Digital mira instant diagnostic chip, analysis device and method

Abstract

The application belongs to the technical field of microfluidic chip, and specifically provides a digital MIRA instant diagnosis chip, an analysis device and a method, wherein the digital MIRA instant diagnosis chip comprises a top glass slide, a micro-pattern layer and a bottom glass slide arranged in sequence from top to bottom; a water-soluble film is embedded at the matching position of the micro-pattern layer and the water inlet; and the sample inlet is adapted to inject reaction reagents and sealing oil in sequence. Through the chip structure design without outlet, when the reaction reagents are introduced from the sample inlet, all the reaction reagents enter the microchambers simultaneously, equally and uniformly, and the reaction reagents are separated into the microchambers in the form of oil sealing, so that the sample has the advantage of 100% sample discretization, and there is no loss of any sample and reagent. At the same time, since each portion of the reaction reagent is accurately separated into the microchambers and cannot be pushed out of the chip, each molecule enters the corresponding microchamber, thereby meeting the detection requirement of low-concentration templates.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a digital MIRA real-time diagnostic chip, analysis device, and method. Background Technology

[0002] Point-of-care testing (POCT) refers to a testing method that utilizes portable analytical instruments and accompanying reagents to quickly obtain test results at the sampling site. The main criteria for POCT are that it does not require a fixed testing location and only requires simple steps to obtain results. In recent years, due to rapid technological advancements and efficient, fast-paced work methods, POCT, with its miniaturized instruments, simplified procedures, and immediate results, has become increasingly popular in various medical settings.

[0003] POCT testing requires the use of a digital MIRA point-of-care diagnostic chip, which is a microfluidic chip that integrates sample pretreatment and multiplex digital recombinase polymerase amplification detection. The chip's design allows the entire process from sample dispensing to nucleic acid amplification and detection to be completed on a single platform.

[0004] In related technologies, digital MIRA point-of-care diagnostic chips only allow a portion of the reagent to enter the microchamber for amplification when introduced; the remainder in the microchannels is ejected from the chip, resulting in reagent loss. If the reagent contains only two molecules, it's possible that no molecules will enter the microchamber. Therefore, traditional digital nucleic acid detection chips are insufficient for detecting low-concentration templates.

[0005] How to avoid wasting samples from digital MIRA point-of-care diagnostic chips in order to meet the detection needs of low-concentration templates is a technical problem that urgently needs to be solved.

[0006] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0007] This disclosure provides at least one digital MIRA real-time diagnostic chip, analysis device, and method.

[0008] In a first aspect, embodiments of this disclosure provide a digital MIRA point-of-care diagnostic chip, which has a top slide, a micropattern layer and a bottom slide arranged sequentially from top to bottom; The micropatterned layer is connected to the sample injection port and water injection port of the top glass slide; A water-soluble film is embedded at the fitting point between the micropattern layer and the water inlet; The injection port is suitable for sequentially injecting reaction reagents and sealing oil.

[0009] In one optional implementation, the micropatterned layer includes a flow channel module, a micro-chamber array module, and a vacuum storage battery module; The micro-chamber array module and the vacuum storage battery module are arranged side by side; One end of the flow channel module is connected to the sample injection port of the top glass slide, and the other end is connected to the microchamber array module; The water-soluble film is disposed above the vacuum storage battery module.

[0010] In one alternative implementation, the microcell array module includes multiple microcells with fractal symmetry structures.

[0011] In one optional implementation, the flow channel module is a T-shaped branch channel network, with the end of each branch channel connected to the corresponding microchamber; Furthermore, the length of the microchannel from the injection port to the microchamber is the same for each of the branch channels.

[0012] In one alternative embodiment, the vacuum storage battery module includes: a series of batteries connected in multiple stages; The adjacent batteries are independent and not connected, and a thin wall is provided between the adjacent batteries, which is suitable for discharging the gas in the space where the battery is located from the thin wall into the flow channel module, discharging the chip, and forming a vacuum in the space where the battery is located. The water-soluble film is disposed above the battery adjacent to the microcell array module.

[0013] In one optional embodiment, the sample inlet and water inlet of the top glass slide are respectively connected to a liquid storage tank and a water storage tank.

[0014] In one alternative embodiment, both the top and bottom glass slides are made of an incompressible, airtight, and thermally conductive transparent material.

[0015] In one alternative implementation, the micropatterned layer is made of a compressible, internally porous, transparent material.

[0016] Secondly, embodiments of this disclosure also provide an analysis apparatus, comprising: The device itself, the smart terminal, and the digital MIRA real-time diagnostic chip as described above; The device body is equipped with a chip holder and a coaxial light source inside. The top of the device body is provided with a fixing groove suitable for fixing a smart terminal; The digital MIRA real-time diagnostic chip is placed on the chip holder; The smart terminal is fixed on the fixing slot and positioned directly above the coaxial light source; The chip holder is positioned directly below the coaxial light source.

[0017] Thirdly, this disclosure also provides an analysis method, the method comprising: Place the digital MIRA real-time diagnostic chip in a vacuum environment and degas it under preset negative pressure for 2-6 hours; Open the vacuum packaging and remove the digital MIRA point-of-care diagnostic chip; The reaction reagent and sealing oil are added to the storage tank in sequence. Under the action of vacuum and atmospheric pressure, the reaction reagent enters the microchannel rapidly in the order of reaction reagent first and sealing oil last. After N fractal structures, the reagent will fill each microchamber in equal and uniform amounts, and the reaction reagent will be separated into each microchamber by the sealing oil. Injecting deionized water into the water storage tank dissolves the water-soluble film, allowing the deionized water to fill part of the battery in the vacuum storage battery module. The digital MIRA point-of-care diagnostic chip is placed on a heating pack to perform a digital amplification reaction; The amplified digital MIRA point-of-care diagnostic chip is placed on the chip holder of the analysis device for analysis.

[0018] The beneficial effects of this invention are that the digital MIRA point-of-care diagnostic chip, analysis device, and method, through an outlet-free chip structure design, ensure that when reaction reagents are introduced from the sample injection port, all reaction reagents simultaneously, equally, and uniformly enter each microchamber. Furthermore, an oil-sealing method is used to separate the reaction reagents into each microchamber, providing the advantage of 100% sample discretization during digital nucleic acid detection, with no sample or reagent loss. Simultaneously, because each reaction reagent is precisely separated into its respective microchamber, it will not be ejected from the chip, ensuring that each molecule enters its corresponding microchamber, thus meeting the detection requirements for low-concentration templates.

[0019] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained in accordance with the structures particularly pointed out in the description, claims and drawings.

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a structural diagram of the digital MIRA instant diagnostic chip provided in an embodiment of this disclosure.

[0023] Figure 2 This is a schematic diagram of the structure of the micropatterned layer provided in an embodiment of this disclosure.

[0024] Figure 3 This is a partial structural diagram of the analysis device provided in an embodiment of this disclosure.

[0025] Figure 4 This is a schematic diagram showing the injection of reaction reagents and sealing oil into the microchamber according to embodiments of this disclosure.

[0026] Figure 5 A schematic diagram of an oil seal provided in an embodiment of this disclosure.

[0027] Figure 6 This is a schematic diagram illustrating the principle of preventing evaporation of deionized water as provided in an embodiment of this disclosure.

[0028] In the figure: 110, bottom slide; 120, micropatterned layer; 121, flow channel module; 122, microchamber array module; 123, vacuum storage battery module; 130, water-soluble film; 140, top slide; 141, sample inlet; 142, water inlet; 151, liquid storage tank; 152, water storage tank; 2, device body; 21, chip holder; 22, coaxial light source; 23, fixing groove. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Definitions: Multienzyme Isothermal Rapid Amplification (MIRA) is a rapid isothermal nucleic acid amplification technique that relies on multiple functional proteins ( helicaseRecombinase, Single-strand binding protein , DNA polymerase (etc.) synergistic effect, to achieve rapid nucleic acid amplification at room temperature.

[0031] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0032] See Figure 1 , Figure 1 The diagram shows the structure of a digital MIRA point-of-care diagnostic chip. The chip includes, from top to bottom, a top slide 140, a micropattern layer 120, and a bottom slide 110. The micropattern layer 120 is connected to the sample inlet 141 and water inlet 142 of the top slide 140. A water-soluble film 130 is embedded at the interface between the micropattern layer 120 and the water inlet 142. The sample inlet 141 is suitable for sequentially injecting reaction reagents and sealing oil. Through a chip structure design without outlets, when reaction reagents are introduced from the sample inlet 141, all reagents enter each microchamber simultaneously, in equal amounts, and uniformly. The oil seal separates the reagents into each microchamber, providing the advantage of 100% sample discretization during digital nucleic acid detection, with no sample or reagent loss. Furthermore, because each reagent is precisely separated into its microchamber, it will not be ejected from the chip, ensuring that each molecule enters its corresponding microchamber, thus meeting the detection requirements for low-concentration templates.

[0033] The micropatterned layer 120 is sealed to the bottom glass slide 110 by means of heat-resistant transparent tape, thermal bonding, heat-resistant adhesive, hot pressing, or air plasma treatment bonding.

[0034] Please continue reading. Figure 1 In some embodiments, the sample inlet 141 and water inlet 142 of the top slide 140 are respectively connected to a liquid storage tank 151 and a water storage tank 152. The liquid storage tank 151 and the water storage tank 152 are sealed to the body of the top slide 140 by means of heat-resistant transparent tape, heat bonding, heat-resistant adhesive, heat-press sealing, or air plasma treatment bonding.

[0035] Both the top slide 140 and the bottom slide 110 are made of an incompressible, airtight, and thermally conductive transparent material, such as high-temperature resistant glass. When the digital MIRA point-of-care diagnostic chip is heated, the top slide 140 and the bottom slide 110 conduct heat to the reaction reagents within the microchamber array module 122, achieving temperature control. The micropattern layer 120 is made of a compressible, internally porous transparent material, such as polydimethylsiloxane.

[0036] Please see Figure 2 The structure of the micropatterned layer 120 is described below. In some embodiments, the micropatterned layer 120 includes a flow channel module 121, a microchamber array module 122, and a vacuum storage battery module 123. The microchamber array module 122 and the vacuum storage battery module 123 are arranged side by side. One end of the flow channel module 121 is connected to the sample injection port 141 of the top slide 140, and the other end is connected to the microchamber array module 122. The water-soluble film 130 is disposed above the vacuum storage battery module 123. The reaction reagent flows through the sample injection port 141 of the top slide 140, passes through the flow channel module 121, and enters the microchamber array module 122 for sample dispersion. The vacuum storage battery module 123 provides driving force for the reaction reagent.

[0037] Specifically, the microchamber array module 122 includes multiple microchambers with fractal symmetry structures. The flow channel module 121 is a T-shaped branch channel network, with the end of each branch channel communicating with the corresponding microchamber; and the length of the microchannel from the sample injection port 141 to the microchamber is the same for each branch channel. The T-shaped branch channel network adopts a fractal structure, which is easy for chip expansion. At the same time, the length of the microchannel from the sample injection port 141 to the microchamber is the same for each branch channel, thereby ensuring uniform distribution of the reaction reagents and template molecules.

[0038] For example, with 10 branch channels, the number of microchambers can reach 1024. When the number of branch channels reaches 20, the total number of microchambers can reach 1,048,576. Compared with megapixel digital nucleic acid detection chips, the digital MIRA point diagnostic chip provided in this embodiment is easier to apply to the amplification of millions of single molecules without any complex control system.

[0039] It should be noted that the detection range can be further adjusted by designing different microwell sizes for different pathogens. The total detection time can be as short as 25 minutes (5 minutes for sample discretization + 20 minutes for isothermal amplification), which is lower in cost and faster in readout compared to digital nucleic acid detection instruments in related technologies.

[0040] Please continue reading. Figure 2In some embodiments, the vacuum storage battery module 123 includes: multi-stage series-connected batteries; adjacent batteries are independent and not interconnected, and a thin wall is provided between adjacent batteries, which is suitable for discharging gas in the battery space from the thin wall into the flow channel module 121, discharging it from the chip, and forming a vacuum in the battery space; the water-soluble film 130 is disposed above the battery adjacent to the microchamber array module 122. After deionized water is injected into the water inlet 142, the sacrificial valve of the water-soluble film 130 dissolves upon contact with water, becoming permanently open, and under the action of the vacuum in the battery space, the deionized water fills the space of the battery connected to the water-soluble film 130. This avoids the reaction reagent from turning into gas when heated during the reaction reagent amplification process, and entering the vacuum battery through the thin wall around the microchamber, effectively preventing the reaction reagent from evaporating and ensuring the normal progress of the amplification reaction.

[0041] like Figure 3 As shown, at least one embodiment also provides an analysis device, including: a device body 2, a smart terminal, and a digital MIRA instant diagnostic chip as described above; the device body 2 has a chip holder 21 and a coaxial light source 22 disposed inside; the top of the device body 2 has a fixing slot 23 suitable for fixing the smart terminal; the digital MIRA instant diagnostic chip is placed on the chip holder 21; the smart terminal is fixed on the fixing slot 23 and is positioned directly above the coaxial light source 22; the chip holder 21 is positioned directly below the coaxial light source 22. The coaxial light source 22 ensures the uniformity and consistency of the excitation light during image acquisition. During fluorescence imaging, the smart terminal is vertically fixed directly above the coaxial light source 22, and the digital MIRA instant diagnostic chip is placed directly below the coaxial light source 22.

[0042] It should be noted that this analytical device can operate for extended periods using a 30V power supply provided by an external adapter.

[0043] At least one embodiment also provides an analysis method, the method comprising: S110: Place the digital MIRA real-time diagnostic chip in a vacuum environment and degas it under preset negative pressure for 2-6 hours.

[0044] Specifically, during the degassing process, air in the series-connected batteries enters the fluid channel through the internal loose and porous thin wall and is discharged from the chip, thereby storing a vacuum in the battery. The preset negative pressure is -80 kPa.

[0045] S120: Open the vacuum packaging and remove the digital MIRA instant diagnostic chip.

[0046] S130: Add the reaction reagent and sealing oil to the storage tank 151 in sequence. Under the action of vacuum and atmospheric pressure, the reaction reagent enters the microchannel quickly in the order of reaction reagent first and sealing oil last. After N fractal structures, the reagent will fill each microchamber in equal and uniform amounts, and the reaction reagent will be separated into each microchamber by the sealing oil.

[0047] Specifically, the schematic diagram of the sample introduction of the reaction reagents and sealing oil into the microchamber is shown below. Figure 4 As shown in the diagram. The schematic diagram of the oil seal is as follows. Figure 5 As shown.

[0048] S140: Inject deionized water into the water storage tank 152 to dissolve the water-soluble film 130, so that the deionized water fills part of the battery in the vacuum storage battery module 123.

[0049] like Figure 6 As shown, specifically, after deionized water is injected into the water inlet 142, the sacrificial valve of the water-soluble membrane 130 dissolves upon contact with the water, becoming permanently open. Under the vacuum effect of the space containing the battery, the deionized water fills the space connected to the water-soluble membrane 130. This prevents the reaction reagents from evaporating into gas during amplification and entering the vacuum battery through the thin wall surrounding the microchamber, effectively preventing reagent evaporation and ensuring the normal progress of the amplification reaction.

[0050] S150: The digital MIRA point-of-care diagnostic chip is placed on a heating pack for digital amplification reaction.

[0051] S160: Place the amplified digital MIRA instant diagnostic chip on the chip holder 21 of the analysis device for analysis.

[0052] In summary, this invention provides a digital MIRA point-of-care diagnostic chip, analysis device, and method. The digital MIRA point-of-care diagnostic chip includes, from top to bottom, a top slide 140, a micropattern layer 120, and a bottom slide 110. The micropattern layer 120 is connected to the sample injection port 141 and the water injection port 142 of the top slide 140. A water-soluble film 130 is embedded at the mating point between the micropattern layer 120 and the water injection port 142. The sample injection port 141 is suitable for sequentially injecting reaction reagents and sealing oil. Through the chip structure design without outlets, when reaction reagents are introduced from the sample injection port 141, all reaction reagents enter each microchamber simultaneously, in equal amounts, and uniformly. The oil seal separates the reaction reagents into each microchamber, providing the advantage of 100% sample discretization during digital nucleic acid detection, with no sample or reagent loss. Meanwhile, because each reaction reagent is precisely separated into each microchamber, it will not be pushed out of the chip, ensuring that each molecule enters the corresponding microchamber, thus meeting the detection requirements of low-concentration templates.

[0053] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A digital MIRA point-of-care diagnostic chip, characterized in that, From top to bottom, there are a top slide, a micro-patterned layer, and a bottom slide. The micropatterned layer is connected to the sample injection port and water injection port of the top glass slide; A water-soluble film is embedded at the fitting point between the micropattern layer and the water inlet; The injection port is suitable for sequentially injecting reaction reagents and sealing oil; The micropatterned layer includes a flow channel module, a micro-chamber array module, and a vacuum storage battery module; The micro-chamber array module and the vacuum storage battery module are arranged side by side; One end of the flow channel module is connected to the sample injection port of the top glass slide, and the other end is connected to the sample inlet of the microchamber array module. The water-soluble film is disposed above the vacuum storage battery module; The vacuum storage battery module includes: a multi-stage series-connected battery; The adjacent batteries are independent and not connected, and a thin wall is provided between the adjacent batteries, which is suitable for discharging the gas in the space where the battery is located from the thin wall into the flow channel module, discharging the chip, and forming a vacuum in the space where the battery is located. The water-soluble film is disposed above the battery adjacent to the microcell array module.

2. The digital MIRA real-time diagnostic chip as described in claim 1, characterized in that, The micro-chamber array module includes multiple micro-chambers with fractal symmetry structures.

3. The digital MIRA real-time diagnostic chip as described in claim 2, characterized in that, The flow channel module is a T-shaped branch channel network, with the end of each branch channel connected to the corresponding microchamber; Furthermore, the length of the microchannel from the injection port to the microchamber is the same for each of the branch channels.

4. The digital MIRA real-time diagnostic chip as described in claim 1, characterized in that, The sample inlet and water inlet of the top glass slide are respectively connected to the liquid storage tank and the water storage tank.

5. The digital MIRA real-time diagnostic chip as described in claim 1, characterized in that, Both the top and bottom glass slides are made of an incompressible, airtight, and thermally conductive transparent material.

6. The digital MIRA real-time diagnostic chip as described in claim 1, characterized in that, The micropatterned layer is made of a compressible, internally porous, transparent material.

7. An analytical apparatus, characterized in that, include: The device body, the smart terminal, and the digital MIRA real-time diagnostic chip as described in any one of claims 1-6; The device body is equipped with a chip holder and a coaxial light source inside. The top of the device body is provided with a fixing groove suitable for fixing a smart terminal; The digital MIRA real-time diagnostic chip is placed on the chip holder; The smart terminal is fixed on the fixing slot and positioned directly above the coaxial light source; The chip holder is positioned directly below the coaxial light source.

8. An analytical method, characterized in that, The method includes: The digital MIRA instant diagnostic chip as described in any one of claims 1-6 is placed in a vacuum environment and degassed under a preset negative pressure for 2-6 hours; Open the vacuum packaging and remove the digital MIRA point-of-care diagnostic chip; The reaction reagent and sealing oil are added to the storage tank in sequence. Under the action of vacuum and atmospheric pressure, the reaction reagent enters the microchannel rapidly in the order of reaction reagent first and sealing oil last. After N fractal structures, the reagent will fill each microchamber in equal and uniform amounts, and the reaction reagent will be separated into each microchamber by the sealing oil. Injecting deionized water into the water storage tank dissolves the water-soluble film, allowing the deionized water to fill part of the battery in the vacuum storage battery module. The digital MIRA point-of-care diagnostic chip is placed on a heating pack to perform a digital amplification reaction; The amplified digital MIRA point-of-care diagnostic chip is placed on the chip holder of the analysis device for analysis.

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