MSRE enzyme digestion treatment and digital detection integrated micro-fluidic chip and application thereof

Abstract

The invention discloses an integrated micro-fluidic chip which comprises a chip main body consisting of a reaction layer, a channel layer and a detection layer which are sequentially sealed from top to bottom in the vertical direction, the chip main body internally comprises a first fluid channel network and a second fluid channel network; the first fluid channel network comprises a first reaction chamber, a first mixing unit, a first transfer sample injection unit, a first digital detection unit and a fluid channel for connecting the structural units, which are communicated in sequence; the first fluid channel network and the second fluid channel network have the same structure and size, and the first fluid channel network and the second fluid channel network are arranged in a mirror symmetry mode along the central symmetry axis of the chip body. According to the chip disclosed by the invention, methylation sensitive incision enzyme digestion treatment, reagent mixing and digital amplification detection can be integrated on a closed micro-fluidic platform, and'sample input-result output 'is realized for the first time aiming at DNA methylation quantitative detection.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and particularly to an integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection, and its application in DNA methylation detection. Background Technology

[0002] DNA methylation modifications (such as CpG island methylation) are a core component of epigenetic research and have been widely recognized as important biomarkers for early cancer screening and prognostic assessment. For example, abnormal hypermethylation in the promoter region of the RASSF1A gene is closely related to the development and progression of various malignant tumors. Therefore, regular methylation biomarker testing in high-risk populations is of great significance for early disease warning and prevention.

[0003] Currently, the DNA methylation detection methods commonly used in clinical diagnosis and laboratory research mainly rely on bisulfite treatment. However, the bisulfite treatment process involves complex steps such as high-temperature denaturation and long-term chemical modification, which can easily lead to DNA degradation and breakage. Moreover, the operation process is cumbersome and difficult to automate in on-site testing scenarios.

[0004] As an alternative, methods based on methylation-sensitive restriction enzymes (MSREs) utilize the specific cleavage of unmethylated sequences by these enzymes, offering advantages such as mild reaction conditions and no need for chemical modification. While MSRE-qPCR technology, combined with real-time quantitative polymerase chain reaction (qPCR), simplifies the process, it is essentially a relative quantification method, dependent on a standard curve, and has limited sensitivity for small sample volumes, typically requiring expensive thermal cycling equipment.

[0005] To achieve more accurate methylation analysis, it is necessary to simultaneously obtain the ratio of methylated copy number to total copy number in the sample. Digital isothermal amplification (DAVA), such as recombinase polymerase amplification coupled with CRISPR-Cas12a (RPA-Cas12a), has become an ideal tool for solving this problem due to its absolute quantification capability at the single-molecule level. However, most existing digital detection platforms only focus on the amplification step and lack integration of the front-end enzyme digestion step. In practice, researchers still need to manually complete the enzyme digestion, inactivation, and mixing with amplification reagents in the tube before transferring the sample to the chip for injection. This step-by-step operation is not only time-consuming and prone to operational errors and aerosol contamination, but also fails to meet the demand for rapid on-site detection with "sample in - result out". Summary of the Invention

[0006] To address the aforementioned issues, this invention discloses an integrated microfluidic chip that combines MSRE enzyme digestion with digital detection. This chip integrates methylation-sensitive restriction enzyme digestion, reagent mixing, and digital amplification detection on a closed microfluidic platform. It achieves rapid, accurate, and absolute quantitative analysis of methylated DNA and total DNA in samples without relying on bisulfite treatment or standard curves. For the first time, it realizes "sample in - result out" for DNA methylation quantitative detection, greatly simplifying the operation process and significantly shortening the detection time, making it particularly suitable for on-site testing scenarios.

[0007] The specific technical solution is as follows:

[0008] An integrated microfluidic chip combining MSRE enzyme digestion and digital detection, comprising a chip body:

[0009] The chip body comprises, from top to bottom, a reaction layer, a channel layer, and a detection layer, which are sequentially sealed along the vertical direction.

[0010] The chip body includes a first fluid channel network and a second fluid channel network, which are disposed in the reaction layer, the channel layer and the detection layer;

[0011] The first fluid channel network, from upstream to downstream, includes the following sequentially connected channels:

[0012] The first reaction chamber is used for MSRE digestion and inactivation of the test sample;

[0013] And, the first mixing unit, used to mix the MSRE digestion product and the RPA-Cas12a reagent;

[0014] And, the first intermediate sample injection unit, used to separate the mixed sample and the separating medium into liquid phases by using density difference;

[0015] And, the first digital detection unit, used for single-molecule fluorescence detection;

[0016] And, fluid channels for connecting the above-mentioned structural units;

[0017] The second fluid channel network, from upstream to downstream, includes the following sequentially connected channels:

[0018] The second reaction chamber is used for control treatment of the sample to be tested;

[0019] And, the second mixing unit, used to mix the product after control treatment with RPA-Cas12a reagent;

[0020] And, the second intermediate sample injection unit, used to separate the mixed sample and the separating medium by density difference into liquid phases;

[0021] And, a second digital detection unit, used for single-molecule fluorescence detection;

[0022] And, fluid channels for connecting the above-mentioned structural units;

[0023] The first fluid channel network and the second fluid channel network have exactly the same structure and size, and the first fluid channel network and the second fluid channel network are arranged in a mirror symmetry along the central axis of symmetry of the chip body.

[0024] The microfluidic chip disclosed in this invention, through its innovative chip structure design and external temperature control and fluid actuation devices, can perform variable-temperature enzyme digestion, inactivation, mixing, and isothermal amplification processes. This invention eliminates the need for bisulfite treatment and achieves integrated absolute quantitative detection from sample in to result out through optimized flow path design. It offers advantages such as full enclosure, contamination prevention, and ease of operation, making it particularly suitable for rapid on-site detection. Furthermore, this invention ensures that the test samples react in parallel under completely consistent temperature control, fluid environment, and amplification system by designing a physically symmetrical dual-fluidic channel network of enzyme digestion and control channels within the chip. This internal reference design minimizes chip variations and operational errors, resulting in higher quantitative accuracy.

[0025] Preferred:

[0026] The chip body also includes a sample introduction unit, through which the sample to be tested is evenly distributed to the first reaction chamber and the second reaction chamber.

[0027] Further preferably, the sample introduction unit includes a sample injection port and a diversion channel communicating with the sample injection port. The sample to be tested is specifically distributed evenly to the first reaction chamber and the second reaction chamber via the diversion channel.

[0028] Preferably, the first reaction chamber is disposed within the reaction layer; the first reaction chamber is pre-filled with MSRE reaction reagent. More preferably, the MSRE reaction reagent is covered with light mineral oil to prevent evaporation.

[0029] Preferably, the second reaction chamber is disposed within the reaction layer; a non-enzyme buffer solution is pre-placed within the second reaction chamber for use as a total assay control. More preferably, the non-enzyme buffer solution is covered with light mineral oil to prevent evaporation.

[0030] Preferably, a temperature control device I is provided outside the first reaction chamber and the second reaction chamber. More preferably, the temperature control device I is selected from flexible heating devices, such as PI heating films.

[0031] The first and second reaction chambers are located in the reaction layer to facilitate independent temperature control.

[0032] In this invention, the first mixing unit is located downstream of the first reaction chamber. Preferably, the first mixing unit includes a reagent chamber and a first injection piston assembly that moves within the chamber in a sealed manner. The reagent chamber is pre-filled with RPA-Cas12a amplification detection reagent. The first injection piston assembly is capable of performing axial pulling and pressing actions to draw upstream enzyme digestion products into the chamber for mixing and to push the mixture downstream.

[0033] In a further preferred embodiment, the first injection piston assembly includes a push rod and a sealing plug. The push rod is embedded in the chip body, and the bottom end of the push rod is sealed to the reagent chamber. By pulling or pressing the push rod with an external driving force, the effective volume of the reagent chamber can be changed, thereby realizing the aspiration and mixing of fluids and quantitative injection.

[0034] Preferably, the reagent chamber is disposed within the channel layer, and the first injection piston assembly penetrates the reaction layer and extends into the reagent chamber.

[0035] Preferably, the second mixing unit includes a reagent chamber and a second injection piston assembly that moves within the chamber under a seal; the reagent chamber is pre-filled with RPA-Cas12a amplification detection reagent. The second injection piston assembly is capable of performing axial pulling and pressing actions to draw the upstream control-treated product into the chamber for mixing and to push the mixture downstream.

[0036] Further preferably, the second injection piston assembly also includes a push rod and a sealing plug.

[0037] Preferably, the reagent chamber is disposed within the channel layer, and the second injection piston assembly penetrates the reaction layer and extends into the reagent chamber.

[0038] In this invention, the first transfer and injection unit is located downstream of the first mixing unit. Preferably, the first transfer and injection unit includes a first sealing film, a cap with a puncture needle, and a first transfer and storage chamber sealed by the two.

[0039] The first transit storage chamber is located within the channel layer; the first sealing film is located between the channel layer and the detection layer.

[0040] Preferably, the first sealing film can be made of plastic film or plastic tape, such as common types like PC film and PP film.

[0041] Preferably, the first transfer storage chamber is pre-filled with a separating medium, such as light mineral oil; the volume of the first transfer storage chamber is configured to accommodate the injected reaction mixture and allow the reaction mixture to settle below the separating medium by density difference to form stratification.

[0042] The second transfer injection unit includes a second sealing film, a cap with a puncture needle, and a second transfer storage chamber sealed by the two.

[0043] The second transfer storage chamber is located within the channel layer; the second sealing film is located between the channel layer and the detection layer.

[0044] Preferably, the second sealing film can be made of plastic film or plastic tape, such as common types like PC film and PP film.

[0045] Preferably, the second transfer storage chamber is pre-filled with a separating medium, such as light mineral oil; the volume of the second transfer storage chamber is configured to accommodate the pushed-in reaction mixture and allow the reaction mixture to settle below the separating medium by density difference to form stratification.

[0046] Once the sealing membrane is punctured, the pressure difference can be used to drive the stratified fluid into the downstream digital detection unit at the moment of connection.

[0047] In this invention, a valve assembly for coordinated control of fluid transfer is provided in the fluid channel network.

[0048] Preferred:

[0049] A first valve I is provided on the fluid channel between the first reaction chamber and the first mixing unit to block the upstream flow path during mixing and sample injection.

[0050] A first valve II is provided on the fluid channel between the first mixing unit and the first transfer injection unit to control the opening and closing of the channel for the mixed liquid to enter the first transfer injection unit.

[0051] A second valve I is provided on the fluid channel between the second reaction chamber and the second mixing unit to block the upstream flow path during mixing and sample injection.

[0052] A second valve II is provided on the fluid channel between the second mixing unit and the second transfer injection unit to control the opening and closing of the channel for the mixed liquid to enter the second transfer injection unit.

[0053] Preferred:

[0054] The first digital detection unit includes a microcavity array; the first digital detection unit is disposed within the detection layer.

[0055] Preferred:

[0056] The second digital detection unit includes a microcavity array;

[0057] The second digital detection unit is disposed within the detection layer;

[0058] The digital detection unit has a pre-placed separating oil phase at the inlet, and the microcavity array is in a pre-vacuum state to assist the mixed fluid in entering the microcavity and achieving droplet separation.

[0059] Preferably, the first digital detection unit and the second digital detection unit are equipped with a temperature control device II.

[0060] In this invention, the reaction layer is located on the top layer of the chip body, corresponding to the reaction chamber upstream of the chip. Its independent protruding structure design allows the temperature control device I to be tightly wrapped around the perimeter and top of the reaction chamber, thereby independently meeting the specific temperature variations required for MSRE enzyme digestion and inactivation reactions, ensuring heat conduction efficiency and uniformity, and avoiding thermal interference to other layers.

[0061] The channel layer is located in the middle layer of the chip body and is mainly made of PDMS. This layer integrates a complex microfluidic channel network, valve control chambers, and hybrid structures, playing a crucial role in fluid transport and logic control.

[0062] The detection layer is located at the bottom of the chip body and mainly contains digital detection units.

[0063] Preferably, a substrate layer is disposed beneath the chip body. The substrate layer is made of a material with good optical transmittance (such as quartz glass or optical-grade plastic) to provide rigid support for the microcavity array and meet the optical requirements for subsequent fluorescence signal acquisition.

[0064] Preferably, the detection layer is directly attached to the substrate layer to ensure the flatness of the bottom of the microcavity array, so as to facilitate the adaptation of the temperature control device II at the bottom for isothermal amplification.

[0065] The microfluidic chip disclosed in this invention can be operated in conjunction with an external temperature control device, a fluid actuator, and an optical detection device. The external temperature control device is used for independent temperature control of the microfluidic chip in different zones. Temperature control device I can control the heating and cooling of the first and second reaction chambers to achieve cooling of the products after enzyme digestion and inactivation. Temperature control device II can control the isothermal amplification of the digital detection unit. The fluid actuator (or manual operation) works in conjunction with the injection piston assembly and valve assembly on the chip to apply axial force to the injection piston assembly to change the chamber volume and apply pressure to the valve assembly to control the flow path opening and closing. The optical detection device is located on one side of the digital detection unit of the microfluidic chip and is used to collect the fluorescence signal of the microcavity array.

[0066] This invention also discloses a method for detecting DNA methylation, employing the integrated microfluidic chip that combines MSRE enzyme digestion and digital detection, comprising:

[0067] a. Sample introduction and splitting: The nucleic acid sample to be tested is injected into the chip body through the sample introduction unit and split evenly into the first reaction chamber and the second reaction chamber;

[0068] b. Differentiated processing: Temperature control is performed by temperature control device I to ensure that the nucleic acid samples to be tested in the first reaction chamber and the second reaction chamber complete the enzyme digestion reaction and the control reaction, respectively, and then the temperature is raised to inactivate and then cooled.

[0069] c. Closed-loop mixing: Within the first fluid channel network, open the first valve I, close the first valve II, and pull the first injection piston assembly upward. Utilize the generated negative pressure to draw the cooled reaction product into the reagent chamber, where it is mixed with the pre-placed RPA-Cas12a amplification detection reagent to obtain a mixed solution.

[0070] The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network.

[0071] d. Stratification and transfer: Within the first fluid channel network, close the first valve I, open the first valve II, press down on the first injection piston assembly to push the mixture into the first intermediate transfer injection unit, where it is stratified with the pre-set separating medium using density difference;

[0072] The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network.

[0073] e. Negative pressure injection: In the first fluid channel network, the sealing cap with the puncture needle pierces the first sealing film, and the mixture is driven into it by the preset negative pressure in the first digital detection unit;

[0074] The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network.

[0075] f. Absolute quantitative detection: Within the first fluid channel network, the first digital detection unit is heated at a constant temperature using temperature control device II to initiate the amplification reaction and collect fluorescence signals;

[0076] The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network.

[0077] Preferably, in step b, the specific sequence of temperature control is as follows: controlling the first reaction chamber to be kept at 37°C for a predetermined time for enzyme digestion, while controlling the second reaction chamber to be kept at 37°C for the same time for control reaction; raising the temperature to 65°C and keeping it for a predetermined time for enzyme inactivation; and then actively lowering the temperature (e.g., to below 40°C) to prevent high temperature from damaging the activity of the downstream RPA-Cas12a amplification detection reagent.

[0078] Preferably, in step f, based on the Poisson distribution principle, the number of methylated DNA copies is calculated according to the number of positive microcavities in the corresponding channel of the first reaction chamber, and the total number of DNA copies is calculated according to the number of positive microcavities in the corresponding channel of the second reaction chamber. Then, the methylation level of the nucleic acid sample to be tested is obtained by the ratio method.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] 1. This invention is the first to achieve a fully integrated "sample in, result out" process in DNA methylation detection, significantly reducing the risk of cross-contamination. Through an innovative microfluidic chip structure design, this invention integrates methylation-sensitive restriction enzyme digestion, enzyme inactivation, reagent mixing, fluid stratification, and digital isothermal amplification into a single, enclosed chip. Compared to traditional step-by-step operations, this invention avoids cumbersome manual pipetting, centrifugation, and transfer operations, effectively isolating aerosol contamination through a fully enclosed flow path, greatly improving the reliability of the detection results.

[0081] 2. This invention eliminates systematic errors and ensures the accuracy of methylation level calculations through a dual-channel parallel design within the chip. The invention designs physically symmetrical enzyme digestion and control channels on the chip, ensuring that the test samples undergo parallel reactions under completely consistent temperature control conditions, fluid environment, and amplification system. This internal chip reference design minimizes inter-well differences and operational errors, resulting in higher quantitative accuracy of the detection method.

[0082] 3. The microfluidic chip provided by this invention has a high degree of integration, reducing reliance on external supporting equipment and exhibiting strong field adaptability. Because the chip integrates the key physical structures required for fluid control and reaction, it does not require large and complex professional laboratory instruments during use. It only needs basic temperature control and auxiliary actuation devices (compatible with manual operation and portable heating devices) to complete the detection. This low-barrier characteristic makes the chip ideal for early cancer screening and genetic disease diagnosis in primary healthcare institutions, community clinics, or resource-constrained field environments.

[0083] 4. The detection method provided by this invention does not rely on bisulfite treatment or a standard curve, achieving precise absolute quantification of DNA methylation levels. Compared to the traditional methylation-specific polymerase chain reaction (MSP) method, this invention employs a mild enzymatic digestion method, avoiding DNA degradation and breakage caused by bisulfite treatment and preserving sample integrity. Simultaneously, single-molecule counting is performed using a digital detection unit (microcavity array) integrated on the chip, directly calculating the absolute copy number of methylated DNA and total DNA without the need to construct a standard curve, effectively solving the problem of insufficient accuracy of qPCR relative quantification in low-abundance sample detection.

[0084] 5. This invention, based on an optimized purification-free process and a unique chip structure, significantly simplifies the operation process and improves detection efficiency. Utilizing an optimized reaction system, the enzyme digestion products can directly enter the downstream amplification reaction without undergoing complex nucleic acid extraction and purification steps. Combined with the chip's unique injection piston assembly and intermediate sample injection unit design, the mechanical structure enables rapid mixing of enzyme digestion products and amplification reagents in a closed environment, along with natural liquid-phase separation and injection, reducing the originally complex process that took several hours to approximately 1.5 hours. Attached Figure Description

[0085] Figure 1 This is a schematic diagram of the overall structure of the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention.

[0086] Figure 2 This is a schematic diagram of the layered exploded structure of the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention.

[0087] Figure 3 This is a three-dimensional perspective schematic diagram of the fluid channel orientation and flow path connectivity within the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention.

[0088] Figure 4This is a side cross-sectional view of the reagent preloading position and the layout of the external temperature control components in the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention.

[0089] Figure 5 This is a cross-sectional schematic diagram of the working principle of the valve component in the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention; wherein, (a) is the valve open state, (b) is the valve closed state under pressure, (c) is the closed state of the sealing film in the first transfer and injection unit before puncture, and (d) is the conductive state of the sealing film in the first transfer and injection unit after puncture under pressure.

[0090] Figure 6 This is a schematic diagram of the process for fluid manipulation and reaction using the integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection disclosed in this invention.

[0091] Figure label:

[0092] 1-Sample introduction unit; 2-First reaction chamber; 3-Second reaction chamber; 4-First valve I; 5-Second valve I; 6-First mixing unit; 7-Second mixing unit; 8-First valve II; 9-Second valve II; 10-First transfer and injection unit; 11-Second transfer and injection unit; 12-First digital detection unit; 13-Second digital detection unit; 14-Fluid channel; 15-Temperature control device I; 16-Temperature control device II;

[0093] 10 - Reaction layer; 20 - Channel layer; 30 - Detection layer; 40 - Base layer;

[0094] 61-Reagent chamber; 62-Injection piston assembly;

[0095] 101 - Transfer storage chamber; 102 - Sealing membrane; 103 - Hole cap with puncture needle; Detailed Implementation

[0096] To further understand the present invention, the present invention will be described in detail below with reference to the accompanying drawings and embodiments. However, the present invention is not limited to these embodiments. Non-essential improvements and adjustments made by those skilled in the art under the core guiding principles of the present invention are still within the scope of protection of the present invention.

[0097] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0098] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0099] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0100] It should be noted that, unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. Where the terms "first," "second," etc., are used throughout the text, they are used only to distinguish similar objects and should not be construed as indicating or implying their relative importance, order of precedence, or implicitly specifying the number of technical features indicated. It should be understood that the data in the descriptions of "first," "second," etc., can be interchanged where appropriate.

[0101] Example

[0102] Reference Figures 1-5 This invention discloses an integrated microfluidic chip that integrates MSRE enzyme digestion and digital detection, comprising a chip body and a base layer 40.

[0103] The chip body has a vertically layered structure, including a reaction layer 10, a channel layer 20 and a detection layer 30 from top to bottom; the layers are tightly connected by oxygen plasma bonding or adhesive bonding processes.

[0104] The chip body includes a first fluid channel network and a second fluid channel network. The first fluid channel network and the second fluid channel network have the same structure and size. Furthermore, the first fluid channel network and the second fluid channel network are arranged in a mirror symmetrical manner along the central axis of symmetry of the chip body.

[0105] Since the first fluid channel network and the second fluid channel network have the same structure and size and are completely symmetrical in position, the following description will only focus on the first fluid channel network. The second fluid channel network is an identical design and will not be described in detail.

[0106] The first fluid channel network is arranged sequentially from upstream to downstream of the chip body, including a first reaction chamber 2, a first valve I 4, a first mixing unit 6, a first valve II 8, a first transfer and injection unit 10, and a first digital detection unit 12. The above structure is arranged in the reaction layer 10, the channel layer 20, and the detection layer 30, and is connected through the fluid channel 14.

[0107] The first reaction chamber 2 is located in the reaction layer 10; it is pre-filled with MSRE reaction reagents for MSRE digestion and inactivation of the nucleic acid sample to be tested.

[0108] To complement the function of the first reaction chamber 2, the topmost reaction layer 10 adopts a raised structure design so that the external flexible temperature control device I15 (such as a PI heating film) can tightly wrap around the perimeter and top of the reaction chamber, thereby individually meeting the specific temperature variation conditions (37℃~65℃) required for MSRE enzyme digestion and inactivation reactions, and ensuring heat conduction efficiency and uniformity, avoiding thermal interference to other layers.

[0109] The second reaction chamber 3 is exactly the same as the first reaction chamber 2, except that the second reaction chamber 3 is pre-filled with a non-enzyme buffer solution, which is used as a total control.

[0110] The first valve I4 is located in the channel layer 20, downstream of the first reaction chamber 2. The first valve I4 adopts an elastic blocking design. During the reaction, the valve is closed to prevent fluid escape caused by the gas pressure generated during high-temperature inactivation; after the reaction is completed, it opens to allow fluid to pass through.

[0111] The first mixing unit 6 is located downstream of the first valve I4. The first mixing unit 6 integrates a reagent chamber 61 and an injection piston assembly 62. The reagent chamber 61 is pre-filled with RPA-Cas12a amplification detection reagent for amplifying MSRE digestion products. The reagent chamber 61 is disposed within the channel layer 20, and the injection piston assembly 62 penetrates the reaction layer 10 and extends into the reagent chamber. The injection piston assembly 62 further includes a push rod and a sealing plug, configured to reciprocate axially within the chamber. When pulled upwards, a negative pressure is generated to draw in the upstream product and mix it within the chamber using turbulent flow; when pressed downwards, a positive pressure is generated to drive the mixture downstream.

[0112] The first valve II8 has the same structure as the first valve I4, and is located in the channel layer 20, downstream of the first mixing unit 6. It is used to precisely control the unidirectional flow of fluid in conjunction with the pull / press operation of the piston assembly.

[0113] The first transfer and injection unit 10 is located between the first mixing unit 6 and the first digital detection unit 12. The first transfer and injection unit 10 includes a sealing film 102, a sealing cap 103 with a puncture needle, and a transfer and storage chamber 101 formed by the sealing of the two; wherein, the transfer and storage chamber 101 is disposed within the channel layer 20, and the sealing film 102 is a polypropylene film located between the channel layer 20 and the detection layer 30.

[0114] The intermediate storage chamber 101 is pre-filled with a separating medium (such as light mineral oil) with a density less than that of the reaction mixture. When the aqueous phase of the reaction mixture is pushed into this chamber, it will naturally settle below the oil layer, forming a stable oil-water stratified structure, thus preparing the liquid phase for subsequent digital injection. Before digital injection, the initial sealing film 102 is intact, maintaining downstream vacuum; during digital detection, the sealing cap 103 with a puncture needle punctures the sealing film 102, triggering negative pressure injection.

[0115] The channel layer 20 is mainly made of PDMS and is located in the middle layer. This layer integrates a complex microfluidic channel network, valve control chamber and hybrid structure, and plays a role in fluid transmission and logic control.

[0116] The first digital detection unit 12, located at the downstream end of the chip body and disposed within the detection layer 30, contains tens of thousands of micron-sized reaction chambers. This unit is pre-vacuumed and sealed before the chip leaves the factory. When the sealing film 102 is punctured, the layered reaction mixture and the separating medium are sequentially drawn into the microcavity array using a pre-set negative pressure driving force, achieving single-molecule-level droplet separation and digital amplification detection.

[0117] The detection layer 30 is located below the channel layer 20 and directly attached to the substrate layer 40 to ensure the flatness of the bottom of the microcavity array, so as to adapt to the bottom temperature control device II 16 (such as a flat plate heating device) for isothermal amplification.

[0118] The substrate 40 is located at the bottom of the chip body and is made of a material with good optical transmittance (such as quartz glass or optical-grade plastic) to provide rigid support for the microcavity array and meet the optical requirements for subsequent fluorescence signal acquisition.

[0119] The chip body also includes a sample introduction unit 1, which includes a sample injection port and a flow channel connected to the sample injection port. The sample to be tested is evenly distributed to the first reaction chamber 2 and the second reaction chamber 3 via the flow channel.

[0120] Application examples

[0121] DNA methylation detection was performed using the integrated microfluidic chip combining MSRE enzyme digestion and digital detection as described in the above embodiments. The chip body disclosed in this embodiment has two parallel fluid channel networks internally. After entering from the sample introduction unit 1, the fluid is automatically and evenly distributed to the two parallel reaction branches, where it undergoes differential processing: the enzyme digestion reaction occurs in the first reaction chamber 2, and the control reaction occurs in the second reaction chamber 3. Subsequently, they converge to the mixing unit for mixing, and finally, the fluid enters the terminal detection unit via the intermediate sample injection unit.

[0122] Before the detection operation, the microfluidic chip is in a pre-loaded state, as shown in Figure 4. All the reagents required for the reaction are pre-loaded in each functional unit:

[0123] The first reaction chamber 2 is pre-filled with MSRE enzyme digestion reagent; the second reaction chamber 3 is pre-filled with control buffer and covered with light mineral oil to prevent evaporation.

[0124] Magnesium acetate initiator is pre-placed in the downstream channels of the first reaction chamber 2 and the second reaction chamber 3, respectively.

[0125] The amplification reagent chamber 61 is pre-filled with RPA-Cas12a reaction reagent;

[0126] The transit storage chamber 101 is pre-filled with light mineral oil as a separating medium;

[0127] The first digital detection unit 12 and the second digital detection unit 13 have been pre-vacuumed and sealed with a sealing film.

[0128] A schematic diagram of the step-by-step detection operation process is shown below. Figure 6 As shown, the specific process is as follows:

[0129] Step a: Sample injection and splitting. The nucleic acid sample to be tested is injected through sample introduction unit 1. As shown by the arrow in Figure 6(a), under the action of fluid pressure, the sample is automatically and equally distributed into the first reaction chamber 2 and the second reaction chamber 3. This process takes about 15 seconds.

[0130] Step b: Enzyme digestion reaction. For example... Figure 6 As shown in (b), temperature control device I15 is activated to maintain the temperature of the first reaction chamber 2 at 37°C. During this period, the MSRE enzyme specifically cleaves the unmethylated DNA in the sample. This process takes approximately 15 minutes.

[0131] Step c: Enzyme inactivation. For example... Figure 6 As shown in (c), the temperature control device I15 is heated to 65°C and maintained for a certain period of time to completely inactivate the restriction endonuclease. After inactivation, this area of ​​the chip is allowed to cool naturally or actively (to below 40°C). This process takes about 5 minutes.

[0132] Step d: Negative pressure mixing. (e.g.) Figure 6 As shown in (d), a downward external force F is applied to the first valve II8 to close it, blocking the downstream pathway. Then, the first valve I4 is opened, and the injection piston assembly 62 is pulled upwards (external force F upwards). This action generates negative pressure in the amplification reagent chamber 61, drawing in the upstream enzyme digestion product and magnesium acetate from the channel, achieving thorough mixing with the RPA-Cas12a reagent through turbulence. This process takes approximately 15 seconds.

[0133] Step e: Fluid transfer. For example... Figure 6 As shown in (e), the external force on the first valve II8 is removed to open it; simultaneously, a downward external force F is applied to the first valve I4 to block the upstream backflow path. Then, the injection piston assembly 62 is pressed downwards (external force F downwards), driving the reaction mixture into the first intermediate transfer injection unit 10. The mixture settles below the light mineral oil here, forming stratification. This process takes approximately 15 seconds.

[0134] Step f: Preparation for puncture and sample injection. (e.g.) Figure 6 As shown in (f), a downward external force F is applied to the first valve II8 again to close the upstream passage and prevent backflow. At the same time, a downward external force F is applied to the sealing cap 103 with the puncture needle to drive the puncture needle to puncture the sealing film 102 and open the flow path.

[0135] Step g: Automatic negative pressure sample injection. (e.g.) Figure 6 As shown in (g), driven by a pre-set negative pressure inside the first digital detection unit 12, the lower layer of reaction reagent in the first intermediate transfer and injection unit 10 is preferentially drawn in, followed by the upper layer of mineral oil, thus achieving droplet generation and physical separation in the microcavity array. This process takes about 2 minutes.

[0136] Step h: Isothermal amplification. For example... Figure 6 As shown in (h), the temperature control device II16 is activated to heat the digital detection unit area to 39°C and maintain this temperature for isothermal amplification of RPA-Cas12a. This process takes approximately 44 minutes. After the reaction is complete, fluorescence signal readings and quantitative analysis are performed.

[0137] The integrated device provided in this application achieves seamless integration of nucleic acid extraction and digital detection. Through a fully enclosed flow path design, it not only significantly simplifies the operation process and automates the "sample in—result out" process, but also effectively eliminates aerosol contamination during the experiment, ensuring biosafety and the reliability of the test results.

[0138] In summary, this invention, with its integrated structural design and precise digital detection capabilities, can meet the clinical needs for rapid and accurate screening of infectious pathogens. It is also applicable to fields such as genetic disease diagnosis and tumor liquid biopsy, and has significant clinical application value and broad market prospects.

[0139] The above-disclosed embodiments are preferred embodiments, but the scope of protection of the present invention is not limited thereto. Those skilled in the art can easily understand the spirit of the present invention based on the above embodiments and make different extensions and variations, but as long as they do not depart from the spirit of the present invention, they are all within the scope of protection of the present invention.

Claims

1. An integrated microfluidic chip combining MSRE enzyme digestion and digital detection, comprising a chip body, characterized in that: The chip body comprises, from top to bottom, a reaction layer, a channel layer, and a detection layer, which are sequentially sealed along the vertical direction. The chip body includes a first fluid channel network and a second fluid channel network, which are disposed in the reaction layer, the channel layer and the detection layer; The first fluid channel network includes the following sequentially connected components: The first reaction chamber is used for MSRE digestion and inactivation of the test sample; And, the first mixing unit, used to mix the MSRE digestion product and the RPA-Cas12a reagent; And, the first intermediate sample injection unit, used to separate the mixed sample and the separating medium into liquid phases by using density difference; And, the first digital detection unit, used for single-molecule fluorescence detection; And, fluid channels for connecting the above-mentioned structural units; The second fluid channel network includes the following sequentially connected components: The second reaction chamber is used for control treatment of the sample to be tested; And, the second mixing unit, used to mix the product after control treatment with RPA-Cas12a reagent; And, the second intermediate sample injection unit, used to separate the mixed sample and the separating medium by density difference into liquid phases; And, a second digital detection unit, used for single-molecule fluorescence detection; And, fluid channels for connecting the above-mentioned structural units; The first fluid channel network and the second fluid channel network have exactly the same structure and size, and the first fluid channel network and the second fluid channel network are arranged in a mirror symmetry along the central axis of symmetry of the chip body.

2. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: The chip body also includes a sample introduction unit, through which the sample to be tested is evenly distributed to the first reaction chamber and the second reaction chamber.

3. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: The first reaction chamber is disposed within the reaction layer; The first reaction chamber is pre-filled with MSRE reaction reagent; The second reaction chamber is disposed within the reaction layer; The second reaction chamber is pre-filled with a non-enzyme buffer solution. Temperature control device I is installed outside the first reaction chamber and the second reaction chamber.

4. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: The first mixing unit includes a reagent chamber and a first injection piston assembly that moves within the chamber under a seal; The reagent chamber is pre-filled with RPA-Cas12a amplification detection reagent; The reagent chamber is disposed within the channel layer, and the first injection piston assembly penetrates the reaction layer and extends into the amplification reagent chamber; The second mixing unit includes a reagent chamber and a second injection piston assembly that moves within the chamber under a seal; The reagent chamber is pre-filled with RPA-Cas12a amplification detection reagent; The reagent chamber is disposed within the channel layer, and the second injection piston assembly penetrates the reaction layer and extends into the reagent chamber.

5. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: The first transfer injection unit includes a first sealing film, a cap with a puncture needle, and a first transfer storage chamber sealed by the two. The first transit storage chamber is pre-filled with a separating medium and is disposed within the channel layer; The first sealing film is located between the channel layer and the detection layer; The second transfer injection unit includes a second sealing film, a cap with a puncture needle, and a second transfer storage chamber sealed by the two. The second transfer storage chamber is pre-filled with a separating medium and is located within the channel layer; The second sealing film is located between the channel layer and the detection layer.

6. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: A first valve I is provided on the fluid channel between the first reaction chamber and the first mixing unit; A first valve II is provided on the fluid channel between the first mixing unit and the first intermediate sample injection unit; A second valve I is provided on the fluid channel between the second reaction chamber and the second mixing unit; A second valve II is provided on the fluid channel between the second mixing unit and the second intermediate sample injection unit.

7. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that: The first digital detection unit includes a microcavity array; The first digital detection unit is disposed within the detection layer; The second digital detection unit includes a microcavity array; The second digital detection unit is disposed within the detection layer; Both the first digital detection unit and the second digital detection unit are equipped with a temperature control device II.

8. The integrated microfluidic chip combining MSRE enzyme digestion and digital detection according to claim 1, characterized in that, A base layer is disposed beneath the chip body.

9. A method for detecting DNA methylation, characterized in that, The integrated microfluidic chip according to any one of claims 1 to 8, comprising: a. Sample introduction and splitting: The nucleic acid sample to be tested is injected into the chip body through the sample introduction unit and split evenly into the first reaction chamber and the second reaction chamber; b. Differentiated processing: Temperature control is performed by temperature control device I to ensure that the nucleic acid samples to be tested in the first reaction chamber and the second reaction chamber complete the enzyme digestion reaction and the control reaction, respectively, and then the temperature is raised to inactivate and then cooled. c. Closed-loop mixing: Within the first fluid channel network, open the first valve I, close the first valve II, and pull the first injection piston assembly upward. Utilize the generated negative pressure to draw the cooled reaction product into the reagent chamber, where it is mixed with the pre-placed RPA-Cas12a amplification detection reagent to obtain a mixed solution. The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network. d. Stratification and transfer: Within the first fluid channel network, close the first valve I, open the first valve II, press down on the first injection piston assembly to push the mixture into the first intermediate transfer injection unit, where it is stratified with the pre-set separating medium using density difference; The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network. e. Negative pressure injection: In the first fluid channel network, the sealing cap with the puncture needle pierces the first sealing film, and the mixture is driven into it by the preset negative pressure in the first digital detection unit; The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network. f. Absolute quantitative detection: Within the first fluid channel network, the first digital detection unit is heated at a constant temperature using temperature control device II to initiate the amplification reaction and collect fluorescence signals; The same operations as those in the first fluid channel network are performed synchronously within the second fluid channel network.

10. The DNA methylation detection method according to claim 9, characterized in that, The methylated DNA copy number is calculated based on the number of positive microcavities in the corresponding channel of the first reaction chamber in step f, and the total DNA copy number is calculated based on the number of positive microcavities in the corresponding channel of the second reaction chamber in step f. The methylation level of the nucleic acid sample to be tested is then obtained by the ratio method.

Images