CN113275049A

CN113275049A - Preparation method of biochip, biochip and detection device

Info

Publication number: CN113275049A
Application number: CN202110539106.9A
Authority: CN
Inventors: 罗欣莹; 赵子健; 丁丁; 黄东升
Original assignee: BOE Technology Group Co Ltd
Current assignee: BOE Technology Group Co Ltd
Priority date: 2021-05-18
Filing date: 2021-05-18
Publication date: 2021-08-20

Abstract

The invention discloses a preparation method of a biochip, the biochip and a detection device, wherein the preparation method comprises the following steps: providing a substrate base plate; carrying out epoxy modification on the substrate base plate to form an epoxy group; and (3) taking the epoxy group as a coupling anchor point, providing a mercapto group to be crosslinked with the epoxy group, and forming a functional molecular layer of a monomolecular layer to seal the surface of the biochip, wherein the functional molecular layer takes a carboxyl group as a coupling group. The invention has the beneficial effects of reducing cost and reducing nonspecific adsorption.

Description

Preparation method of biochip, biochip and detection device

Technical Field

The invention relates to the field of chips, in particular to a preparation method of a biochip, the biochip and a detection device.

Background

The biochip technology is based on the principle of specific interaction between molecules and integrates discontinuous analysis process in life science field into micro biochemical analysis system on the chip surface to realize accurate, fast and large information amount detection of cell, protein, gene and other biological components.

The accuracy and sensitivity of biochip detection are closely related to sample prepayment rate and background noise, which are often dependent on the efficiency of chip surface modification.

At present, the main biochip modification method is to plate precious metals on the surface, which is high in cost and easy to cause nonspecific adsorption.

Disclosure of Invention

In view of the above problems, the present invention has been made to provide a method of manufacturing a biochip, a biochip and a detection apparatus that overcome the above problems or at least partially solve the above problems.

In a first aspect, a method for preparing a biochip includes:

providing a substrate base plate;

carrying out epoxy modification on the substrate base plate to form an epoxy group;

and (3) taking the epoxy group as a coupling anchor point, providing a mercapto group to be crosslinked with the epoxy group, and forming a functional molecular layer of a monomolecular layer to seal the surface of the biochip, wherein the functional molecular layer takes a carboxyl group as a coupling group.

Optionally, the epoxy modifying the substrate base plate to form an epoxy group includes: and performing epoxy modification on the substrate by adopting propyl trimethoxy silane to form an epoxy group with a Si-O-Si bond.

Optionally, the providing mercapto groups cross-linked with the epoxy groups comprises: a mercapto group is provided with 12-mercaptododecanoic acid to crosslink with the epoxy group.

Optionally, the coupling group has the formula:

optionally, the providing a substrate base plate includes: providing an initial base plate, preparing a silicon dioxide layer on the surface of the initial base plate, and forming the substrate base plate.

Optionally, after forming the silicon dioxide layer on the surface of the initial substrate, the method further includes: and patterning the silicon dioxide layer to form a microfluidic channel, wherein the microfluidic channel is a drainage channel of the functional molecular layer.

Optionally, the substrate base plate is a glass base plate.

In a second aspect, there is provided a biochip comprising:

a substrate base plate;

the functional molecular layer is arranged on the substrate base plate and is a monomolecular layer, wherein the functional molecular layer comprises a coupling group, and the coupling group is a carboxyl group.

Optionally, the coupling group is of the formula:

optionally, the substrate base plate includes: an initial substrate and a layer of silicon dioxide, the layer of silicon dioxide being located between the initial substrate and the layer of functional molecules.

Optionally, a microfluidic channel is formed in the silicon dioxide layer, wherein the microfluidic channel is a drainage channel of the functional molecular layer.

Optionally, the substrate base plate is a glass base plate.

In a third aspect, there is provided a detection apparatus comprising the biochip of the second aspect.

The technical scheme provided by the embodiment of the invention at least has the following technical effects or advantages:

according to the preparation method of the biochip, the biochip and the detection device provided by the embodiment of the invention, the substrate is subjected to epoxy modification to form the epoxy group, then the epoxy group is used as a coupling anchor point to provide crosslinking between the mercapto group and the epoxy group, so that a dense monomolecular functional molecular layer can be formed, the surface of the biochip is effectively sealed, the nonspecific adsorption of small molecules and various impurities on the surface of the biochip can be effectively avoided, the nonspecific adsorption is reduced, the false positive rate of the detection of the biochip is reduced, and the sensitivity and the signal-to-noise ratio are improved. Furthermore, the formed functional molecular layer takes carboxyl as a coupling group, so that the subsequent sealing and coupling efficiency is effectively improved. In addition, the surface of the chip is not required to be modified by consuming precious metals, so that the cost is effectively reduced.

The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1 is a flow chart showing a method for preparing a biochip according to an embodiment of the present invention;

FIG. 2 is a first schematic diagram of a method for preparing a biochip according to an embodiment of the present invention;

FIG. 3 is a second schematic diagram of a method for preparing a biochip according to an embodiment of the present invention;

FIG. 4 is a third schematic view of a method of preparing a biochip according to an embodiment of the present invention;

FIG. 5 is a fourth schematic view of a method of preparing a biochip according to an embodiment of the present invention;

FIG. 6 is a schematic view of a microfluidic channel according to an embodiment of the present invention;

FIG. 7 is a data diagram of test results in an embodiment of the present invention;

fig. 8 is a schematic diagram of a detection device in an embodiment of the invention.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Referring to fig. 1, a flow chart of a method for preparing a biochip according to an embodiment of the present invention includes:

step S101, providing a substrate base plate;

step S102, carrying out epoxy modification on the substrate base plate to form an epoxy group;

step S103, using the epoxy group as an anchor point of coupling, providing a mercapto group to crosslink with the epoxy group, and forming a functional molecular layer of a monomolecular layer to seal the surface of the biochip, wherein the functional molecular layer uses a carboxyl group as a coupling group.

In step S101, as shown in fig. 2, the substrate 1 provided may be a substrate purchased by a chip manufacturer, or a substrate manufactured by the chip manufacturer, which is not limited herein. The substrate base plate 1 can be a glass base plate, and the glass base plate contains silicon dioxide so as to provide silicon ions and oxygen ions for forming an epoxy group subsequently; the substrate 1 may be a semiconductor substrate, and is not limited thereto.

As shown in fig. 3, it is also possible to use a glass substrate or a semiconductor substrate as the initial substrate 11, and prepare the silicon dioxide layer 12 on the initial substrate 11, so as to form the substrate 1 with the silicon dioxide layer 12 on the surface, so as to provide richer and more uniform silicon ions and oxygen ions through the silicon dioxide layer 12 to form epoxy groups. Specifically, the silicon dioxide layer 2 may be formed on the surface of the starting substrate 11 by a vapor deposition process to have a uniform thickness of several tens nm to several hundreds nm.

In step S102, the substrate base 1 is epoxy-modified with a chemical substance to form an epoxy group. Wherein the chemical substance can be propyl trimethoxy silane GPTMS, or 3-aminopropyl triethoxy silane APTES. Specifically, taking the substrate 1 as a glass substrate as an example, performing epoxy modification on the glass substrate by adopting GPTMS can provide epoxy groups more efficiently, because physisorbed water and silicon hydroxyl on the surface of the glass substrate can react with functional groups of an epoxy silane coupling agent in GPTMS in the presence of water to efficiently generate covalent bonds, thereby efficiently providing epoxy groups.

The reaction principle of epoxy modification of the surface of the glass substrate by GPTMS is as follows: firstly, in an aqueous solution system, namely in an environment of soaking or scouring the surface of a glass substrate by adopting a GPTMS solution, methoxy in GPTMS molecules firstly reacts with water molecules to hydrolyze to generate Si-OH, namely hydrolysis reaction shown in the following chemical formula is carried out:

the Si-OH generated by the hydrolysis reaction and the Si-OH on the surface of the SiO2 undergo a glycidyl reaction to form an Si-O-Si bond, namely, a bonding reaction shown by the following chemical formula is carried out:

wherein, Si-O-Si bonds generated by the bonding reaction are epoxy groups.

In step S103, the biochip is modified with a blocking activation layer. That is, the generated epoxy group is used as an anchor point of coupling, and a thiol group and an epoxy group are crosslinked, as shown in fig. 4, a monolayer functional molecular layer 2 is formed to block the surface of the biochip, wherein the functional molecular layer 2 uses a carboxyl group as a coupling group. Wherein, the chemical substance for providing the sulfydryl can be 12-mercaptododecanoic acid, also can be thioglycolic acid and the like.

Specifically, 12-mercaptododecanoic acid is adopted to provide sulfydryl, and due to the fact that sulfydryl is rich and is arranged closely, a dense monomolecular functional molecular layer 2 can be formed after the sulfydryl and epoxy groups are crosslinked, so that the surface of a chip can be sealed more effectively, nonspecific adsorption is further reduced, the accuracy of detection by adopting a biochip is improved, background noise is reduced, and the noise reduction performance requirement on instrument equipment is lowered. And after the 12-mercaptododecanoic acid is reacted, more abundant carboxyl-COOH can be provided as a coupling group, so that the subsequent coupling of various antibody or polypeptide molecular probes is facilitated, and the coupling efficiency is effectively improved. Wherein the chemical formula of the formed carboxyl-COOH coupling group is as follows:

in a specific embodiment, the specific process steps of using 12-mercaptododecanoic acid to perform the modification of the blocking activation layer on the biochip surface can be set as required. For example, the epoxy-modified biochip obtained in step S102 may be washed first, and the washing may be performed by soaking in purified water. Then, the biochip was placed in 1M aqueous solution of 12-mercaptododecanoic acid to perform a crosslinking reaction between thiol and epoxy groups, and the biochip was allowed to soak for 24 hours to achieve a sufficient reaction, thereby forming a dense monolayer of functional molecule layer 2. And then, deeply cleaning the reacted biochip by sequentially adopting 10x PBS (phosphate buffer solution), 1xPBS and ultrapure water to finish modification of the closed activation layer.

After step S102 and step S103, the functional molecular layer 2 formed on the surface of the regenerated chip is a monolayer, and a carboxyl group is used as a coupling group. When the biochip is used for detection in the subsequent process, the biochip can be used for detection after being activated, subjected to anti-spotting and subjected to protein sealing treatment.

It should be noted that the biochip can be divided into a biochip with a control device and a biochip without a control device based on the control requirement of the biochip.

In the biochip with a control device, as shown in fig. 5, a device layer 13 (for disposing a control device such as a transistor), a protective layer 14(PVX), and a structural layer (i.e., a silicon dioxide layer 12 in the present application) may be further prepared on the provided initial substrate 11 in this order from bottom to top. The foregoing steps S102-S103 are then used to prepare a layer 2 of functional molecules for blocking and providing coupling groups. When the biochip is used for detection, the biochip is activated, subjected to anti-spotting and protein blocking treatment to form a protein layer 3.

For a biochip without a control device, it is sufficient to prepare a functional molecule layer 2 on a substrate base plate 1 as shown in fig. 4. When the biochip is used for detection, the biochip is activated, subjected to anti-spotting and protein blocking treatment to generate a protein layer.

Further, in order to ensure that the target liquid to be detected can completely react with the functional molecule layer 2, the silicon dioxide layer 12 may be patterned by performing photolithography and etching (dry etching or wet etching) on the silicon dioxide layer to form a microfluidic channel 121 as shown in fig. 6. Wherein, the functional molecule layer 2 is arranged in the microfluidic channel 121, so that the target liquid to be detected can be drained to the functional molecule layer 2 (i.e. the reaction region) through the microfluidic channel 121, thereby ensuring the accuracy of the biochip test. In an implementation, the microfluidic channel 121 may include an elongated flow channel region 1211 and a relatively large receiving region 1212 as shown in fig. 6, and the functional molecule layer 2 may be formed in the receiving region 1212 to increase a reaction area.

Of course, in the implementation process, the microfluidic channel is not limited to be prepared on the silicon dioxide layer 12, and the microfluidic channel may also be prepared by a splicing process. For example, the base substrate 1 and the functional molecular layer 2 are formed as the lower lid plate using steps S101 to S103. And preparing a micro-flow channel on the upper cover plate, wherein the micro-flow channel comprises a flow channel and a hollow area. And bonding the upper cover plate and the lower cover plate to form the biochip. Wherein, the functional molecule layer 2 is exposed in the hollow-out area, so that the target liquid to be detected can be drained to the functional molecule layer 2 (namely, the reaction area) through the microfluidic channel, and the detection accuracy of the biochip can be ensured.

Of course, in the implementation process, the implementation manner of the microfluidic channel 121 is not limited to the above two, and is not limited herein. And the biochip can also be not provided with a microfluidic channel, the prepared functional molecule layers 2 are arranged on the surface of the biochip according to an array shape, and non-flowing static detection is realized by soaking or covering the target liquid to be detected.

By adopting the preparation method of the biochip provided by the embodiment of the application, the coupling efficiency can be improved, and the cost is reduced. And a dense monolayer functional molecular layer can be formed, so that the effective sealing of the surface of the chip is realized, and the nonspecific adsorption of small molecules and various impurities on the surface of the chip can be effectively avoided, so that the nonspecific adsorption is reduced, the false positive rate of the detection of the biochip is reduced, and the sensitivity and the signal-to-noise ratio are improved. Specific experimental data are provided below to verify the significant effects of the preparation method of the biochip provided in this example:

three groups of chips were prepared: the first group is a blank group chip; the second group is an experimental group of chips, i.e. the chips prepared by the method provided by the embodiment; the third group is a control group chip, namely a chip modified by adopting a conventional precious metal plating method.

The preparation method of the blank group chip comprises the following steps: biological chip sets without any modification were directly coated with Epidermal Growth Factor Receptor (EGFR) antibodies as blank chip sets.

The preparation method of the experimental group chip comprises the following steps: the chip set prepared by the method provided by the embodiment of the application is cleaned, and particularly, 10xPBS, 1xPBS and ultrapure water can be used for deep cleaning. Then, carboxyl group activation was performed on the chip using a mixed solution of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 1: 1. Next, the chip was subjected to EGFR antibody spotting again and washing was performed after sufficient incubation coupling was performed. And finally, placing the chip in skimmed milk powder with the concentration of 2.5% for full immersion and sealing, and then carrying out deep cleaning on the chip by using 10xPBS, 1xPBS and ultrapure water to finish the preparation of the experimental group chip.

The preparation method of the control group chip comprises the following steps: soaking the chip modified by the conventional precious metal plating method in a mixed solution of concentrated sulfuric acid and hydrogen peroxide with the volume ratio of 1:1 at 70 ℃ for 4 hours. And then fully soaking the cleaned and dried chip in a mixed solution of concentrated hydrochloric acid and absolute ethyl alcohol at the temperature of 37 ℃ in a volume ratio of 1: 1. Then soaking the cleaned and dried chip in 0.5mol/L absolute ethanol solution of 3-aminopropyl triethoxysilane (APTES) at 50 ℃ for 24 h. Next, the chip was subjected to EGFR antibody spotting again and washing was performed after sufficient incubation coupling was performed. Finally, the chip is placed in skimmed milk powder with the concentration of 2.5% to be fully soaked and sealed, and then the biochip is deeply cleaned by 10xPBS, 1xPBS and ultrapure water, so that the preparation of the control group chip is completed.

The following tests were performed on the three groups of chips: PBS solution of fluorescence labeling human serum albumin (FITC-HSA) is prepared, and the PBS solution is respectively introduced into the blank chip, the experimental chip and the control chip at the flow rate of 2 uL/s. Fluorescence signals were collected once per minute for each of the three sets of chips to obtain a fluorescence test result chart as shown in FIG. 7. In fig. 7, the abscissa is time (in min), the ordinate is fluorescence intensity, the triangle is the fluorescence detection data point of the test group chip, the circle is the fluorescence detection data point of the control group chip, and the square is the fluorescence detection data point of the blank group chip.

As can be seen from FIG. 7, the fluorescence signal intensity of the blank chip and the control chip increases with time, indicating that the nonspecific adsorption is strong and the background noise is strong. The fluorescence signal intensity of the experimental group chip is obviously lower than that of other two groups, which shows that the functional molecular layer 2 of the experimental group chip can efficiently reduce the nonspecific adsorption on the surface of the chip, avoid the impurity adsorption to a greater extent and reduce the background noise fluorescence, thereby improving the detection sensitivity and the signal-to-noise ratio.

Based on the same inventive concept, the embodiment of the present invention further provides a biochip, as shown in fig. 4, including:

a base substrate 1; and the functional molecular layer 2 is arranged on the substrate base plate 1, wherein the functional molecular layer 2 is a monomolecular layer, the functional molecular layer 2 comprises a coupling group, and the coupling group is a carboxyl group.

It should be noted that the biochip may be a biochip with a control device or a biochip without a control device; the biochip can be a microfluidic biochip or a microarray biochip, which is not limited herein.

In some embodiments, the coupling group is of the formula:

in some embodiments, the substrate base plate 1 is as shown in fig. 3, and includes: a starting substrate 11 and a layer of silicon dioxide 12, the layer of silicon dioxide 12 being located between the starting substrate 11 and the layer of functional molecules 2. A microfluidic channel may also be formed in the silicon dioxide layer 12, wherein the microfluidic channel is a drainage channel of the functional molecule layer 2.

The substrate 1 may be a glass substrate, which saves cost and provides silicon dioxide for epoxy modification.

Since the biochip according to the embodiment of the present invention is prepared by the method for preparing a biochip according to the embodiment of the present invention, and the specific principle, structure and effect of the method are described in the process of describing the method for preparing the biochip, those skilled in the art can understand the specific structure and modification of the biochip based on the method for preparing the biochip according to the embodiment of the present invention, and thus the details are not described herein. The biochip prepared by the method for preparing the biochip of the embodiment of the invention is within the protection scope of the invention.

Based on the same inventive concept, the embodiment of the present invention further provides a detection apparatus, as shown in fig. 8, including the biochip 800 provided in the embodiment of the present application.

The algorithms and displays presented herein are not inherently related to any particular computer, virtual machine, or other apparatus. Various general purpose systems may also be used with the teachings herein. The required structure for constructing such a system will be apparent from the description above. Moreover, the present invention is not directed to any particular programming language. It is appreciated that a variety of programming languages may be used to implement the teachings of the present invention as described herein, and any descriptions of specific languages are provided above to disclose the best mode of the invention.

In the description provided herein, numerous specific details are set forth. It is understood, however, that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Similarly, it should be appreciated that in the foregoing description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. However, the disclosed method should not be interpreted as reflecting an intention that: that the invention as claimed requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Those skilled in the art will appreciate that the modules in the apparatus of an embodiment may be adaptively changed and disposed in one or more apparatuses other than the embodiment. The modules or units or components of the embodiments may be combined into one module or unit or component, and furthermore they may be divided into a plurality of sub-modules or sub-units or sub-components. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and all of the processes or elements of any method or apparatus so disclosed, may be combined in any combination, except combinations where at least some of such features and/or processes or elements are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the following claims, any of the claimed embodiments may be used in any combination.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word "comprising" does not exclude the presence of elements or steps not listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the unit claims enumerating several means, several of these means may be embodied by one and the same item of hardware. The usage of the words first, second and third, etcetera do not indicate any ordering. These words may be interpreted as names.

Claims

1. A method for preparing a biochip, comprising:

providing a substrate base plate;

2. The method of claim 1, wherein the epoxy modifying the substrate base plate to form an epoxy group comprises:

and performing epoxy modification on the substrate by adopting propyl trimethoxy silane to form an epoxy group with a Si-O-Si bond.

3. The method of claim 1, wherein said providing mercapto groups to crosslink with said epoxy groups comprises:

a mercapto group is provided with 12-mercaptododecanoic acid to crosslink with the epoxy group.

4. The method of claim 1, wherein the coupling group has the formula:

5. the method of claim 1, wherein the providing a substrate base plate comprises:

providing an initial base plate, preparing a silicon dioxide layer on the surface of the initial base plate, and forming the substrate base plate.

6. The method of claim 5, further comprising, after forming a silicon dioxide layer on the initial substrate surface:

and patterning the silicon dioxide layer to form a microfluidic channel, wherein the microfluidic channel is a drainage channel of the functional molecular layer.

7. The method of any one of claims 1-6, wherein the substrate base plate is a glass base plate.

8. A biochip, comprising:

a substrate base plate;

9. The biochip of claim 8, wherein the coupling group is of the formula:

10. the biochip of claim 8, wherein the substrate base plate comprises:

an initial substrate and a layer of silicon dioxide, the layer of silicon dioxide being located between the initial substrate and the layer of functional molecules.

11. The biochip of claim 10, wherein:

and the silicon dioxide layer is provided with a micro-flow channel, wherein the micro-flow channel is a drainage channel of the functional molecular layer.

12. The biochip according to any one of claims 8 to 11, wherein the substrate base is a glass base.

13. A detection apparatus comprising the biochip according to any one of claims 8 to 12.