WO2025086145A1 - Microfluidic chip and preparation method for microfluidic chip - Google Patents

Abstract

Disclosed is a microfluidic chip and a preparation method for a microfluidic chip. The microfluidic chip comprises reaction modules; each reaction module comprises a sample loading flow channel, sample separation flow channels, and reaction pool; a sample inlet is formed at one end of each of the sample loading flow channels; there are a plurality of sample separation flow channels, and all the sample separation flow channels are sequentially connected to the sample loading flow channels in the direction of fluid motion within the sample loading flow channels; in the direction of fluid motion within the sample loading flow channels, the inner channel lengths of the sample separation flow channels connected to the sample loading flow channels gradually shorten; and a sample outlet of each sample separation flow channel is provided with a reaction pool. In the microfluidic chip provided by the invention, one end of each of the sample loading flow channels is provided with a sample inlet, there are a plurality of sample separation channels, and in the direction of fluid motion within the sample loading flow channels, the inner channel lengths of the sample separation flow channels connected to the sample loading flow channels gradually shorten, so that a liquid in the sample loading flow channels nearly synchronously enters the reaction pools via the sample separation flow channels, thus realizing multiple reactions being synchronously carried out. This further improves the detection throughput of the microfluidic chip.

Description

Microfluidic chip and method for preparing the same Technical Field

The present invention relates to the field of microfluidic technology, and in particular to a microfluidic chip and a method for preparing the microfluidic chip.

Background Art

Microfluidic chips are also called Miniaturized Total Analytical Systems or Micro Total Analysis Systems (MTAS). They can control the flow of reaction liquids in micron-scale channels and cavities, thereby achieving reaction closure. Microfluidic chip technology involves knowledge in disciplines and fields such as chemistry, fluid mechanics, biology, medicine, microelectronics, and new materials. For example, molecular detection technology based on polymerase chain reaction (PCR) is widely used in medicine, agriculture, food and other fields. PCR reaction can amplify trace amounts of target DNA in vitro, and has the characteristics of strong specificity, high sensitivity, and relatively simple operation. Currently, the widely used PCR technologies mainly include real-time fluorescence quantitative PCR technology, digital PCR technology, isothermal amplification PCR technology, etc.

Microfluidic chip technology can concentrate multiple steps of sample detection on a single chip, integrate functional units through the combined design of flow channels, microvalves, cavities, etc., and ultimately achieve miniaturization and automation of chip devices. Through the microfluidic network, the samples to be tested can be diverted to multiple independent high-throughput and high-efficiency reaction units at the same time, so that the same sample can be tested in parallel in multiple tasks as needed. However, traditional microfluidic chips have defects such as low detection throughput and low integration, which makes it difficult to promote the use of microfluidic chips.

Therefore, how to improve the detection throughput of microfluidic chips is a technical problem that needs to be solved urgently by those skilled in the art.

Summary of the invention

The object of the present invention is to provide a microfluidic chip, the detection throughput of the microfluidic chip is improved.

To achieve the above object, the present invention provides a microfluidic chip, including a reaction module, wherein the reaction module includes:

An injection channel, one end of which is provided with an injection port;

The sample splitting flow channel includes a plurality of sample splitting flow channels, all of which are sequentially connected to the sample inlet flow channel along the direction of fluid movement in the sample inlet flow channel; the length of the flow channel in the sample splitting flow channel connected to the sample inlet flow channel gradually shortens along the direction of fluid movement in the sample inlet flow channel;

A reaction pool, wherein the sample outlet of each sample distribution channel is respectively provided with the reaction pool.

Optionally, in the above-mentioned microfluidic chip, the sample inlet channel includes a first channel and a second channel connected to the first channel, the sample inlet is arranged on the first channel, the sample splitting channel is connected to the second channel, and the second channel is an arc-shaped channel.

Optionally, in the above-mentioned microfluidic chip, the sample distribution channel is radially arranged along the length direction of the second channel.

Optionally, the above-mentioned microfluidic chip further includes a substrate, the reaction modules include multiple ones, and all the reaction modules are arranged on the substrate.

Optionally, in the above-mentioned microfluidic chip, 8 reaction modules are provided on the substrate, and each reaction module is provided with 32 sample distribution channels.

Optionally, in the above microfluidic chip, the reaction modules are symmetrically distributed on opposite sides of the center line of the substrate.

Optionally, in the above-mentioned microfluidic chip, the injection port is located at one end of the injection channel close to the center line of the substrate.

Optionally, in the above-mentioned microfluidic chip, the height of the reaction liquid in the reaction pool is lower than the height of the bottom end of the sample distribution channel.

A method for preparing a microfluidic chip, used to prepare the above-mentioned microfluidic chip, comprises the steps of:

The sampling channel, sample distribution channel and reaction pool of the microfluidic chip were preliminarily drawn using computer 3D drawing software to form an initial model diagram of the microfluidic chip;

The injection channel is designed to be arc-shaped;

In the three-dimensional simulation software, the injection volume and the length of the sampling channel of each reaction pool of a single reaction module are obtained by combining the fluid state, rated pressure and the same injection time conditions;

The fluid state is, in the three-dimensional simulation process, the flow state of the fluid in the microfluidic chip is calculated according to the Navier-Stokes equation (a) under the state of conservation of kinetic energy (b);

The variables in formula (a) are:

ρ represents the fluid density

u represents the fluid velocity

Represents a constant function

I represents the turbulence variable

K represents incompressible liquid

F represents the identity matrix

The variables in formula (b) are:

ρ represents the fluid density

u represents the fluid velocity

Represents a constant-valued function.

In the microfluidic chip preparation method provided in the present application, the reaction module is processed by etching.

In the above technical scheme, the microfluidic chip provided by the present invention includes a reaction module, the reaction module includes an injection channel, a sample splitting channel and a reaction pool, and an injection port is provided at one end of the injection channel; the sample splitting channel includes multiple sample splitting channels, and all the sample splitting channels are connected to the injection channel in sequence along the direction of fluid movement in the injection channel; the length of the flow channel in the sample splitting channel connected to the injection channel along the direction of fluid movement in the injection channel is gradually shortened; and the sample outlet of each sample splitting channel is respectively provided with a reaction pool.

It can be seen from the above description that in the microfluidic chip provided in the present application, one end of the sample inlet flow channel is provided with a sample inlet; the sample splitting flow channel includes a plurality of sample splitting flow channels, and along the direction of fluid movement in the sample inlet flow channel, the length of the flow channel in the sample splitting flow channel connected to the sample inlet flow channel is gradually shortened, so that the liquid in the sample inlet flow channel passes through the sample splitting flow channel almost synchronously. The samples enter the reaction pool through the channel, realizing multiple reactions simultaneously, thereby increasing the detection throughput of the microfluidic chip.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

FIG1 is a schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present invention;

FIG. 2 is a schematic diagram showing a simulation of the injection volume of the microfluidic chip provided in an embodiment of the present invention for different sample splitting channel lengths 20 milliseconds after injection.

In Figure 1: 1-substrate, 2-inlet, 3-injection channel, 4-sample splitting channel, 5-reaction cell.

DETAILED DESCRIPTION

The core of the present invention is to provide a microfluidic chip, the detection flux of the microfluidic chip is improved.

In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation modes.

Please refer to Figure 1 and Figure 2.

In a specific embodiment, the microfluidic chip provided in a specific embodiment of the present invention includes a reaction module, and the reaction module includes an injection channel 3, a sample splitting channel 4 and a reaction pool 5. One end of the injection channel 3 is provided with an injection port 2, and the injection channel 3 can be one or a combination of a straight channel, a curved channel or a broken line channel. The injection channel 3 and the sample splitting channel 4 are based on achieving automatic flow of fluids, and the specific dimensions are determined according to actual needs, and this application does not make specific limitations. The injection channel 3 and the sample splitting channel 4 are specifically capillary structures.

The sample splitting flow channel 4 includes a plurality of sample splitting flow channels, all of which are sequentially connected to the sample feed flow channel 3 along the fluid movement direction in the sample feed flow channel 3. Specifically, the specific lengths of two adjacent sample splitting flow channels 4 may be equal or unequal. The length of the flow channel in the sample splitting flow channel 4 connected to the sample splitting flow channel 3 in the direction of fluid movement in the sample splitting flow channel 3 is gradually shortened; and a reaction pool 5 is provided at the sample outlet of each sample splitting flow channel 4 .

Specifically, primers and corresponding probes used to detect sample target DNA may be contained in the reaction pool 5. The type of primers depends on actual needs and is not specifically limited in this application.

It can be seen from the above description that in the microfluidic chip provided in the specific embodiment of the present application, one end of the sample inlet channel 3 is provided with a sample inlet 2; the sample splitting channel 4 includes multiple sample splitting channels, and along the direction of fluid movement in the sample inlet channel 3, the length of the flow channel in the sample splitting channel 4 connected to the sample inlet channel 3 is gradually shortened, so that the liquid in the sample inlet channel 3 passes through the sample splitting channel 4 almost synchronously into the reaction pool 5, and multiple reactions are carried out synchronously. In this way, the detection throughput of the microfluidic chip is improved.

In a specific embodiment, the sample inlet channel 3 includes a first channel and a second channel connected to the first channel, the sample inlet 2 is arranged on the first channel, the sample splitting channel 4 is connected to the second channel, and the second channel is an arc-shaped channel. As shown in FIG1 , the first channel can be an L-shaped channel, and the first channel and the second channel are connected. During processing, preferably, the sample inlet channel 3 is integrally formed.

The sample splitting channel 4 is radially arranged along the length direction of the second channel. Specifically, the second channel is preferably a linear channel, so that the fluid can flow smoothly to the reaction pool 5.

The microfluidic chip also includes a substrate 1, and the reaction modules include multiple ones, and all the reaction modules are arranged on the substrate 1 to improve the integration of the reaction modules. Specifically, the number of reaction modules on the same substrate 1 can be 4-10. Each reaction module is provided with 10-40 sample distribution channels 4.

The substrate 1 is provided with 8 reaction modules, and each reaction module is provided with 32 sample distribution channels 4. The chip is configured in this way to carry out the detection of 256 targets for one sample, or 32 targets for each of 8 samples. DNA primers and corresponding probes for detecting targets are embedded in each reaction pool 5, and corresponding detection equipment and nucleic acid amplification reaction reagents are provided. The microfluidic chip provided in this application provides a high-throughput detection method for multiple detection fields such as genetically modified component screening, crop strain identification, food inspection, and medical diagnosis.

In order to make the layout of the reaction modules regular and facilitate the loading of samples into each reaction module, the reaction modules are preferably symmetrically distributed on opposite sides of the center line of the substrate 1. Of course, the reaction modules can be evenly distributed in the circumferential direction with the center of the substrate 1 as the center.

Furthermore, the injection port 2 is located at one end of the injection channel 3 close to the center line of the substrate 1. The distance traveled by the injection needle between each injection port 2 is reduced, and the injection speed is further improved.

The height of the reaction liquid in the reaction pool 5 is lower than the height of the bottom of the sample distribution channel 4. This arrangement prevents the overflow of the reaction liquid during the sample addition process from causing mutual contamination of the detection reaction. Specifically, the volume of the microfluidic chip reaction pool 5 should be larger than the total volume of the detection experiment reaction liquid in actual application.

The present application provides a method for preparing a microfluidic chip, which is used to prepare any of the above-mentioned microfluidic chips, comprising the steps of:

The sampling channel 3, the sample distribution channel 4 and the reaction pool 5 of the microfluidic chip are preliminarily drawn using computer three-dimensional drawing software to form an initial model diagram of the microfluidic chip;

Under the premise that the reaction pool 5 of the microfluidic chip achieves the same injection volume under the conditions of rated injection pressure and the same injection time, the injection channel 3 adopts an arc design;

In the three-dimensional simulation software, the injection volume of each reaction pool 5 and the length of the sample distribution channel 4 of a single reaction module are obtained under the conditions of fluid state, rated pressure and equal injection time;

Specifically, the collective model of the microfluidic chip can be modified and improved according to the simulation calculation results for the preparation of the microfluidic chip.

The fluid state is calculated according to the Navier-Stokes equation (a) under the condition of conservation of kinetic energy (b) during the three-dimensional simulation process.

The variables in formula (a) are:

ρ represents the fluid density

u represents the fluid velocity

Represents a constant function

I represents the turbulence variable

K represents incompressible liquid

F represents the identity matrix

The variables in formula (b) are:

ρ represents the fluid density

u represents the fluid velocity

Represents a constant-valued function.

The relationship between the injection time, injection pressure and injection volume of the microfluidic chip was simulated by three-dimensional simulation software to determine the injection time and injection pressure of the microfluidic chip. As shown in Figure 2, the color on the right represents the volume fraction of the solution in the flow channel. The volume fraction gradually decreases from top to bottom. After the preset injection time, the injection volume at the reaction pool position is almost the same.

The geometric model of the microfluidic chip is modified and improved according to the simulation calculation results for the preparation of the microfluidic chip.

Specifically, the reaction module is processed by etching, and the etching of the microfluidic chip is completed by using a laser etching method according to the size of each component in the drawing of the microfluidic chip.

In specific use, the proportion of each component in the single target detection system is determined by extracting the DNA of the sample to be detected, and the detection reaction premix is prepared according to the number of detection targets and the proportion of the components in the reaction system. Then, the volume proportion of each component in the single detection target detection system is determined.

According to the total volume of the reaction, the injection pressure and injection time are selected, and the sample is dispensed into each reaction pool 5 by using the capillary pressure injection method. The premixed liquid and the pre-embedded target primers and probes mix the reaction system under the action of the injection pressure.

The microfluidic chip after sample injection is placed in a device equipped with a temperature control and fluorescence signal detection unit to perform nucleic acid amplification reaction and real-time detection of results. After amplification, the fluorescence signal analysis is used to determine whether the detection target is detected.

The present invention is used for the detection and identification of new varieties of biological breeding industrialization and related products, and has the following advantages:

The product has high detection throughput, wide application range, high integration and portability.

Specifically, during the specific processing, the sample injection channel 3, the sample distribution channel 4 and the reaction pool on the substrate 1 are sealed by a sealing film. The processed microfluidic chip has strong sealing performance, which can reduce the impact of manual operation and environment on the detection results, and the detection is more accurate.

The microfluidic chip provided in this application can be used for polymerase chain reaction technology, mainly referring to real-time fluorescence PCR technology, the purpose of which is to use in vitro amplification to obtain sufficient results in a short period of time in vitro. Characterize the target DNA fragment. At this time, the DNA primer for detecting the sample target is a DNA sequence designed and synthesized based on the specific sequence of the target DNA to be detected and combined with the characteristics of the polymerase chain reaction. The corresponding probe is designed and synthesized based on the fragment amplified by the primer DNA sequence and is fluorescently labeled to facilitate the characterization of subsequent test results. The present invention coats the target primer DNA and the fluorescently labeled DNA probe in the reaction pool 5 of the microfluidic chip by freeze coating and freezes them.

The DNA detection template used in this application is to extract the genomic DNA of the sample to be detected by cell lysis, DNA separation and precipitation.

For ease of understanding, the present application is described below in conjunction with specific embodiments:

Example 1

(1) Application: Used for screening of genetically modified ingredients in crops and related products.

(2) Preparation process: The microfluidic chip is etched according to the size of the drawn microfluidic chip, mainly including an injection port 2, an injection channel 3, a sample splitting channel 4, and a reaction pool 5, with a total of 8 reaction modules, which can meet the detection of 8 samples, 32 targets in each sample, or 256 targets in one sample. Primer DNA and corresponding probes for the detection target are embedded in each reaction pool 5. The source of the DNA primer sequence of the detection target is mainly the related sequences in the qualitative PCR method of herbicide-resistant, insect-resistant and herbicide-resistant corn transformants, herbicide-resistant, insect-resistant and quality-improved soybean transformants, insect-resistant, insect-resistant and herbicide-resistant rice transformants, insect-resistant and herbicide-resistant cotton transformants and their derivative varieties involved in the national standards of the People's Republic of China, as well as the common transgenic and product component detection genes bar, pat, CaMV35S promoter, FMV 35S promoter, NOS promoter, NOS terminator and CaMV35S, etc., totaling 256 detection targets.

(3) Application method: Genomic DNA from crops and related products is extracted by cell lysis, DNA separation and precipitation. It is diluted as a detection template, and a premix is prepared according to the proportion of each component in the reaction solution and the number of test samples. The reaction solution is filled into the reaction pool 5 by capillary pressure, and the reaction system is mixed under the action of pressure injection.

Place the sampled microfluidic chip in a heating and fluorescence signal detection device, set the reaction temperature, reaction time and fluorescence signal acquisition time. After the amplification is completed, determine whether relevant transgenic components are detected by fluorescence signal analysis.

Example 2

(1) Application: Used for identification of crop varieties.

(2) Preparation process: According to the size of the drawn microfluidic chip, the microfluidic chip is etched, mainly including an injection port 2, an injection channel 3, a sample splitting channel 4, and a reaction pool 5, with a total of 8 reaction modules, which can meet the detection of 8 samples, 32 targets per sample, or 256 targets per sample. According to the SNP markers of the genome of crops such as corn, wheat, soybean, rice, and cotton, the identification primers and probes are designed, and the typing and identification of species SNP sites are carried out based on the fluorescent probe method, and the amplification primers and probes are embedded in the reaction pool 5. The detection of SNP sites in the genome of crops such as corn, wheat, soybean, rice, and cotton is carried out.

(3) Application method: Use a DNA extraction kit to extract the genomic DNA of the sample to be tested, and dilute the DNA to an appropriate concentration as a test template. Prepare a premix according to the proportion of each component in the reaction solution and the number of test samples, use capillary pressure to fill the reaction solution into the reaction pool 5, and mix the reaction system under the action of pressure injection.

The microfluidic chip with sample loading is placed in a heating and fluorescence signal detection device, and the reaction temperature, reaction time and fluorescence signal acquisition time are set. After the amplification is completed, the fluorescence signal analysis is used to determine whether relevant genetically modified components are detected.

Example 3

(1) Application: Used for the detection of foodborne pathogens such as Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Bacillus cereus in food.

(2) Preparation process: According to the size of the drawn microfluidic chip, the microfluidic chip is etched, mainly including an injection port 2, an injection channel 3, a sample splitting channel 4, and a reaction pool 5, with a total of 8 reaction modules, which can meet the detection of 8 samples, 32 targets per sample, or 256 targets per sample. Primers and probes are designed according to the nucleic acid sequence characteristics of pathogenic bacteria and embedded in the reaction pool 5.

(3) Application method: The genomic DNA of the sample to be tested is extracted by cell lysis, DNA separation and precipitation, and diluted as a test template. The premix is prepared according to the proportion of each component in the reaction solution and the number of test samples. The reaction solution is filled into the reaction pool 5 using capillary pressure, and the reaction system is mixed under the action of pressure injection.

The microfluidic chip with sample loading is placed in a heating and fluorescence signal detection device, and the reaction temperature, reaction time and fluorescence signal acquisition time are set. After the amplification is completed, the fluorescence signal analysis is used to determine whether relevant genetically modified components are detected.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

A microfluidic chip, characterized in that it comprises a reaction module, wherein the reaction module comprises: An injection channel (3), wherein one end of the injection channel (3) is provided with an injection port (2); A sample splitting flow channel (4), wherein the sample splitting flow channel (4) comprises a plurality of sample splitting flow channels, all of which are sequentially connected to the sample inlet flow channel (3) along the direction of fluid movement in the sample inlet flow channel (3); the flow channel length of the sample splitting flow channel (4) connected to the sample inlet flow channel (3) along the direction of fluid movement in the sample inlet flow channel (3) gradually shortens; A reaction pool (5), wherein each sample outlet of the sample distribution channel (4) is provided with the reaction pool (5). The microfluidic chip according to claim 1 is characterized in that the sample inlet channel (3) includes a first channel and a second channel connected to the first channel, the sample inlet (2) is arranged on the first channel, the sample splitting channel (4) is connected to the second channel, and the second channel is an arc-shaped channel. The microfluidic chip according to claim 2 is characterized in that the sample distribution channel (4) is arranged radially along the length direction of the second flow channel. The microfluidic chip according to claim 1 is characterized in that it also includes a substrate (1), the reaction modules include a plurality, and all the reaction modules are arranged on the substrate (1). The microfluidic chip according to claim 4 is characterized in that 8 reaction modules are provided on the substrate (1), and each reaction module is provided with 32 sample distribution channels (4). The microfluidic core according to claim 4 is characterized in that the reaction modules are symmetrically distributed on opposite sides of the center line of the substrate (1). The method for preparing a microfluidic chip according to claim 6, characterized in that the injection port (2) is located at one end of the injection channel (3) close to the center line of the substrate (1). The microfluidic chip according to any one of claims 1 to 7, characterized in that the height of the reaction liquid in the reaction pool (5) is lower than the height of the bottom end of the sample distribution channel (4). A method for preparing a microfluidic chip, characterized in that it is used to prepare the microfluidic chip as claimed in claims 1-8 The microfluidic chip according to any one of the above items comprises the steps of: Using computer three-dimensional drawing software to preliminarily draw the sample injection channel (3), sample distribution channel (4) and reaction pool (5) of the microfluidic chip to form an initial model diagram of the microfluidic chip; The sample inlet channel (3) is designed to be arc-shaped; In the three-dimensional simulation software, the injection volume of each reaction pool (5) and the length of the sample distribution channel (4) of a single reaction module are obtained under the conditions of fluid state, rated pressure and equal injection time; The fluid state is, in the three-dimensional simulation process, the flow state of the fluid in the microfluidic chip is calculated according to the Navier-Stokes equation (a) under the state of conservation of kinetic energy (b);
The variables in formula (a) are: ρ represents the fluid density u represents the fluid velocity Represents a constant function I represents the turbulence variable K represents incompressible liquid F represents the identity matrix
The variables in formula (b) are: ρ represents the fluid density u represents the fluid velocity Represents a constant-valued function. The method for preparing a microfluidic chip according to claim 9 is characterized in that the reaction module is processed by etching.