TW200808963A - Biochip - Google Patents

Abstract

The present invention relates to a biochip used in nucleic acid hybridization. The biochip of this invention comprises a cavity like hybridization chamber installed in the biochip, a substrate with holes is clipped and installed in the hybridization chamber, and at least a first flow channel and at least a second flow channel are installed and connected to the hybridization chamber. The reaction solution may flow into at least one of the flow channel, then through holes inside the substrate, and after that discharge through at least one of the rest of the flow channel; wherein the reaction area for the reaction solution may be increased by holes inside the substrate, the sensitivity of the reaction may be improved, the diffusion distance for reaction molecules may be decreased by the restriction of the space inside the substrate, and the hybridization time may be reduced.

Description

200808963 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a biochip, and more particularly to a biochip for use in nucleic acid hybridization (Hybridization). [Prior Art] Using a probe nucleic acid to hybridize with a nucleic acid to be analyzed, and confirming whether the sample DNA has a desired gene or a nucleic acid fragment. Conventional hybridization reaction analysis mainly utilizes the method of printing and pulsing The method transfers the nucleic acid to be analyzed to, for example, a substrate of a film, and then performs a hybridization pairing reaction with a probe nucleic acid having a spedfleity, and further proceeds to a color by the display molecule labeled by the probe nucleic acid. The cold light or radioactive display shows a hybridization reaction filament. " ” ^Responsible for the analysis of the nucleic acid to be analyzed if the nucleic acid is converted by electrophoresis colloid and then hybridized with the probe, called the southern smasher, the eye IS coffee bk> gt) The hybridization reaction of the RNA will be blGtting for the northern willow. Other direct ^: the nucleic acid is dropped on the _, and the fine-grained and block-like printing method is called by the drip-distribution (-·/-· /_制ng). After the electric IS: striated and other printing method 'Because the nucleic acid to be analyzed does not need to analyze the y 1 ° direct transfer step, it can shorten the operation of the second and because of its pasteless electrophoresis and phase The solution test is relatively economical, so it is commonly used in the qualitative analysis or 5 200808963. The method is to discriminate the nucleic acid to be analyzed by using a heating or ultraviolet light. Crosslinking on the surface of the film, so as not to be washed away after the subsequent washing reaction with the probe nucleic acid. Under the conventional reaction conditions, since the nucleic acid to be analyzed is dropped on the surface of a wet film, It is diffused on the wet surface and adsorbed on On the surface, therefore, most of the

The nucleic acid to be analyzed is fixedly attached to the surface of the film and to the pores adjacent to the surface, and the number of nucleic acid molecules to be analyzed which are fixed is limited, and the nucleic acid molecule is produced.

Health (the intensity of the heterogeneous signal will also be weak, so the sensitivity of the reaction will be greatly reduced for the nucleic acid to be analyzed with a small sample size or a long molecule. In addition, when adding a conventional blocker (bl〇cking reagem) The probe nucleic acid solution is subjected to hybridization and anti-correction, and the probe is also like the nucleic acid to be analyzed, which can only be used for the diffusion of the surface of the thin county, and the movement of the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The hybridization reaction needs to be taken care of, and the number of green f long Xuan Xu and above ^ Day Shoujian 'for _ more eager to know the test results of the test, that is, can not be short 70 ▲ In addition, for some simple nucleic acid qualitative test, if still It is quite uneconomical to spend more time and reagents on the test. Because of the second::t speed, it can simplify the steps and time required for the analysis of the break, and take into account the low background miscellaneous marks or the method. For - (4) == batch detection, it can effectively shorten the cost of materials required to reduce the cost of the same day. [Summary of Contents] 200808963

When the Hbf is known to carry out the hybridization reaction, the analysis or the probe nucleic acid is inclined, and the time required for the movement and the base pairing and the e'乂 reaction by the natural Brownian motion is as long as more than ten hours. The time-consuming problem of 'while making more energy to be analyzed_fixed on the surface/inside of the substrate increases the number of pairing molecules to increase the sensitivity of the reaction, and the present invention provides a hybrid anti-red biocrystal, By the limitation of the internal space of the organism, the detection of the hybridization reaction is carried out into the matrix, so that the nucleic acid or the probe nucleic acid molecule to be analyzed can be completely diffused in the pores in a very short time and complete. Hybridization reaction. In addition, in the case of the grasping body plus >1, the nucleic acid or probe nucleic acid molecule to be analyzed can be rapidly mobilized, and because it can flow inside the matrix, the attachment or reaction area is amplified, so that the pairing can be increased on the one hand. The number of nucleic acid molecules increases the sensitivity of detection. On the other hand, by means of the movement of the flow, the base pairing is accelerated, and the cleaning solution is also penetrated into the interior of the matrix, and the unpaired needle nucleic acid inside the matrix is washed away. Improve the cleanliness of the cleaning reaction while reducing the background value of the reaction. Based on the above object, the present invention provides a biochip, comprising: a hybrid chamber in a cavity shape in the biochip, wherein the hybrid chamber is provided with a matrix of holes, and the hybrid chambers are respectively provided with at least one a flow port and at least one second flow port, whereby the reaction solution is injected from at least one of the flow ports and flows through the inside of the substrate to flow out from the remaining at least one flow port. The bio-wafer may be formed by an upper substrate and a lower substrate. The upper substrate and the lower substrate are vertically connected to each other, and the hybridization chamber is formed therebetween, and the hybrid chamber is provided with a hole. The matrix, the hybrid 7 200808963

There is no special restriction on the shape and thickness of the mixed parent room. It can be combined with the matrix (4) dragon to make the whole, and the side of the matrix seaming room can be left - the scent of the predetermined degree: 'the solution of the solution is obtained from the matrix. The sides enter the matrix. Further, the first flow port may be connected to the upper side of the center of the hybrid chamber, but the position and number of the opening are not limited thereto. In addition, the first flow port, the second flow port and the hybridization chamber can be connected to each other to form a micro flow channel, so as to apply pressure to the reaction solution through the micro flow channel, so that the reaction solution can quickly pass through the inside of the matrix, and the impurities should be scaled. The substrate can be dry on the reverse side, so that when the solution is injected, it can be quickly entered into the carbene by the attraction of capillary action. The pores of the Kibe's hole are about 5 〇, so that the nucleic acid is transparent. 'But the test is not limited to this, and the material (4) can be nylon membrane or nitril fiber paper (nitr〇ceUul〇se) Membrane), but not limited to this. With the biochip of the present invention, the nucleic acid solution to be analyzed can be introduced into the hybridization chamber by any one or several flow ports for adsorption by the matrix, and since the nucleic acid to be analyzed can enter the inside of the matrix, the number of the immobilized nucleic acid molecules to be analyzed can be increased. And increase its test sensitivity. After the nucleic acid to be analyzed is immobilized, the probe nucleic acid solution may be fed from any one or several flow ports to allow the probe nucleic acid to enter the matrix of the hybridization chamber and perform base pairing with the nucleic acid to be analyzed. Since the probe nucleic acid can easily move through the tiny holes in the matrix, the pairing step can be completed quickly. Thereafter, the cleaning solution can be further injected into the cleaning solution by any one or several flow ports, and the probe nucleic acid can be easily moved in the microscopic pores of the matrix. Thoroughly complete the cleaning = fines and react to the daytime and reduce the background noise value. The invention also provides a biochip, which comprises an upper substrate and a lower substrate, wherein the upper substrate and the lower substrate are vertically connected to each other, and the two are formed with a hybridization chamber, and the hybridization chamber is provided with upper and lower clips. a substrate having a hole, a plurality of small protrusions protruding from a bottom edge of the upper substrate, and the ends of the small protrusions are in contact with the surface of the substrate disposed in the hybridization chamber, and The hybrid chamber is connected to the upper substrate with at least one first flow port, and the hybrid chamber is connected to at least one second flow port on the side, and the second flow port is connected to the space between the small protrusions. . The second flow port is in communication with the space between the small protrusions, so that the solution injected from the second flow port can enter the matrix through the space between the small columns, and the solution solution can enter the area of the substrate. This in turn increases the efficiency and speed at which the solution penetrates the substrate, making the reaction more rapid and complete. The biochip may be composed of an upper substrate and a lower substrate and a lower substrate, in addition to the upper substrate and the lower substrate. a single-layer lower substrate or a double-layer lower substrate composed of a top substrate and a bottom substrate, and a third flow port may be connected through the hybridization chamber, and the third flow port may be connected to a second micro flow channel, the second micro flow The circuit is connected to a fourth flow port, so that the reaction solution also enters from below the substrate, and can be utilized in the test of different reaction processes. The embodiments of the present invention will be further described in conjunction with the following drawings. The following examples are set forth to illustrate the invention, and are not intended to limit the scope of the present invention, which is known to those skilled in the art. In the meantime, it is possible to make some changes and _, and therefore the protection of the present invention is subject to the definition of the scope of the patent application. & circumference [Embodiment]

Look at the third-intention of the first-A ®, the county. The biochip of the embodiment of the invention comprises an upper substrate, a 2G and a substrate 3A. The upper substrate 1 () and the lower substrate 2 () are connected in abundance, and the 3 (4) is mounted in the hybrid 61 provided in the upper substrate ig. Please also refer to the figures -A to -c, the upper substrate 1G, which is provided with a disk chamber-shaped hybridization chamber n, and the size and thickness of the hybridization chamber n are not particularly limited. It can also be a quadrilateral hollow. A first flow port 12 is disposed at a center point of the hybridization chamber 11. The position of the first flow port 12 is not limited by the financial system, and the position of the flow port can be adjusted to adjust the flow path of the reaction solution. There is no particular limitation on the number of the first flow ports 12 provided throughout the entire interior of the substrate 30. The first flow port 12 can be further connected to a micro flow channel or a flow port (not shown) to facilitate the injection of the solution or the arrangement between the micro flow channels. A first microchannel 14 is connected to the side of the parent compartment 11, and the first microchannel is connected to the second orifice 13. The number and position of the first microchannels 14 are not particularly limited, and can be adjusted according to the flow path of the reaction solution. Further, between the substrate 3 and the side wall of the hybridization chamber η, a gap 15 of a predetermined width is left, and the gap 15 is between about 〇5 0 0. 2 mm 200808963, wherein o.l mm is preferred. Continuing to refer to Figures -A and -B, the hybrid chamber n, the first microchannel 14 and the second flow σ 13 are ultimately formed between the substrate 104 and the lower substrate 2 of the substrate 10 14 . Therefore, the positions of the hybrid chamber 11, the first microchannel 14 and the second flow port 13 are not limited to the upper substrate 10, and may be disposed on the lower substrate 20 or on the upper substrate 10 and the lower substrate 20, respectively. When the corresponding chamber, micro flow channel or flow port is formed, the upper substrate 10 is connected to the lower substrate 20 to form the desired configuration. The upper substrate 10 and the lower substrate 20 may be separately formed as described above, and may be formed into a single plate, and the hybrid chamber η, the first micro flow channel 14 and the second flow port 13 are formed in the interior thereof during fabrication. . The biochip is a microfluidic wafer of the present invention, but it is not limited thereto. It may also include a nanofluidic wafer having a smaller flow channel size, or a wafer which can form the configuration of the present invention. In the present embodiment, the microfluidic wafer is made of a material (for example, quartz or glass), and a wet etching method is used to etch a capillary-like microfluid and then overlying it. A layer of quartz or (4) can be used to complete a wafer with a seal = microchannel or chamber. It can also utilize a hard polymer, such as P〇lymethyl methacrylate (hereinafter referred to as polycarbonate), which is firstly engraved in a square or a pattern; The mold is then formed into a micro flow channel on the PMMA or PC by hot pressing (_(10) fine). Finally, the substrate is coated with the same material force. In addition, the biochip of the present invention can also be prepared by using a soft polymer D. P〇lydimethyl siloxane (hereinafter referred to as PDMS), which is a soft polymer, has a good fluidity and does not require a pressurizing master during the manufacturing process, thereby reducing the chance of harming the master mold. The PDMS is a better substrate material, but it is not limited to this. The Kibe 30' can be nyl〇nmeinbrane or stone Films such as nitrocellulose membranes, but not limited to them. Ni Yue I film can be positively or uncharged, and the pores of nylon membrane and nitrocellulose paper

/The same diameter can be from 0·1 to 50 μπια ', depending on the molecular weight of the nucleic acid to be analyzed, the pore size is selected, and the pore size used in the recovery of the nuclear complex is also large, of which 〇.2 μιη and 0 45 Μιη is preferred. In addition, the film can be dried so that the core to be analyzed can be quickly adsorbed onto the film. "Please refer to the -D ® 'This is a schematic diagram of the solution flow direction when the hybridization reaction is carried out in the embodiment of the present invention. Before the hybridization is reversed, the solution τ can be injected from the first flow port ,, and the nucleic acid solution to be analyzed is entered into the human hybridization chamber. After u, the center point of the substrate 30 enters the inside of the substrate 3〇, flows to the outer edge of the substrate 3〇, and finally flows out into the gap 环绕 around the side of the base f 3G, and then passes through the first-micro flow channel 14 The second flow port 13 is discharged. If the mass 3〇 is dry, the nucleic acid solution τ to be analyzed is more rapidly formed by the capillary phenomenon generated by the second fine pores of the matrix, and the pores of the matrix 3〇 are more quickly entered. Thereafter, the method of drying or riding the "outside light" method can be used to analyze the core of the Q to be analyzed and the inside thereof. Thereafter, the probe nucleic acid solution P is injected from the first flow port 12 to perform a hybridization pairing reaction. The display nucleic acid is labeled with a display molecule to detect the result of the reaction of the hybrid 12 200808963, and the display molecule may be a coloring matter, an enzyme, a fluorescent substance, or a radiation element, but is not limited thereto. The probe nucleic acid solution p after the addition, like the flow path of the nucleic acid solution T to be analyzed, can be spread over the entire matrix. If the substrate is also a dry bear before the addition of the probe nucleic acid solution p, the probe nucleic acid solution P can also rapidly enter the interior of the matrix. After the probe nucleic acid solution P is added, the biochip is placed at an appropriate temperature (for example, 4 〇 to 48 C) for about several minutes, and the probe nucleic acid is searched for the paired nucleic acid to be analyzed for annealing. Base pairing hybridization reaction m ° ~ After the parental pairing reaction, the unpaired probe nucleic acid must be washed, and the rinsed day can be injected into the cleaning solution w by the second flow port 13, and the cleaning is performed after the first micro The flow path 14 enters the hybridization chamber η, which enters the impurity = ns first fills the entire gap 15' and then gradually occupies from the side of the substrate 30, f 3G flows, and is finally discharged by the first flow enthalpy 12. By month washing %, the cleaning solution W is from the outside of the entire matrix Deng into the internal rush

This is a quick and easy way to transfer the probe nucleic acid of a smaller molecule from the matrix, and then it can reduce the background noise value and shorten the display molecule labeled by the flushing By conducting a debt test, the result of the hybridization reaction can be obtained. The tea is read in the second diagram, which is a three-dimensional diagram of the second embodiment of the present invention: Fig. The biochip in the embodiment of the present invention comprises an upper substrate, a substrate 20 and a substrate. The upper substrate 4A and the lower substrate 2 are connected to each other, and the substrate 3() is mounted on the upper substrate 4G. 13 200808963 The same as 4, the second A to the second c, The substrate 40 is provided with a hybrid chamber 41 in the shape of a disk, and the hybrid chamber 41 has a size of a na[gamma], which may be a quadrangular cavity. The hybrid chamber 41 has a tooth σ, and the first through port 42 has no special restriction on the number and opening position of the first flow port 42, and the reaction can be made according to the position of the other flow port. The flow path of the solution can be distributed throughout the entire substrate 3, and in addition, the first flow π 42 can be connected to the connection - the micro flow channel or the flow port is not shown, and the solution is convenient to reduce the flow between the micro flow channels.

Configuration. A plurality of small studs 4U are protruded from the bottom edge of the upper substrate 40 at the bottom of the hybrid chamber 41. The small studs 411 are disposed on the surface of the substrate 30 disposed in the hybrid chamber 41 after being assembled on the biochip. Touch. The first micro flow path is connected to the side of the 41, and the first micro flow path 44 is connected to the H pass π 43 . The first microfluid channel 44 has no limitation on the f/muth position and the position. Similarly, the n-pass σ 43 and the first micro-channel 44 and the stud 411 can be arranged according to the reaction solution. The spaces of the spaces are connected. Further, between the substrate 30 and the side walls of the two chambers 41, a gap 45 of a predetermined width is left, and the gap 45 is preferably between 0.05 and 〇 2 戸 戸 其中, wherein 0.1 mm is preferable. Please refer to the second A diagram and the second (four), the chamber 4, the circulation D43', and finally the system is formed on the biochip. The substrate substrate 2G m is the hybrid chamber 4

The flow channel 44 and the second flow D may be disposed on the lower substrate Lrt and are not limited to the upper substrate 40 or the corresponding substrate, the micro flow channel or the lower substrate 40 and the lower substrate 34 200808963 respectively. After the flow port is connected, the upper substrate 40 and the lower substrate 20 are connected to form a desired configuration. The production of the bio-wafer can be made by the above-mentioned conventional methods, but is not limited thereto. The material, shape and pore size of the substrate 3 can also be as described above, and are not particularly limited. Please refer to the second D diagram. This figure is a schematic diagram of the flow direction of the solution in the hybridization reaction of the second embodiment of the present invention. Before the hybridization is reversed, the Wei solution τ can be analyzed by the second art channel 4 on the left side, and the solution solution is flowed through the first microchannel 44 into the hybridization chamber 4 in the hybridization chamber 41, and the nucleic acid to be analyzed is analyzed. The solution τ - fills the entire gap 45, from the side of the base f 3 () into the matrix 3 〇 _, and on the other hand flows into the space between the small protrusions 411, and enters from the upper surface of the substrate 30 to the substrate 3 〇 The bottom flow is diffused, and finally discharged through the first microfluid 44 from the second flow port 43 on the right side and the first flow port 42. The silky 3 〇 is filamentous, and the lining analysis of the solution rapidly attracts the capillary phenomenon generated by the micropores of the matrix 30, and • quickly enters the pores inside the matrix 3 。. Thereafter, the nucleic acid to be analyzed may be immobilized on the exposed surface of the substrate and the inside thereof by means of drying or sensitization in the form of external light. Thereafter, the probe nucleic acid solution p is injected from the first flow port 42 to carry out a hybridization pairing reaction. The probe nucleic acid solution p after the addition, as in the flow path of the nucleic acid solution T to be analyzed in the first example, can cover the entire substrate 3〇 If the substrate is also in a dry state before being added to the probe nucleic acid solution p, the same needle The nucleic acid solution p quickly enters the inside of the 3G. After adding the probe ^ > Valley liquid P, the biochip is placed at an appropriate temperature (for example: 4〇~ suppression. C meaning) 15 200808963, the force is the clock, so that the plate nucleic acid is searched for the fine nucleic acid to be analyzed. Pairing can complete the base pairing hybridization reaction step. After the hybridization pairing reaction, the cleaning solution w can be injected from the second flow port 43 on the left side, and the cleaning solution w flows into the hybridization chamber 41 through the first microchannel 44, and flows from the side and the upper surface of the substrate 3〇 as described in the crucible. The substrate flows, such as the center and the bottom, and is finally discharged by the first-flow π 42 and the second flow port 43 on the right side. Since the cleaning solution w is washed by the entire substrate such as the side and the upper surface during cleaning, it is possible to more quickly and easily separate the smaller probe-only probe from the hole of the base f 3G, thereby reducing Its moon view noise value and shorten the time required for flushing. . The second A diagram, which is a schematic diagram of the stereoscopic decomposition of the third embodiment of the present invention. The biochip in the embodiment of the present invention comprises an upper substrate 50, a substrate 2 and a substrate 30 formed by the top substrate 201 and the bottom substrate 2〇2. The upper substrate 50 is vertically connected to the top substrate 201 and the bottom substrate 202, and the substrate 3G is mounted in the hybrid chamber 51 provided in the upper substrate 5 (). Please also refer to '三Α ϋ to the third c-figure, the upper substrate 5 (), which is provided with a hybrid chamber 51 having a disk-shaped cavity, and the hybrid chamber 51 is provided in a shape and size, which is not prepared by the Han Dynasty. It can be a quadrilateral hollow. The hybrid chamber 51 has a first flow port 52 in the mouth, and the number of the first flow port 52 and the position of the opening port are not particularly limited, and the reaction solution can be made according to the position of the other flow port. The flow path can be spread throughout the entire substrate. In addition, the first-flow π 52 can be connected to the micro-channel or the * port (the towel is not) to facilitate the injection of the solution or between the micro-fluids. 200808963 Configuration. The bottom edge of the upper substrate 50 protrudes from the hybridization chamber with a plurality of small protrusions 511 m;}: the main 511 end is disposed on the surface of the substrate 3 after being assembled in the biochip, Pick up. The side of the hybridization chamber 51 is connected to the second first to micro flow passages 54, and the first micro flow passages 54 are connected to the second flow passages 53 respectively. The number and position of the first microchannel 54 are not particularly limited, and the same can be configured according to the flow path of the reaction solution. The second flow port Μ and the first micro flow channel are connected to the space between the small protrusions 511. Further, the matrix 30 and the side walls of the dopants 51 are tied with a gap % of a predetermined width, and the gap 55 is between about 0.05 mm and 0.2 mm, with 〇 lmn^ being preferred. In the present embodiment, the lower substrate 20 is formed by superposing the top substrate 2〇1 and the base substrate 2〇2 on top of each other. The top substrate 2〇1 has a third flow port 2011 therethrough, and the bottom substrate 202 is provided with a third flow port 2021 corresponding to the third flow port 2〇11. The third flow port 2〇11 or 2〇21 may be disposed only in the lower substrate 20 of the single layer, or may be disposed in the double lower substrate 2 of the top substrate 2〇1 and the bottom substrate 202 as in the present embodiment. The third flow port 2021 can be connected to a second micro flow channel 2022, and the second micro flow channel 2022 can be connected to a fourth flow port 2023. Referring to FIGS. 3A and 3B, the hybridization chamber 51, the first microchannel 54 and the second flow port 53 are finally formed between the substrate 50 and the lower substrate 20 on the biochip. Therefore, the positions of the hybrid chamber 51, the first microchannel 54 and the second flow port 53 are not limited to the upper substrate 5, but may be disposed on the lower substrate 20' or the upper substrate 5 and the lower substrate, respectively. 17 200808963 A corresponding chamber, micro flow channel or flow port is formed on the plate 20, and then the upper substrate 50 = and the lower substrate 20 are connected to form the desired configuration. In the same situation, the third flow port, the second micro flow channel 2〇22 and the fourth flow port 2023 may be disposed only on the top substrate 201 or the bottom substrate 202, or the corresponding top substrate 2〇. 1. The bottom substrate is between 2 and 2. The production of the bio-wafer can be made by the above-mentioned conventional methods, but the material, the shape and the hole size of the base 30 are not limited thereto as described above, and are not particularly limited. _ Refer to the third D diagram, which is a schematic diagram of the flow direction of the solution in the hybridization reaction of the third embodiment of the present invention. Before the hybridization is reversed, the nucleic acid solution τ to be analyzed may be injected from the fourth flow through 2023, and the nucleic acid solution τ to be analyzed is introduced into the hybridization chamber 51 via the second microchannel 2022 and the third flow port 2021 and 2011. In the hybridization chamber 51, the nucleic acid solution τ to be analyzed enters the crotch portion of the matrix 30 from the bottom center point of the substrate 3, and flows to the outer edge of the substrate 30, some of which flow out from the space between the small studs 511, and finally concentrate. The ring is wound around the substrate 3 in the gap 55, and is discharged from the first to the micro flow passages 54 and the second flow port 53 and the first flow port 52 on the upper and lower sides, respectively. If the base λ 30 is dry, the τ of the nucleic acid solution to be analyzed is more rapidly attracted by the capillary phenomenon generated by the micropores of the matrix, and it is faster to enter the pores inside the matrix grandchild. Thereafter, the nucleic acid to be analyzed U can be set on the exposed surface of the base #3G and the inside thereof by means of dry mode or irradiation in the form of ultraviolet light. Thereafter, the probe nucleic acid solution p is injected from the first flow port 52 to carry out a chain-pairing reaction. The probe nucleic acid solution p after the addition, as in the flow path of the nucleic acid solution T to be analyzed in the first embodiment, can be spread over the entire matrix. 18 200808963 If the substrate is also in a dry state prior to the addition of the probe nucleic acid solution P, the probe nucleic acid solution P can also be quickly introduced into the interior of the matrix. After adding the probe nucleic acid solution, the biochip is placed at an appropriate temperature (for example, 40~48 〇c) for about several minutes to complete the base pairing by pairing the nucleic acids to be analyzed for the probe nucleic acid to be paired. Hybridization reaction step. After the mixed reaction of the parent, the cleaning solution can be injected into the fourth microchannels 2023 through the second microchannels 54, the third flow ports 2021 and 2011, and enter the hybridization chamber 51, as described above. The solution w may enter the interior of the substrate 3 from the center point of the bottom of the substrate 30 and flow to the outer edge of the substrate 3, some of which flow out of the space between the small protrusions 511, and finally concentrate on the gaps 55 around the sides of the substrate 30. The first microchannel 54 and the second flow port 53 and the first flow port 52 are respectively discharged from the upper and lower sides. Since the cleaning solution w is washed from the lower surface of the entire substrate 3 into the inner surface by the entire substrate 3, and through the outward diffusion and the space between the small protrusions 511, the probe nucleic acid of the smaller molecule can be easily used from the substrate. The 30 holes rush out, reducing the background noise value and shortening the time required for flushing. Please refer to FIG. 4A, which is a schematic diagram of the stereoscopic decomposition of the fourth embodiment of the present invention. The biochip in the embodiment of the present invention comprises an upper substrate, and a substrate 2 and a substrate 30 which are formed by the top substrate 201 and the bottom substrate 2〇2. The upper substrate 60 is superposed on the top substrate 2 and the bottom substrate 2〇2, and the shim 30 is mounted in the hybrid chamber 61 provided in the upper substrate 6(). Please refer to (4) four A ® to fourth c, the upper substrate 6 (), which is set to 200808963 with a disk chamber-shaped hybrid chamber 61, the size of the hybrid chamber 61 is set, and there is no special restriction. It can also be a quadrilateral hollow. The hybrid chamber 61 is provided with a first flow port 62. The number and opening position of the first flow port 62 are not particularly limited, and can be adjusted according to the position of the other flow port to make the reaction solution flow. The radial energy extends throughout the interior of the substrate 30. In addition, the first flow port 62 can be further connected to a micro flow channel or a flow port (not shown) to facilitate the injection of the solution or the arrangement between the micro flow channels. The first microfluid channel 64 is connected to the side of the m hybrid chamber 61, and the first microchannel 64 is connected to the second flow port 63. The number and position of the first microchannels 64 are not particularly limited, and can be adjusted according to the flow path of the reaction solution. Further, a gap 65 of a predetermined width is left between the substrate 3 and the side wall of the hybridization chamber 61, and the gap 65 is approximately between 〇5 and 〇.2 mm, wherein 〇·1 mm is preferable. In the present embodiment, the lower substrate 20 is formed by superposing the top substrate 2〇1 and the bottom substrate 2〇2. The top substrate 201 is provided with a third flow port. The bottom substrate 202 is provided with a second flow port 2021 corresponding to the third flow port 2〇11. The third flow port 2G11 or 2() 21 may be disposed only under the single layer lower substrate 2 〇 + or, as in the present embodiment, the double bottom substrate 2 〇 + disposed on the top substrate 2〇1 and the silk plate 202. The third flow channel can be connected to a second microchannel 2, and the second microchannel 2022 is connected to the fourth flow port 2023. Please continue to refer to the fourth A picture and the fourth B picture, the hybrid chamber 6 first micro flow channel 64 and the second flow port a, and the 63μ〆 encounter 63 is formed on the biochip 20 200808963, board 60 Between the lower substrate 20. Therefore, the position of the hybridization chamber 61, the first micro-stage 64 and the second flow port 63 is not limited to the upper substrate (9), and may be disposed on the lower substrate 20 or on the upper substrate 6 and the lower substrate 20, respectively. The corresponding substrate, micro flow channel or flow port is formed on the upper substrate, and then the upper substrate and the lower substrate 20 are connected to form the desired configuration. In the same state, the second flow port 2〇11, 2〇21, the second micro flow channel and the fourth hung channel 2〇23 may be disposed only on the top substrate 2〇1 or the bottom substrate, or Corresponding between the top substrate 201 and the base substrate 2〇2. • The production of money chips can be made using the above-mentioned conventional methods, but not limited to them. The material, shape and pore size of the substrate 30 can also be as described above, and are not particularly limited. Please refer to the fourth D diagram, which is a schematic diagram of the flow direction of the solution in the hybridization reaction of the fourth embodiment of the present invention. Before the hybridization is reversed, the nucleic acid solution τ to be analyzed may be injected from the fourth flow through 2023, and the nucleic acid solution to be analyzed passes through the second microchannel 2022, the third flow port 2021 and 2〇11 to enter the hybridization = 61. In the hybridization chamber 61, the nucleic acid solution to be analyzed enters the interior of the matrix 30 from the bottom of the matrix 3, and flows to the base f30: most concentrated in the gap 65 around the side of the substrate 30, respectively From the upper and lower sides, the first-micro flow channel 64 and the second flow port 63 and the first flow port are illusory. If the substrate 30 is dry, the nucleic acid solution to be analyzed is more rapidly attracted to the capillary phenomenon generated by the micropores of the matrix 30, and is introduced into the pores inside the matrix 30. Thereafter, the nucleic acid to be analyzed may be fixed to the substrate 3 () by using a drying method or by ultraviolet light = ",, the inch surface and the inside thereof. The surface of the table 21 200808963 about several minutes" causes the probe to be searched for the phase. After the nucleic acid to be analyzed is paired, the hybridization reaction step of base pairing can be completed. ★ After that, the probe nucleic acid solution P is injected into the flow port 62 to perform the heterogeneous pairing reaction. The probe nucleic acid solution p after the addition, as in the flow path of the nucleic acid solution T to be analyzed in the first embodiment, can be spread over the entire substrate %. If the substrate is also in a dry state before the addition of the reincarnation p, then the probe nucleic acid solution P quickly enters the interior of the base f 3Q. After adding the probe nucleic acid solution P, the biochip is placed at an appropriate temperature (for example, HC)

After the hybridization pairing reaction, the cleaning solution W can be injected from the fourth flow σ, and the cleaning solution is passed through the second microchannel 64, the third flow port 2〇21 and 201 into the human hybrid chamber 61, and the solution is washed as described above. w may enter the interior of the substrate 30 from the center point of the bottom of the substrate 30, and flow and diffuse toward the outer edge of the substrate 3, and finally concentrate in the gap 65 around the side of the substrate 30, and the first microchannels 64 and the upper and lower sides respectively The two flow ports 63 are discharged, and the portions are discharged from the first flow port 62. Since the cleaning solution is cleaned from the lower surface of the entire substrate 30 during cleaning, the probe nucleic acid of the smaller molecule can be easily flushed out of the pores of the matrix 30, thereby reducing the background noise value and shortening the flushing. Time required. According to the well-known P〇lymerase chain reaction (PCR), the pairing time of the primer and the nucleic acid to be analyzed can be completed in less than one minute. Therefore, under the structure of the present invention, after the probe nucleic acid enters or touches the substrate, it can diffuse completely inside or part of the surface in a relatively short period of time, and complete base pairing with the paired nucleic acid to be analyzed, thereby enabling Significantly shortened the familiar hybrid 22 200808963

It takes ten hours and is completed in a few minutes. Another - the steam + ^ lack of judging and the analysis of the marrow to be paired Xian # rapid, so =, ',,: acid in a very short mixed reaction time and not easy to adhere to the substrate can go - and smart wash 'liquid rinse When the 'cleaning solution' is washed into the interior of the matrix, it is easy to remove the mass-transformation and the molecular weight of the probe, and the hybridization reaction of the low background noise. In addition, the above-mentioned T, t material wafers are _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Injecting people, the rest of the circulation is out. BRIEF DESCRIPTION OF THE DRAWINGS FIG. A is a perspective exploded view of an embodiment of the present invention. The first B diagram is a schematic cross-sectional view of the embodiment of the present invention. The first C diagram is a schematic top view of the embodiment of the present invention after being assembled. The first D is an illustration of the flow of the solution in the hybridization reaction of the examples of the present invention. Figure 2A is a perspective exploded view of a second embodiment of the present invention. The second B is a schematic cross-sectional view of the second embodiment of the present invention. The second C is a schematic plan view of the second embodiment of the present invention after being assembled. The first D diagram is not intended to flow the solution in the hybridization reaction of the second embodiment of the present invention. The third A is a perspective exploded view of the third embodiment of the present invention. 23 200808963 The third B is a schematic cross-sectional view of the third embodiment of the present invention. The third C diagram is a schematic plan view of the third embodiment of the present invention after being assembled. The third D is a schematic view showing the flow direction of the solution in the hybridization reaction of the third embodiment of the present invention. Figure 4A is a perspective exploded view of a fourth embodiment of the present invention. The fourth B is a schematic cross-sectional view of the fourth embodiment of the present invention. The fourth C is a schematic plan view of the fourth embodiment of the present invention after being assembled. The fourth D diagram is a schematic diagram showing the flow direction of the solution in the hybridization reaction of the fourth embodiment of the present invention. [Main component symbol description] 10 Upper substrate 11 Hybridization chamber 12 First flow port 13 Second flow port 14 First micro flow path 15 Void 20 Lower substrate 201 Top substrate 2011 Third flow port 202 Base substrate 2021 Third flow port 2022 Second micro flow channel 2023 fourth flow port 30 substrate 40 upper substrate 41 hybridization chamber 411 small stud 24 200808963 42 first flow port 43 44 first micro flow channel 45 50 upper substrate 51 hybridization chamber 511 52 first flow port 53 54 first microchannel 55 60 upper substrate 61 hybridization chamber 62 63 second flow port 64 65 gap T nucleic acid solution to be analyzed P probe nucleic acid solution W cleaning solution second flow port gap small stud second flow port gap first Flow port first micro flow channel 25

Claims (1)

200808963 X. Patent application scope: 1. A biochip comprising: a hybrid chamber in a cavity shape in the biochip, wherein the hybrid chamber is provided with a matrix of holes, and the hybrid chambers are respectively connected with at least one a first flow port and at least one second flow port, wherein the reaction solution is injected from at least one of the flow ports and then flows through the interior of the substrate to flow from the remaining at least one flow port. 2, as described in claim 1 a biochip comprising an upper substrate and a lower substrate, wherein the upper substrate and the lower substrate are vertically connected to each other; the hybridization chamber is formed therebetween, and the substrate is provided with a hole in the hybrid chamber. The hybridization chamber is connected to the upper substrate with at least one of the first flow ports. The hybridization chamber is connected to at least one of the second flow ports at the side, and the biochip according to claim 2, wherein A gap of a predetermined width is anchored between the substrate and one of the sidewalls of the hybridization chamber. 10 4. If f is the organism mentioned in the second item, the hybrid chamber is in the shape of a round helmet, and the first circulation port is connected above the center of the hybrid chamber. 5. The biochip according to claim 2, wherein the hybrid chamber is a quadrangular cavity, and the first flow port is connected above a center point of the hybrid chamber. The biochip according to the second aspect of the invention, wherein the second flow port (four) is connected to the first microfluidic channel in a stepwise communication manner. The biochip of claim 2, wherein the substrate has a hole diameter of 0.1 to 50 μm. 8. The biochip of claim 2, wherein the substrate is in a dry state. 9. The biochip of claim 2, wherein the substrate is a nylon membrane. The biochip according to the second or seventh aspect of the patent application, wherein the substrate is a nitrulized fiber paper (nitr〇cellul〇se). 11. The biochip of claim 2, wherein the hybrid chamber further communicates with the lower substrate with at least one third flow port. 12. The biochip of claim n, wherein the lower substrate is further formed by a top substrate and a bottom substrate stacked one above another, wherein the third flow port is formed on the top substrate. Between the base substrate and the base substrate. 13. The biochip according to claim 12 or 12, wherein the first f flow port is further connected with a second micro flow channel, and the second micro flow channel is connected to have a fourth Circulation. 14. The organism of claim 2, wherein the first flow port is connected to the hybrid-step-connected third micro flow channel. 15. A biochip comprising: The substrate and the -T substrate 'the upper substrate_lower substrate are superposed on each other, and a hybridization chamber is formed therebetween. The hybrid chamber is provided with a substrate of holes 27 200808963, and the bottom edge of the upper substrate is And a plurality of small protrusions protruding from the hybrid chamber, wherein the small protrusion ends are in contact with the surface of the substrate disposed in the hybrid chamber, and further, the hybrid chamber is connected to the upper substrate by at least one The first flow port has at least one second flow port connected to the side of the hybrid chamber, and the second flow port is in communication with a space between the small protrusions. The biochip of claim 15, wherein the substrate and the sidewall of one of the hybrid chambers are each provided with a gap of a predetermined width. 7. The biochip of claim 15, wherein the second flow port and the hybridization chamber are in communication with the first-microchannel. 18. The biochip of claim 15, wherein the hybridization chamber is further connected to the lower substrate with at least a third flow port. 19. The biochip according to claim 18, wherein the lower substrate is stepped by a top substrate and a bottom substrate, and the fifth channel is formed. Between the top substrate and the base substrate. The biochip according to claim 18 or 19, wherein the third flow port is further connected to a second micro flow channel, and the second micro flow channel is connected and provided with a gift π. . The biological wafer of claim 18 or 19, wherein the heterogeneous system is in the form of a cavity, and the third flow port is connected to the lower side of the center of the intersection. The biochip of claim 18 or 19, wherein the hybrid chamber is in the shape of a quadrilateral cavity, and the third flow port is connected below the center of the hybrid chamber. The biochip according to claim 15, wherein a third microchannel is further connected between the first flow port and the hybrid chamber. 24. According to claim 15 The biochip, wherein the substrate has a pore diameter of 0.1 to 50 μm. 25. The biochip according to claim 15 or claim 24, wherein the substrate is in a dry form. 26, as claimed in claim 15 or 24. The biochip of the present invention, wherein the substrate is a nylon film. The biochip of claim 15 or claim 24, wherein the substrate is a nitrocellulose paper.