling portion 110 of the upper plate 100 .

In addition, when the water pump 360 is operated, the reaction completion reagent or washing reagent inside the well 204 is removed through the suction nozzle 132 of each well 204, and at the same time, the washing reagent injection nozzle 131a The washing reagent is dispensed through the , so that the washing reagent continuously flows to enable washing of the inside of the well 204 .

In addition, after attaching the analysis sample to the cover glass surface of the lower plate 200 and covering the upper plate 100, the operating time of the diaphragm-type micro water pump 300 is set according to the immunoassay protocol to be performed, and the reagent is dispensed. Immunoassay experiments that require reagent processing such as , incubation, and washing can be automated.

As described above, a function for automating the immunoassay experiment was added to the existing microplate cover, which had no special function during the immunoassay experiment performed on the microplate. A microfluidic passage 120 and a nozzle 130 for dispensing and sucking a reagent are present in the microplate cover (top plate 100) itself. After closing the cover (upper plate 100) on the plate (lower plate 200) on which the sample is prepared, dispensing, incubation, and washing of reagents in the microplate well 204 without opening the cover (upper plate 100) in all experimental procedures can be processed. Therefore, it is possible to reduce the required time and labor compared to the existing manual method, and to reduce the difference in experimental results according to the skill of the experimenter. In addition, by using the microplate cover (top plate 100) itself as an automated device for experiments, the volume is small compared to existing equipment such as ELISA workstations and micro plate washers, and the cover (top plate 100) is used throughout the sample processing process. ))), it is possible to conduct an immunoassay without opening the sample, so it is possible to reduce the contamination of the sample.

100: upper plate, 110: tubing coupling part, 111a, 111b, 111c, 111d, 111e: reagent injection tubing coupling part, 112: reagent suction tubing coupling part, 120: microfluidic passageway, 121a, 121b, 121c, 121d, 121e: microfluidic passage for reagent injection, 122: microfluidic passage for reagent suction, 130: nozzle, 131a, 131b, 131c, 131d, 131e: nozzle for reagent injection, 132: microfluidic passage for reagent suction, 200: lower plate, 200a: upper surface, 200b: lower surface, 201: step, 202: groove, 203: hole, 204: well, 300: pump, 301: inlet, 302: outlet, 310, 320, 330, 340, 350: reagent injection pump, 360: reagent drain pump, 400: reagent vessel, 410, 420, 430, 440, 450: reagent storage vessel, 460: reagent drain vessel, 500, 510: tubing

Claims (11)

a lower plate in the form of a microplate and having a plurality of wells;
An upper plate in the form of a microplate cover placed on the upper part of the lower plate and with an added automation function, a reagent injection nozzle disposed on the lower part of the upper plate and injecting reagents into each well of the lower plate, and a reagent injection nozzle disposed on the lower part of the upper plate and sucking reagents from each well an upper plate having a reagent suction nozzle, a microfluidic passage disposed inside the upper plate and connected to each nozzle, and a tubing coupling part disposed on the front surface of the upper plate and connected to the microfluidic passage;
a pump connected to the tubing coupling part of the upper plate through tubing; and
An assay automation device comprising a pump and reagent vessels connected via tubing. According to claim 1,
The lower plate is disposed on the lower surface and has a groove into which a photocurable adhesive is injected, and an assay automation device capable of bonding to a cover glass through a photocurable adhesive. According to claim 1,
The lower plate is formed around the well and has a stepped area having an inclined surface, and the reagent injection nozzle is disposed in the step area. According to claim 1,
The lower plate has a hole formed on one side of the well, and a reagent suction nozzle is inserted into the hole. According to claim 1,
A plurality of reagent injection nozzles are arranged per well, and at least one reagent aspiration nozzle is arranged per well, and each nozzle is each individual reagent injection and reagent suction through microfluidic passageways for individual reagent injection and reagent suction. An assay automation device that is connected to the tubing connector for use. 6. The method of claim 5,
Automated assay in which a set of tubing coupling portions having the same number as a set of nozzles disposed per well are formed as a single set in the center of the upper plate, and microfluidic passages are branched from the single set of tubing couplings by the number of wells Device. 7. The method of claim 6,
A plurality of microfluidic passages branched from one tubing coupling part have the same length and structure from the tubing coupling part to the nozzle, so that the same amount of reagent is injected or sucked into each well. 7. The method of claim 6,
The upper plate is manufactured through 3D printing, and by increasing or decreasing the number of branches of the microfluidic passage starting from a single set of tubing joints, the assay automation device can be applied to the lower plate with different number of wells. 7. The method of claim 6,
The pump is a diaphragm-type micro water pump, the number of each of the pumps and containers is equal to the number of tubing coupling parts, and the pump is divided into a reagent injection pump and a reagent discharge pump each having an inlet and an outlet, and the container includes a reagent storage container and It is divided into a reagent discharging vessel, and the inlet of the reagent injection pump and the outlet of the market storage vessel and reagent infusion pump and the reagent injection tubing coupling are respectively connected by tubing, and the discharging portion of the reagent discharging pump and the market discharging vessel and reagent discharge An assay automation device in which the inlet of the pump and the coupling part of the reagent suction tubing are respectively connected by tubing. 10. The method of claim 9,
Reagents are divided into reaction reagents and washing reagents, and the reaction completion reagent or washing reagent inside each well is removed through the suction nozzle of each well by the operation of the reagent discharge pump, and the washing reagent is simultaneously operated by the reagent injection pump An assay automation device that dispenses wash reagents through an injection nozzle to enable rinsing of the inside of the wells with a continuous flow of wash reagents. According to claim 1,
After attaching the analyte sample to the surface of the cover glass and bonding the cover glass to the lower surface of the lower plate, cover the upper plate on the lower plate and operate the pump, by setting the operation time of the pump according to the assay protocol to be performed, dispensing and incubating the reagent and an assay automation device that automatically handles the washing process.

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