Patent Description:
Enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA") is a method of measuring the amount of antigen or antibody using an antigen-antibody reaction with an enzyme as a marker.

ELISA is widely used as an analysis tool for quantitative investigation of antigens/antibodies inside an analyte in the fields of clinical medicine, environment, food contamination monitoring, etc. ELISA is comprised of simple processes, and is an efficient multi-analysis tool with high sensitivity and selectivity.

The process of the conventional ELISA is performed using a <NUM>-well plate that has ninety six densely, horizontally and vertically formed insertion holes into which each of ninety six sample beakers or tubes are inserted respectively, so that the data that can be read in colors is provided after a relatively long period of time. In addition, since the required amount of sample should be sufficient to fill a beaker or a tube to a certain level, it costs a significant price to perform the process, especially in the case of an expensive sample. Therefore, it is extremely difficult to apply the conventional method due to a problem that it takes a long time, and a cost problem especially when immediate diagnosis is required in the field.

In the conventional ELISA, a <NUM>-well plate is utilized, and it is based on a basic immunological concept in which a single antigen is bound to a specific antibody for that single antigen. So, in order to perform an immunoassay that requires to take several steps, each reaction step requires to wash away the substances other than the necessary substances out of reactants, and needs a process of transporting and injecting reagents with a pipette, and it takes a long time to react such sufficient amount of sample that may be observed with the naked eyes.

However, antibody immunoreactions are widely used not only for academic experiments in research institutes, but also for urgent prescriptions in a wide range of hospital sites, so they need to be carried out quickly for urgent treatment. But, since the conventional ELISA process takes considerable time, cost, and effort, it cannot meet the needs of on-site medical care so that many patients cannot receive treatment through accurate diagnosis in a timely manner.

Reaction process with ELISA includes a process in which a measurement sample such as blood or plasma is supplied to a substrate on which the capture antibody is immobilized and the target antigen present in the sample is bound to the capture antibody as shown in <FIG>, a process of binding the labeled antibody to the capture antibody-antigen conjugate in the form of a sandwich, and a process of finally bonding the enzyme substrate to the labeled antibody. At this time, in each reaction step, it is required to inject a sample such as an antigen or an antibody and wait for reaction for a long time, and it also requires a process of washing and removing unnecessary reactants in the middle of each reaction. Therefore, it consumes an enormous amount of time for one immunoreaction, and when a conventional <NUM>-well plate is used, a considerable amount of expensive sample is used so that the reaction can be visually determined, resulting in considerable cost.

Therefore, there is a request for a technology for a reaction device that enables antibody immunoreaction analysis much faster than the <NUM>-well plate used in the conventional ELISA, and requires much cheaper cost as the analysis can be performed with a very small amount of sample, and performs a plurality of reaction processes that include intermediate washing so that the intermediate washing is carried out automatically, thereby reducing the labor of the experimenter.

Accordingly, the present invention provides a microfluidic connection device that enables antibody immunoreaction analysis much faster than the <NUM>-well plate used in the conventional ELISA, and requires much lower cost as the analysis can be performed with a very small amount of sample, and performs a plurality of reaction processes that include intermediate washing so that the intermediate washing is carried out automatically, thereby reducing the labor of the experimenter.

The microfluidic connection device according to the present invention is defined in appended claim <NUM>.

In an embodiment of the present invention, a microfluidic connection device according to an embodiment of the present invention comprises: a microvalve comprising microtubes in form of a micro tube, and a fluid transfer unit that flows or stops samples filled in the microtubes; a plate-shaped plate; reaction zones installed on the upper surfaces of the plates, and having observation windows through which chemical reactions between samples supplied to therein through the microtubes are observed; supply tubes connecting the microtubes to the reaction zones; and a reaction plate comprising discharge tubes for discharging reactants from the reaction zones.

The reaction zones are formed at the upper ends of the reagent lifting columns installed on the upper surface of the plate in form of columns, and the areas where the supply tubes and the discharge tubes are connected to the reaction zones are elongated in a vertical direction in the reagent lifting columns.

The reaction zones are formed on the upper surfaces of the reagent lifting columns with a predetermined area, the reaction zones are protected by a sealing covers that seal the upper parts of the reagent lifting columns, and the observation windows are formed in the centers of the sealing covers.

Preferably, the reagent lifting columns, the reaction zones, and the sealing covers are formed in parallel on the upper surface of the plate, and a plurality of microtubes are installed to correspond to the number of the reagent lifting columns.

In this case, desirably, the microvalve comprises: a plate-shaped body; a plurality of microtubes embedded in parallel to each other in the body; a plurality of bumpers installed in longitudinal direction over some sections of the area where the microtubes are embedded; a pressure roller bar being a member in form of a rod having a circular cross section, installed on the upper parts of the bumpers so that its longitudinal direction crosses the bumpers, and for pressurizing a plurality of the bumpers at the same time by moving while rolling along the upper parts of the bumpers; and a drive unit for moving the pressure roller bar or the body along the direction of the microtubes, wherein the body has a plurality of microchannels formed in parallel to each other therein, wherein the microtubes are embedded in the microchannels, wherein the body and the bumpers are made out of elastic material, so that when either the pressure roller bar or the body is moved due to the drive unit, the pressure roller bar rolls along the upper portions of the bumpers to press the bumpers, thereby transferring samples filled in the microtubes by movement of compression areas formed by pressing the bumpers, the microchannel and the microtube with the pressure roller bar.

The microfluidic connection device according to the present invention may further comprise: a suction pump installed at the discharge tube, for taking in a plurality of samples filled in the microtubes under the pressure release sections so as to transfer a plurality of the samples toward the reaction zones when the pressure roller bar reaches the upper part of the pressure release sections, wherein the pressure release sections in which the bumpers are disconnected, are formed on the upper part of the microchannels, wherein the pressure release sections are formed to deviate from each other for each of a plurality of the microchannels, wherein when the pressure roller bar that is rolling on the upper part of the bumpers reaches the upper parts of the pressure release sections, the microtubes and the microchannels are released from compression.

Furthermore, the pressure release sections may be formed to meet the pressure roller bar sequentially from the microchannel at one end to the microchannel at the other end among a plurality of the microchannels while the pressure roller bar is rolling to advance.

Preferably, all discharge tubes each of which is provided for each of the reaction zones are connected to the one discharge hole, and the suction pump is installed to be connected to one discharge hole.

On the other hand, preferably, the microvalve further comprises: a base installed under the body; a rotation support bracket fixedly installed on both sides of the base, having bearings therein, and coupled to both ends of the pressure roller bar so as to rotatably fix the pressure roller bar; and a linear motor for advancing or reversing the microvalve between the bearing and the pressure roller bar.

Furthermore, a microfluidic connection device according to the present invention enables antibody immunoreaction analysis much faster than the <NUM>-well plate used in the conventional ELISA, and requires much lower cost as the analysis can be performed with a very small amount of sample, and performs a plurality of reaction processes that include intermediate washing so that the intermediate washing is carried out automatically, thereby reducing the labor of the experimenter.

Specific structural or functional descriptions presented in the embodiments of the present invention are exemplified for the purpose of describing the embodiments according to the concept of the present invention only. The embodiments according to the concept of the present invention may be implemented in various forms. In addition, it should not be construed as limited to the embodiments described in the present specification, and should be understood to include all modifications, equivalents, and substitutes that belong to the spirit and scope of the present invention.

The microfluidic connection device according to the present invention comprises a reaction plate <NUM> as shown in <FIG>.

The reaction plate <NUM> comprises a plate-shaped plate <NUM>, reaction zones <NUM> where a chemical reaction occurs between samples supplied through the microtubes <NUM>, which have observation windows for observing the chemical reaction and are installed on the upper surface of the plate, supply tubes <NUM> connecting the reaction zones <NUM> with the microtubes <NUM>, and discharge tubes <NUM> for discharging the reactants from the reaction zones <NUM>.

Here, column-shaped reagent lifting columns <NUM> are installed on the upper surface of the plate <NUM>, and the reaction zones <NUM> are formed at the upper ends of the reagent lifting columns <NUM>. The supply tubes <NUM> and the discharge tubes <NUM> are both elongated in a vertical direction in the reagent lifting columns <NUM> to be connected to the reaction zones <NUM> formed at the upper ends of the reagent lifting columns <NUM>.

Since the reagent lifting columns <NUM> are manufactured in column shapes as shown in <FIG>, they have the same shape as beakers or microbeakers that are densely inserted into a <NUM>-well plate used in a conventional antibody immunoreaction experiment. Therefore, it can be used together with equipment required for analysis experiments using various <NUM>-well plates such as ovens or cyclers manufactured to process conventional <NUM>-well plates.

However, since the reaction zones <NUM> are installed on the upper surfaces of the reagent lifting columns <NUM>, the reactions proceeds only in spaces of thin volumes formed on the upper surfaces of the reagent lifting columns <NUM>. So, it enables the analysis experiment which has been carried out in a conventional <NUM>-well plate, even if there is only a very small amount of reagent or the object to be measured since the required amount of the object to be measured is remarkably less than conventional <NUM>-well plate.

In addition, the reaction zones <NUM> are formed on the upper surfaces of the reagent lifting columns <NUM>, and the supply tubes <NUM> for supplying reagents or objects to be measured to the reaction zones <NUM> and the discharge tubes <NUM> for discharging the reactants from the reaction zones <NUM> are installed in the vertical direction in the inner spaces under the reagent lifting columns <NUM>. So, although the amount of the required sample is extremely small, the area where the reaction occurs are located in the reaction zones <NUM> which are the top portions most easily observed, so that the reaction is more easily observed compared to the conventional <NUM>-well plate.

As shown in <FIG> and <FIG>, an observation window is formed on the top of the reaction zone <NUM> which is formed on the upper surface of the reagent lifting column <NUM> for easy observation. However, in <FIG> and <FIG>, the observation window is so transparent that it may not be visible, but there is an observation window at the top of the reaction zone <NUM>. In particular, the observation window is formed in the center of the upper surface of the sealing cover <NUM> that seals the upper portion of the reagent lifting column <NUM>.

The sealing cover <NUM> seals the upper part of the reagent lifting column <NUM> as well as the reaction zone <NUM> as shown in <FIG>. The sealing cover <NUM> is particularly detachably coupled to the reagent lifting column <NUM>. So, If it is urgently needed to collect the reactants, the sealing cover <NUM> is separated from the reagent lifting column <NUM> so that the reactants can be collected directly from the reaction zone <NUM>. In addition, even when disinfection and precise cleaning of the reaction zone <NUM> are required, disinfection and cleaning are possible by removing the sealing cover <NUM> from the reagent lifting column <NUM>. At this time, it is also possible to clean the conventional <NUM>-well plate with specialized equipment commonly used.

The reaction zone <NUM> is formed at an area where the inner ceiling of the sealing cover <NUM> and the upper surface of the reagent lifting column <NUM> meet. The space formed by the reaction zone <NUM> is specifically a space formed by processing the upper surface of the reagent lifting column <NUM> to have a pattern having a certain shape such as the long hexagonal shape shown in <FIG>. Therefore, when the sealing cover <NUM> is coupled to the upper surface of the reagent lifting column <NUM>, the processed pattern-shaped space becomes the reaction zone <NUM>.

At this time, the capture antibody is immobilized on the inner ceiling of the sealing cover <NUM>, that is, the lower surface of the observation window. Immobilization of the capture antibody is the same as the commercially available product in which a certain capture antibody is immobilized inside the well in the case of a <NUM>-well used in a typical immunoreaction experiment.

The conventional <NUM>-well comprises twelve rows of wells. Each row of wells comprises eight wells and are manufactured to be connected side by side with each other. At this time, a row of eight wells can be used as eight sealing covers <NUM> connected to each other as shown in <FIG>.

In particular, since the commercially available sealing covers <NUM> comprises eight wells shown in <FIG> have capture antibodies that have already been immobilized to the inner ceilings, a commercially available <NUM>-well can be immediately used as the sealing covers <NUM> of the present invention without separately manufacturing sealing covers or separately immobilizing the capture antibodies to the manufactured sealing covers in the present invention.

When the commercially available sealing covers <NUM> comprising eight wells are coupled to the upper portions of the eight reagent lifting columns <NUM>, the upper surfaces of the reagent lifting columns <NUM> are sealed due to the eight wells, and the reaction zones <NUM> are sealed. So, an antigen-antibody reaction can be performed with the capture antibodies.

As shown in <FIG>, the reaction zone <NUM> receives a sample or an object to be measured from the supply tube <NUM>, and discharges the substance remaining after the reaction or the substance washed away after intermediate washing through the discharge tube <NUM>. The reaction zone <NUM> has a planar shape in the form of a hexahedron as shown in <FIG>, but there is no particular limitation on the shape of the reaction zone <NUM> and may be formed in a circular shape as in <FIG>. The reaction zone <NUM> has a certain area, but the distance between the observation window and the reaction zone <NUM>, that is, the height of the space that makes up the reaction zone <NUM> is very small, so the reaction can be performed with only a very small amount of sample or object to be measured. Therefore, the microfluidic reaction device according to the present invention requires such extremely small amount of sample for the reaction that only a small amount is required even for an expensive sample. So, compared to the prior art, the required cost is significantly reduced in the present invention.

The supply tube <NUM> and the discharge tube <NUM> are connected to the injection hole <NUM> and the discharge hole 18a, respectively, as shown in <FIG>. The injection hole <NUM> and the discharge hole 18a may be in the form of a simple hole as shown in <FIG>, and it may also include a short tube protruding above the plate <NUM> as shown in <FIG> and <FIG>.

The supply tube <NUM> and the discharge tube <NUM> are embedded horizontally in plate <NUM> in the section from the lower end of the reagent lifting column <NUM> to the injection hole <NUM> or the discharge hole 18a, as shown in <FIG>.

Furthermore, the reagent lifting column <NUM> may be formed on the bottom surface of the plate <NUM> as shown in <FIG>. At this time, the injection hole <NUM> and the discharge hole 18a are formed on the upper surface of the plate <NUM>. This configuration is made because a conventional <NUM>-well can be used as a sealing cover, and when a fluid material is embedded in the <NUM>-well, the reaction can be observed due to the combination and configuration as shown in <FIG>.

Any material that is inexpensive and has good chemical resistance can be selected for the plate <NUM>. Materials having good transparency, very strong durability, and good workability, such as PDMS (polydimethylsiloxane) may be selected as the material of the plate <NUM>.

The plate <NUM> may be manufactured by overlapping two plates <NUM> and <NUM>. At this time, the horizontal portions of the supply tube <NUM> and the discharge tube <NUM> can be made by inserting tubes between the two plates constituting the plate <NUM>, that is, the upper plate <NUM> and the lower plate <NUM>, or by forming horizontal grooves on the bottom surface of the upper plate <NUM> or on the upper surface of the lower plate <NUM>. The bottom surface of the upper plate <NUM> and the upper surface of the lower plate <NUM> are the contact portions that the upper plate <NUM> and the lower plate <NUM> contact each other.

In addition, a microvalve <NUM> for supplying a sample to the reaction plate <NUM> may be connected to the reaction plate <NUM>.

The microvalve <NUM> includes microtubes <NUM> in the form of microtubes, and a fluid transfer unit for moving the sample when the microtubes <NUM> are filled with samples. The microvalve <NUM> will be described in detail later with reference to <FIG>.

In the present invention, as in the embodiment shown in <FIG>, a plurality of reagent lifting columns <NUM> are arranged side by side on the upper surface of the plate <NUM>. Accordingly, as in a conventional <NUM>-well plate, a plurality of related reaction experiments can be performed simultaneously. At this time, each of a plurality of microtubes <NUM> for connecting the microvalve <NUM> and the reaction plate <NUM> are also connected to each of the reagent lift columns <NUM> as many as the number of reagent lift columns <NUM> as shown in <FIG>.

As shown in <FIG>, the microvalve <NUM> comprises: a plate-shaped body <NUM>; a plurality of microtubes <NUM> embedded in parallel to each other in the body <NUM>; a plurality of bumpers <NUM> installed in longitudinal direction over some sections of the area where the microtubes <NUM> are embedded; a pressure roller bar <NUM> which is a member in the form of a rod having a circular cross section, installed on the upper parts of the bumpers <NUM> so that its longitudinal direction crosses the bumpers <NUM>, and pressurizes a plurality of the bumpers <NUM> at the same time by moving while rolling along the upper parts of the bumpers <NUM>; and a drive unit (not shown) for moving the pressure roller bar <NUM> or the body <NUM> along the direction of the microtubes <NUM>.

According to the embodiment shown in <FIG>, the body <NUM> in which the microtubes <NUM> are embedded may be formed by overlapping the upper plate <NUM> and the lower plate <NUM>. Here, the microchannels <NUM> are formed on the bottom surface of the upper plate <NUM>, and the upper plate <NUM> is formed of an elastic material. So, when a pressure is applied to the bumpers <NUM> above microchannels <NUM>, the pressure is transmitted through the upper plate <NUM>, so the microchannels <NUM> are deformed flat as shown on the right side of <FIG>. When the microchannels <NUM> are flattened under pressure, the microtubes <NUM> embedded in the microchannels <NUM> are also deformed flat and clogged as shown in <FIG>. Hereinafter, the area at which the microtubes <NUM> become flat will be referred to as 'compression areas'.

Since the 'compression areas' are formed in the vertical lower part of the pressure roller bar <NUM>, when the pressure roller bar <NUM> moves along the upper parts of the bumpers, the compression areas also move together with the pressure roller bar <NUM>. However, the pressure roller bar <NUM> may either move or only rotate in place and the body <NUM> in which the microtubes <NUM> are embedded may move instead. The direction of movement is the longitudinal direction of the bumpers <NUM> formed along the longitudinal direction of the microtubes <NUM>.

As shown in <FIG> and <FIG>, the bumpers <NUM> transmit the pressure of the pressure roller bar <NUM> to the microtubes <NUM> so as to seal certain areas of the microtubes <NUM>. The microtubes <NUM> are sealed at the very compression areas, and the compression areas prevent the reagents or the objects to be measured filled in the microtubes <NUM> from moving when the pressure roller bar <NUM> and the body <NUM> are both stopped.

However, if only when one of the pressure roller bar <NUM> or the body <NUM> moves, a samples or objects to be measured (hereinafter, referred to as 'sample or the like') filled in the microtubes <NUM> are moved, then the control of the moving distance of a fine sample or the like depends only on the pressure roller bar <NUM>, so that a plurality of microtubes <NUM> are arranged in parallel as shown in <FIG> and the sample or the like is filled at different location corresponding to a desired reaction time in each microtube <NUM>. So, the samples or the like moving inside the microtubes <NUM> arrive at a different time. Due to the error during movement, however, the moving speed or the order of the sample or the like inside each microtube <NUM> may vary.

Therefore, in the present invention, a pressure release sections <NUM> and a suction pump <NUM> are provided in order that the samples or the like filled in the microtubes <NUM> may reach the reaction zones <NUM> sequentially in order according to the experiment plan.

The pressure release sections <NUM> are sections where the bumpers <NUM> are disconnected as shown in <FIG>. The sections where the bumpers <NUM> are disconnected are not crushed because the microchannels <NUM> and the microtubes <NUM> are not subjected to pressure even when the pressure roller bar <NUM> presses the sections from the top as shown on the right side of <FIG>. Therefore, when air is blown or sucked from either side of the microtubes <NUM>, the samples or the like get ready to be moved accordingly.

The suction pump <NUM> is connected to the discharge hole 18a formed in the reaction plate <NUM> as shown in <FIG>. One discharge hole 18a is formed in the reaction plate <NUM>, and a plurality of the discharge tubes 18b which are connected to each of a plurality of the reaction zones <NUM> and discharge the reaction residue from the reaction zones <NUM> are all connected to one discharge hole 18a. It is not only because if the suction pump <NUM> is installed for each of a plurality of the discharge tubes 18b, the cost will also increase but also because if it is necessary to control a plurality of suction pumps <NUM> to control the speed of the sample or the like that reaches each reaction zone <NUM>, a control system for sequentially controlling the suction pumps <NUM> is also required, thereby increasing the cost required for this too.

Accordingly, in the present invention, one suction pump <NUM> is provided for one discharge hole 18a and the pressure release sections <NUM> are sequentially formed in the bumpers <NUM> in a desired reaction order. And, when the relative motion between the pressure roller bar <NUM> and the body <NUM> occurs and the pressure release sections <NUM> formed on the upper portions of microtubes <NUM> and the pressure roller bar <NUM> meet, the pressure is released at the microtubes <NUM>. Here, when the suction pump <NUM> is operated, the samples or the like are moved only within the microtubes <NUM> where the pressure is released.

The pressure release sections <NUM> are also formed sequentially in a desired reaction order. As shown in <FIG>, since the suction pump <NUM> is installed, the reactions proceed at the reaction zones15 connected to the pressure release sections <NUM> in the order of meeting the pressure release sections <NUM> during the pressure roller bar <NUM> moves.

Referring to <FIG>, all of the microtubes <NUM> other than the microtubes <NUM> where the pressure release sections <NUM> are formed are compressed by the pressure roller bar <NUM>. Since only the microtubes <NUM> where the pressure release sections <NUM> are located under the pressure roller bar <NUM> are opened, the samples or the like are moved only in the opened microtubes <NUM> and they enter the reaction zones <NUM> when the suction pump <NUM> is operated.

In addition, since the suction pump <NUM> is installed, even if the pressure roller bar <NUM> does not move several times to push the samples or the like, the samples or the like were made to reach the reaction zones <NUM> at once by the suction force as soon as the pressure roller bar <NUM> is positioned above the pressure release sections <NUM>. Therefore, the entire reaction analysis process can be quickly performed due to the interaction between the suction pump <NUM> and the pressure release sections <NUM>.

On the other hand, when the samples or the like move toward the reaction zones <NUM> by the operation of the suction pump <NUM>, the residues other than the substances required after any one reaction need to be discharged in order for a series of two or more reactions to occur in one reaction zone 15d. Therefore, as shown in <FIG>, the first washing solution b-<NUM> and the second washing solution b-<NUM> are filled between the first reagent a-<NUM> and the second reagent a-<NUM>, and between the second reagent a-<NUM> and the third reagent a-<NUM> respectively, so as to wash the reaction zone <NUM>. So, the next reaction can be prepared. As an example in which such a series of reactions occurs in one place, there may be mentioned the ELISA described with reference to <FIG> in the section BACKGROUND OF THE INVENTION. In this case, the first antibodies are coated on the reaction zones <NUM> in advance, the first reagent a-<NUM> is the target antigen, the second reagent a-<NUM> is a second antibody bound in a sandwich form, and the third reagent a-<NUM> becomes an enzyme substrate.

Since such a series of reactions occur at the bottom of the beaker in a conventional <NUM>-well plate, observation was rather difficult even though a significant amount of expensive sample was required, but in the present invention, reactions can be observed at the optimal observation position even with a very small amount of sample.

In addition, there may be a case where the position of the pressure release sections <NUM> need to change or adjust between the microchannels <NUM> according to the needs for the experiment. In preparation for this case, the bumpers <NUM> are separately manufactured to be separated from the upper plate <NUM> so that the pressure release sections <NUM> can be formed in the desired sections as shown in <FIG>. And, the bumpers <NUM> are manufactured in the form of a module with a certain length and a bumper gripping protrusion <NUM> to which the bumpers <NUM> are detachably fixed may be formed on the upper surface of the upper plate <NUM>.

The bumper gripping protrusion <NUM> may be installed so that its one end and other end are fixed in the longitudinal direction of the bumpers <NUM> as shown in <FIG>. Here, the bumper gripping protrusion <NUM> is also made out of an elastic material. Therefore, when the pressure roller bar <NUM> passes, the bumper gripping protrusion <NUM> does not hinder the bumpers <NUM> from contracting, and the bumpers <NUM> and the bumper gripping protrusion <NUM> may be compressed together.

In the process of pressing the bumpers <NUM> while the pressure roller bar <NUM> moves along the upper parts of the bumpers <NUM>, the pressure roller bar <NUM> itself may be moved along the longitudinal direction of the bumpers <NUM>, or the body <NUM> constituting the microvalve <NUM> may be itself moved while the pressure roller bar <NUM> is fixed in place and rotated only.

In the embodiment shown in <FIG>, the bumpers <NUM> is rotated in place and the body <NUM> moves. For this operation, the microvalve <NUM> further comprises: a base <NUM> installed under the body <NUM>; a rotation support bracket <NUM> fixedly installed on both sides of the base <NUM> and having bearings (not shown) therein and coupled to both ends of the pressure roller bar <NUM> to rotatably fix the pressure roller bar <NUM>; a linear motor <NUM> for advancing or reversing the body <NUM> between the bearing and the pressure roller bar <NUM>. In this case, when the reaction plate <NUM> and the body <NUM> constituting the microvalve <NUM> are fixedly coupled together and move, the microtubes <NUM> are prevented from being pulled out of the injection holes <NUM> and separated from the injection holes <NUM> due to the applied tension even if the microtubes <NUM> are not made manufactured longer than necessary.

Owing to the configuration in this way, the process of pulling the body <NUM> only once by the linear motor <NUM> completes all series of reactions for each reaction zones <NUM>, and all the reactions in the reaction zones <NUM> can be sequentially completed in the desired order. At this time, the linear motor <NUM> stops when the pressure roller bar <NUM> is positioned at any one pressure release section <NUM>. From then on, the suction pump <NUM> collects the samples a-<NUM>, a-<NUM> and a-<NUM> and the washing liquids b-<NUM> and b-<NUM> sequentially through the microtubes <NUM> with the pressure release sections <NUM> open. All sequential reactions are performed in the reaction zones <NUM> connected to the microtubes <NUM> where the pressure roller bar <NUM> is located at the upper portions of the pressure release sections <NUM>, and the linear motor <NUM> is stopped during this time.

This process is shown in the photograph of <FIG>. In <FIG>, the reaction proceeds in the order of (a), (b), (c), (d), (e) and (f).

On the other hand, the amount of sample required for the reaction to proceed clearly is shown in the photograph of <FIG>. The eight pictures shown in <FIG> are taken from the above of the reaction zones <NUM>, and each picture shows the degree of reaction of a different amount of sample administered by the intensity of the color.

The photographs of <FIG> is a color change of the reaction zones when the reaction of the same procedure as the actual ELISA procedure for quantitatively detecting cardiac tro-ponin I (cTnI) protein is performed in the microfluidic reaction device according to the present invention. Here, the first antibodies are coated in the reaction zones <NUM>. And, <NUM>µL cTnI antigen (cTnI protein, Enzo Biosemic, USA), <NUM>µL washing solution, and secondary antibody conjugated with <NUM>µL HRP substrate were sequentially passed through here first, and the antigen binding reaction, washing, and secondary antibody binding reaction proceed in order.

In this case, the color change corresponding to the eight different types of the concentrations of cTnI in the <NUM>µL cTnI antigen is shown in <FIG>. At this time, a clear detection reaction was observed at the concentration of <NUM> pg/mL. At concentrations above <NUM> pg/mL, the color is the same even if the concentration of cTnI is increased. So, even if a clear reaction observation is desired, it can be seen that a successful experimental process can be performed only with cTnI at a concentration of <NUM> pg/mL.

Meanwhile, the graph of <FIG> shows the reaction time of the enzyme substrate and the amount of the produced reactant with a series of points. As shown in <FIG>, it can be seen that the amount of the produced reactant increases as the reaction time increases until a certain point but the amount of the produced reactant keeps constant even after a certain time passes. That is, it can be seen that the smaller the amount of sample or the like is, the shorter the required reaction time is as in the present invention.

The graphs shown in <FIG> show the amount of the product according to the concentration of cTnI, and are graphs corresponding to the photographs shown in <FIG>. It can be seen that the amount of the product has a linear relationship with the concentration of cTnI. However, since the concentration at which a clear reaction result can be obtained is <NUM> pg/mL as previously seen, there is no need to increase the concentration of cTnI more than that.

The table shown in <FIG> is a comparison of the amount of sample and the reaction time required in the ELISA reaction process using the conventional <NUM>-well plate and in the ELISA reaction process using the microfluidic reaction device according to the present invention. The leftmost data is the amount and time required in the case of the present invention, and it can be seen that the amount of the sample and reaction time required in the present invention are significantly smaller than that of the prior art.

As described so far, in the present invention, owing to the reaction section formed with an extremely small height, the immunoreaction test is possible even with a small amount of sample, so that the required cost and time are drastically reduced. And, furthermore, due to the interaction between the suction pump and the pressure release sections, a plurality of channels can react independently and sequentially. In addition, it is automatically and conveniently performed without any preparatory procedures such as supplying reagents with a dropper every time for a series of reactions in one reaction zone and washing a microbeaker between reactions and reactions. Therefore, there is no need for the labor of performing an immunoreaction test with concentration for a long time, and the time required for the entire reaction process is further shortened.

Therefore, the examination apparatus according to the present invention can perform an examination on the spot even in an emergency situation requiring an urgent examination, thereby enabling a dramatic improvement in the quality of medical services.

Claim 1:
A microfluidic connection device comprising:
a plate-shaped plate (<NUM>);
reaction zones (<NUM>) being microchambers installed at predetermined areas of the plate (<NUM>), and having observation windows through which chemical reactions between samples supplied to therein are observed;
supply tubes (<NUM>) connected to the reaction zones (<NUM>) so as to supply reagents to the reaction zones (<NUM>); and
discharge tubes (<NUM>) for discharging reactants from the reaction zones,
wherein the supply tubes (<NUM>) and the discharge tubes (<NUM>) are elongated in a vertical direction therein on one side of the plate (<NUM>), so that reagent lifting columns (<NUM>) in the form of columns through which reagents are lifted are formed,
wherein the reaction zones (<NUM>) are formed at the ends of the reagent lifting columns (<NUM>), and the supply tubes (<NUM>) and the discharge tubes (<NUM>) are connected to the reaction zones (<NUM>), and
characterized in that
the reagent lifting columns (<NUM>) are coupled to sealing covers (<NUM>), so that the reaction zones (<NUM>) are formed to be closed spaces due to the inner surfaces of the sealing covers (<NUM>), and immobilized capture antibodies are provided on the inner surfaces of the sealing cover (<NUM>).