Abstract:
Disclosed is a micro-fluidic chip for high-throughput screening and high-throughput assay, in which its structure is improved, thereby enhancing the efficiency of high-throughput screening and high-throughput assay. The micro-fluidic chip includes a well for isolating a specimen. The well can be arranged in a one- or two-dimension. A specimen-isolating means is disposed above the well and is movable upwards and downwards. An opening and closing means for moving the specimen-isolating means upwards and downwards is disposed above the specimen-isolating means. An inlet for injection the specimen and an outlet for discharging an excess of the injected specimen are provided. A reagent-injecting passage for injecting a reagent and a reagent-discharging passage for discharging the reagent are also provided.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a micro-fluidic chip, particularly to such a chip for high-throughput screening and high-throughput assay, in which its structure is improved, thereby enhancing the efficiency of high-throughput screening and high-throughput assay. 
     2. Background of the Related Art 
     In general, a well plate has been used for biological and chemical experiments. There are a 16-well plate, a 48-well plate or a 96-well plate depending on the number of wells. Recently, a plate having more than 1536 wells has been introduced for a high-throughput assay. 
     Further, the advancement in micro-machining technique facilitates the development of micro-fluidic chips for a high-throughput assay. U.S. Pat. No. 6,235,520 B1 discloses a micro-fluidic chip having a high degree of integration. However, only the highly integrated structure would not help injecting a reagent into each individual well independently. 
     Also, an article, “Microfluidic device for single-cell analysis” A. R. Wheeler; Analytical Chemistry, Vol. 75, pp. 3581-3586, 2003, has proposed a chip in which a cell is immobilized at a desired place, and a reagent is injected through a fluid passage. However, it has disadvantages in that the cell cannot be stably held in place in absence of fluid flow and many numbers of cells cannot be analyzed at the same time due to its one-dimensional configuration. 
     The above-described conventional micro-fluidic chip has increased only its degree of integration, and thus additional control devices are required for loading a specimen and reagent into the highly-integrated wells. Furthermore, in the case where the specimen and reagent are a fluid, its amount is reduced due to the high integration and thus easily evaporated. 
     SUMMARY OF THE INVENTION 
     Therefore, the present invention has been made in view of the above problems occurring in the prior art, and it is an object of the present invention to provide a micro-fluidic chip, in which different reagents can be injected into respective different wells. 
     Another object of the present invention is to provide a micro-fluidic chip, in which wells can be arranged in a two-dimensional pattern and thus a degree of integration can be improved. 
     Another object of the present invention is to provide a micro-fluidic chip, in which a specimen or reagent can be isolated inside the well and be prevented from evaporating outside the well. 
     Another object of the present invention is to provide a micro-fluidic chip, in which a pair of electrode is disposed inside the well, thereby enabling an easy injection of reagent into the well by means of dielectrophoresis phenomena. 
     Another object of the present invention is to provide a micro-fluidic chip, in which an electric reaction can be detected using an electrode disposed inside the well. 
     To accomplish the above objects, according to one aspect of the present invention, there is provided a micro-fluidic chip for high-throughput screening and high-throughput assay. The micro-fluidic chip of the invention comprises: a) a well for isolating a specimen, the well being capable of being arranged in a one- or two-dimension; b) a specimen-isolating means disposed above the well and movable upwards and downwards; c) an opening and closing means disposed above the specimen-isolating means and for moving the specimen-isolating means upwards and downwards; d) an inlet for injection the specimen and an outlet for discharging an excess of the injected specimen; and e) a reagent-injecting passage for injecting a reagent and a reagent-discharging passage for discharging the reagent. 
     According to the invention, the well is highly integrated in the micro-fluidic chip in a one- or two-dimensional fashion. In addition, the specimen-isolating means isolates the specimen and reagent inside the well and prevents them from evaporating. The opening and closing means opens and closes the specimen-isolating means. Furthermore, the reagent-injecting passage is formed at each well such that the reagent is selectively injected. In this way, the micro-fluidic chip of the invention can perform high-throughput screening and high-throughput assay more efficiently. 
     According to another aspect of the invention, there is also provided a micro-fluidic chip for high-throughput screening and high-throughput assay. The micro-fluidic chip of the invention comprises: a) a well for isolating a specimen, the well being capable of being arranged in a one- or two-dimension; b) a specimen-isolating means disposed above the well and movable upwards and downwards; c) an opening and closing means disposed above the specimen-isolating means and for moving the specimen-isolating means upwards and downwards; d) an inlet for injection the specimen and an outlet for discharging an excess of the injected specimen; e) a reagent-injecting passage for injecting a reagent and a reagent-discharging passage for discharging the reagent; and f) a pair of metal electrode formed inside the well and causing a dielectrophoresis phenomena. 
     According to the invention, a pair of electrodes is disposed inside of each well, so that the specimen can be easily entrapped into the inside of the well, using the dielectrophoresis phenomena. Furthermore, the electrode can be used for detecting the electric reaction of the specimen. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and other objects, features and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments of the invention in conjunction with the accompanying drawings, in which: 
         FIG. 1   a  is a plan view schematically showing a micro-fluidic chip according to a first embodiment of the invention; 
         FIG. 1   b  is a cross-sectional view schematically showing a micro-fluidic chip according to a first embodiment of the invention; 
         FIG. 2  is a perspective exploded view of the micro-fluidic chip according to the first embodiment of the invention; 
         FIG. 3  shows an operation of the micro-fluidic chip according to the first embodiment of the invention; 
         FIG. 4  shows a cross-section of a micro-fluidic chip according to a second embodiment of the invention; and 
         FIG. 5  is a photograph showing a micro-fluidic chip fabricated according to the first embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Reference will now made in detail to the preferred embodiment of the present invention with reference to the attached drawings. 
       FIGS. 1   a  and  1   b  are respectively a plan view and a cross-sectional view schematically showing a micro-fluidic chip according to a first embodiment of the invention. 
     As shown in  FIGS. 1   a  and  1   b , the micro-fluidic chip comprises a well  10 , a specimen isolating means  20 , an opening and closing means  30 , an inlet  40 , an outlet  50 , a reagent-injecting passage  60 , and a reagent-discharging passage  70 . 
     The well  10  is arranged in one- or two-dimension, in which a specimen such as a cell, a bead or a solution is placed. Also, a reagent is injected thereto in order to detect a reaction with the specimen. 
     In addition, the wells  10  may be extended endlessly in a two-dimensional plane. Furthermore, the two-dimensional arrangement of the wells  10  may be carried out in a patterned or non-patterned fashion. 
     Also, the amount or number of isolated specimen to be injected into the well  10  can be varied with the size of the well  10 . 
     The specimen-isolating means  20  is disposed above the well  10 , and movable upwards and downwards to thereby be able to isolate the specimen placed inside the well  10 . The specimen and reagent are isolated inside the well  10  so that they are prevented from escaping or evaporating outside the well and entering neighboring wells. 
     The opening and closing means  30  is disposed at the upper end of the specimen-isolating means  20 , and opens or closes the well  10  by moving upwards and downwards the specimen-isolating means  20 . The opening and closing means  30  is provided with a space  90  formed therein to place the specimen-isolating means upwardly. 
     According to the first embodiment of the invention, the opening and closing means  30  is provided with a pneumatic passage  80 , and the specimen-isolating means  20  is opened and closed by an air pressure through the pneumatic passage  80 . In  FIG. 1 , the pneumatic passage  80  is disposed centrally at the left side of the opening and closing means  30 , but may be disposed at any position of the opening and closing means  30 . The opening and closing means  30  having the pneumatic passage  80  will be hereinafter described in greater detail. 
     The specimen is injected into the inside of the micro-fluidic chip from the inlet  40 , and a certain amount of the specimen is entered into the inside of the well. Then, the remaining excessive portion of the injected specimen is discharged through the outlet  50 . 
     The reagent-injecting passage  60  is a passageway through which a reagent is injected into the wells  10 . The reagent-injecting passage  60  is communicated with each well  10  through different channels. Therefore, the same or different reagents can be injected through the channels to the respective wells from the reagent-injecting passage  60 . 
     The superfluous reagent excepting a certain amount required for the reaction is discharged through the reagent-discharging passage  70 , also through which the reagent used for the reaction is discharged. In  FIG. 1   a , the reagent-discharging passage  70  is formed as the same channel, but may be embodied in different channels. 
       FIG. 2  is a perspective exploded view of the micro-fluidic chip according to the first embodiment of the invention. As shown in  FIG. 2 , the micro-fluidic chip of this embodiment is formed of four substrates combined with each other. 
     A first substrate  210  is provided as a base of the micro-fluidic chip. 
     Above the first substrate  210  is provided a second substrate  220 . The second substrate  220  is provided with wells  10  arranged in a one- or two-dimensional pattern, and, at the lower end, with a reagent-injecting passage  60  and a reagent-discharging passage  70  connected to the well  10 . 
     Above the second substrate  220  is disposed a third substrate  230 . Here, the third substrate  230  has the form of a thin cover  231  and serves as the specimen-isolating means  20 . 
     A fourth substrate  240  is disposed above the third substrate  230 . The fourth substrate  240  is provided with an empty space thereinside. In addition, the fourth substrate  240  is constructed such that the third substrate  231  can be lifted or descended by an air pressure through the pneumatic passage  80 . That is, the fourth substrate  240  functions as an opening and closing means  30 . 
     Here, the micro-fluidic chip of the invention may be fabricated by forming four or more substrates and combining them, or any two or more of the above substrates may be formed of one unitary substrate and combined into the micro-fluidic chip of the invention. 
     The micro-fluidic chip of the invention may be fabricated using the semi-conductor process or the microelectro-mechanical Systems (MEMS) technique. 
     The respective substrates described above may be formed of various materials, such as silicon, glass, PDMS (polydimethilsiloxane), silicon rubber, or other polymers. 
       FIG. 3  shows an operation of the micro-fluidic chip according to the first embodiment of the invention. As shown in  FIG. 3(   a ), first, the cover  231  as a specimen-isolating means has a downwardly convex shape. In this way, the cover  231  is pressed downwardly by a certain magnitude of force due to its convexity so that the respective wells  10  can be isolated. 
     Afterwards, as shown in  FIG. 3(   b ), an air is suctioned through the pneumatic passage  80  and transferred into the empty space  90  above the cover  231 . 
     Then, a specimen is injected through the inlet  40 . The injected specimen is discharged towards the outlet  50 , and simultaneously in part flows towards the reagent-discharging passage  70 . Here, since the size of the reagent-discharging passage  70  is smaller than that of the specimen, the specimen such as cells, beads, or the like cannot be discharged through the reagent-discharging passage  70 , but trapped inside the well  10 . 
     When a certain amount of the specimen  310  is injected into the inside of the well  10 , the reagent-discharging passage  70  is more or less blocked such that the amount of the specimen exiting therethrough is reduced. Therefore, after that, the specimen entering through the inlet  40  can no longer flow towards the well containing a specimen, but flows out towards the outlet  50 . 
     Here, the amount or number of specimen  310  to be injected and isolated inside the well  10  may be varied with the size of the well  10 . 
     Next, as shown in  FIG. 3(   c ), an air is injected through the pneumatic passage  80  into the opening and closing means  30  to again move the cover  231  downwardly. That is, the specimen  310  inside the well  10  is isolated by blocking the upper portion of the well  10 . 
     Then, a reagent  320  needed for reaction is injected through the reagent-injecting passage  60 , and thus, a desired experiment can be carried out inside the well  10 . Here, the injected reagent  320  can be prevented from entering neighboring wells since the upper portion thereof is blocked. Also, different reagents can be injected into different wells through different channels of the reagent-injection passage  60 . The injected reagent  320  can only be discharged through the reagent-discharging passage  70 . 
     As described above, in the first embodiment of the invention, the opening and closing means is provided with a pneumatic passage, and the isolating means is configured to be opened and closed by an air pressure through the pneumatic passage  80 . 
     In addition, the specimen-isolating means and the opening and closing means are provided with a metal electrode so that the isolating means can be opened and closed by an electric field, which is electrically controlled. 
     Furthermore, the specimen-isolating means and the opening and closing means are provided with a conductor line formed therein, such that the isolating means can be opened and closed by an electromagnetic field, which is electrically controlled. 
     Also, the opening and closing means is provided with an electrode formed therein, and the specimen isolating means is provided with a piezoelectric device, so that the isolating means can be opened and closed by an application of external voltage. 
       FIG. 4  shows a cross-section of a micro-fluidic chip according to a second embodiment of the invention. As shown in  FIG. 4 , similar to the first embodiment, the micro-fluidic chip of this embodiment comprises a well  10 , a specimen isolating means  20 , an opening and closing means, an inlet  40 , an outlet  50 , a reagent-injecting passage  60 , and a reagent-discharging passage  70 . In addition, each of the wells is provided with a pair of electrodes  410  formed thereinside. 
     Application of voltage to the pair of electrodes enables the specimen to be attracted into the well or to be pushed away therefrom. This action is caused by dielectrophoresis phenomena. 
     The dielectrophoresis phenomena can be described by that a particle such as a cell and a bead is forced into an area of dense electric field, or towards a region of weak electric field. Here, the mobility of particle varies with the type of the solution or the particles, and also can be controlled by varying the magnitude and frequency of applied electric field. Using the dielectrophoresis phenomena, only required cell or bead from the specimen can be introduced into the inside of a particular well  10 . 
     In addition, the metal electrode  410  may exert an electric stimulation to the specimen inside the well. For example, in case where the specimen is a cell, a study on a particular disease such as a nervous disorder can by performed by observing the reaction of the cell to an electric stimulation externally applied. Also, a hole can be formed in a cell by applying externally an electric field, and through the hole a reagent, DNA or the like can be introduced. 
     Furthermore, the metal electrode  410  may detect an electric signal of the reaction produced inside the well. For example, a membrane potential can be detected by the metal electrode. Also, an oxidation and reduction method can be used in that the metal electrode applies an appropriate voltage or current and detects a signal in response to the applied voltage or current. 
     Here, the metal electrode according to the second embodiment of the invention may be extended into the outside thereof to thereby form a pad  420 , through which an electric signal can be applied or detected. 
     The metal electrode  410  is preferred to be formed of one of gold, silver, platinum, aluminum, semiconductor material or conductive polymer. 
     The second embodiment of the invention can obtain the same effects as in the first embodiment, by applying in the same way a two-dimensional arrangement, the amount or number of specimen according to the size of the well, the channel of the reagent-injection passage, the reagent to be injected, and the empty space of the isolating means. The detailed explanation of the above application is previously made in conjunction with the first embodiment of the invention, and thus will not be repeated here. 
     In addition, similar to the first embodiment, the second embodiment may employ various types of opening and closing means, such as by an air pressure, an electric field, an electromagnetic field, or a piezoelectric element. 
       FIG. 5  is a photograph showing an actual micro-fluidic chip according to the first embodiment of the invention.  FIG. 5  is a photograph of a real micro-fluidic chip of the invention, which is fabricated using a semiconductor processing and MEMS technique (International micro TAS conference 2003). 
     As shown in  FIG. 5 , the micro-fluidic chip has sixteen wells of 4×4 array, each of which has a CHO(Chinese hamster ovary) cell isolated therein. 
     As described above, the micro-fluidic chip of the invention has various effects as follows: 
     First, using a semiconductor processing and MEMS technique the wells can be highly integrated into a chip, thereby performing many experiments and analyses at the same time. 
     Second, a specimen-isolating means is provided above the wells, thereby enabling a stable isolation of the specimen such as a cell, a bead or the like inside the well. In addition, a cover for the specimen-isolating means is disposed above the wells, so that a reagent flown into the respective wells can be prevented from leaking into neighboring wells. 
     Third, reagent-injecting passages connected to the respective wells are provided, so that different reagents can be flown into different wells, thereby carrying out, at the same time, different experiments in different wells. 
     Fourth, a pair of metal electrodes is disposed inside each well, so that the specimen can be attracted inward the well or pushed away therefrom by the dielectrophoresis phenomena. In addition, an electric stimulation can be exerted to the specimen inside the well through the electrode to thereby be able to electrically detect the reaction being occurred inside the well. 
     While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.