Abstract:
The present invention relates to a biochip for nucleic acid hybridization. The biochip of the present invention comprises a hybridization chamber which is in the form of a cavity, a porous membrane pressed in the hybridization chamber; and at least one first circulation hole and at least one second circulation hole which are communicated with the hybridization chamber so that the reaction solution flows in the at least one first circulation hole and flows out the at least one second circulation hole through the pores of the porous membrane. The hybridization reaction area is increased by flowing the reaction solution through the pores of the membrane, which enable the reaction sensitivity to be increased. The diffusion distance for the reaction molecules is decreased due to the limited inside space of the membrane, and thereby the hybridization time is shortened.

Description:
[0001]    This is a continuation-in-part of U.S. patent application Ser. No. 11/675,637, filed Feb. 16, 2007, which is incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to a biochip, and particularly relates to a biochip for nucleic acid hybridization and a method for accelerating the rate of hybridization between a target nucleic acid and a nucleic acid probe. 
         [0004]    2. The Prior Arts 
         [0005]    The hybridization method in which the nucleic acid probe is hybridized to the target nucleic acid is one of the most common analytical techniques to confirm whether the target DNA has the desired gene or nucleic acid sequence or not, wherein the nucleic acid probe is a nucleic-acid fragment that is complementary to another nucleic-acid sequence and thus, when labeled in some manner, as with a radioisotope, can be used to identify complementary segments present in the nucleic-acid sequences of various nucleic acids. Conventionally, the blotting process used in hybridization analysis is to transfer the target nucleic acid to a substrate such as membrane, and then the nucleic acid probe with specificity is applied for hybridization, and then the color, chemiluminescence, or radioactivity exhibited by the labeled molecules in the nucleic acid probe is detected whereby it is possible to judge whether a target base sequence is present in the target nucleic acid or not. 
         [0006]    One of the hybridization techniques used today is the “Southern blotting”, in which the target DNA is transferred from an electrophoresis gel to a membrane, and then hybridized with a probe. When used with RNA target the method is called “Northern blotting”. In the other methods, in accordance with the dropping area the target nucleic acid is directly dropped onto the membrane by dot blotting, slot blotting, or spot blotting. In the dot blotting, slot blotting, and spot blotting method, the nucleic acid can be directly blotted onto a substrate without the transfer process of electrophoresis. Therefore, the analysis time for target nucleic acid is reduced. The Blotting method can be used in the qualitative analysis by batch. 
         [0007]    In the conventional blotting assay, after the target nucleic acid is dropped onto the surface of the membrane, the nucleic acid is permanently attached to the membrane by cross-linking using heating or UV radiation so that the target nucleic acid will remain on the film when being washed after the step of hybridization with a probe. Because the target nucleic acid is dropped onto a wet surface of a membrane, and then diffuses and adsorbs on the wet surface under the known working conditions. Most of the target nucleic acid is only firmly absorbed on the membrane surface and the nearby pores thereof, and thereby the numbers of nucleic acid molecules firmly absorbed are limited, and thus the produced hybridization signal is relatively weak. As a result, if the amount of the nucleic acid sample is not enough or the nucleic acid sample has a long molecular chain, the reaction sensitivity will be greatly reduced. When the probe hybridization solution containing the blocking reagent is added to the membrane, the nucleic acid probe can only diffuse on the surface of the membrane as the target nucleic acid does, and find out the target nucleic acid for hybridization in Brownian movement. Therefore, the nucleic acid hybridization reaction takes more processes to be accomplished, and the reaction time is more than 10 hours. Therefore, the hybridization results cannot be obtained in a short time. In addition, it is not economical for the qualitative analysis of the nucleic acids if the hybridization assay takes long time and needs lots of reagents. Therefore, there is a need for the development of a simple blotting device and a blotting method to reduce the process steps and time for hybridization assay and to reduce the background noise, and thus such a blotting device and such a blotting method can be applied to the detection for simple or batch process. 
       SUMMARY OF THE INVENTION 
       [0008]    In order to solve the time-consuming problem in hybridization assay when the nucleic acid probe is base-paired with the target nucleic acid by Brownian motion, and in order to increase the amount of target nucleic acid firmly absorbed on the surface and the inside of the pores of the membrane for increasing base pairing probabilities and reaction sensitivity, the present invention provides a biochip for nucleic acid hybridization assays. In the present invention, the inside space of the biochip is limited so that the target nucleic acid and the nucleic acid probe can rapidly diffuse into the micropores of the substrate in a very short time and are base paired with each other when the hybridization solution enters the inside of the substrate. Furthermore, under the condition of pressurizing the fluid, the target nucleic acid and the nucleic acid probe can move rapidly, and because the adsorption and reaction area of the target nucleic acid and the nucleic acid probe are enlarged when they flow into the inside of the membrane, the number of base pairing is increased, which can enhance the detection sensitivity. Meanwhile, the base pairing between the target nucleic acid and the nucleic acid probe is speeded up due to the flowing movement of the nucleic acid molecules. Moreover, because the washing solution can be deeply flushed into the inside of the membrane and washes away the probe molecules unspecifically bound to the membrane, and thereby the cleanness is improved and the reaction background level is reduced. 
         [0009]    In order to achieve the above objectives, the present invention provides a biochip, comprising: a hybridization chamber which is in the form of a cavity, a porous membrane pressed in the hybridization chamber; and at least one first circulation hole and at least one second circulation hole which are communicated with the hybridization chamber so that the reaction solution flows in the at least one first circulation hole and flows out the at least one second circulation hole through the pores of the porous membrane. The biochip includes an upper substrate and a lower substrate, wherein the upper substrate and the lower substrate are stacked together one on top of the other to form the hybridization chamber therein, and the porous membrane is provided in the hybridization chamber, and at least one first circulation hole located at the upper substrate and at least one second circulation hole, which are communicated with the hybridization chamber, are provided on the top and the side of the hybridization chamber, respectively. 
         [0010]    There is no specific limitation on the shape and the thickness of the hybridization chamber, and its shape and thickness can be changed with the porous membrane structure. An interstice with predetermined width is left between the porous membrane and the sidewall of the hybridization chamber so that the reaction solution can enter the porous membrane from the side thereof. In addition, the central top of the hybridization chamber is provided with a first circulation hole. There is no specific limitation on the position and the number of the first circulation hole. The first circulation hole, the second circulation hole, and the hybridization chamber can be further communicated with a microchannel, and the reaction solution can rapidly pass through the porous membrane by pressurizing the reaction solution via the microchannel, and thereby the reaction rate is increased. If the porous membrane is in a dry state before the reaction, the reaction solution can rapidly enter the inside of the porous membrane due to the capillary attraction of the pores of the membrane. The porous membrane has a pore diameter of 0.1 μm to 50 μm. The porous membrane can be a nylon membrane, a nitrocellulose membrane, or any other suitable membrane. 
         [0011]    In the biochip of the present invention, the target nucleic acid can enter the hybridization chamber via one or more circulation holes and can be absorbed by the membrane therein. Because the target nucleic acid can enter the inside of the membrane, the number of the target nucleic acid molecules firmly adsorbed by the membrane is increased, which can enhance the detection sensitivity. After the target nucleic acid molecules are firmly adsorbed by the membrane, the nucleic acid probe solution enters the membrane pressed in the hybridization chamber via one or more circulation holes and anneals with the target nucleic acid. Because the nucleic acid probe can easily move in the pores of the membrane, it can anneal with the target nucleic acid in a very short time. Furthermore, the washing solution is flushed into the membrane via one or more circulation holes for washing. Because the nucleic acid probe can easily move in the pores of the membrane, the nucleic acid probe molecules unspecifically bound to the membrane can be easily and rapidly flushed out of the membrane. Therefore, the washing process is rapid and complete, the reaction time is shortened, and the background noise level is reduced. 
         [0012]    The present invention provides a biochip, comprising an upper substrate and a lower substrate, which are stacked together one on top of the other. A hybridization chamber is provided between the upper substrate and the lower substrate, and a porous membrane is provided in the hybridization chamber. A plurality of little pillars protrude from an interface between a bottom of the upper substrate and the hybridization chamber wherein the ends of the little pillars are in contact with the surface of the porous membrane pressed in the hybridization chamber. In addition, at least one first circulation hole located at the upper substrate and at least one second circulation hole are provided on a top and a side of the hybridization chamber, respectively, wherein the second circulation hole is communicated with the interspace among the little pillars, so that the reaction solution is able to fill up the interspace among the little pillars via the second circulation hole and then enters the membrane. By using such little pillars, the area occupied by the reaction solution in the membrane is enlarged, and thereby the rate of the reaction solution that enters the membrane and its efficiency are increased. As a result, the reaction is rapid and complete. 
         [0013]    The present invention provides a biochip comprising an upper substrate and a lower substrate, and the lower substrate can be a single-layer lower substrate or can be composed of a top substrate and a bottom substrate. The third circulation hole which is communicated with hybridization chamber can be provided in a single-layer lower substrate or in a lower substrate composed of a top substrate and a bottom substrate. The third circulation hole is further communicated with the second microchannel, and the second microchannel is further communicated with the fourth circulation hole so that the reaction solution can enter the membrane from the bottom of the lower substrate. Therefore, the reaction solution can enter the membrane from different flow paths. 
         [0014]    Moreover, in order to solve the time-consuming problem in hybridization assay when the nucleic acid probe is base-paired with the target nucleic acid by Brownian motion, and in order to increase the amount of target nucleic acid firmly absorbed on the surface and the inside of the pores of the fiber substrate for increasing base pairing probabilities and reaction sensitivity, the present invention also provides a method for hybridizing a nucleic acid probe to a target nucleic acid, comprising the following steps: providing a fiber substrate having a plurality of pores; transferring the target nucleic acid to the fiber substrate by means of a first liquid flow, the first liquid flow being accelerated from an inlet side to an exit side of the fiber substrate, wherein the target nucleic acid is captured by the fiber substrate, and stretched and wound around fibers of the fiber substrate; fixing the target nucleic acid on the fiber substrate; transferring the nucleic acid probe to the fiber substrate by means of a second liquid flow, the second liquid flow being accelerated from the inlet side to the exit side of the fiber substrate, wherein the nucleic acid probe is captured by the fiber substrate, and is stretched and wound around fibers of the fiber substrate; and hybridizing the target nucleic acid with the nucleic acid probe on the fiber substrate for a time period sufficient for base-pairing the nucleic acid probe with the target nucleic acid and forming a hybridization product. 
         [0015]    Other objects, advantages, and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1A  is an exploded view of the biochip according to the first embodiment of the present invention; 
           [0017]      FIG. 1B  is a cross-section view after the biochip is assembled according to the first embodiment of the present invention; 
           [0018]      FIG. 1C  is a top view after the biochip is assembled according to the first embodiment of the present invention; 
           [0019]      FIG. 1D  is a schematic view of the solution flowing direction during the hybridization reaction according to one preferred embodiment of the present invention; 
           [0020]      FIG. 1E  is a schematic view of the solution flowing direction during the hybridization reaction according to another embodiment of the present invention; 
           [0021]      FIG. 1F  is a top view of another biochip in square shape derived from the embodiment of the present invention; 
           [0022]      FIG. 2A  is an exploded view of the biochip according to an embodiment of the present invention; 
           [0023]      FIG. 2B  is a cross-section view after the biochip is assembled according to the embodiment of the present invention; 
           [0024]      FIG. 2C  is a top view after the biochip is assembled according to the embodiment of the present invention; 
           [0025]      FIG. 2D  is a schematic view of the solution flowing direction during the hybridization reaction according to the embodiment of the present invention; 
           [0026]      FIG. 3A  is an exploded view of the biochip according to an embodiment of the present invention; 
           [0027]      FIG. 3B  is a cross-section view after the biochip is assembled according to the embodiment of the present invention; 
           [0028]      FIG. 3C  is a top view after the biochip is assembled according to the embodiment of the present invention; 
           [0029]      FIG. 3D  is a schematic view of the solution flowing direction during the hybridization reaction according to the embodiment of the present invention; 
           [0030]      FIG. 4A  is an exploded view of the biochip according to an embodiment of the present invention; 
           [0031]      FIG. 4B  is a cross-section view after the biochip is assembled according to the embodiment of the present invention; 
           [0032]      FIG. 4C  is a top view after the biochip is assembled according to the embodiment of the present invention; and 
           [0033]      FIG. 4D  is a schematic view of the solution flowing direction during the hybridization reaction according to the embodiment of the present invention. 
           [0034]      FIG. 5  is the fluorescent microscope image of the fluorescent nucleic acid probes present in the porous membrane according to one embodiment of the present invention. 
           [0035]      FIG. 6  is the fluorescent microscope image of the fluorescent nucleic acid probes present in the porous membrane according to another embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0036]      FIG. 1A  is an exploded view of the biochip of the first embodiment of the present invention. With reference to  FIG. 1A , the biochip of this embodiment comprises an upper substrate  10 , a lower substrate  20 , and a porous membrane  30 , wherein the upper substrate  10  and the lower substrate  20  are stacked together, and the porous membrane  30  is provided in the hybridization chamber  11 . The porous membrane  30  having an upper surface which is tightly contacted with a lower surface of the upper substrate  10 , and having a lower surface which is tightly contacted with an upper surface of the lower substrate  20 , an interstice  15  is defined between the porous membrane  30  and a sidewall of the hybridization chamber  11 . 
         [0037]    With reference to  FIG. 1A ,  FIG. 1C , and  FIG. 1E , the upper substrate  10  has a hybridization chamber  11 , which is in the form of a disk-shaped cavity. However, the shape, the size and the thickness of the hybridization chamber  11  have no restriction, and the hybridization chamber  11  can be a tetrahedral cavity (as shown in  FIG. 1E ). The center of hybridization chamber  11  is provided with a first circulation hole  12 . There is no specific limitation on the position of the first circulation hole  12 , and the position of the first circulation hole  12  can be changed with the position of another circulation hole so that the reaction solution can flow over the whole inside of the porous membrane  30 . As  FIG. 1A  shows, the first circulation hole  12  is located at the upper substrate and on the top of the hybridization chamber  11 , whereas in  FIG. 1E  the first circulation hole  12 ′ may either be located on the side of the hybridization chamber  11 . Besides, there is also no specific limitation on the number of the first circulation holes  12 . The first circulation hole  12  can be further communicated with a microchannel (such as the microchannel  17  communicated with one side of the hybridization chamber  11  shown in  FIG. 1E ) or another circulation hole (not shown in the drawings) for facilitating solution injection. 
         [0038]    The first microchannel  14  is communicated with one side of the hybridization chamber  11 , and the first microchannel  14  is further communicated with the second circulation holes  13 . There is no specific limitation on the number and the positions of the first microchannels  14 , and the number and the positions thereof can be changed with the flow path of the reaction solution. In addition, an interstice  15  with predetermined width is left between the porous membrane  30  and the sidewall of the hybridization chamber  11 . The interstice  15  has a width of 0.05 to 0.2 mm, and preferably 0.1 mm. In another example, two interstices  15 ′ with predetermined width, as shown in  FIG. 1E , are respectively left between the porous membrane  30 ′ and the sidewall of the hybridization chamber  11 , and not communicated. 
         [0039]    With reference to  FIG. 1A  and  FIG. 1B , the hybridization chamber  11 , the first microchannel  14 , and the second circulation hole  13  are formed between the upper substrate  10  and the lower substrate  20 . The hybridization chamber  11 , the first microchannel  14 , and the second circulation holes  13  are not limited to be located on the upper substrate  10 , but they may be located on the lower substrate  20 , or on both the upper substrate  10  and the lower substrate  20  (divided into male and female halves). The upper substrate  10  and the lower substrate  20  may be separately manufactured, or integrally manufactured, and the hybridization chamber  11 , the first microchannel  14 , and the second circulation hole  13  will be formed inside the substrates while manufactured. 
         [0040]    The biochip of this embodiment includes, but not limited to, a microfluidic chip, a nanofluidic chip, or any other structure which is suitable to the present invention. The quartz, glass, or the like can be used as the substrate of the microfluidic chip, and the capillary microchannels are formed by wet etching, and a layer of quartz, or glass covers the tops of the capillary microchannels, and then the chip with the closed microchannels or cavities is produced. Alternatively, the substrate of the biochip is made of the hard polymer, for example, polymethyl methacrylate (PMMA), polycarbonate (PC). First, the mother mold is made by wet-etching the quartz, and then the microchannels are formed on the PMMA or PC material by the embossing method, and then the tops of the microchannels is covered with the same material as the substrate. The substrate of the biochip of the present invention can also be made of soft polymer, for example, polydimethyl siloxane (PDMS). Because of good flowing ability, the thermal compression is not needed so that the mother mold will not be easily damaged, and the biochips can be manufactured in large scale. This makes PDMS a preferable material to be used. 
         [0041]    The porous membrane  30  can be a fiber membrane, such as a nylon membrane, a nitrocellulose membrane, or any other suitable membrane fitted in shape for the hybridization chamber  11 . The nylon membrane is positive charged or neutral. The nylon membrane and nitrocellulose membrane have a pore diameter of 0.1 μm to 0.5 μm. The proper porous diameter is selected based on the molecular weight of the target nucleic acid, and the larger the nucleic acid, the larger the porous diameter is used. The pore diameter is preferably 0.2 μm to 0.45 μm. Moreover, the porous membrane can be in a dry state so that the injected target nucleic acid can be adsorbed on the porous membrane rapidly. Furthermore, the target nucleic acid can be DNA or RNA. 
         [0042]      FIG. 1D  is the schematic view showing the flow direction of the hybridization solution according to one preferred embodiment of the present invention. With reference to  FIG. 1D , the target nucleic acid solution T is injected into the second circulation hole  13 , and then flows through the first microchannel  14  and enters the interstice  15  surrounding the periphery of the porous membrane  30 . After entering the interstice  15 , the interstice  15  is firstly filled with the target nucleic acid solution T, and then the target nucleic acid solution T diffuses toward the center of the porous membrane  30  from the periphery of the porous membrane  30 , and finally is discharged to the outside via the first circulation hole  12 . Furthermore, if the porous membrane  30  is in a dry state, the target nucleic acid solution T can rapidly enter the inside of the porous membrane  30  due to the capillary attraction of the micropores of the porous membrane  30 . Moreover, the target nucleic acid solution T which enters the porous membrane  30  can be permanently attached to the surface and the inside of the porous membrane  30  by heating or UV irradiation. 
         [0043]    Afterwards, the nucleic acid probe solution P is also injected via the second circulation hole  13  for hybridization reaction. The added nucleic acid probe solution P will take the same flow path as the target nucleic acid solution T, and distribute over the whole porous membrane  30 . The nucleic acid probe is labeled with chromogenic molecules in order to detect the results of hybridization reaction. The nucleic acid probes can be detectably labeled, for example, with a radioisotope, a fluorescent compound, or an enzyme. If the porous membrane  30  is in a dry state before the nucleic acid probe solution P is added, the nucleic acid probe solution P can also rapidly enter the inside of the porous membrane  30 . After the nucleic acid probe solution P is added, the biochip is placed at a proper temperature (such as 40 to 48° C.) for 2 to 5 minutes to allow the nucleic acid probe to base-pair with the target nucleic acid, and thus the process of hybridization is completed. 
         [0044]    After the process of hybridization, the unhybridized nucleic acid probes are washed away. During the washing process, the washing solution W is flushed into the hybridization chamber  11  via the first circulation hole  12 , and enters the inside of the porous membrane  30  from the center thereof. After entering the inside of the porous membrane  30 , the washing solution W diffuses across the porous membrane  30  to the interstice  15  which surrounds the periphery of the porous membrane  30 . Then, the washing solution W flows through the first microchannel  14 , and is discharged to the outside via the second circulation hole  13 . Because the washing solution W is flushed from the outside of the porous membrane  30  to the inside of the porous membrane  30 , the nucleic acid probe, which is a relatively small molecule, can be easily and rapidly flushed out of the pores of the porous membrane  30 . Therefore, the background noise level is reduced, and the time for flushing is shortened. Then, the nucleic acid probes labeled with chromogenic molecules are detected, and the results of hybridization reaction are obtained and thereby the complementary sequence of the nucleic acid sequence of the nucleic acid probe present in the sequence of the target nucleic acid is determined. 
         [0045]      FIG. 1E  is the schematic view showing the flow direction of the hybridization solution. With reference to  FIG. 1E , the target nucleic acid solution T is injected into the hybridization chamber  11  via the first circulation hole  12 , and enters the inside of the porous membrane  30  from the center thereof. After entering the inside of the porous membrane  30 , the target nucleic acid solution T diffuses across the porous membrane  30  to the interstice  15  which surrounds the periphery of the porous membrane  30 . Then, the target nucleic acid solution T flows through the first microchannel  14 , and is discharged to the outside via the second circulation hole  13 . If the porous membrane  30  is in a dry state, the target nucleic acid solution T can rapidly enter the inside of the porous membrane  30  due to the capillary attraction of the micropores of the porous membrane  30 . Moreover, the target nucleic acid solution T which enters the porous membrane  30  can be permanently attached to the surface and the inside of the porous membrane  30  by heating or UV irradiation. 
         [0046]    Afterwards, the nucleic acid probe solution P is injected into the hybridization chamber  11  via the first circulation hole  12  for hybridization reaction. The nucleic acid probe is labeled with chromogenic molecules in order to detect the results of hybridization reaction. The nucleic acid probes can be detectably labeled, for example, with a radioisotope, a fluorescent compound, or an enzyme. The added nucleic acid probe solution P will take the same flow path as the target nucleic acid solution T, and distribute over the whole membrane  30 . If the porous membrane  30  is in a dry state before the nucleic acid probe solution P is added, the nucleic acid probe solution P can also rapidly enter the inside of the porous membrane  30 . After the nucleic acid probe solution P is added, the biochip is placed at a proper temperature (such as 40 to 48° C.) for several minutes to allow the nucleic acid probe to anneal with the target nucleic acid, and thus the process of base pairing is completed. 
         [0047]    After the process of base pairing, the unhybridized nucleic acid probes are washed away. The washing solution W is injected via the second circulation hole  13  during the washing process. The washing solution W is flushed into the hybridization chamber  11  through the first microchannel  14 . When the washing solution W is flushed into the hybridization chamber  11 , the interstice  15  is firstly filled with the washing solution, and then the washing solution W diffuses toward the center of the porous membrane  30  from the edge of the porous membrane  30 , and finally is discharged to the outside via the first circulation hole  12 . Because the washing solution W is flushed from the outside of the porous membrane  30  to the inside of the porous membrane  30 , the nucleic acid probe, which is a relatively small molecule, can be easily and rapidly flushed out of the pores of the porous membrane  30 . Therefore, the background noise level is reduced, and the time for flushing is shortened. Then, the nucleic acid probes labeled with chromogenic molecules are detected, and the results of hybridization reaction are obtained. 
         [0048]    A test for the flow directions of the nucleic acid probe solution is done. In one case, the fluorescent nucleic acid probe (such as 20-mer DNA oligomers) solution P is injected into the second circulation hole  13 , and then flows through the first microchannel  14 , enters the interstice  15 , and diffuses toward the center of the porous membrane  30  from the periphery of the porous membrane  30 , and finally is discharged to the outside via the first circulation hole  12 , wherein the fluorescent nucleic acid probe solution P is accelerated from the second circulation hole  13  to the first circulation hole  12  due to a reduction in the flow area (by considering mass conservation). Then, the porous membrane  30  with the fluorescent nucleic acid probes is dried. The dried porous membrane  30  with the fluorescent nucleic acid probes is observed using a fluorescent microscope, and the fluorescent microscope image obtained is shown in  FIG. 5 . In another case, the fluorescent nucleic acid probe solution P is injected into the hybridization chamber  11  via the first circulation hole  12 , and enters the inside of the porous membrane  30  from the center thereof. After entering the inside of the porous membrane  30 , the target nucleic acid solution T diffuses across the porous membrane  30  to the interstice  15 , and then flows through the first microchannel  14 , and is discharged to the outside via the second circulation hole  13 . Then, the porous membrane  30  with the fluorescent nucleic acid probes is dried. The dried porous membrane  30  with the fluorescent nucleic acid probes is observed using a fluorescent microscope, and the fluorescent microscope image obtained is shown in  FIG. 6 . 
         [0049]    From the above flow direction test, it surprisingly found that when the nucleic acid probe solution P diffuses toward the center of the porous membrane  30  from the periphery of the porous membrane  30 , the nucleic acid probe can be captured by the porous membrane  30 , and stretched and wound around the fibers of the porous membrane  30  due to the change in strain. In this case, it is noted that the nucleic acid probe solution P is accelerated from the periphery (the inlet side) to the center (the exit side) of the porous membrane  30  due to the mass conservation law so that the length of the nucleic acid probe becomes more stretched towards the center than in the periphery of the porous membrane  30 . As a result, a lot of the nucleic acid probe molecules are present in the porous membrane  30  (see  FIG. 5 ). By contrast, when the nucleic acid probe solution P diffuses toward the periphery of the porous membrane  30  from the center of the porous membrane  30 , the nucleic acid probe cannot be captured by the porous membrane  30 . This is because the nucleic acid probe solution P is decelerated from the center (the inlet side) to the periphery (the exit side) of the porous membrane  30  that the length of the nucleic acid probe cannot be stretched. As a result, much less of the nucleic acid probe molecules are present in the porous membrane  30  (see  FIG. 6 ). By considering mass conservation, the same results for the nucleic acid probe solution P should be also applied to the target nucleic acid (such as large molecules of DNA) solution T. 
         [0050]      FIG. 2A  is an exploded view of the biochip of the second embodiment of the present invention. With reference to  FIG. 2A , the biochip of this embodiment comprises an upper substrate  40 , a lower substrate  20 , and a membrane  30 , wherein the upper substrate  40  and the lower substrate  20  are stacked together one on top of the other, and the porous membrane  30  is provided in the hybridization chamber  41  located on the upper substrate  40 . 
         [0051]    With reference to  FIG. 2A  to  FIG. 2C , the upper substrate  40  has a hybridization chamber  11 , which is in the form of a disk-shaped cavity. However, the shape, the size and the thickness of the hybridization chamber  41  have no restriction, and the hybridization chamber  41  can be a tetrahedral cavity. The center of hybridization chamber  41  is provided with a first circulation hole  42 . There is no specific limitation on the numbers and the positions of the first circulation hole  42 , and the positions of the first circulation hole  42  can be changed with the position of another circulation hole so that the reaction solution can flow over the whole inside of the porous membrane  30 . In addition, the first circulation hole  42  can be further communicated with a microchannel or another circulation hole (not shown in the drawing) for facilitating the solution injection. 
         [0052]    A plurality of little pillars  411  protrude from the interface between the bottom of the upper substrate  40  and the hybridization chamber  41 . The ends of these little pillars  411  are in contact with the surface of the porous membrane  30  located in the hybridization chamber after the biochip is assembled. 
         [0053]    A pair of the first microchannel  44  are respectively communicated with the hybridization chamber  41 , and the pair of the first microchannel  44  are further respectively communicated with a pair of the second circulation holes  43 . There is no specific limitation on the number and the positions of the first microchannel  44 , and the number and the positions thereof can be changed with the flow path of the reaction solution. The second circulation holes  43  and the first microchannels  44  are communicated with the interspace among the little pillars  411 . In addition, an interstice  45  with predetermined width is left between the porous membrane  30  and the sidewall of the hybridization chamber  41 . The interstice  45  has a width of 0.05 to 0.2 mm, and preferably 0.1 mm. 
         [0054]    With reference to  FIG. 2A  and  FIG. 2B , the hybridization chamber  41 , the first microchannels  44 , and the second circulation hole  43  are formed between the upper substrate  40  and the lower substrate  20 . The hybridization chamber  41 , the first microchannels  44 , and the second circulation holes  43  are not limited to be located on the upper substrate  40 , but they may be located on the lower substrate  20 , or on the upper substrate  40  and the lower substrate  20  (divided into male and female halves). Once the upper substrate  40  and the lower substrate  20  are stacked together one on top of the other, the desired structures of the hybridization chamber, the microchannels, and the circulation holes will be formed. 
         [0055]    The biochips can be fabricated by the conventional method. There is no specific limitation on the material, the shape, and the pore size of the porous membrane  30 . 
         [0056]      FIG. 2D  is the schematic view showing the flow direction of the hybridization solution according to the second embodiment. With reference to  FIG. 2D , before the hybridization reaction, the target nucleic acid solution T is injected into the hybridization chamber  41  from the second circulation hole  43  on the left side of the hybridization chamber through the first microchannels  44 . The target nucleic acid solution T firstly fills up the interstice  45 , and then enters the inside of the porous membrane  30  from the lateral side of the porous membrane  30 . While entering the porous membrane  30  from the lateral side thereof, the target nucleic acid solution T fills up the interspace among the little pillars  411  and then diffuses from the top side the porous membrane  30  to the bottom side thereof. Finally, the target nucleic acid solution T flows through first microchannels  44  on the right side of the hybridization chamber, and is discharged to the outside via the second circulation hole  43 . and the first circulation hole  42 . If the porous membrane  30  is in a dry state, the target nucleic acid solution T can rapidly enter the inside of the porous membrane  30  due to the capillary attraction of the fine pores of the porous membrane  30 . Moreover, the target nucleic acid solution T which enters the porous membrane  30  can be permanently attached to the surface and the inside of the porous membrane  30  by heating or UV irradiation. 
         [0057]    Afterwards, the nucleic acid probe P is injected into the hybridization chamber  41  via the first circulation hole  42  for hybridization reaction. After the nucleic acid probe solution P is added, the added nucleic acid probe solution P will take the same flow path as the target nucleic acid solution T as exemplified in the first embodiment, and distribute over the whole membrane  30 . If the porous membrane  30  is in a dry state before the nucleic acid probe solution P is added, the nucleic acid probe solution P will rapidly enter the inside of the porous membrane  30 . After the nucleic acid probe solution P is added, the biochip is placed at a proper temperature (such as 40 to 48° C.) for several minutes to allow the nucleic acid probe to anneal with the target nucleic acid, and thus the process of base pairing is completed. 
         [0058]    After the process of base pairing, the unhybridized nucleic acid probes are washed away. The washing solution W is flushed into the hybridization chamber  41  from the second circulation hole  43  on the left side through the first microchannel  44 . When the washing solution W is flushed into the hybridization chamber  41 , the washing solution W diffuses toward the center and the bottom of the porous membrane  30  from the lateral side and the top side of the porous membrane  30 , respectively, and finally is discharged to the outside through the first circulation hole  42  and the second circulation hole  43  on the right side. Because the washing solution W is flushed from the lateral side and the top side of the porous membrane  30  to the inside of the porous membrane  30 , the nucleic acid probe, which is a relatively small molecule, can be easily and rapidly flushed out of the pores of the porous membrane  30 . Therefore, the background noise level is reduced, and the time for flushing is shortened. 
         [0059]      FIG. 3A  is an exploded view of the biochip of the third embodiment of the present invention. With reference to  FIG. 3A , the biochip of this embodiment comprises an upper substrate  50 , a lower substrate  20  composed of a top substrate  201  and a bottom substrate  202 , and a membrane  30 , wherein the upper substrate  50 , the top substrate  201 , and the bottom substrate  202  are stacked together one on top of the other, and the porous membrane  30  is provided in the hybridization chamber  41  located on the upper substrate  50 . 
         [0060]    With reference to  FIG. 3A  to  FIG. 3C , the upper substrate  40  has a hybridization chamber  51 , which is in the form of a disk-shaped cavity. However, the shape, the size and the thickness of the hybridization chamber  41  have no restriction, and the hybridization chamber  41  can be a tetrahedral cavity. The hybridization chamber  41  is provided with a first circulation hole  52 . There is no specific limitation on the numbers and the positions of the first circulation hole  52 , and the positions of the first circulation hole  52  can be changed with the position of another circulation hole so that the reaction solution can flow over the whole inside of the porous membrane  30 . In addition, the first circulation hole  52  can be further communicated with a microchannel or another circulation hole (not shown in the drawing) for facilitating solution injection. 
         [0061]    A plurality of little pillars  511  protrude from the interface between the bottom of the upper substrate  50  and the hybridization chamber  51 . The ends of these little pillars  511  are in contact with the surface of the porous membrane  30  located in the hybridization chamber after the biochip is assembled. 
         [0062]    A pair of the first microchannel  54  are respectively communicated with the hybridization chamber  51 , and the pair of first microchannels  54  are further respectively communicated with a pair of the second circulation holes  53 . There is no specific limitation on the number and the positions of the first microchannels  54 , and the number and the positions thereof can be changed with the flow path of the reaction solution. The second circulation holes  53  and the first microchannels  54  are communicated with the interspace among the little pillars  411 . In addition, an interstice  55  with predetermined width is left between the porous membrane  30  and the sidewall of the hybridization chamber  51 . The interstice  55  has a width of 0.05 to 0.2 mm, and preferably 0.1 mm. 
         [0063]    The lower substrate  20  is composed of a top substrate  201  and a bottom substrate  202 , which are stacked together one on top of the other. The top substrate  201  has the third circulation hole  2011 , and the bottom substrate  202  has the third circulation hole  2021  corresponding to the third circulation hole  2011 . The third circulation hole  2011  or  2021  can be provided in a single-layer lower substrate  20  or in a lower substrate  20  composed of a top substrate  201  and a bottom substrate  202  as this embodiment. The third circulation hole  2021  is further communicated with the second microchannel  2022 , and the second microchannel  2022  is further communicated with the fourth circulation hole  2023 . 
         [0064]    With reference to  FIG. 3A  and  FIG. 3B , the hybridization chamber  51 , the first microchannels  54 , and the second circulation hole  53  are formed between the upper substrate  50  and the lower substrate  20 . The hybridization chamber  51 , the first microchannels  54 , and the second circulation holes  53  are not limited to be located on the upper substrate  50 , but they may be located on the lower substrate  20 , or on both the upper substrate  50  and the lower substrate  20  (divided into male and female halves). Once the upper substrate  50  and the lower substrate  20  are stacked together one on top of the other, the desired structures of the hybridization chamber  51 , the first microchannels  54 , and the second circulation holes  53  will be formed. Likewise, the third circulation holes  2011 ,  2021 , the second microchannels  2022 , and the fourth circulation holes  2023  are not limited to be located on the top substrate  201 , but they may be located on the bottom substrate  202 , or on both the top substrate  201  and the bottom substrate  202  (divided into male and female halves). 
         [0065]    The biochips can be fabricated by the conventional method. There is no specific limitation on the material, the shape, and the pore size of the porous membrane  30 . 
         [0066]      FIG. 3D  is the schematic view showing the flow direction of the hybridization solution according to the third embodiment. With reference to  FIG. 3D , before the hybridization reaction, the target nucleic acid solution T is injected into the hybridization chamber  51  from the fourth circulation hole  2023  through the second microchannel  2022 , and then the third circulation holes  2021  and  2011 . After entering the hybridization chamber  51 , the target nucleic acid solution T diffuses into the inside of the porous membrane  30  from the bottom center thereof, and continuously diffuses toward the outer edge of the porous membrane  30 . Some of the target nucleic acid solution T flows in the interspace among the little pillars  511 , and is collected in the interstice  55  surrounding the periphery of the porous membrane  30 . Finally, the target nucleic acid solution T is discharged to the outside via the first microchannel  54  and the second circulation holes  53  which are located on the two sides of the hybridization chamber  51 , and the first circulation hole  52 . If the porous membrane  30  is in a dry state, the target nucleic acid solution T can rapidly enter the inside of the porous membrane  30  due to the capillary attraction of the fine pores of the porous membrane  30 . Moreover, the target nucleic acid solution T which enters the porous membrane  30  can be permanently attached to the surface and the inside of the porous membrane  30  by heating or UV irradiation. 
         [0067]    Afterwards, the nucleic acid probe solution P is injected into the hybridization chamber  51  via the first circulation hole  52  for hybridization reaction. After the nucleic acid probe solution P is added, the added nucleic acid probe solution P will take the same flow path as the target nucleic acid solution T as exemplified in the first embodiment, and distribute over the whole membrane  30 . If the porous membrane  30  is in a dry state before the nucleic acid probe solution P is added, the nucleic acid probe solution P will rapidly enter the inside of the porous membrane  30 . After the nucleic acid probe solution P is added, the biochip is placed at a proper temperature (such as 40 to 48° C.) for several minutes to allow the nucleic acid probe to anneal with the target nucleic acid, and thus the process of base pairing is completed. 
         [0068]    After the process of base pairing, the unhybridized nucleic acid probes are washed away. The washing solution W is flushed into the hybridization chamber  51  from the fourth circulation hole  2023  through the second microchannel  2022 , and then the third circulation holes  2021  and  2011 . When the washing solution W is flushed into the hybridization chamber  51 , the washing solution W diffuses into the inside of the porous membrane  30  from the bottom center thereof, and continuously diffuses toward the outer edge of the porous membrane  30 . Some of the washing solution W flows in the interspace among the little pillars  511 , and is collected in the interstice  55  surrounding the periphery of the porous membrane  30 . Finally, the washing solution W is discharged to the outside via the first microchannels  54  and the second circulation holes  53  which are located on the two sides of the hybridization chamber  51 , and the first circulation hole  52 . Because the washing solution W is flushed from the bottom of the porous membrane  30  to the inside thereof, the nucleic acid probe, which is a relatively small molecule, can be easily and rapidly flushed out of the pores of the porous membrane  30  by the outward diffusion of the washing solution W and the guidance of the interspace among the little pillars. Therefore, the background noise level is reduced, and the time for flushing is shortened. 
         [0069]      FIG. 4A  is an exploded view of the biochip of the fourth embodiment of the present invention. With reference to  FIG. 4A , the biochip of this embodiment comprises an upper substrate  60 , a lower substrate  20  composed of a top substrate  201  and a bottom substrate  202 , and a membrane  30 , wherein the upper substrate  60 , the top substrate  201 , and the bottom substrate  202  are stacked together one on top of the other, and the porous membrane  30  is provided in the hybridization chamber  61  located on the upper substrate  60 . 
         [0070]    With reference to  FIG. 4A  to  FIG. 4C , the upper substrate  60  has a hybridization chamber  61 , which is in the form of a disk-shaped cavity. However, the shape, the size and the thickness of the hybridization chamber  61  have no restriction, and the hybridization chamber  61  can be a tetrahedral cavity. The hybridization chamber  61  is provided with a first circulation hole  62 . There is no specific limitation on the numbers and the positions of the first circulation hole  62 , and the positions of the first circulation hole  62  can be changed with the position of another circulation hole so that the reaction solution can flow over the whole inside of the porous membrane  30 . In addition, the first circulation hole  62  can be further communicated with a microchannel or another circulation hole (not shown in the drawing) for facilitating solution injection. 
         [0071]    A pair of the first microchannel  64  are respectively communicated with the hybridization chamber  61 , and the pair of first microchannels  64  are further respectively communicated with a pair of the second circulation holes  63 . There is no specific limitation on the number and the positions of the first microchannels  64 , and the number and the positions thereof can be changed with the flow path of the reaction solution. In addition, an interstice  65  with predetermined width is left between the porous membrane  30  and the sidewall of the hybridization chamber  61 . The interstice  65  has a width of 0.05 to 0.2 mm, and preferably 0.1 mm. 
         [0072]    The lower substrate  20  is composed of a top substrate  201  and a bottom substrate  202 , which are stacked together one on top of the other. The top substrate  201  has the third circulation hole  2011 , and the bottom substrate  202  has the third circulation hole  2021  corresponding to the third circulation hole  2011 . The third circulation hole  2011  or  2021  can be provided in a single-layer lower substrate  20  or in a lower substrate  20  composed of a top substrate  201  and a bottom substrate  202  as this embodiment. The third circulation hole  2021  is further communicated with the second microchannel  2022 , and the second microchannel  2022  is further communicated with the fourth circulation hole  2023 . 
         [0073]    With reference to  FIG. 4A  and  FIG. 4B , the hybridization chamber  61 , the first microchannels  64 , and the second circulation hole  63  are formed between the upper substrate  60  and the lower substrate  20 . The hybridization chamber  61 , the first microchannels  64 , and the second circulation holes  63  are not limited to be located on the upper substrate  60 , but they may be located on the lower substrate  20 , or on both the upper substrate  60  and the lower substrate  20  (divided into male and female halves). Once the upper substrate  60  and the lower substrate  20  are stacked together one on top of the other, the desired structures of the hybridization chamber  61 , the first microchannels  64 , and the second circulation holes  63  will be formed. Likewise, the third circulation holes  2011 ,  2021 , the second microchannels  2022 , and the fourth circulation holes  2023  are not limited to be located on the top substrate  201 , but they may be located on the bottom substrate  202 , or on both the top substrate  201  and the bottom substrate  202  (divided into male and female halves). 
         [0074]    The biochips can be fabricated by the conventional method. There is no specific limitation on the material, the shape, and the pore size of the porous membrane  30 . 
         [0075]      FIG. 4D  is the schematic view showing the flow direction of the hybridization solution according to the fourth embodiment. With reference to  FIG. 4D , before the hybridization reaction, the target nucleic acid solution T is injected into the hybridization chamber  61  from the fourth circulation hole  2023  through the second microchannel  2022 , and then the third circulation holes  2021  and  2011 . After entering the hybridization chamber  61 , the target nucleic acid solution T diffuses into the inside of the porous membrane  30  from the bottom center thereof, and continuously diffuses toward the outer edge of the porous membrane  30 . Subsequently, the target nucleic acid solution T is collected in the interstice  65  surrounding the periphery of the porous membrane  30 . Finally, the target nucleic acid solution T is discharged to the outside via the first microchannels  64  and the second circulation holes  63  which are located on the two sides of the hybridization chamber  51 , and the first circulation hole  62 . If the porous membrane  30  is in a dry state, the target nucleic acid solution T can rapidly enter the inside of the porous membrane  30  due to the capillary attraction of the fine pores of the porous membrane  30 . Moreover, the target nucleic acid solution T which enters the porous membrane  30  can be permanently attached to the surface and the inside of the porous membrane  30  by heating or UV irradiation. 
         [0076]    Afterwards, the nucleic acid probe solution P is injected into the hybridization chamber  61  via the first circulation hole  62  for hybridization reaction. After the nucleic acid probe solution P is added, the added nucleic acid probe solution P will take the same flow path as the target nucleic acid solution T as exemplified in the first embodiment, and distribute over the whole membrane  30 . If the porous membrane  30  is in a dry state before the nucleic acid probe solution P is added, the nucleic acid probe solution P will rapidly enter the inside of the porous membrane  30 . After the nucleic acid probe solution P is added, the biochip is placed at a proper temperature (such as 40 to 48° C.) for several minutes to allow the nucleic acid probe to anneal with the target nucleic acid, and thus the process of base pairing is completed. 
         [0077]    After the process of base pairing, the unhybridized nucleic acid probes are washed away. The washing solution W is flushed into the hybridization chamber  61  from the fourth circulation hole  2023  through the second microchannel  2022 , and then the third circulation holes  2021  and  2011 . When the washing solution W is flushed into the hybridization chamber  61 , the washing solution W diffuses into the inside of the porous membrane  30  from the bottom center thereof, and continuously diffuses toward the outer edge of the porous membrane  30 . Subsequently, the washing solution W is collected in the interstice  65  surrounding the periphery of the porous membrane  30 . Finally, the washing solution W is discharged to the outside via the first microchannels  64  and the second circulation holes  63  which are located on the two sides of the hybridization chamber  61 , and the first circulation hole  52 . Because the washing solution W is flushed from the bottom of the porous membrane  30  to the inside thereof, the nucleic acid probe, which is a relatively small molecule, can be easily and rapidly flushed out of the pores of the porous membrane  30 . Therefore, the background noise level is reduced, and the time for flushing is shortened. 
         [0078]    According to polymerase chain reaction (PCR), the annealing of the primer and the target nucleic acid only took one minute to be completed. By using the biochip of the present invention, the nucleic acid probe can effectively diffuse on part of the surface and in the inside of the membrane and forms a base pair with the target nucleic acid in a very short time after the nucleic acid probe enters the hybridization chamber and contacts with the membrane. The hybridization reaction of the present invention only takes several minutes instead of over 10 hours for the prior art. On the other hand, the nucleic acid probe cannot easily stick to the membrane because the nucleic acid probe can form a base pair with the target nucleic acid in a very short time. Therefore, when the washing solution W is flushed into the inside of the porous membrane  30 , the small nucleic acid probe molecules unspecifically bound to the membrane can be easily and rapidly flushed out of the membrane. As a result, the background noise level is reduced. It is also worth noting that the flow directions of the target nucleic acid solution and the nucleic acid probe solution play a very important role in the present invention. Moreover, the structures of the flow-in and the flow-out circulation holes for the target nucleic acid solution T, the nucleic acid probe solution P, and the washing solution W described in the above embodiments are just exemplified, therefore, those are not limited to the described ones. 
         [0079]    It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Thus, it is intended that the present invention cover the modifications and the variations of this invention provided they come within the scope of the appended claims and their equivalents.