Biochip manufacturing method, product thereof, and detection method using the same

Abstract

The present application provides a method for manufacturing a biochip, including: manufacturing a substrate; forming a plurality of observation holes penetrating the substrate; installing a plurality of fixing members on the bottom surface of the substrate surrounding each of the plurality of observation holes; attaching a plurality of reaction sheets to the bottom surface corresponding to each of the plurality of observation holes; and fixing the plurality of reaction sheets with the plurality of fixing members. The present application also provides a biochip manufactured by the above method and a method for detecting a sample using the biochip.

Description

[Technical Field]

[0001] The present disclosure relates to a method for manufacturing a biochip, specifically, to a method for manufacturing a biochip in which a plurality of types of reaction sheets are attached to a substrate and fixed with a fixing member of the substrate. [Background technology]

[0002] Biochips originated in the 1980s as various electronic industries miniaturized their products, while related industries such as life sciences and bioinformatics recognized the advances that could be made by miniaturizing biochemical assays.

[0003] In a broad sense, a biochip refers to a device that utilizes principles of molecular biology, analytical chemistry, biochemical reactions, etc. to miniaturize reaction components for analyzing samples and immobilize them on a substrate made of glass, silicon, plastic, etc. The objects to be analyzed may be genes, proteins, cell tissues, etc. By miniaturizing the reaction components, the accuracy, speed, and completeness of the analysis results can be improved, and the amounts of sample and reagents required are far smaller than those of conventional analysis methods.

[0004] Specifically, biochips can be broadly divided into two types: microarrays, which have a high density of bioprobes arranged on a small area of ​​the chip, and microprocessing chips, which process biological samples and perform reaction analysis on the chip. Microarray manufacturing methods can be divided into (1) on-chip synthesis, in which probes are synthesized directly on a support, and (2) spotting, in which pre-synthesized probes are placed on a support. On-chip synthesis is used to produce high-density DNA chips, but its cost is high, its efficiency is limited, and its range of applications is relatively limited. In comparison, spotting is relatively inexpensive and has a broad range of applications, making it suitable for use in academic organizations or general companies.

[0005] However, currently available biochips have a relatively high density, are difficult to manufacture, and are relatively expensive, making their widespread use difficult. Furthermore, specialized equipment is required for their storage, use, and interpretation. Therefore, there is a need for a biochip and its manufacturing method that can improve the accuracy, speed, and completeness of analytical results, require less sample and reagent amounts than conventional analytical methods, and are also low-cost. Summary of the Invention

[0006] To achieve the above object, the present application provides a method for manufacturing a biochip, including manufacturing a substrate, forming a plurality of observation holes penetrating the substrate, installing a plurality of fixing members on the bottom surface of the substrate surrounding each of the plurality of observation holes, attaching a plurality of reaction sheets to the bottom surface in correspondence with the plurality of observation holes, and fixing the plurality of reaction sheets with the fixing members.

[0007] Preferably, the method further comprises deforming the plurality of fixing members to fix the plurality of reaction sheets.

[0008] Preferably, the deformation is carried out by heating and melting the plurality of fixing members.

[0009] Preferably, the method further comprises placing the reaction components on a reaction mother sheet and cutting the reaction mother sheet into a plurality of reaction sheets.

[0010] Preferably, each of the reaction sheets contains two or more reaction components.

[0011] Preferably, the minimum distance between the centres of each of the reaction sheets is 1 to 10 mm.

[0012] Preferably, the fixing member is an L-shaped pillar, a cylindrical body, a polygonal pillar, a cone, a cylinder, a sphere, a polyhedron, a ring, or a combination thereof.

[0013] To achieve another object of the present application, a biochip is provided, which is manufactured by the above-mentioned method for manufacturing a biochip.

[0014] Preferably, each of the observation holes is surrounded by at least one corresponding fixing member.

[0015] Preferably, the substrate is made of silicon, glass, a polymeric material or ceramic.

[0016] Preferably, the fixing member is made of plastic, silica gel or rubber.

[0017] To achieve another object of the present application, there is provided a method for detecting a sample using the aforementioned biochip, which comprises adding the sample to the reaction sheet through the observation holes respectively, and observing the result through the observation holes.

[0018] Due to the above technical features, the biochip manufacturing method provided by the present application can produce biochips at low cost and with simple manufacturing methods. In addition, by using pre-cut reaction sheets and fixing members, different types of reaction sheets can be arranged on a single substrate, and different reaction components can be combined according to different needs. This further reduces manufacturing costs and makes the use of biochips more flexible.

[0019] In the following detailed description, for purposes of explaining the present application, numerous specific details are provided to provide a thorough understanding of the disclosed embodiments. However, it will be apparent that one or more embodiments may be practiced without the specific details. In other instances, known structures and processes are generally shown to simplify the drawings. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a flow chart of a method for manufacturing a biochip according to an embodiment of the present application. [Figure 2]FIG. 2 is a schematic diagram of a substrate used in the method for producing a biochip according to the first embodiment of the present application. [Figure 3] FIG. 3 is a schematic diagram showing an observation hole formed in a substrate in a biochip manufacturing method according to a first embodiment of the present application. [Figure 4] FIG. 4 is a perspective view of a biochip including a cone-shaped fixing member according to a first embodiment of the present application. [Figure 5] FIG. 5 is a schematic side view based on a cross section taken along line AA' of the substrate in FIG. [Figure 6] FIG. 6 is a schematic side view of a cross section of a substrate with a reaction sheet attached thereto in a method for producing a biochip according to a first embodiment of the present application. [Figure 7] FIG. 7 is a schematic diagram showing how the reaction sheet is further fixed by dissolving the fixing member in the method for producing a biochip according to the first embodiment of the present application. [Figure 8] FIG. 8 is a perspective view of a biochip including mushroom-shaped fixation members according to a second embodiment of the present application. [Figure 9] FIG. 9 is a schematic side view based on a cross section taken along the cutting line BB' of the substrate in FIG. [Figure 10] FIG. 10 is a schematic side view of a cross section of a substrate with a reaction sheet attached thereto in a method for producing a biochip according to a second embodiment of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following detailed description of the embodiments is provided with reference to the accompanying drawings. However, these embodiments may be realized in various forms and are not the only forms for implementing or utilizing the specific embodiments of the present application, and therefore should not be construed as limiting the above examples. The embodiments encompass features of multiple specific embodiments, as well as steps and sequences of methods for constructing and operating these specific embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent functions and sequences of steps. On the contrary, these examples are provided to thoroughly and completely disclose the present specification and fully and completely convey the gist of the present application to those skilled in the art to which the present application pertains. Similar element reference numerals in the figures refer to similar elements. In the following description, known functions or structures will not be described in detail, and unnecessary details in the embodiments will not be described.

[0022] Unless otherwise defined, all technical and specialized terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In the case of conflict, the present specification, including definitions, will control.

[0023] Where not contradicted by the context, as used herein, the singular term "a" or "an" includes the plural of that term and the plural term "a" or "an" includes the singular. In addition, as used herein and in the claims, the expressions "at least one" and "one or more" have the same meaning and both refer to the inclusion of one, two, three, or more.

[0024] Although the numerical ranges and parameters used to define the broad scope of this application are all approximations, the relevant numerical values ​​in the specific examples are presented as precisely as possible. However, all numerical values ​​inherently contain standard deviations resulting from individual measurement methods. Herein, "about" typically means that the actual numerical value is within plus or minus 10%, 5%, 1%, or 0.5% of the specified numerical value or range. Alternatively, the term "about" indicates that the actual numerical value is within an acceptable standard error of the mean, as determined by one skilled in the art to which this application pertains. Except in the examples, or unless otherwise expressly stated, all ranges, quantities, values, and percentages used in this specification (e.g., used to describe amounts of materials, lengths of time, temperatures, operating conditions, ratios of quantities, and other similar terms) should be understood to be modified by "about." Therefore, unless otherwise stated to the contrary, all numerical values ​​and parameters disclosed in this specification and claims are approximations and may vary as needed. At a minimum, these numerical values ​​and parameters should be understood to be numerical values ​​obtained by directly using the number of significant digits shown and conventional carry techniques. Numerical ranges are expressed herein as ranging from one endpoint to another endpoint, or between two endpoints. Unless otherwise specified, all numerical ranges described herein include the endpoints.

[0025] Please refer first to Figure 1. Figure 1 is a flow chart of a method for manufacturing a biochip according to an embodiment of the present application.

[0026] The embodiments according to the present application provide a method for manufacturing a biochip. S1: A process for manufacturing a substrate; S2: forming a plurality of observation holes in the substrate, the observation holes penetrating the substrate; S3: installing a plurality of fixing members on the bottom surface of the substrate so as to surround each of the plurality of observation holes; S4: A step of attaching a plurality of reaction sheets to the bottom surface side so as to correspond to the plurality of observation holes, respectively; S5: fixing the reaction sheets with the fixing members; Includes:

[0027] Another embodiment of the present application further includes a step of melting the fixing member after attaching multiple reaction sheets to the bottom surface of the substrate to more stably fix the reaction sheets to the bottom surface of the substrate. The method of melting the fixing member is not limited to, but includes heating, and any method that can deform the fixing member and prevent the reaction sheets from falling off can be used. Specifically, for example, a metal piece can be heated and then pressed flat with the fixing member. In another embodiment of the present application, after attaching the reaction sheets to the bottom surface of the substrate, they can be further fixed with resin or adhesive to improve the robustness or sealing of the reaction sheets.

[0028] In an embodiment of the present application, the method for manufacturing a biochip can further include placing reaction components (e.g., bioprobes) on a reaction mother sheet and cutting it into multiple reaction sheets. The material of the reaction mother sheet can be paper or plastic, preferably plastic, which is elastic and easy to attach. Methods for placing reaction components on the reaction mother sheet can include a photomask method, a pin method, an inkjet method, and a piezo method. A suitable method can be used to place the reaction components based on different needs. Methods for cutting the reaction mother sheet can include cutting with a template cutter or laser cutting, and can be adjusted based on different types of bioprobes required.

[0029] In an embodiment of the present application, the reaction sheet may be circular, square, rectangular, or hexagonal, and may be arbitrarily adjusted based on needs, with a circular shape being preferred. In an embodiment of the present application, the reaction sheet may be circular with a diameter of 1 to 3 mm, preferably 1.5 mm. In another embodiment of the present application, the reaction sheet may be square with a side length of 1 to 3 mm, preferably 1.5 mm.

[0030] In an embodiment according to the present application, the fixing member may be an L-shaped pillar, a cylindrical body, a polygonal pillar, a cone, a cylinder, a sphere, a polyhedron, a ring, or a combination thereof. Preferably, the fixing member may be a cone. In another embodiment according to the present application, the portion of the fixing member that contacts the bottom surface of the substrate may be a cylinder or a polygonal pillar, and the portion that does not contact the substrate may be a sphere or a polyhedron.

[0031] In the examples of the present application, the reaction components may be, but are not limited to, protein probes, such as antigens or antibodies, or nucleic acid probes, such as DNA or RNA probes. Those skilled in the art can arrange the necessary or desired reaction components based on their common knowledge in the art. By arranging different reaction components (reaction sheets) on a single biochip, different steps of measurement of the same or multiple samples can be integrated on the same biochip, making it more convenient for users to set up the reactions they want to perform on a single biochip and observe the results.

[0032] A biochip can be manufactured using the above-described biochip manufacturing method. In an embodiment of the present application, the observation hole of the biochip can be surrounded by at least one fixing member, preferably four fixing members. In addition, the substrate of the biochip can be made of silicon, glass, a polymer material, or ceramic, preferably a polymer material, such as plastic. In an embodiment of the present application, the fixing member can be made of plastic, silica gel, or rubber. Specifically, any material that is easy to mold and can be attached to a substrate can be used as the fixing member, preferably plastic. In another embodiment of the present application, after the reaction sheet is attached to the fixing member, the fixing member is deformed to further fix the reaction sheet. For example, the fixing member can be deformed by heating and melting, or by applying pressure.

[0033] An embodiment of the present application provides a method for detecting a sample using the biochip prepared by the above-described method, which comprises dropping a sample into an observation hole on the opposite side of the bottom surface where the immobilizing member is installed, bringing the sample into contact with the reaction sheet to react, and observing the result through the observation hole with the naked eye or an instrument.

[0034] Hereinafter, the biochip manufacturing method, biochip and sample detection method provided by the present application will be described in detail with reference to specific examples and drawings.

[0035] Please refer to Figures 2 to 6. Figure 2 is a schematic diagram of a substrate used in the biochip manufacturing method according to the first embodiment of the present application. Figure 3 is a schematic diagram of an observation hole formed in the substrate in the biochip manufacturing method according to the first embodiment of the present application. Figure 4 is a perspective view of a biochip including a conical fixing member according to the first embodiment of the present application. Figure 5 is a schematic side view based on a cross section taken along the cutting line A-A' of the substrate in Figure 4. Figure 6 is a schematic side view of a cross section in which a reaction sheet is attached to a substrate in the biochip manufacturing method according to the first embodiment of the present application. Figure 7 is a schematic diagram of a biochip manufacturing method according to the first embodiment of the present application in which the fixing member is dissolved to further fix the reaction sheet.

[0036] A first example based on the present application includes preparing a substrate 1 as shown in Figure 2. In this example, the substrate 1 is formed using plastic. Then, as shown in Figure 3, a plurality of observation holes 2 are formed in the substrate 1, penetrating the substrate 1. In this example, the diameter of the observation holes 2 is 1.1 mm, and the distance between two adjacent observation holes is 1.54 mm.

[0037] Please refer to Figure 4. The bottom of Figure 4 is the surface of the substrate 1, and the top is the bottom of the substrate 1. In the first embodiment, a plurality of conical fixing members 3 are formed on the bottom side of the substrate 1, and the fixing members 3 are made of plastic. Furthermore, when a sample is dropped, it is added from the surface of the biochip, so to facilitate sample addition, the cross section of the observation hole 2 is narrow at the top and wide at the bottom, as shown in Figure 4. Furthermore, this arrangement prevents the reaction sheet 4 from falling into the observation hole 2 when it is attached. In this embodiment, the inner diameter (bottom side) of the observation hole 2 is 1.1 mm, the outer diameter (top side) is 1.2 mm, the center-to-center distance between the observation holes 2 in the same row or column is 1.54 mm, and the center-to-center distance between the conical fixing members 3 in the same row or column is 1.54 mm. The substrate 1 with the conical fixing members 3 installed is shown in Figure 5.

[0038] Next, refer to Figure 6. A reaction sheet 4 is attached to the bottom surface of the substrate 1. In this example, the reaction sheet 4 is a circular thin piece with a diameter of 1.4 mm, made of a plastic sheet, and the reaction component placed on top is a protein probe. After the reaction sheet 4 is attached, the tip of the conical fixing member 3 made of plastic is melted and flattened with an iron, resulting in a fixing portion 5 as shown in Figure 7. The position of the reaction sheet 4 is fixed between the four conical fixing members 3 and between the fixing portion 5 and the substrate 1.

[0039] A second embodiment of the present application will be described below with reference to Figs. 8 to 10. Fig. 8 is a perspective view of a biochip including a mushroom-shaped fixing member according to the second embodiment of the present application. Fig. 9 is a schematic side view of a cross section taken along the cutting line B-B' of the substrate in Fig. 8. Fig. 10 is a schematic side view of a cross section in which a reaction sheet is attached to a substrate in a method for manufacturing a biochip according to the second embodiment of the present application.

[0040] The biochip manufacturing method in the second embodiment of the present application does not share the same features as the first embodiment. The only difference is that the fixing members 3a are mushroom-shaped, as shown in Figures 8 and 9. The mushroom-shaped fixing members 3a allow the reaction sheet 4 to be more easily inserted and fixed therein. In the second embodiment, the reaction sheet 4 is made of a slightly elastic plastic sheet. The biochip after the reaction sheet 4 is attached is shown in Figure 10.

[0041] In the third embodiment of the present application, the structures of the substrate, observation hole, and fixing member are the same as those in the first and second embodiments. The difference is that in the third embodiment, bioprobes are placed on a paper-made reaction mother sheet using an inkjet method, and then the reaction mother sheet is cut into circles with a diameter of 1.4 mm using a laser to obtain the reaction sheet 4. The back side of the reaction sheet 4 without the bioprobes is then suction-attached using a mounting device, and the reaction sheet is fixed between the fixing members 3 and 3a. Different types of reaction sheets 4 can be attached to the same biochip based on experimental needs, achieving cost savings. In this embodiment of the present application, the fixing member 3 is flattened to form the fixing member 5, which fits snugly together, and the reaction sheet 4 is sealed between the fixing member 5 and the substrate 1. When observing results through the observation hole 2, an external light source from the surface of the substrate 1 can assist in observing the results. In another embodiment according to the present application, if the fixing members 3 do not fit together tightly after being flattened to create the fixing portions 5, the observation can be aided by adding a light source to the bottom surface of the substrate 1 when observing the results through the observation hole 2.

[0042] The fourth embodiment of the present application provides a method for detecting a sample using the biochip obtained above. In this embodiment, the biochip obtained in the previous embodiment is placed face up, and a sample is added to the biochip manually or automatically. After the sample and bioprobe react, the results are read visually or by instrument.

[0043] Due to the above technical features, the biochip manufacturing method of the present application allows the reaction sheet to be fixed using a simple fixing member, and during manufacturing, the reaction sheet can be attached (positioned) by adsorbing its back surface, preventing damage to the bioprobes that can occur during conventional manufacturing processes when the surface is adsorbed. The resulting biochip is inexpensive, and reaction sheets with different reaction components can be attached as needed, further reducing costs. For example, two or more reaction sheets with different bioprobes (and reaction components) can be prepared and placed on the same biochip. The detection method using the biochip disclosed in the present application is easy to operate, the experimental process is easy to interpret, and it is easier to use than known detection methods. In addition, the dimensions of the biochip of the present application are convenient, so only a portion of the reaction sheets can be used as needed, thereby saving costs.

[0044] The foregoing description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations made without departing from the spirit and scope of this application should be included in the scope defined by the claims. [Explanation of symbols]

[0045] 1 board 2 Observation hole 3 Fixing member 3a Fixing member 4 Reaction Sheet 5 Fixed part S1 process S2 process S3 process S4 process S5 process

Claims

1. Fabricating a substrate; creating a plurality of observation holes in the substrate that extend through the substrate; installing a plurality of fixing members on the bottom surface of the substrate so as to surround each of the plurality of observation holes; attaching a plurality of reaction sheets to the bottom surface side so as to correspond to the plurality of observation holes, respectively; fixing the reaction sheets with the fixing members, respectively; characterized by: A method for manufacturing a biochip.

2. The method further comprises deforming the plurality of fixing members to fix the plurality of reaction sheets. The method of claim 1.

3. The deformation is performed by heating and melting the plurality of fixing members. The method of claim 2.

4. The method further comprises disposing reaction components on a reaction mother sheet and cutting the reaction mother sheet into a plurality of reaction sheets. The method of claim 1.

5. Each of the reaction sheets attached to the bottom surface contains a different reaction component. The method of claim 1.

6. The minimum distance between the centers of each of the reaction sheets is 1 to 10 mm. The method of claim 5.

7. The fixing member is an L-shaped pillar, a cylindrical body, a polygonal pillar, a cone, a cylindrical body, a sphere, a polyhedron, a ring, or a combination thereof. The method of claim 1.

8. A biochip manufactured by the method for manufacturing a biochip according to any one of claims 1 to 7.

9. Each of the observation holes is surrounded by at least one corresponding fixing member. The biochip of claim 8.

10. The substrate is made of silicon, glass, polymer material or ceramic. The biochip of claim 8.

11. The fixing member is made of plastic, silica gel or rubber. The biochip of claim 8.

12. adding a sample to the reaction sheet through an observation hole; observing the results from the observation hole; characterized by: A method for detecting a sample using a biochip according to any one of claims 8 to 11.

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