TWI841195B - Manufacturing method of biochip, product thereof, and detection method using the same - Google Patents

Abstract

The present disclosure provides a method for manufacturing a biochip, including manufacturing a substrate; generating a plurality of observation holes through the substrate; arranging a plurality of fixing parts on the bottom surface of the substrate around each of the plurality of observation holes; arranging a plurality of reaction sheets corresponding to the plurality of observation holes on the side of the bottom surface; and the plurality of reaction sheets are fixed by the plurality of fixing parts. At the same time, the present disclosure also provides a biological chip made by the aforementioned method and a sample detection method using the biochip.

Description

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

The present disclosure relates to a method for manufacturing a biochip, and more specifically, to a method for manufacturing a biochip in which a plurality of reaction sheets are arranged on a substrate and the reaction sheets are fixed by a fixing member on the substrate.

Biochips originated in the 1980s. While various electronic industries were miniaturizing their products, related industries such as life sciences and bioinformatics also realized the progress that could be brought about by miniaturizing biochemical analysis.

In a broad sense, biochip refers to the use of molecular biology, analytical chemistry, biochemical reactions and other principles to miniaturize the reaction elements used to analyze samples and fix them on a substrate made of glass, silicon or plastic to form a biochip. The objects of analysis can be genes, proteins, cell tissues, etc. By miniaturizing the reaction elements, the accuracy, speed and integrity of the analysis results can be improved, and the amount of samples and reagents required are much smaller than traditional analysis methods.

Specifically, biochips can be divided into two categories: microarray chips that grow high-density biological probes on a small area of the chip, and microprocessor chips that process biological samples and perform reaction analysis on the chip. Among them, the production methods of microarray chips are divided into: (1) in situ synthesis method, that is, direct synthesis on a carrier; and (2) placement method, that is, placing pre-synthesized probes on a carrier. The in situ synthesis method is suitable for the production of high-density DNA chips, but it is expensive and has limited efficiency, and its application range is relatively limited. In comparison, the placement method is relatively inexpensive and has a wider application range, suitable for use by academic institutions or general enterprises.

However, the currently used biochips have a high density, and are difficult and costly to manufacture, making them difficult to popularize. In addition, their preservation, use, and interpretation also require special instruments. Therefore, a biochip and its manufacturing method that can improve the accuracy, speed, and integrity of the analysis results, require less sample and reagent than traditional analysis methods, and take into account low costs is needed.

To achieve the above-mentioned purpose, the present invention provides a method for manufacturing a biochip, comprising: manufacturing a substrate; generating a plurality of observation holes penetrating the substrate on the substrate; arranging a plurality of fixing members on a bottom surface of the substrate around each of the plurality of observation holes; arranging a plurality of reaction plates on one side of the bottom surface respectively corresponding to the plurality of observation holes; and fixing the plurality of reaction plates by the plurality of fixing members.

Preferably, the method further comprises deforming the plurality of fixing elements to fix the plurality of reaction sheets.

Preferably, the deformation is performed by melting the plurality of fixing elements by heating.

Preferably, it further comprises arranging reaction elements on a reaction master sheet, and cutting the reaction master sheet into a plurality of reaction sheets.

Preferably, each of the reaction sheets comprises two or more reaction elements.

Preferably, the minimum distance between the centers of the reaction plates is 1 to 10 mm.

Preferably, the fixing member is in the shape of an L-shaped column, a cylinder, a polygonal column, a cone, a cylinder, a sphere, a polyhedron, a ring or a combination thereof.

In order to achieve another purpose of the present invention, a biochip is provided, which is manufactured using the aforementioned biochip manufacturing method.

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

Preferably, the substrate is made of silicon, glass, polymer material or ceramic.

Preferably, the fixing member is made of plastic, silicone or rubber.

To achieve another object of the present invention, a method for detecting a sample using the aforementioned biochip is provided, comprising: adding a sample to a reaction chip through an observation hole; and observing the result from the observation hole.

By virtue of the above technical features, the biochip manufacturing method provided by the present invention can manufacture biochips with low cost and simple manufacturing method. In addition, through the pre-cut reaction pieces and fixing parts, different types of reaction pieces can be arranged on a single substrate, and different reaction element combinations can be provided based on different needs, thereby further saving manufacturing costs and the flexibility of the use of biochips.

The following is a detailed description of the embodiments with reference to the relevant drawings. However, these embodiments can be implemented in different forms, but this is not the only form of implementing or applying the specific embodiments of the invention claimed in this case, and should not be understood as a limitation on the above-mentioned embodiments. The implementation method covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences. On the contrary, these embodiments are provided so that this specification can be thoroughly and completely disclosed, so as to fully express the spirit of the invention claimed in this case to those with ordinary knowledge in the technical field to which the invention belongs. Similar component symbols in the drawings refer to similar components. In the following description, well-known functions or structures will not be described in detail to avoid unnecessary details of the embodiments.

Unless otherwise defined, all technical terms and terminology used herein have the same meaning as commonly understood by those with ordinary knowledge in the art to which the present invention belongs. In the event of a conflict, this specification including the definitions shall prevail.

In the absence of a conflict with the context, a singular term used in this specification includes the plural form of the term; and a plural term used in this specification also includes the singular form of the term. In addition, in this specification and the scope of the patent application, the expressions "at least one" and "one or more" have the same meaning, both of which represent one, two, three or more.

Although the numerical ranges and parameters used to define the broader scope of the claimed invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person of ordinary skill in the art to which the invention belongs. Except for the embodiments, or unless otherwise expressly stated, it is understood that all ranges, quantities, values and percentages used herein (for example, to describe material usage, time duration, temperature, operating conditions, quantity ratios and the like) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the patent application are approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated number of significant digits and the values obtained by applying the general rounding method. Herein, the numerical range is expressed from one end point to another or between two end points; unless otherwise stated, the numerical range described herein includes the end points.

First, please refer to FIG. 1 , which is a flow chart of a biochip manufacturing method according to an embodiment of the present invention.

According to an embodiment of the present invention, a biochip manufacturing method is provided, comprising: S1: a step of manufacturing a substrate, S2: a step of generating a plurality of observation holes penetrating the substrate on the substrate, S3: a step of setting a plurality of fixing members on a bottom surface of the substrate around each of the plurality of observation holes, S4: a step of setting a plurality of reaction sheets on one side of the bottom surface corresponding to the plurality of observation holes, and S5: a step of fixing the plurality of reaction sheets by the plurality of fixing members.

According to another embodiment of the present invention, after a plurality of reaction sheets are disposed on the bottom surface of the substrate, the step of melting the fixing member is further included to fix the reaction sheets more stably on the bottom surface of the substrate. The method of melting the fixing member includes but is not limited to heating, and any method that can deform the fixing member and prevent the reaction sheets from falling off can be used. Specifically, for example, the metal sheet can be heated and then pressed flat to flatten the fixing member. According to another embodiment of the present invention, after the reaction sheet is disposed on the bottom surface of the substrate, it can be further fixed with resin or glue to increase the firmness or sealing of the reaction sheet.

According to one embodiment of the present invention, the method for manufacturing a biochip may further include the steps of placing a reaction element (such as a biological probe) on a reaction master and cutting it into a plurality of reaction chips. The material of the reaction master may be paper or plastic, preferably plastic, which is elastic and easy to place. The method of placing the reaction element on the reaction master may include a mask type, a pinpoint type, an inkjet type, and a piezoelectric type, and the reaction element may be placed in an appropriate manner according to different requirements. The method of cutting the reaction master may include model tool cutting or laser cutting, etc., which can be adjusted according to the different types of biological probes required.

According to one embodiment of the present invention, the reaction sheet can be round, square, rectangular or hexagonal, and can be adjusted arbitrarily according to needs, preferably round. According to one embodiment of the present invention, the reaction sheet can be round with a diameter of 1 to 3 mm, preferably round with a diameter of 1.5 mm. According to another embodiment of the present invention, the reaction sheet can be a square with a side length of 1 to 3 mm, preferably a square with a side length of 1.5 mm.

According to one embodiment of the present invention, the fixing member may be an L-shaped column, a cylinder, a polygonal column, a cone, a cylinder, a sphere, a polyhedron, a ring or a combination thereof. Preferably, the fixing member may be a cone. According to another embodiment of the present invention, the fixing member may be a cylinder or a polygonal column at a portion in contact with the bottom surface of the substrate, and may be a sphere or a polyhedron at a portion not in contact with the substrate.

According to one embodiment of the present invention, the reaction element may be a protein probe, such as an antigen, an antibody, or a nucleic acid probe, such as a DNA probe or an RNA probe, but is not limited thereto. A person having ordinary knowledge in the technical field to which the present invention belongs may set the reaction element required or intended for use according to the ordinary knowledge in the technical field to which the present invention belongs. By setting different reaction elements (reaction chips) on a single biochip, the tests of the same or multiple samples at different stages can be integrated on the same biochip, making it easier for users to customize the reactions they want to perform on a single biochip and observe the results.

The biochip can be manufactured by the above-mentioned biochip manufacturing method. According to one embodiment of the present invention, the observation hole on the biochip can be surrounded by at least one fixing member, preferably, can be surrounded by four fixing members. On the other hand, the substrate of the biochip can be made of silicon, glass, polymer material or ceramic, preferably made of polymer material, such as plastic. According to one embodiment of the present invention, the fixing member can be made of plastic, silicone or rubber. Specifically, any material that is easy to shape and can be set on the substrate can be suitable for the fixing member, preferably plastic. According to another embodiment of the present invention, after the reaction sheet is set on the fixing member, the fixing member is deformed to further fix the reaction sheet, for example, the fixing member is heated and melted to cause it to deform, or it is deformed by extrusion.

According to one embodiment of the present invention, a method for testing a biochip sample manufactured using the aforementioned method is provided, comprising dripping the sample into an observation hole from the opposite side of the bottom surface where a fixing member is provided, so that the sample contacts the reaction sheet for reaction, and observing the result from the observation hole by naked eyes or an instrument.

The biochip manufacturing method, biochip and sample detection method provided by the present invention are described in detail below based on specific embodiments and drawings.

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 invention. Figure 3 is a schematic diagram of an observation hole generated on a substrate in the biochip manufacturing method according to the first embodiment of the present invention. Figure 4 is an oblique view of a biochip including a conical fixing member according to the first embodiment of the present invention. Figure 5 is a side view schematic diagram of a cross-section along the tangent line A-A’ of the substrate according to Figure 4. Figure 6 is a side view schematic diagram of a cross-section of a reaction plate arranged on a substrate in the biochip manufacturing method according to the first embodiment of the present invention. Figure 7 is a schematic diagram of melting the fixing member to further fix the reaction plate in the biochip manufacturing method according to the first embodiment of the present invention.

According to the first embodiment of the present invention, a substrate 1 as shown in FIG2 is prepared. In this embodiment, the substrate 1 is formed of plastic. Then, as shown in FIG3, a plurality of observation holes 2 are formed on the substrate 1 through the substrate 1. In this embodiment, the aperture of the observation hole 2 is 1.1 mm, and the distance between two adjacent observation holes is 1.54 mm.

Please refer to FIG4 , the lower part of FIG4 is the front side of the substrate 1, and the upper part is the bottom side of the substrate 1. In the first embodiment, a plurality of cone-shaped fixing members 3 are formed on the bottom side of the substrate 1, and the fixing members 3 are made of plastic. In addition, since the sample will be added from the front side of the biochip when dripping, the cross-section of the observation hole 2 is set to be narrow at the top and wide at the bottom as shown in FIG4 to facilitate sample addition. On the other hand, this setting can prevent the reaction sheet 4 from falling into the observation hole 2 when the reaction sheet 4 is arranged. In this embodiment, the inner diameter (bottom side) of the observation hole 2 is 1.1 mm, the outer diameter (front side) is 1.2 mm, the center distance of the observation holes 2 in the same row or column is 1.54 mm, and the center distance of the cone-shaped fixing members 3 in the same row or column is 1.54 mm. The substrate 1 on which the cone-shaped fixing member 3 is disposed is shown in FIG5 .

Next, please refer to FIG6 , a reaction sheet 4 is placed on the bottom surface of the substrate 1 . In this embodiment, the reaction sheet 4 is a circular slice with a diameter of 1.4 mm, and its material is a plastic sheet. The reaction element placed on the top is a protein probe. After the reaction sheet 4 is placed, the tip of the plastic cone-shaped fixing member 3 is melted and pressed flat with an iron to produce a fixing portion 5 as shown in FIG7 , so that the position of the reaction sheet 4 is fixed between the four cone-shaped fixing members 3 and between the fixing portion 5 and the substrate 1 .

The second embodiment of the present invention is described below with reference to FIGS. 8 to 10. FIG. 8 is an oblique view of a biochip including a mushroom-shaped fixing member according to the second embodiment of the present invention. FIG. 9 is a side view schematic diagram of a cross section of the substrate along the tangent line B-B' according to FIG. 8. FIG. 10 is a side view schematic diagram of a cross section of a reaction chip disposed on a substrate in a biochip manufacturing method according to the second embodiment of the present invention.

The biochip manufacturing method of the second embodiment of the present case is similar to the first embodiment and will not be described in detail. The difference is that the fixing member 3a is mushroom-shaped as shown in Figures 8 and 9. The shape of the mushroom-shaped fixing member 3a makes it easier to insert the reaction sheet 4 therein and fix it 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 set is shown in Figure 10.

In the third embodiment of the present invention, the structures of the substrate, the observation hole and the fixing part are as shown in the first and second embodiments. The difference is that in the third embodiment, the biological probe is set on the reaction mother sheet made of paper by inkjet method, and then the reaction mother sheet is cut into a circle with a diameter of 1.4 mm by laser cutting to obtain a reaction sheet 4. Then, the back of the reaction sheet 4 without the biological probe is adsorbed by a placement machine, and the reaction sheet is set and fixed between the fixing parts 3 and 3a. At this time, different types of reaction sheets 4 can be set on the same biochip according to experimental requirements to achieve the effect of cost saving. According to one embodiment of the present invention, the fixing part 3 is pressed flat to produce the fixing part 5, and then they are tightly fitted to each other, so that the reaction sheet 4 is sealed between the fixing part 5 and the substrate 1. At this time, when observing the result through the observation hole 2, the result can be observed with the aid of an external light source from the front surface of the substrate 1. According to another embodiment of the present invention, the fixing member 3 is flattened to produce the fixing portion 5, and the fixing member 3 is not tightly fitted to each other. When observing the result through the observation hole 2, the result can be observed with the aid of a light source applied through the bottom surface of the substrate 1.

According to the fourth embodiment of the present invention, a method for testing a sample using the biochip prepared in the above embodiment is provided. In this embodiment, the biochip prepared in the above embodiment is placed face up, and the sample is applied to the biochip manually or automatically. After the sample reacts with the bioprobe, the result is read by naked eyes or machine.

By virtue of the above-mentioned technical features, the biochip manufacturing method of the present invention can fix the reaction sheet through a simple fixing part, and can be set (placed) by adsorbing the back of the reaction sheet during manufacturing, thereby avoiding damage to the biological probe that may be caused by adsorbing the front side in the traditional process. The produced biochip is low-cost, and reaction sheets with different reaction elements can be set according to needs, thereby further saving costs. For example, 2 or more reaction sheets with different biological probes (and reaction elements) can be prepared and placed on the same biochip. In the detection method using the biochip disclosed in the present invention, the experimental process is easy to operate and the results are easy to interpret, which is easier to use than the known detection method. In addition, since the size of the biochip in this case is moderate, only part of the reaction sheet can be used according to needs, which can also save costs.

The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made thereto without departing from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

1: Substrate

2: Observation hole

3, 3a: Fixing parts

4: Reaction film

5: Fixed part

S1~S5: Steps

In the following detailed description, in order to explain the present invention, many specific details are provided so that the disclosed embodiments can be thoroughly understood. However, it is obvious that one or more embodiments can be implemented without the specific details. In other cases, in order to simplify the drawings, the known structures and processes will be shown in a schematic manner.

FIG. 1 is a flow chart of a biochip manufacturing method according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a substrate used in the biochip manufacturing method according to the first embodiment of the present invention.

FIG3 is a schematic diagram of generating observation holes on a substrate in the biochip manufacturing method according to the first embodiment of the present invention.

FIG. 4 is an oblique view of a biochip including a conical fixing member according to the first embodiment of the present invention.

FIG5 is a schematic side view of a cross section of the substrate along the tangent line A-A’ according to FIG4 .

FIG6 is a schematic side view of a cross section of a reaction chip disposed on a substrate in a method for manufacturing a biochip according to the first embodiment of the present invention.

FIG. 7 is a schematic diagram showing the melting of the fixing member to further fix the reaction chip in the biochip manufacturing method according to the first embodiment of the present invention.

FIG8 is an oblique view of a biochip including a mushroom-shaped fixing member according to a second embodiment of the present invention.

FIG9 is a schematic side view of a cross section of the substrate along the tangent line B-B’ according to FIG8 .

FIG10 is a schematic side view of a cross section of a reaction chip disposed on a substrate in a biochip manufacturing method according to a second embodiment of the present invention.

S1~S5: Steps

Claims (10)

A method for manufacturing a biochip comprises: manufacturing a substrate; forming a plurality of observation holes penetrating the substrate on the substrate; arranging a plurality of fixing members on a bottom surface of the substrate around each of the plurality of observation holes; arranging a plurality of reaction sheets on one side of the bottom surface corresponding to the plurality of observation holes; and fixing the plurality of reaction sheets by deforming the plurality of fixing members; wherein the deformation is performed by heating and melting the plurality of fixing members. The manufacturing method as described in claim 1 further comprises setting a reaction element on a reaction master sheet and cutting the reaction master sheet into a plurality of reaction sheets. The manufacturing method as described in claim 1, wherein each of the reaction sheets disposed on the bottom surface comprises different reaction elements. The manufacturing method as described in claim 3, wherein the minimum distance between the centers of each reaction sheet is 1 to 10 mm. The manufacturing method as described in claim 1, wherein the fixing member is an L-shaped column, a cylinder, a polygonal column, a cone, a cylinder, a sphere, a polyhedron, a ring or a combination thereof. A biochip manufactured by the biochip manufacturing method as described in any one of claims 1 to 5. A biochip as described in claim 6, wherein each of the observation holes is surrounded by at least one corresponding fixing member. The biochip as described in claim 6, wherein the substrate is made of silicon, glass, polymer material or ceramic. A biochip as described in claim 6, wherein the fixing member is made of plastic, silicone or rubber. A method for detecting a sample using a biochip as described in any one of claims 6 to 9, comprising: adding a sample to a reaction plate through an observation hole; and observing the result from the observation hole.

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