Abstract:
An electrochemical detector integrated on a capillary electrophoresis chip according to the present invention includes: a first substrate having a microchannel; a second substrate adapted to mate with the first substrate and having at least one peripheral electrode for conducting electrophoresis of a sample injected along the microchannel of the first substrate, in which a separation channel is formed along the microchannel by bonding the first substrate with the second substrate; a first electrode, made of indium tin oxide (ITO), formed on the first substrate to be positioned over the separation channel; and a second electrode, made of indium tin oxide (ITO), formed on the second substrate to be positioned under the separation channel, and spaced apart from the first electrode at a predetermined interval, wherein the first electrode and the second electrode constitute a detector to measure electrical characteristics of the sample passing along the separation channel. According to the present invention, since the specific characteristics of a sample can be evaluated by measuring the electrical or genetic characteristics of the sample flowing along the microchannel formed in a chip using a detector, a chip for a micro-analysis system having a simple structure can be realized.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates a detector for detecting the electrical or genetic characteristics of samples, integrated on a microfabricated capillary electrophoresis chip, and a method of manufacturing the same, and, more particularly, to a detector for biochips, which is simply configured and efficiently detects samples, and a method of manufacturing the same. 
         [0003]    2. Description of the Related Art 
         [0004]    Recently, with the advancement of genetic engineering techniques, biochips, which can be used to diagnose the symptoms of various diseases, have been actively researched. Most commercially available biochips use pure genetic samples obtained through the separation, refinement, genetic amplification and electrophoresis of blood or cells. Currently, research on PCR (polymerase chain reaction) chips and electrophoresis chips, each of which include flow channels and reaction channels formed on a silicon, glass or polymer substrate using MEMS (microelectromechanical systems), is being actively conducted. Methods of detecting desired pure genes using PCR chips or electrophoresis chips include a fluorescence detection method, a UV/Vis spectrophotometric method, an electrochemical method, and the like. However, these methods have many problems in that large-scaled and high-priced equipment is required and it is difficult to fabricate devices in the form of chips because they are complicated. For instance, the optical detection methods, such as the fluorescence detection method and the UV/Vis spectrophotometric method, are problematic in that various optical parts, such as a microbalance, a microfilter, etc., as well as a laser source and a microscope, are required. Further, the electrochemical method is problematic in that electrodes having complicated structures are used, and detection conditions are not suitable for PCR products. 
         [0005]    As a first conventional technology, U.S. Pat. No. 6,045,676 discloses an electrochemical detector integrated on a microfabricated capillary electrophoresis chip, in which the change in DNA is detected by providing electrodes in an array type hybridization chamber, fixing probe DNA on the electrodes, and then measuring the change in dielectric constant or dielectric loss from before the probe DNA reacts with target DNA to thereafter. However, the first conventional technology is problematic in that, although it is only observed that DNA itself which is fixed on the electrodes in the hybridization chamber for the purpose of the diagnosis of disease, is changed from single-strand DNA to double-strand DNA, DNA floating and moving in fluid in microchannel cannot be detected. 
         [0006]    As a second conventional technology, U.S. Pat. No. 6,169,394 discloses an electrical detector for a micro-analysis system, in which whether or not biomolecules exist is determined by measuring the impedance change when samples, such as cells, biomolecules, ions, etc. flow through microchannel by forming electrodes on both side walls of the microchannel and then applying signal voltage to the electrodes. However, the second conventional technology is problematic in that manufacturing processes are very complicated because electrodes are formed on both side walls of the microchannel. 
         [0007]    As such, in conventional chips, the detection of DNA, etc. depends on optical methods or electrochemical methods. The optical methods are problematic in that, since various optical parts, such as laser light sources, lenses, filters, mirrors, and the like, are required, high expenses are incurred and a large amount of space is necessary, and thus it is very difficult to integrate the optical parts, 
         [0008]    Further, recently, chips provided therein with laser diodes, filters, etc. in a thin film form have been developed. However, since these chips are manufactured at high cost, they are not suitable for disposable chips for micro-analysis systems. 
         [0009]    Furthermore, the electrochemical methods are also problematic in that, since three or more electrodes are required and the material properties of each of the electrodes must vary depending on the purpose in these methods, manufacturing processes are complicated, and particularly, when these methods are conducted using an oxidation-reduction reaction, measurement errors occur due to environmental factors. 
       SUMMARY OF THE INVENTION 
       [0010]    Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art. 
         [0011]    A first aspect of the present invention provides an electrochemical detector integrated on a capillary electrophoresis chip, including: a first substrate having a microchannel; a second substrate adapted to mate with the first substrate and having at least one peripheral electrode for conducting electrophoresis of a sample injected along the microchannel of the first substrate, in which a separation channel is formed along the microchannel by bonding the first substrate with the second substrate; a first electrode, made of indium tin oxide (ITO), formed on the first substrate to be positioned over the separation channel; and a second electrode, made of indium tin oxide (ITO), formed on the second substrate to be positioned under the separation channel, and spaced apart from the first electrode at a predetermined interval, wherein the first electrode and the second electrode constitute a detector to measure electrical characteristics of the sample passing along the separation channel. 
         [0012]    The first substrate may be made of polydimethylsiloxane (PDMS), and the second substrate may be made of glass, quartz, or silicon. More preferably, in the first substrate, the portion having the first electrode formed thereon may be made of glass, quartz or silicon, and the remaining portion may be made of polydimethylsiloxane (PDMS). 
         [0013]    The electrical characteristics of the sample, moving between the first electrode and the second electrode, may include capacitance, dielectric constant, resonance frequency, and impedance. 
         [0014]    A second aspect of the present invention provides a method of manufacturing an electrochemical detector integrated on a capillary electrophoresis chip, including: forming a microchannel on a first substrate made of polydimethylsiloxane (PDMS); forming at least one peripheral electrode used to conduct electrophoresis of a sample injected along the microchannel, and a second electrode, made of indium tin oxide (ITO), used to measure electrical characteristics of the sample moving along the microchannel, on a second substrate; bonding the first substrate with the second substrate to form a separation channel along the microchannel between the first and second substrates; and forming a first electrode, made of indium tin oxide (ITO), on the first substrate to be positioned over the separation channel, wherein electrical characteristics of the sample passing along the separation channel are detected by the first and second electrodes mating with each other. 
         [0015]    The second substrate may be made of glass, quartz, or silicon. 
         [0016]    In the method, the formation of the microchannel on the first substrate may include: forming a pattern corresponding to the microchannel on a silicon wafer using a photoresist; forming a PDMS layer on the patterned silicon wafer; and removing the patterned silicon wafer. 
         [0017]    Further, the formation of the at least one peripheral electrode for electrophoresis and the second electrode on the second substrate may include: forming an ITO layer having a predetermined thickness on a substrate; applying a photoresist on the ITO layer to form a pattern corresponding to the at least one peripheral electrode and the second electrode; and removing the patterned photoresist to form the at least one peripheral electrode and the second electrode. 
         [0018]    Further, the bonding the first substrate with the second substrate may be performed using a UV-Ozone cleaner. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]    The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description, taken in conjunction with the accompanying drawings, in which: 
           [0020]      FIG. 1  is a view showing a capillary electrophoresis chip provided with an electrochemical detector according to the present invention; 
           [0021]      FIGS. 2A and 2B  are views showing the structures of the capillary electrophoresis chips having electrodes formed thereon according to the present invention, respectively; 
           [0022]      FIG. 3  is a sectional view showing a separation channel and a detector provided on the capillary electrophoresis chip according to the present invention; 
           [0023]      FIGS. 4A to 4D  are process views showing a method of forming a microchannel in a first substrate; 
           [0024]      FIGS. 5A to 5D  are process views showing a method of fabricating a second substrate having a second electrode, serving as a reference electrode; and 
           [0025]      FIG. 6  is a schematic sectional view showing an electrophoresis chip completed using the first substrate of  FIGS. 4A to 4D  and the second substrate of  FIGS. 5A to 5D . 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0026]    Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. 
         [0027]    Reference now should be made to the drawings, in which the same reference numerals are used throughout the different drawings to designate the same or similar components. 
         [0028]    Generally, since nucleic acid, protein, DNS, cells, and the like, constituting all living things, are electrically polar, when voltage is applied to samples including them and then the voltage and frequency thereof are changed, the specific electrical characteristics of the samples can be measured. 
         [0029]    In the present invention, the products obtained through PCR (Polymerase Chain Reaction) are analyzed through electrophoresis using such specific electrical characteristics of samples. The principle of electrophoresis is that components included in a sample are separated from each other according to the difference in sizes and characteristics between the components by gelating the sample and then applying voltage thereto. 
         [0030]    In the electrophoresis, sample moves through a capillary separation channel. In the present invention, a pair of electrodes is formed at microchannel of the separation channel using indium tin oxide (ITO), and then a specific voltage and frequency are applied to the pair of electrodes, and thus a detector measures the electrical characteristics of the sample. 
         [0031]    Hereinafter, an electrochemical detector integrated on a capillary electrophoresis chip according to the present invention will be described in more detail with reference to the accompanying drawings. 
         [0032]      FIG. 1  shows a capillary electrophoresis chip provided with an electrochemical detector according to the present invention. 
         [0033]    The capillary electrophoresis chip  100  includes a sample reservoir  110  for storing a sample, a sample waste reservoir  120  for storing sample waste, a buffer reservoir  130  for storing a buffer solution, a separation channel  150  formed of a capillary microchannel, a detector  200  for detecting the electrical characteristics of the sample, and a detected sample reservoir  140  for storing the detected sample. 
         [0034]    All solutions introduced into all of the reservoirs are filtered using a membrane filter having a thickness of about 0.45 μm, and all of the microchannel are deionized and then flushed using purified water. Subsequently, all of the reservoirs and the microchannel are filled with buffering solutions, and then the testing sample is loaded in the sample reservoir  110 . After the test sample is loaded into the sample reservoir  110 , when the sample is injected into the microchannel connected to the sample reservoir  110  by applying an electric field between the sample reservoir  110  and the sample waste reservoir  120 , the sample passes through an intersection and flows in the separation channel  150 . At this time, the detected sample reservoir  140  is grounded, a separation voltage is applied to the buffer reservoir  130 , and other reservoirs are floated. At this time, the sample moves along the separation channel  150 , and the detector  200  measures the electrical characteristics of the sample flowing in the separation channel  150 . 
         [0035]      FIGS. 2A and 2B  show the structures of the capillary electrophoresis chips having electrodes formed thereon according to the present invention, respectively. 
         [0036]      FIG. 2A  shows a structure in which a first electrode  210 , serving as a working electrode of a detector  200 , is formed on a first substrate  300 , and  FIG. 2B  shows a structure in which various electrodes, including a second electrode  220 , serving as a reference electrode, an SB electrode  115 , an SW electrode  125 , a BR electrode  135  and a DR electrode  145 , which are used in electrophoresis, are formed on a second substrate  350 . 
         [0037]    The SB electrode  115  and the SW electrode  125  are electrodes formed to inject a sample, and serve to move a sample to a detector  200  by applying a separation voltage on the BR electrode  135  and the DR electrode  145 , thus causing electrophoresis along a separation channel  150 . 
         [0038]    The detector  200  includes a first electrode  210  formed on a first substrate  300  and a second electrode  220  formed on a second substrate  350 , and measures the electrical characteristics of the sample flowing along the separation channel, indicated by dot lines in the drawings, through the first electrode  210  and the second electrode  220 . In the case where the detector  200  is a three-electrode system, the detector  200  may further include a counter electrode (not shown) on the second substrate  350 . 
         [0039]    Generally, electrodes formed on a chip are composed of gold (Au) or platinum (Pt), thus increasing the cost of manufacturing the chip. However, in the present invention, since they are composed of indium tin oxide (ITO), the disposable chips can be manufactured at low cost. Further, in the present invention, the specific characteristic of the sample can be easily and simply evaluated by applying a specific voltage and frequency to the sample flowing in the separation channel through a pair of electrodes, that is, the first electrode and the second electrode, formed on and beneath the chip, thus measuring the electrical characteristics of the sample. 
         [0040]    Furthermore, as described below, in the detector  200  according to the present invention, since high voltage for electrophoresis extends in a direction toward the separation channel  150 , it is preferred that the detector  200  be configured such that the first electrode  210  and the second electrode  220  are disposed in a direction leading toward the capillary tubes of the separation channel, so that the direction of the measured voltage is perpendicular to the direction of the electric field in electrophoresis. When the detector  200  is configured in this way, the occurrence of noise due to the voltage in electrophoresis is minimized, thus decreasing measurement errors attributable to changes in the external environment. 
         [0041]      FIG. 3  shows a separation channel and a detector provided on the capillary electrophoresis chip according to the present invention. 
         [0042]    The detector  200  includes a first electrode  210  formed on a first substrate  300  and a second electrode  220  formed on a second substrate  350 , and the first electrode  210  and second electrode  220  are symmetrically arranged and spaced apart from each other. 
         [0043]    As shown in  FIG. 3 , a microchannel is formed on the first substrate  300 , and a separation channel  150  is formed along the microchannel by bonding the first electrode  210  and the second electrode  220 . 
         [0044]    The first substrate  300  is chiefly composed of polydimethylsiloxane (PDMS), but the portion thereof on which the first electrode  210  is formed may be composed of glass, quartz, or the like in order to form the first electrode  210  of the detector  200 . 
         [0045]    The second substrate  350  is provided thereon with the second electrode  220  and electrodes for electrophoresis. The sample moves along the separation channel  150  due to the electrophoresis conducted using the electrodes for electrophoresis, and the specific characteristics of the sample are measured by the first electrode  210  and the second electrode  220 , formed in the separation channel  150 . 
         [0046]    Generally, microchips in an ECD system (electrochemical detection system) include a substrate made of glass, quartz, or the like. A microchip including such a glass substrate is difficult to manufacture in a general laboratory because it must be manufactured in a clean room at a high molding temperature. Therefore, in the present invention, the first substrate  300  is fabricated using polydimethylsiloxane (PDMS), which makes it easy to fabricate a delicate first substrate even at low temperatures, and has excellent optical properties and high adhesivity. As such, since a microchannel is formed on the first substrate  300 , made of PDMS, a delicate microchannel can be formed much more easily than at the time of forming a separation channel using glass, quartz, or the like, and, when the first substrate  300  and the second substrate  350  are bonded, the separation channel  150  is formed along the microchannel. 
         [0047]    Here, when the first substrate  300  and the second substrate  350  are cleaned using a UV-Ozone cleaner before the first substrate  300  and the second substrate  350  are sequentially bonded to the separation channel, the bonding strength between the first substrate  300 , made of PDMS, and the second substrate  350 , made of glass, etc. can be increased. 
         [0048]    When an alternating voltage having a specific frequency is applied between the first electrode  210  and the second electrode  220 , an electric field is formed therebetween, and thus charged particles flowing in the separation channel are influenced by the electric field, so as to show specific behavior. 
         [0049]    The detector  200  of the present invention detects the specific characteristic of a sample by measuring the electrical characteristics attributable to the specific behavior of the charged particles. The electrical characteristics may vary depending on the media located between the two electrodes. For example, when dipoles or charged particles exist in the insulation medium located between the first electrode  210  and the second electrode  220 , there is a tendency to increase capacitance, and thus the specific characteristics of the sample can be detected by measuring the change in the capacitance. Further, the specific characteristics of the sample may be detected by measuring the change in dielectric constant directly related with dipole moment and charge amount or the change in resonance frequency greatly influenced by particle size, particle weight and environmental factors. Furthermore, the specific characteristics of the sample may be detected by measuring the change in impedance or admittance related to resistance occurring when an alternating voltage is applied between the first electrode  210  and the second electrode  220 . These measured values also change depending on the ion concentration between the two electrodes, the presence of DNA etc. therebetween, and the distance therebetween. 
         [0050]      FIGS. 4A to 4D  and  5 A to  5 D show methods of manufacturing a capillary electrophoresis chip according to an embodiment of the present invention. 
         [0051]      FIGS. 4A to 4D  show a method of forming a microchannel in a first substrate,  FIGS. 5A to 5D  show a method of fabricating a second substrate having a second electrode, etc., serving as a reference electrode, and  FIG. 6  is a schematic sectional view showing an electrophoresis chip completed using the first substrate of  FIGS. 4A to 4D  and the second substrate of  FIGS. 5A to 5D . 
         [0052]    In order to form a PDMS layer having a microchannel of the first substrate, a photoresist  402 , for example, SR-850, is applied on a silicon wafer  401  using a spin coating method, as shown in  FIG. 4A , and then a pattern  403  corresponding to the microchannel is formed thereon, as shown in  FIG. 4B . In this case, it is preferred that the height of the patterned photoresist be approximately 40 μm, which is the same as the depth of the microchannel in the PDMS layer to be formed later. Subsequently, PDMS is applied on the silicon wafer  401  having the patterned photoresist  403  formed thereon to form a PDMS layer  404 , as shown in  FIG. 4C , and then the PDMS layer  404  is cured and then separated from the silicon wafer  401  having the patterned photoresist  403  formed thereon to form a PDMS layer  405  having the microchannel formed therein, as shown in  FIG. 4D . In this case, the PDMS used in the formation of the PDMS layer may be a mixture in which a silicon elastomer (Sylgard 184) and a curing agent are mixed at a ratio of 10:1, and the PDMS layer  404  formed using this PDMS (Sylgard 184) may be cured at a temperature of about 72□ for about 1 hour. 
         [0053]    Meanwhile, a second substrate is fabricated through a process that is different from the process of fabricating the first substrate. First, as shown in  FIG. 5A , an ITO layer  502  is formed on a glass substrate  501  through R.F. magnetron sputtering. In this case, the ITO layer  502  may have a thickness of about 340 nm and a surface resistance of 10 ohm/sq. In order to form ITO electrodes  505 , as shown in  FIGS. 5B and 5C , a photoresist  503 , for example, AZ 1512, is applied on the ITO layer  502 , patterns corresponding to electrodes to be formed on a second substrate are formed on the glass substrate  501 , and then the patterns formed on the glass substrate  501  are etched to form final ITO electrodes  505 . In the final ITO electrodes, a reference electrode and a counter electrode may have widths of about 100 μm and 200 μm, respectively. 
         [0054]    Further, a work electrode having a width of about 100 μm is formed using the same method as in the fabrication of the second substrate, and is then appropriately cut. Subsequently, as shown in  FIG. 6 , a glass substrate  501 ′, having the work electrode formed thereon, and a PDMS layer  602 , having an opening  601 , are additionally provided, and then the PDMS layer  602  is bonded with the PDMS layer  405  having the microchannel formed therein such that the work electrode faces the reference electrode of the second substrate, and simultaneously the PDMS layer  405  is bonded with the glass substrate  501  having the reference electrode  505  formed thereon using a UV-Ozone cleaner, thereby completing a capillary electrophoresis chip. 
         [0055]    As described above, according to the present invention, since the specific characteristics of a sample can be evaluated by measuring the electrical or genetic characteristics of the sample flowing along the microchannel formed in a chip using a detector, a chip for a micro-analysis system having a simple structure can be realized. Further, according to the present invention, since cheap ITO electrodes are used instead of conventional expensive gold (Au) or platinum (Pt) electrodes, manufacturing costs can be decreased. 
         [0056]    Further, according to the present invention, since the microchannel is formed in a polydimethylsiloxane (PDMS) substrate and the formed microchannel is used as a separation channel, various desired types of microchannel can be easily formed, manufacturing costs are low, and the integration thereof is easy. 
         [0057]    Furthermore, according to the present invention, since the electrical characteristics of a sample are measured by applying a specific voltage and frequency to a pair of electrodes located in a separation channel, the sample is little influenced by environmental factors, and thus accurate measurement values can be obtained. 
         [0058]    The electrochemical detector integrated on a capillary electrophoresis chip according to the present invention can be variously modified and applied within the technical scope and spirit of the present invention, and is not limited to the above embodiment. As described above, although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the present invention as disclosed in the accompanying claims.