KR100764022B1 - Microfluidic biochip for blood typing based on agglutination reaction - Google Patents

Abstract

A microfluidic biochip for blood typing based on agglutination reaction is provided to carry out mixing and reaction of sample with reagents, and determination of agglutination in one apparatus, use small amounts of sample and reagents, preserve blood typing results, and improve portability and manufacturing costs of the biochip. The microfluidic biochip(300) for blood typing based on agglutination reaction comprises: a sample transport micro channel(320) having a sample inlet(310); a reagent transport micro channel(325) having a reagent inlet(315); a micro mixer(340) connected to the rears of the sample and reagent transport micro channels and mixing transported sample and reagents; a first manual micro valve(330) formed between the rears of respective micro channels and the front of micro mixer and controlling flows of sample and reagents; a reaction micro chamber(350) connected to the rear of the micro mixer, storing mixed sample and reagents and inducing reaction of them; a micro filter(370) connected to the rear of reaction micro chamber and filtering agglutinated bodies formed by the agglutination reaction; a second manual micro valve(360) formed between the rear of micro chamber and front of micro filter, and controlling the mixed sample and reagents to be placed in the reaction micro chamber; and an outlet(380) connected to the rear of micro filter and discharging sample and reagents.

Description

Microfluidic biochip for diagnosis of blood type based on coagulation reaction {MICROFLUIDIC BIOCHIP FOR BLOOD TYPING BASED ON AGGLUTINATION REACTION}

1 is a plan view showing the basic structure of a microfluidic biochip for blood type diagnosis according to a first embodiment of the present invention.

FIG. 2A is a perspective view illustrating the concept of a micromixer of a microfluidic biochip for blood type diagnosis according to a first embodiment of the present invention, and FIG. 2B illustrates a mixing behavior in a cross section occurring at a position shown in FIG. 2A. It is a schematic diagram to show.

3 is a plan view illustrating an introduction section shape of a micro filter introduced into a microfluidic biochip for blood type diagnosis according to a first embodiment of the present invention.

4 is a plan view showing a microfluidic biochip for blood type diagnosis having four test lines according to a second embodiment of the present invention.

5 is a perspective view illustrating a microfluidic biochip for blood type diagnosis having four test lines according to a second exemplary embodiment of the present invention.

6A to 6K are flowcharts sequentially illustrating a manufacturing process of a microfluidic biochip for blood type diagnosis according to an embodiment of the present invention, in particular, a manufacturing process through mass production such as injection molding.

7 is a photograph showing a microfluidic biochip for diagnosing blood types actually manufactured according to an injection molding and heat bonding process according to an exemplary embodiment of the present invention.

8A to 8C are photographs showing blood group diagnosis test results actually performed using a microfluidic biochip for diagnosing blood types actually manufactured according to an injection molding and heat bonding process according to an exemplary embodiment of the present invention.

The present invention relates to a point-of-care microfluidic biochip and a method of manufacturing the same, which can be directly carried out where desired.

If a blood transfusion is needed, pre-transfusion blood type diagnosis is necessary for both donors and blood donors in order to prevent a transfusion accident. As pre-transfusion blood group test, cross-matching is performed after ABO blood group and Rh (D) blood group diagnosis is performed. In particular, agglutinin (agglutinin) present in the serum of the ABO system among various blood types can fix the inadequate erythrocytes and induce vascular hemolysis (hemolysis), which is necessary for accurate blood type diagnosis.

Blood group diagnosis is generally confirmed by agglutination reaction of red blood cells and serum, and when aggregation occurs, it is determined that agglutination source (agglutinogen) corresponding to serum aggregates exists in red blood cells. That is, when the reaction occurs with the anti-A serum and the aggregation occurs and the reaction does not occur with the anti-B serum, it can be judged that the sample erythrocytes have only the aggregation source for type A, and the sample erythrocytes can be diagnosed as type A. . At this time, more accurate ABO blood group diagnosis is performed by performing red cell typing (forward typing) and serum typing (serum typing; backward typing). The reason for performing both tests simultaneously is to check the test results between each other. This is to increase the confidence of blood type diagnosis and to detect blood types of subtypes (weak antigen) that may be present in the sample.

In fact, the subtype blood types such as A ₂ , A ₃ , and A _int exist for the blood type A, and the subtype blood types such as B ₃ and B _int exist for the blood type B. These subtype blood types have a weak aggregation reaction in response to the corresponding serum, so the probability of being diagnosed as O type is high. Therefore, effective mixing of red blood cells and serum is essential for more accurate blood type diagnosis, and it is essential to perform blood cell test and serum test at the same time to ensure the reliability of diagnosis.

Conventional blood typing methods include plate testing, gel card testing, automated blood typing diagnostics, and recently reported miniaturized blood typing (S.-J. Lee, H.-W. Kang, Y. Kim, G.-W). Lee, G. Lim and D.-W. Cho, and evelopment of a Micro-Blood-Typing System Using Micro-Stereolithography, Sensors and Materials, Vol. 17, pp. 113-123, 2005) and microchannels, micro There is a blood type test apparatus using a filter (Korean Patent No. 0520896).

In the case of plate inspection, samples and reagents are injected by mixing on a plate such as slide glass, mixed, and agglomerated after a certain reaction time, and most of the process is performed manually. However, if all the processes are made by hand, the determination of the agglomeration depends on the subjectivity of the inspector. Therefore, there may be a lack of objectivity of the test results, and there may be a description error if the number of inspections is increased even in the description of the results. In addition, since the specimen is handled by hand, the risk of infection of the sample blood is inherent.

Recently, automation systems for diagnostic medical tests have been developed and introduced due to automation efforts throughout diagnostic medicine. Among these, the gel card test method is recognized as a semi-automated test method and a test that gives accurate test results. However, the gel card test method is not only expensive, but also requires a tester who can professionally handle machines such as centrifuges, and it takes a long time to settle the blood and makes it difficult to apply in an emergency. Have

Automated blood group diagnostic systems actually provide fully automated blood group diagnostic tests. However, this system is very expensive and the size of the equipment is large, so it is difficult to operate outside a large hospital such as a blood bank.

Recently, Lee et al. Reported a miniaturized blood type diagnostic system that integrates a flow separation channel, a chaotic micromixer, and a reaction chamber using micro-stereolithography. This miniaturized blood type diagnosis system separates the injected blood through the flow separation channel, simultaneously mixes the injected reagent with the chaotic micromixer, and checks the hemagglutination in the reaction chamber. The miniaturized blood type diagnosis system has the advantage of being small in size and easy to carry, so that it can cope with emergencies, but this system has a disadvantage in that it takes much time to manufacture by micro-optic molding technology and high manufacturing cost.

Finally, these conventional methods require about 20 μl of sample volume for blood typing, so there is no significant difference compared to manual, and the inconvenience of repeated sample taking remains.

Korean Patent No. 0520896 proposes a blood type test apparatus using a microchannel and a micro filter. This blood type test apparatus is injecting blood, separated through a microchannel and reacted in a reagent storage chamber to filter out the agglomerated blood through a micro filter, and a small amount of the sample used is 10 μl or less. However, this blood type test device is intended to induce a natural mixing and reaction of the sample and reagents encountered in the microchamber. As mentioned above, effective mixing of the sample and reagent is essential to detect unexpected antibodies that may be present. In fact, in the case of a microchannel or a microchannel, only a mixing effect due to diffusion can be expected due to a significant decrease in the characteristic length, and mixing due to turbulence cannot be expected, so that the mixing performance is significantly reduced. For example, in a microchannel having a width of 100 μm, the natural mixing time by diffusion requires several hours. Therefore, the micro blood test apparatus without the mixing device has its limit in terms of performance. In addition, the micro filter composed of pillars having the same thickness in the longitudinal direction introduced by the blood test apparatus has a limitation that the pillar thickness is too thin to be produced in a mass production method such as injection molding. Lastly, the blood type test apparatus of Korean Patent No. 0520896 has a predetermined reagent to be used, and is designed to perform blood cell tests only, and thus, there is a limit in accurate blood type diagnosis.

The sample and reagent are used in small amounts, and the mixing and reaction of the sample and reagent, reaction and aggregation can be easily read in a single device, enabling the objective diagnosis of the agglutination test for diagnosing blood types and performing the agglutination test results. It is necessary to develop a diagnostic medical flocculation device that can be preserved, yet can be manufactured at low cost and is portable because of its portability.

The objective of the present invention is to solve this problem of the prior art, while using a small amount of both the sample and the reagents, while the effective mixing, reaction and aggregation of the sample and reagents are all implemented on the microfluidic biochip for objective blood type diagnosis To make it possible.

In addition, the present invention is to produce a biochip at a low price by implementing the microfluidic biochip into plastic through a mass production method such as injection molding.

It is also an object of the present invention to enable point-of-care diagnosis and to preserve the result of the aggregation test, which is easy to carry.

As a result, the present invention provides a sample inlet, a reagent inlet, a microchannel system for separating and moving a sample and a reagent, a micromixer for effective mixing of a sample and a reagent, and a reaction of the mixed sample and the reagent. Reaction microchamber, multi-step microfilter for detection of reacted samples and reagents, passive microvalve for control of sample and reagent flow and outlet Implementing on a chip, we propose a microfluidic biochip for point-of-care that can perform blood type diagnosis based on a diagnostic medical flocculation test on a biochip.

In order to achieve the above technical problem, the microfluidic biochip for agglutination reaction based diagnostic test according to the present invention includes: i) a sample transfer microchannel in which a sample inlet is formed, ii) a reagent transfer microchannel in which a reagent inlet is formed, and iii) the sample. A micromixer connected to a transfer microchannel and a rear end of the reagent transfer microchannel, wherein the transferred sample and reagent meet and are mixed together, iv) formed between the rear end of each of the microchannels and the front end of the micromixer, A first passive microvalve for controlling the flow of the sample and the reagent, v) a reaction microchamber connected to a rear end of the micromixer, and storing the mixed sample and the reagent to induce a reaction, vi) of the reaction microchamber It is connected to the rear stage, and filters the aggregate formed by the agglomeration reaction of the mixed sample and reagent Vii) a second manual microvalve formed between the rear end of the microchamber and the front end of the microfilter, the second passive microvalve controlling the mixed sample and reagent to stay in the reaction microchamber, and viii) the microfilter It is connected to the rear of the chamber, and includes an outlet for discharging the tested sample and reagent.

The micro filter has a plurality of rows of micro pillars arranged at regular intervals along the direction of transport of the fluid, and filter spaces are formed between the micro pillars of each row, and each filter space has an inlet wider than the outlet. Can be.

In the micro filter, a plurality of rows of micro filters may be arranged at uniform intervals along the fluid conveying direction.

The micro filter may have a portion in which the filter space of each row decreases stepwise along the fluid transfer direction in the initial section.

The micro filter may have a portion in the initial section in which the size of the micro pillars in each row increases step by step along the fluid transfer direction.

The micro filter may have a portion in which the number of micro pillars in each row increases gradually in the initial section along the fluid transport direction.

The micro pillars may be formed to have a larger front surface in the fluid conveying direction than a rear surface.

The flat cross section of the micro pillar may be made of a trapezoid, a pentagon or a hexagon.

The first manual microvalve may be formed to have a sharply reduced width from the sample transfer microchannel or the reagent transfer microchannel, and the second manual microvalve may be formed to have a rapidly reduced width from the reaction microchamber.

On the other hand, the sample transfer micro-channel is connected to each of the plurality of micro-mixer branched into a plurality of pieces, it is possible to separate the injected sample to be transferred to each of the micro mixer.

The reagent transfer micro channel may be formed in plural and connected to the plurality of micro mixers, respectively, and each reagent transfer micro channel may be provided with a plurality of reagent inlets separated from each other to inject different types of reagents.

The micromixer transfers the transferred sample and reagent through a three-dimensional spiral flow path and combines and divides and rearranges and combines chaotic mixing mechanism of chaotic advection.

The micromixer includes a pair of inlets through which the transferred sample and the reagent are respectively injected, an inlet channel through which the transferred sample and the reagent are joined, an outlet channel through which the transferred sample and the reagent are mixed and discharged, and And a mixing unit including a first mixing unit and a second mixing unit for mixing the transferred sample and the reagent while being connected to each other between the inflow channel and the outflow channel to form a three-dimensional spiral flow path.

In the micromixer, the first mixing unit is branched from the inlet channel and extends toward the first side in the inflow direction of the inflow channel while at least one pair of primarys through which the joined fluid is repartitioned. A split channel and a primary confluence channel disposed in a different layer from the primary split channel and in communication with each end of the primary split channel, where the divided fluid passes through the split channel, wherein the second mixing unit comprises: At least a pair of secondary divided channels branched from the primary confluence channel and extending toward the second side opposite to the first side, through which the joined fluid is repartitioned; A second confluence channel disposed in a different layer and communicating with each end of the secondary division channel to allow the divided fluids to merge; and the secondary confluence channel is connected to the outlet channel. It leads.

The primary divided channel may include a main channel extending in a direction parallel to the inflow channel, and a branch channel formed to be bent in a direction substantially perpendicular to a traveling direction of the main channel toward the first side.

The secondary divided channel includes a main channel extending in a direction parallel to the inflow channel, and a branch channel formed to be bent in a direction substantially perpendicular to a direction in which the main channel extends toward the second side.

The mixing unit may be provided by alternately repeating the first mixing unit and the second mixing unit which are arranged in succession.

The primary splitting channel of the first mixing unit is formed in a different layer from the secondary splitting channel of the second mixing unit, and the primary joining channel of the first mixing unit is the secondary joining channel of the second mixing unit. And may be formed of different layers.

The primary confluence channel of the first mixing unit may be formed in the same layer as the secondary division channel of the second mixing unit.

The primary and secondary division channels and the primary and secondary confluence channels are each formed to move substantially the same distance while the fluid divided through the respective division channels is transferred through the respective confluence channels to the recombination point. Can be.

Blood test is performed by injecting red blood cells of test blood into the sample, injecting standard serum into the reagent, or performing blood cell test by injecting serum of test blood into the sample, and performing serum test by injecting standard red blood cells into the reagent. Diagnosis can be made.

The method of manufacturing a microfluidic biochip for agglutination based diagnostic tests may include: i) the sample and reagent transfer microchannel, the first layer portion of the micromixer, the first and second passive microvalve, and the reaction microchamber. Fabricating a first plate comprising a first layer portion of and a first groove having a respective shape of the micro filter; ii) a second layer portion of the micromixer, and a second layer portion of the reaction microchamber. Manufacturing a second plate comprising a second groove having a shape, and iii) bonding the first plate and the second plate to each other.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like elements throughout the specification.

First, the structure and operation concept of the point-of-care microfluidic biochip for blood type diagnostic tests based on the aggregation reaction proposed in the present invention will be described with reference to FIG. 1. 1 is a plan view showing the basic structure of a microfluidic biochip for blood type diagnosis according to a first embodiment of the present invention.

Referring to FIG. 1, the blood type diagnostic microfluidic biochip 100 according to the present embodiment includes a sample inlet 110, a reagent inlet 115, microchannels 120 and 125 for sample and reagent transfer, sample and reagent flow. First manual microvalve (130, 135) for control, chaotic micromixer (140) for effective mixing of samples and reagents, reaction microchamber (150) for storing mixed samples and reagents and inducing a reaction, mixing A second manual microvalve 160 for allowing the sampled and reagents to remain in the reaction microchamber, a microfilter 170 for filtering erythrocyte aggregates formed by agglutination of the mixed sample and reagents and the finished test It is composed of an outlet 180 for discharging the specimen and reagent.

Red cell typing (forward typing) is a standard serum through the reaction of red blood cells in the test blood with standard serum such as Anti-A, Anti-B, Anti-AB, Anti-A1, Anti-H, Anti-D, etc. When agglutination occurs, a blood group diagnosis is made by determining that agglutinants corresponding to standard serum are present in red blood cells. For example, in the case of erythrocytes that cause an aggregation reaction with Anti-A, it can be determined that there is a type A aggregation source (blood type A or AB type). Therefore, in the case of blood cell test, the sample becomes red blood cells of the test blood, and the reagent becomes standard serum.

Serum typing (backward typing) is the reaction of test blood serum with standard erythrocytes such as Test cell A1, Test cell A2, Test cell B, and Test cell O. A blood group diagnosis is made by determining that agglutinates correspond to the presence in the serum. For example, serum that causes agglutination with test cell A may be determined to have agglutinates of type A (blood type B or AB). Therefore, in the case of serum test, the sample is serum of test blood, and the reagent is standard red blood cell.

According to each test, the sample and the reagent are respectively injected by about 1 to 3 μl through the sample inlet 110 and the reagent inlet 115. In particular, the sample inlet 110 and the reagent inlet 115 is present separately it is possible to use a variety of different types of reagents that can be used in the blood cell test or serum test to suit the test. Injected samples and reagents are passed through the first manual microvalve 130, 135 through each conveying microchannel 120, 125 by a driving force given by an external pressure transfer device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. Injection).

The first passive microvalve (130, 135) has a shape that suddenly decreases in width, this shape induces a sudden change in the surface tension to induce the effect of stopping the sample and reagent fluid flow in the microvalve. In particular, such microvalve may increase the surface tension effect by making the hydrophobic or hydrophilic surface depending on the plastic surface quality. The first passive microvalve 130 and 135 thus introduced are simultaneously introduced into the chaotic micromixer 140 followed by the injected sample and the reagent fluid flow so as to effectively mix. The sample and reagent fluid flows stopped at the first manual microvalve (130, 135) are simultaneously chaos micromixer 140 by the driving force given by an external pressure transmission device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. ) And effectively mix with each other.

In particular, the chaos micromixer 140 is a spiral lamination chaos micromixer in which an F-shaped mixing unit is arranged on the lower and upper plates periodically to effectively combine the two chaotic mixing mechanisms of split / recombination and chaotic advection. Mix sample and reagent effectively. This effective mixing not only significantly increases blood type diagnostic detection performance, but also helps to effectively detect unexpected blood types that may be present. In addition, the spiral lamination chaos micro mixer 140 has a simple shape and can be manufactured in mass production. Samples and reagents mixed by the spiral lamination chaos micromixer 140 are injected into the reaction microchamber 150 followed by a driving force given by an external pressure transfer device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. do.

The reaction microchamber 150 is patterned in the upper and lower layers like the spiral lamination chaos micromixer 140 and is designed to increase the capacity of the chamber. At this time, the injected sample and reagent mixture is maintained in the reaction microchamber 150 for the reaction time (approximately 1 to 3 minutes) of the sample and the reagent by the second manual microvalve 160. At this time, when there is a coagulation source and agglomerate in the sample and the reagent, an aggregation reaction occurs between red blood cells.

The second passive microvalve 160 also has a shape such that the first passive microvalve 130 and 135 suddenly decrease in width, which induces a sudden change in the surface tension to mix the sample and the reagent in the reaction microchamber. Induces the effect of fluid flow stopping at the microvalve. In particular, such a microvalve may be made hydrophobic or hydrophilic, depending on the plastic surface quality, to increase the surface tension effect. After a certain reaction time of the sample and reagent mixture for 1 to 3 minutes, it is injected into the micro filter 170 followed by a driving force given by an external pressure transmission device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like.

The micro filter 170 introduced to the microfluidic biochip 100 for diagnosing blood type according to the present embodiment has a filter space that is reduced in multiple stages, so that the aggregated red blood cells in which the aggregation reaction occurs can be effectively filtered. In particular, the filter space is larger than the size of the normal red blood cells, and the red blood cells that do not cause the aggregation reaction easily exit the micro filter 170, and the aggregated red blood cells that have increased in size due to the aggregation reaction are easily filtered through the micro filter 170. .

Finally, the sample and reagent reactants after the reaction time are discharged through the outlet 180 through the micro filter 170. In this case, when the aggregation reaction between the sample and the reagent occurs, the coagulation erythrocytes are filtered through the micro filter 170, and when the coagulation reaction does not occur, the erythrocytes exit all of the micro filters 170 so that the coagulation reaction easily occurs with the eyes. It is possible to determine whether the blood type diagnosis is possible based on this.

2A and 2B illustrate the basic concept of the structure and mixing of the micromixer, which is a part of the microfluidic biochip for blood type diagnosis according to the first embodiment. FIG. 2A is a perspective view illustrating the concept of the micromixer according to the present embodiment, and FIG. 2B is a schematic diagram showing the mixing behavior in the cross section occurring at the position indicated in FIG. 2A.

The micromixer 10 according to the present exemplary embodiment includes a pair of inlets 12a and 12b through which different fluids, ie, samples and reagents, transferred from the transfer microchannel are introduced, and an inflow channel through which the samples and reagents are joined ( 12) and an outlet channel 13 through which the sample and the reagent are mixed and discharged, and a mixing unit 15 formed between the inlet channel 12 and the outlet channel 13 to connect the sample and the reagent while connecting them. It is composed. The mixing unit 15 includes a first mixing unit 20 and a second mixing unit 30 which are arranged successively.

The first mixing unit 20 includes a pair of primary split channels 21 and 22 branched from the inlet channel 12, and ends of the primary split channels 21 and 22. And a primary confluence channel 23 in communication. The primary split channels 21 and 22 extend toward the first side with respect to the fluid transfer direction in the inflow channel 12 while the mixed fluid of the sample and the reagent joined in the inflow channel 12 is repartitioned. The primary confluence channel 23 is disposed in a different layer from the primary partition channels 21 and 22, and the mixed fluids divided in the primary partition channels 21 and 22 are joined.

The second mixing unit 30 includes a pair of secondary split channels 32 and 34 branched from the primary confluence channel 23 and each of the secondary split channels 32 and 34. And a secondary confluence channel 35 in communication with the end. The secondary split channels 32 and 34 extend toward the second side piece opposite to the first side piece while the mixed fluid joined in the primary confluence channel 23 is repartitioned. The secondary confluence channel 35 is disposed in a different layer from the secondary partition channels 32 and 34, and the mixed fluid divided in the secondary partition channels 32 and 34 joins.

In the present exemplary embodiment, the primary divided channels 21 and 22 are formed to face the left side with respect to the advancing direction of the inflow channel 12, and the secondary divided channels 32 and 34 are the inflow channel 12. It is formed to face the right side with respect to the traveling direction of. In this case, the primary split channels 21 and 22 are formed by being bent in a direction substantially perpendicular to the flow direction of the fluid in the inflow channel 12, and the secondary split channels 32 and 34 are also inflow channels. It is formed by bending in the direction substantially perpendicular to the direction of fluid flow in (12). However, the present invention is not limited thereto, and the first divided channels 21 and 22 may face the right side, and the second divided channels 32 and 34 may face the left side.

The primary split channels 21 and 22 are main channels extending in a direction parallel to the inflow channel 12 and branch channels formed to be substantially perpendicular to the traveling direction of the main channel toward the first side. Can be divided into By providing a pair of branching channels, the splitting channel of this embodiment has an F-shape. When the number of split channels increases, the main channel has a longer length, and the number of branch channels increases. Similarly, the secondary divided channels 32 and 34 also have a main channel extending in a direction parallel to the inflow channel 12 and a branch formed by bending substantially perpendicularly to the traveling direction of the main channel toward the second side. Can be divided into channels.

The mixing unit 15 may be provided with a plurality of repeated alternating structures of the first mixing unit 20 and the second mixing unit 30 arranged in succession. In the mixing unit 15 illustrated in FIG. 2A, the structures of the first mixing unit 20 and the second mixing unit 30 are repeated twice each up to the outlet channel 13. At this time, the final confluence channel 51 is connected to the outflow channel 13 to flow out the sample and reagent homogeneously mixed.

On the other hand, in the mixing unit arranged in succession, the split channel and the confluence channel are formed by changing the layers. That is, the primary splitting channels 21 and 22 of the first mixing unit 20 are formed in different layers from the secondary splitting channels 32 and 34 of the second mixing unit 30 and the first mixing unit 20. The primary confluence channel 23 of) is formed in a different layer from the secondary confluence channel 35 of the second mixing unit 30. The primary confluence channel 23 is formed on the same layer as the secondary division channels 32 and 34 and connected to each other, and the secondary confluence channel 35 is connected to the primary division channels 21 and 22. Is formed on the same layer. The three-dimensional spiral flow path is formed by the first mixing unit 20 and the second mixing unit 30 provided as described above, and the transferred sample and the reagent are transferred through the chaotic mixing mechanism and chaotic advection. The chaotic mixing mechanism of the chaotic advection can be combined and mixed homogeneously.

In this case, the primary and secondary split channels 21, 22, 32, and 34 and the primary and secondary join channels 23 and 35 may be formed by mixing fluids divided through the respective divided channels. Each of which is moved substantially the same distance during transport to the recombination point through.

In addition, the expansion unit 31 may be formed at the time points of the primary confluence channel 230 and the secondary division channels 32 and 34 branched therefrom to smoothly flow the mixed fluid.

Hereinafter, a process of dividing the injected mixed fluid will be described with reference to FIG. 2B.

When the fluid flow of the sample and the reagent injected through the pair of inlets 12a and 12b and joined in the inlet channel 12 meets the first mixing unit 20, the primary split channels 210 and 220 are separated. Split into two flows through, and recombine again in the primary confluence channel (230). That is, the specimens 17 and the reagents 18 injected through the injection holes 12a and 12b, respectively, meet as shown in FIG. 2B in section A of FIG. 2A, and are divided in the primary partition channels 210 and 220, respectively. It is divided as B and C of FIG. 2B. The divided sample 17 and the reagent 18 are combined in the thickness direction due to the arrangement of the first mixing unit 20 according to the present embodiment in the primary confluence channel 230. The same lamination is made. This mixing mechanism causes the stacking in the thickness direction in D, G, J, and K of FIG. 2A to exponentially increase the interface of the fluid to induce chaotic mixing.

In this case, Schonfeld et al. (F. Schonfeld, V. Hessel and C. Hofmann, "An Optimized Split-and-Recombine Micro-mixer with Uniform 'Chaotic Mixing", Lab on a Chip , vol. 4, pp. 65 As shown in (69, 2004), the ideal stacking of FIG. 2B is difficult to expect when there is no separation wall in the recombination section. However, in the micromixer 10 according to the present embodiment, the flow path of the entire microchannel is a three-dimensional spiral shape. Since it has a (3-dimensional serpentine), when the fluid flow proceeds toward the outlet channel 13 of the micromixer 10, the chaotic advection is induced, so that the rotational momentum in each of the confluence channels 23 and 35. This results in the ideal lamination shown in FIG. 2B.

Therefore, the micromixer 10 according to the present embodiment induces an ideal lamination even without a separation wall, thereby increasing the interface between the two fluids exponentially, leading to more effective chaos mixing. In particular, since there is no separation wall, it can be manufactured at a low price through a mass production method such as injection molding. In addition, the micro mixer 10 according to the present embodiment is laminated in a thin direction in a thickness direction for a shape in which the width of the channel is considerably larger than the thickness, which is characteristic of micro channels having a general rectangular cross section manufactured through a general MEMS process. This has the great advantage of reducing the diffusion length of the fluid more effectively. Finally, the continuously arranged first and second mixing units 20 and 30 of the micro mixer 10 according to the present embodiment have the same flow path lengths of the flows flowed in and divided so that the mixing does not occur at all. It also has the advantage of being eliminated.

Meanwhile, FIG. 3 illustrates an example of the shape of the micro filter 170 that is a part of the microfluidic biochip for blood type diagnosis according to the first embodiment. 3 shows the initial section 200 of the micro filter 170 introduced into the microfluidic biochip, in particular for blood type diagnosis according to this embodiment.

The micro filter 170 has a plurality of rows of micro pillars 210 arranged at regular intervals along the transport direction of the fluid, and a filter space 220 is formed between the micro pillars of each row. At this time, each filter space 220 is formed in the inlet is wider than the outlet, the initial section 200 has a portion in which the filter space 220 of each row decreases stepwise along the fluid transport direction. In addition, the size of the micro pillars 210 in each row of the initial section 200 not only has a portion that increases step by step along the fluid transport direction, but also the number of micro pillars 210 in each row increases in steps along the fluid transport direction. It has a losing part. The micro pillar 210 has a larger surface (ie, a surface facing the inlet) located at a front side (ie, a side facing the outlet) along a fluid transfer direction, and a flat cross section is trapezoidal. , Pentagon or hexagon.

That is, the initial section 200 of the micro filter has a multi-stage filter space 220, the micro pillars 210 having the same trapezoidal, pentagonal or hexagonal flat cross section is periodically arranged in the vertical direction of the flow In the vertical direction of the flow it has the same filter space 220. In particular, the pillars having the trapezoidal, pentagonal or hexagonal flat sections of the initial section 200 of the microfilter according to the present embodiment have a wide inlet and narrow outlet for fluid flow due to their shape. ). In the direction of flow of the micro pillars 210 having the same or different trapezoidal, pentagonal or hexagonal cross-section of the same or different shape with a predetermined space 230 is arranged periodically, filters of various widths in the longitudinal direction It may have a space 220. In addition, in the microfluidic biochip for diagnosing the actual blood type, the minimum filter space 220 and the minimum micro pillars are considered in consideration of the size of red blood cells having a size of about 10 μm in the radial direction and the moldability of injection molding, which is a general mass production process. 210) The width may be 50 μm or more.

As described above, for a more accurate blood type diagnosis, a blood cell test and a serum test should be performed at the same time to compare the results. In this case, the standard serum, which is a reagent used for red blood cells of the same test blood, may be Anti-A, Anti-B, Anti-AB, Anti-A1, Anti-H, and Anti-D. The above inspection line is needed simultaneously. In addition, in the case of a serum test, a test cell A1, a test cell A2, a test cell B, or a test cell O may be used as a standard red blood cell, a reagent used for serum of the same test blood.

4 and 5 are a plan view (FIG. 4) and a perspective view (FIG. 4) and a perspective view (FIG. 5) of a microfluidic biochip for blood type diagnosis having four test lines according to a second embodiment of the present invention so that various tests can be simultaneously performed. ).

4 and 5, the microfluidic biochip 300 for blood type diagnosis according to the second embodiment of the present invention includes a sample inlet 310, a reagent inlet 315, and a sample for simultaneously performing various tests. Flow separation micro channel 320 responsible for separation and micro channel 325 for reagent transfer, first manual micro valve 330 for sample and reagent flow control, spiral lamination chaos micro for effective mixing of samples and reagents A mixer 340, a reaction microchamber 350 for storing the mixed sample and reagents to induce a reaction, a second manual microvalve 360 for allowing the mixed sample and reagents to stay in the reaction microchamber, a mixed sample And a micro filter 370 for filtering erythrocyte aggregates formed by agglutination of reagents and an outlet 380 for discharging the tested samples and reagents. Consists of. Except for the flow separation microchannel 320 from which the sample is separated, each test line is a microfluidic structure (sample and reagent inlet, microchannel) of the microfluidic biochip according to the first embodiment of the present invention described with reference to FIG. 1. , First passive microvalve, helical lamination micromixer, reaction microchamber, second passive microchannel, microfilter, outlet).

4 and 5, the operation concept of the microfluidic biochip for blood type diagnosis according to the present embodiment will be briefly described based on the description of FIG.

According to the blood cell and serum test, the sample and reagents are injected by about 1-3 μl through the sample inlet 310 and the reagent inlets 315, respectively. In particular, the sample inlet 310 and the reagent inlet 315 are present separately, and a plurality of reagent inlets 315 are present (four in the present embodiment), so that multiple coagulation reactions can be tested at the same time. It is possible to use many different kinds of reagents that can be used for test purposes. The injected sample is separated by the flow separation microchannel 320 by a driving force given by an external pressure transmission device such as a syringe pump, surface tension, gravity generated by shaking the chip, etc., and introduced into each inspection line to be introduced into each test line. It is injected up to the valve 330. Reagents are also injected by the driving force through the transfer microchannel 325 to the first manual microvalve 330.

The first passive microvalve 330 has a shape in which the width suddenly decreases, and this shape induces a sudden change in the surface tension, thereby inducing the effect of stopping the sample and reagent fluid flow in the microvalve 330. In particular, the micro valve 330 may increase the surface tension effect by making a hydrophobic or hydrophilic surface according to the plastic surface quality. The sample and reagent fluid flows stopped at the first manual microvalve 330 are simultaneously helical lamination chaos micromixers by a kind of driving force given by an external pressure transmitter such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. Are injected into the field 340 and are effectively mixed with each other.

This effective mixing not only significantly increases blood type diagnostic detection performance, but also helps to effectively detect unexpected blood types that may be present. In addition, in this embodiment, the spiral lamination chaos micro mixer 340 has a simple shape, which helps to manufacture the microfluidic biochip for blood type diagnosis in mass production. The mixture of sample and reagent mixed by the spiral lamination chaos micromixer 340 is transferred to the reaction microchambers 350 by a driving force given by an external pressure transfer device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. Is injected.

The reaction microchamber 350 is patterned in the upper and lower layers like the spiral lamination chaos micromixer 340 and is designed to increase the capacity of the chamber. In this case, the injected sample and reagent mixtures stay in each reaction micro chamber 350 for the reaction time (about 1 to 3 minutes) of the sample and the reagent by the second manual microvalve 360. At this time, when there is a coagulation source and agglomerate in the sample and the reagent, an aggregation reaction occurs between red blood cells.

The second passive microvalve 360 also has a shape such that the width of the first passive microvalve 330 suddenly decreases, and this shape induces a sudden change in the surface tension, thereby causing a sample and a reagent in the reaction microchamber 350. The effect of stopping the mixed fluid flow at the microvalve 360 is induced. In particular, the microvalve 360 may increase the surface tension effect by making the hydrophobic or hydrophilic surface according to the plastic surface quality. After a certain reaction time of the sample and reagent mixture of 1 to 3 minutes, it is injected into the microfilters 370 followed by a driving force given by an external pressure transmission device such as a syringe pump, surface tension, gravity generated by shaking the chip, and the like. .

The micro filter 370 introduced to the microfluidic biochip 300 for blood type diagnosis according to the present exemplary embodiment has a filter space that is reduced in multiple stages, so that the aggregated erythrocytes in which the aggregation reaction has occurred can be effectively filtered. In particular, since the filter space is larger than the size of the general red blood cells, the red blood cells that do not cause the aggregation reaction easily exit the micro filter 370, and the aggregated red blood cells that have increased due to the aggregation reaction are easily filtered through the micro filter 370. .

Finally, the sample and reagent reactants after the reaction time are discharged through the outlets 380 through the micro-filters 370. In this case, when the aggregation reaction between the sample and the reagent occurs, the coagulation erythrocytes are filtered through the micro filter 370. In particular, in the case of the microfluidic biochip 300 for blood type diagnosis of FIGS. 4 and 5, it is possible to simultaneously check four types of tests, and thus blood type diagnosis is possible.

Now, the manufacturing process of the microfluidic biochip for blood type diagnosis according to an embodiment of the present invention will be described with reference to FIGS. 6A to 6K. In describing the present embodiment, when it is determined that the detailed description of the related known technology or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. In addition, terms to be described below are terms defined in consideration of functions in the present invention, which may vary according to the intention or custom of a user or an operator. Therefore, the definition should be made based on the contents throughout the specification.

6A to 6K are flowcharts sequentially illustrating a manufacturing process of a microfluidic biochip for blood type diagnosis according to an embodiment of the present invention, in particular, a manufacturing process through mass production such as injection molding.

First, FIGS. 6A to 6E sequentially illustrate a top plate manufacturing process of the microfluidic biochip for blood type diagnosis according to the present embodiment.

First, the substrate (for example, a nickel metal substrate, 410) is washed through a surface cleaning process, and then a photoresist 420 such as SU-8 is applied to the sample inlet, the reagent inlet, and the sample flow separation microchannel as shown in FIG. 5A. UV light through the reagent transport microchannel, the first manual microvalve, the F-shaped mixing unit of the spiral lamination chaos micromixer, the reaction microchamber, the second manual microvalve, the microfilter and the outlet shape first groove 430 It is formed through a photolithography process.

Next, as shown in FIG. 6B, a sample inlet, a reagent inlet, a sample flow separation microchannel, a reagent transfer microchannel, a first manual microvalve, and a spiral lamination are applied to the substrate through a process such as electroplating or electroforming. A metal 440 such as nickel, copper, or the like is formed in the first groove 430 having the F-shaped mixing unit, the reaction micro chamber, the second manual micro valve, the micro filter, and the outlet shape of the chaotic micro mixer.

Next, if the photosensitive material 420 such as SU-8 formed as shown in Figure 6c is removed through a process such as etching, the mold insert for manufacturing the top plate of the microfluidic biochip for blood type diagnosis according to the present embodiment ( 450).

Next, as shown in Figure 6d through a mass production method such as injection molding, hot embossing, UV-molding, casting, COC (cyclic olefin copolymer), PMMA (polymethylmethacrylate), PS (polystyrene), PC (polycarbonate) The polymer 460 is molded, such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (Teflon), and polyvinylchloride (PVC).

Next, as shown in FIG. 6E, when the molded polymer 460 is taken out, the top plate 470 of the microfluidic biochip for blood type diagnosis according to the present embodiment may be obtained.

6F to 6J sequentially show a lower plate manufacturing process of the microfluidic biochip for diagnosing blood type according to an embodiment of the present invention.

First, the substrate (for example, a nickel metal substrate; 415) is washed through a surface cleaning process, and then F-shaped mixing of the spiral lamination micromixer is applied by applying a photosensitive material 425 such as SU-8 as shown in FIG. 6F. The second groove 435 having the shape of the unit and the reaction microchamber is formed through a general ultraviolet photolithography etching process.

Next, as shown in FIG. 6G, the second groove 435 having the shape of the F-shaped mixing unit of the spiral lamination micromixer and the reaction microchamber is formed on the substrate through a process such as electroplating or electroforming. Metals 445 such as nickel and copper are formed on the substrate.

Next, if the photosensitive material 425 such as SU-8 formed as shown in Figure 6h is removed through a process such as etching, the mold insert for manufacturing the lower plate of the microfluidic biochip for blood type diagnosis according to the present embodiment ( 480 may be formed.

Next, as shown in FIG. 6i, through a mass production method such as injection molding, hot embossing, UV-molding, casting, etc., COC (cyclic olefin copolymer), PMMA (polymethylmethacrylate), PS (polystyrene), and PC (polycarbonate) Polymers 465 such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (Teflon), and polyvinylchloride (PVC) are molded. Now, when the molded polymer 465 is taken out, the lower plate 490 of the microfluidic biochip for blood type diagnosis according to the present embodiment can be obtained.

Finally, the microfluidic biochip for the blood type diagnosis according to the present embodiment is prepared through a bonding method selected from thermal bonding, bonding through a bond material, lamination, and ultrasonic bonding as shown in FIG. 6K. When the upper plate 470 and the lower plate 490 are bonded to each other, a microfluidic biochip 400 for blood type diagnosis may be manufactured.

That is, mold inserts 450 and 480 for manufacturing the microfluidic biochip 400 for blood type diagnosis according to the present embodiment may be manufactured through FIGS. 6C and 6H, as seen through FIGS. 6E and 6I. An upper plate 470 and a lower plate 490 for manufacturing the microfluidic biochip 400 for diagnosing blood type according to the embodiment may be manufactured. Finally, as shown in FIG. 6K, the microfluidic biochip 400 for diagnosing blood type according to the present embodiment may be manufactured by bonding the upper plate 470 and the lower plate 490.

However, the manufacturing method of the microfluidic biochip of the present invention is not limited to the above-described method, and the microfluidic biochip for diagnosing blood types described in this embodiment is formed directly on the polymer through a precision molding process such as milling. It is also possible. In addition, the sample inlet, reagent inlet, sample flow separation microchannel, reagent transfer microchannel, first manual microvalve, spiral lamination chaos micromixer of microfluidic biochip for blood type diagnosis using photosensitive material without molding method The F-shaped mixing unit, the reaction microchamber, the second passive microvalve, the microfilter and the outlet space may be patterned or fabricated by directly etching a substrate such as silicon. Finally, it is also possible to use the patterned photosensitive material directly as a mold insert by patterning the photosensitive material on a substrate.

Now, the results of blood type diagnosis experiments using biochips together with microfluidic biochips for blood type diagnosis based on the basic concept and manufacturing method described above will be described.

FIG. 7 is a photograph showing a microfluidic biochip for diagnosing blood types actually manufactured according to an injection molding and heat bonding process according to an exemplary embodiment of the present invention.

Referring to FIG. 7, the actually manufactured microfluidic biochip 500 for blood type diagnosis has two basic units of the biochip for blood type diagnosis according to the second embodiment of the present invention shown in FIG. 3. Able to know. This is because the manufactured microfluidic biochip 500 for blood type diagnosis is designed to simultaneously perform a blood cell test and a serum test as a microfluidic biochip for one blood type diagnosis.

In addition, the microfluidic biochip 500 for blood type diagnosis shown in FIG. 7 includes a sample inlet 510, a reagent inlet 515, a flow separation microchannel 520 that is responsible for separation of a sample to simultaneously perform various tests, and Micro channel 525 for reagent transfer, first manual microvalve 530 for sample and reagent flow control, spiral lamination chaos micromixer 540 for effective mixing of samples and reagents, storing mixed samples and reagents Reaction microchamber 550 for inducing a reaction, second manual microvalve 560 for allowing the mixed sample and reagent to remain in the reaction microchamber, and red blood cells formed by agglutination of the mixed sample and reagent It can be seen that it is composed of a micro filter 570 for filtering the aggregates and an outlet 580 for discharging the tested sample and reagent.

8A to 8C show the results of blood type diagnosis experiments actually performed using the microfluidic biochip for blood type diagnosis according to the present invention actually manufactured according to the injection molding and heat bonding process according to one embodiment of the present invention. In particular, FIG. 8A shows the experimental results of blood type A, FIG. 8B shows the experimental results of blood type B, and FIG. 8C shows the experimental results of blood type AB.

The actual red blood cells were injected into the sample inlet, and the reagents of Anti-A and Anti-B were injected into the reagent inlet. The injected sample erythrocytes were separated through two flows and met with reagents of Anti-A and Anti-B and mixed through a spiral lamination chaos micromixer. Mixed samples and reagents were filtered through a micro filter after reaction in each reaction micro chamber.

In fact, in the case of type A blood type, as shown in FIG. In addition, in the case of blood type B, as shown in FIG. 8B, it can be confirmed that the aggregated red blood cells are filtered in the micro-filter of the test line into which the Anti-B is injected. Finally, as shown in FIG. 8C, it can be seen that the coagulated erythrocytes were filtered through the micro-filters in both test lines in which Anti-A and Anti-B were injected. Through this, it was confirmed that blood type diagnosis can be successfully performed through the microfluidic biochip for blood type diagnosis according to an embodiment of the present invention.

In the above, the present invention has been described with reference to the presently considered embodiments, but the present invention should not be construed as being limited to the above embodiments. Rather, it should be construed as including all modifications of the range which are easily changed by those skilled in the art from the above-described embodiment of the present invention and considered equivalent.

As described above, the microfluidic biochip for diagnosing blood types according to the present invention is a plastic chip including a sample inlet, a reagent inlet, a separate microchannel, a transfer microchannel, a chaos micromixer, a reaction microchamber, a microfilter, a manual microvalve, and an outlet. Implementing the above, it becomes possible to perform blood type diagnosis based on agglutination reaction which is frequently used as a qualitative diagnostic method in diagnostic test medicine, simply and accurately objectively at a desired position using a biochip.

Using the microfluidic biochip for diagnosing blood types according to the present invention has the advantage of increasing the accuracy and objectivity of blood type diagnosis by comparing the results of each other by simultaneously performing blood cell test and serum test. In addition, by placing the injection port of the sample and reagent separately, it is possible to perform a variety of flocculation test reaction through a variety of reagents, it is possible to perform a variety of blood tests at the same time because the sample is separated through a separate micro-channel.

In particular, the spiral lamination chaos micromixer introduced in the microfluidic biochip for blood group diagnosis according to the present invention effectively mixes the sample and the reagents to promote the aggregation reaction to increase the accuracy of the diagnosis and to detect the unexpected blood types that may exist. Also makes it possible. The first manual microvalve helps to introduce the sample and reagent into the micromixer at the same time, and the second manual microvalve allows the mixed sample and reagent to stay in the reaction microchamber for the reaction time. Finally, the microfilter, which consists of trapezoidal micro pillars and has a multi-stage filter space, effectively filters the aggregated red blood cells, improving the efficiency of the diagnostic test.

In addition, the sample inlet, the reagent inlet, the separation microchannel, the transfer microchannel, the chaos micromixer, the reaction microchamber, the microfilter, the manual microvalve and the outlet introduced into the microfluidic biochip for blood group diagnosis according to the present invention are in the shape thereof. This simple has the advantage that can be easily manufactured through mass production methods such as injection molding to secure the price competitiveness of biochips.

The microfluidic biochip for blood type diagnosis through the present invention may be applied to various clinical medical diagnostic tests based on agglutination tests.

Claims (35)

A sample transfer micro channel in which a sample inlet is formed; A reagent delivery micro channel in which a reagent inlet is formed; A micromixer connected to a rear end of the sample transfer microchannel and the reagent transfer microchannel, wherein the transferred sample and reagent meet and are transferred together; A first manual microvalve formed between a rear end of each of the microchannels and a front end of the micromixer, the first manual microvalve controlling the flow of the sample and the reagent; A reaction microchamber connected to a rear end of the micromixer and storing the mixed sample and reagents to induce a reaction; A micro filter connected to a rear end of the reaction micro chamber and filtering out aggregates formed by agglomeration of the mixed sample and reagents; A second manual microvalve formed between a rear end of the microchamber and a front end of the microfilter, the second manual microvalve controlling the mixed sample and reagent to stay in the reaction microchamber; And A microfluidic biochip for agglutination reaction based diagnostic tests connected to a rear end of the micro filter, the outlet including a discharged sample and a reagent outlet. The method of claim 1, The micro filter has a plurality of rows of micro pillars are arranged at periodic intervals along the conveying direction of the fluid, the filter space is formed between the micro pillars of each row, Wherein each filter space has an inlet formed wider than an outlet. The method of claim 2, In the micro filter, a plurality of rows of micro filters are arranged at uniform intervals along the fluid conveying direction. The method of claim 1, The micro filter is a microfluidic biochip for agglutination reaction based diagnostic test having a portion in which the filter space of each row is gradually reduced along the fluid transport direction in an initial section. The method of claim 4, wherein The micro filter is a microfluidic biochip for agglutination reaction based diagnostic test having a portion in which the size of the micro pillars of each row increases gradually in the initial section along the fluid transport direction. The method of claim 4, wherein The micro filter is a microfluidic biochip for agglutination reaction based diagnostic test having a portion in which the number of micro pillars in each row increases gradually in the initial section along the fluid transport direction. The method of claim 2, The micro-pillar is a microfluidic biochip for agglutination reaction based diagnostic test, wherein the front surface of the micro pillar is formed larger than the rear surface. The method of claim 7, wherein Flat section of the micro-pillar is a microfluidic biochip for agglutination reaction-based diagnostic test consisting of trapezoid, pentagon or hexagon. The method of claim 1, The first manual microvalve is a microfluidic biochip for agglutination reaction based diagnostic test which is formed such that its width is rapidly reduced from the sample transfer microchannel or the reagent transfer microchannel. The method of claim 1, And the second passive microvalve is rapidly reduced in width from the reaction microchamber. The method of claim 1, The sample transport microchannel is connected to each of a plurality of micro-mixers are divided into a plurality of micro-mixers, the microfluidic biochip for agglutination reaction-based diagnostic test to separate the injected samples to each micromixer. The method of claim 11, The reagent transfer micro channel is formed in plural and connected to the plurality of micro mixers, respectively, and each reagent transfer micro channel is formed with a plurality of reagent inlets separated from each other to inject different types of reagents. Microfluidic biochip for diagnostic testing based The method of claim 1, The micromixer is a microfluidic biochip for agglutination reaction based diagnostic tests for transferring the transferred sample and reagents through a three-dimensional spiral flow path and combining and mixing the splitting and rearrangement and the chaos mixing mechanism of the chaotic type. The method of claim 13, The micro mixer, An inlet channel having a pair of inlets through which the transferred sample and the reagent are respectively injected, and wherein the transferred sample and the reagent are joined together; An outlet channel through which the transferred sample and reagent are mixed and discharged; And Agglomeration reaction base including a mixing unit having a first mixing unit and a second mixing unit for mixing the transported sample and the reagent while being arranged in series and connected to each other between the inlet and outlet channels to form a three-dimensional spiral flow path Microfluidic biochip for diagnostic testing of blood. The method of claim 14, The first mixing unit comprises: at least a pair of primary split channels branched from the inlet channel and extending toward the first side with respect to the inflow channel traveling direction, through which the joined fluid is repartitioned; A first confluence channel disposed in a different layer from the first division channel and in communication with each end of the first division channel to allow the divided fluid to merge; The second mixing unit includes at least one pair of secondary split channels branched from the primary confluence channel and extending toward the second side piece in a direction opposite to the first side piece, through which the joined fluid is subdivided. And a secondary confluence channel disposed in a different layer from the secondary partition channel and in which fluids divided in communication with each end of the secondary partition channel join. The secondary confluence channel is a microfluidic biochip for diagnostic tests based on aggregation reaction leading to the outlet channel. The method of claim 15, The primary splitting channel may include a main channel extending in a direction parallel to the inflow channel, and a branching channel bent in a direction substantially perpendicular to a direction in which the main channel extends toward the first side. Microfluidic biochip for diagnostic testing of blood. The method of claim 15, The secondary divided channel may include a main channel extending in a direction parallel to the inflow channel, and a branch channel formed to be bent in a direction substantially perpendicular to a direction in which the main channel extends toward the second side. Microfluidic biochip for diagnostic testing of blood. The method of claim 15, The mixing unit is a microfluidic biochip for a diagnostic test based on agglutination reaction in which a plurality of first and second mixing units arranged in succession are alternately provided. The method of claim 15, The primary splitting channel of the first mixing unit is formed in a different layer from the secondary splitting channel of the second mixing unit, and the primary joining channel of the first mixing unit is the secondary joining channel of the second mixing unit. And microfluidic biochips for agglutination based diagnostic tests formed of different layers. The method of claim 15, The first confluence channel of the first mixing unit is a microfluidic biochip for agglutination reaction based diagnostic test is formed in the same layer as the secondary split channel of the second mixing unit. The method of claim 15, The primary and secondary division channels and the primary and secondary confluence channels are each formed to move substantially the same distance while the fluid divided through the respective division channels is transferred through the respective confluence channels to the recombination point. Microfluidic biochip for diagnostic tests based on aggregation reaction. The method of claim 1, A microfluidic biochip for agglutination reaction-based diagnostic tests capable of diagnosing blood types by injecting red blood cells of test blood into the sample and injecting standard serum into the reagents to perform blood cell tests. The method of claim 1, A microfluidic biochip for agglutination reaction-based diagnostic tests capable of diagnosing blood types by injecting serum of test blood into the sample and injecting standard red blood cells into the reagent to perform serologic tests. 24. A method of manufacturing a microfluidic biochip for agglutination based diagnostic tests according to any one of claims 1 to 23, The sample and reagent transfer microchannel, the first layer portion of the micromixer, the first and second passive microvalve, the first layer portion of the reaction microchamber, and a first groove having each shape of the microfilter. Manufacturing a first plate; Manufacturing a second plate comprising a second layer portion of the micromixer and a second groove having a respective shape of the second layer portion of the reaction microchamber; And Bonding the first plate and the second plate to each other Microfluidic biochip manufacturing method for agglutination-based diagnostic tests comprising a. The method of claim 24, Joining the first plate and the second plate against each other, A method of manufacturing a microfluidic biochip for agglutination reaction based diagnostic tests, comprising any one of thermal bonding, bonding through a bond material, lamination, and ultrasonic bonding. The method of claim 24, The step of manufacturing each plate, Forming a mold insert having the first groove or the second groove shape; Molding a polymer on the mold insert; And Taking out the molded polymer Microfluidic biochip manufacturing method for agglutination-based diagnostic tests comprising a. The method of claim 26, Forming the mold insert, Applying a photosensitive material to the substrate to form the first groove or the second groove through a photolithography process, to form a metal in the first groove or the second groove on the substrate, and then remove the photosensitive material from the substrate A microfluidic biochip manufacturing method for agglutination reaction-based diagnostic tests forming a mold insert. The method of claim 27, Forming a metal in the first groove or the second groove, A method for producing a microfluidic biochip for agglutination based diagnostic tests formed through electroplating or electroforming. The method of claim 26, The process of molding the polymer, A method for producing a microfluidic biochip for agglutination based diagnostic tests, comprising any one of injection molding, hot embossing, UV-molding, and casting. The method of claim 26, The polymer is a microfluidic biochip manufacturing method for agglutination reaction-based diagnostic test made of a plastic or curable polymer resin. The method of claim 26, The polymer is any one selected from the group consisting of COC (cyclic olefin copolymer), PMMA (polymethylmethacrylate), PS (polystyrene), PC (polycarbonate), PDMS (polydimethylsiloxane), Teflon (Polytetrafluoroethylene), PVC (polyvinylchloride) Microfluidic biochip manufacturing method for agglutination based diagnostic tests. The method of claim 26, Forming the mold insert, A microfluidic biochip manufacturing method for agglutination based diagnostic tests formed through a micromilling process. The method of claim 26, Forming the mold insert, A method of manufacturing a microfluidic biochip for agglutination based diagnostic tests using a photosensitive material on the substrate as a mold insert. The method of claim 24, The step of manufacturing each plate, A method of manufacturing a microfluidic biochip for agglutination reaction based diagnostic tests for directly forming a pattern of the first groove or the second groove in a polymer or a metal through a precision molding process. The method of claim 24, The step of manufacturing each plate, A method of manufacturing a microfluidic biochip for agglutination based diagnostic tests by directly etching a substrate to form a pattern of the first groove or the second groove.

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