Multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technologies and application thereof

Abstract

The invention discloses a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technologies and application thereof. The multi-target full-process nucleic acid detection chip comprises a carbon-doped PC substrate, an EWOD electrode layer, a first Teflon hydrophobic layer, a supporting layer, a second Teflon hydrophobic layer and an ITO glass layer which are sequentially arranged from bottom to top, a magnetic bead nucleic acid extraction area, a nucleic acid isothermal amplification area and a CRISPR detection area which are communicated in sequence are formed between the first Teflon hydrophobic layer and the second Teflon hydrophobic layer; wherein the surface of the EWOD electrode is hydrophilic by controlling the EWOD electrode to be electrified, and liquid drops move in the magnetic bead nucleic acid extraction area, the nucleic acid constant-temperature amplification area and the CRISPR detection area, so that nucleic acid amplification and CRISPR detection are realized. The device integrates magnetic bead method nucleic acid extraction, EWOD droplet driving and isothermal amplification, has the advantages of full-process detection, low cost and small size, and has a good application prospect in the field of nucleic acid detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic chips, and more particularly to a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology and application thereof. BACKGROUND

[0002] Current nucleic acid detection methods for pathogens mainly include conventional RT-PCR / PCR, qPCR, and LAMP, RPA and other isothermal amplification methods. However, these existing pathogen detection technologies have deficiencies or defects, such as PCR-based molecular methods requiring PCR instruments and professional technicians, long detection time, and the defects of low specificity and high false positive rate of LAMP, RPA and other isothermal amplification methods. In recent years, the CRISPR / Cas system has been widely used in the field of nucleic acid detection. Clustered regularly interspaced short palindromic repeats (CRISPR) is an immune mechanism widely existing in prokaryotes to resist invasion. CRISPR and its associated proteins (CRISPR associated proteins, Cas) are called CRISPR / Cas system. Researchers have developed SHERLOCK, HOLMESv2, DETECTR and other detection systems by combining RPA, LAMP and CRISPR family nucleases, effectively overcoming the false positive defects of RPA, LAMP and other isothermal amplification technologies, and shortening the nucleic acid detection time. They are used for high-sensitivity and specific nucleic acid detection. However, these detection systems often have the problems of multiple operation steps and aerosol pollution in the process of pipetting. The full-process nucleic acid detection chip integrates sample processing, nucleic acid extraction, isothermal amplification and detection, and other steps, and can realize the integration and automation of nucleic acid detection. However, in order to meet the detection requirements of the full process, the existing full-process nucleic acid detection chip has a complex structure design, many external controller devices, a complex device structure, a large device size, and a high cost. Therefore, it is urgent to develop a low-cost, ultra-fast and full-process nucleic acid detection chip suitable for RPA, CRISPR / Cas system, to meet the demand for on-site rapid multi-target detection.

[0003] Medium electro-wetting (EWOD) technology has emerged in the field of nucleic acid detection PCR due to its fast characteristics of driving droplets by electrically controlling surface tension. The principle is to change the wettability of the medium film and the surface liquid by applying electric potential on the micro electrode array, so as to accurately and quickly drive the droplets. The typical EWOD device is a three-layer structure, and the controlled droplets are located between the upper and lower electrode plates. The lower plate is composed of a substrate, a micro electrode array, a dielectric layer and a hydrophobic layer, which can ensure the smooth and stable movement of the droplets. The upper and lower plates are filled with air or silicone oil, which can reduce the driving damping of the droplets, reduce evaporation and reduce the driving voltage. However, the traditional EWOD chip is prepared by using PCB, MEMS or flexible circuit technology, which has the disadvantages of high cost, thick electrode (minimum about 35 to 50 microns), etc., which is not conducive to the movement of droplets and is not suitable as a disposable consumable, so it is urgent to develop a low-cost EWOD chip to meet the demand of nucleic acid detection. SUMMARY

[0004] The purpose of the present application is to provide a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology and its application, so as to solve the problems of long detection time, complex chip structure, high cost, large equipment size, easy aerosol pollution in the process of constant temperature amplification reaction and other problems in the prior art full-process nucleic acid detection.

[0005] To solve the above problems, the present application adopts the following technical solutions:

[0006] According to the first aspect of the present application, a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology is provided, which comprises, from bottom to top, a carbon-doped PC substrate, an EWOD electrode layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer; the first Teflon hydrophobic layer and the second Teflon hydrophobic layer form a magnetic bead nucleic acid extraction area, a nucleic acid constant temperature amplification area and a CRISPR detection area which are sequentially connected; the magnetic bead nucleic acid extraction area comprises, in sequence, a lysis cavity, a first washing cavity, a second washing cavity and an elution cavity; the magnetic bead nucleic acid extraction area, the nucleic acid constant temperature amplification area and the CRISPR detection area are obtained by etching the second Teflon hydrophobic layer and the ITO film on the ITO glass layer by a dry etching process; wherein the EWOD electrode layer comprises an EWOD driving electrode prepared by screen printing, and the EWOD driving electrode is aligned with the elution cavity, the nucleic acid constant temperature amplification area and the CRISPR detection area; by controlling the power supply of the EWOD electrode, the surface of the EWOD electrode presents hydrophilicity, and the droplets move in the magnetic bead nucleic acid extraction area, the nucleic acid constant temperature amplification area and the CRISPR detection area, realizing nucleic acid amplification and CRISPR detection.

[0007] Preferably, the PC substrate is PC material doped with 30-50% carbon elements. According to the research of the present application, it is found that by doping 30-50% carbon elements, the substrate prepared thereby not only has high thermal conductivity, but also has more uniform heat conduction, thereby better realizing constant temperature amplification. Most preferably, the PC substrate is PC material doped with 40% carbon elements.

[0008] Preferably, the PC substrate is prepared by hot pressing or injection molding.

[0009] Preferably, the thickness of the support layer is 0.3-0.5 mm, providing a storage space for the droplets.

[0010] Preferably, by changing the structure of the screen printing plate, the manipulation of droplets of multiple paths and the parallel design of multiple paths are realized, thereby realizing the simultaneous detection of multiple targets.

[0011] According to the second aspect of the present application, a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology is provided for application in full-process nucleic acid detection, including the following steps: S1, adding a detection sample to a lysis chamber, destroying the cell structure by chemical and acoustic lysis, and releasing the nucleic acid in the cell; S2, after lysis is completed, controlling a magnetic bead to enter the lysis chamber by an external magnetic rod, mixing by moving the magnetic rod, and then moving the magnetic bead through the first washing chamber and the second washing chamber in sequence by controlling the magnetic rod; S3, connecting a power supply to an EWOD driving electrode, adding a nucleic acid extraction solution in an elution chamber, controlling the magnetic rod to move the magnetic bead to the elution chamber for elution, and then controlling the magnetic rod to move the magnetic bead back to the second washing chamber, and then controlling the EWOD driving electrode to be powered on, moving the reaction reagent in the elution chamber to a nucleic acid constant temperature amplification area, performing nucleic acid amplification, after completion, controlling the EWOD driving electrode to be powered on and powered off, moving the droplet to a CRISPR detection area, and realizing detection of the fluorescence signal; S4, performing nucleic acid detection by means of fluorescence photography.

[0012] Preferably, the application includes application in the detection of multiple rice pathogenic bacteria.

[0013] It should be known that the existing EWOD technology for nucleic acid detection generally adopts PCB, flexible circuit or MEMS process, which has high processing cost, while the present application adopts the way of multi-layer screen printing conductive silver paste to prepare the EWOD driving electrode, which has lower cost. The present application integrates RPA, CRISPR / Cas technology and EWOD droplet driving technology, so that it can realize full-process automatic detection. Through the combination of the above technologies, the present chip can complete the full-process nucleic acid detection in a high-sensitivity, multi-path and portable manner.

[0014] The application point of the application mainly lies in that the lysis technology, the magnetic bead nucleic acid extraction technology, the EWOD droplet driving technology and the RPA and CRISPR / Cas technology are integrated, dependence on large instruments, especially a heating module, can be reduced, and detection of multiple rice pathogenic bacteria can be simultaneously realized. By controlling power-on and power-off of the EWOD driving electrode, the droplet is controlled to be separated, moved and stayed in the RPA constant temperature amplification area and the CRISPR detection area, and the whole detection time is not more than 30 min.

[0015] Another application point of the application lies in that the PC material doped with 30-50% carbon is used as the substrate. By doping the carbon element, the thermal conductivity of the substrate is improved, which is beneficial to rapid transmission and stability of temperature. In addition, the substrate can be prepared by an injection molding process and a hot pressing process, batch preparation can be realized, and the substrate preparation cost of the chip can be effectively reduced.

[0016] Another application point of the application also lies in that the hydrophilic droplet fixing area is prepared by etching and removing the second Teflon coating and the ITO layer on the ITO glass plated with the second Teflon coating, and serves as a lysis cavity, a first washing cavity and a second washing cavity. Therefore, the magnetic bead nucleic acid extraction module is integrated on the EWOD chip. The cavities are filled with mineral oil, so that the magnetic beads can freely move between the cavities. The module is characterized in that the droplet can be fixed by modification of the hydrophilic site, and the cross talk between the cavities due to the droplet being dragged by the moving magnetic beads can be prevented.

[0017] Compared with the prior art, the superiority of the application mainly lies in that the whole process detection based on RPA, CRISPR / Cas and multiple targets is completed on the same chip, the droplet is quickly driven, and the amplification time is controlled within 30 min. The application reduces the cost by a low-cost screen printing process, and can integrate the whole lysis, elution, extraction and nucleic acid translation and detection process on one chip. Another advantage of the application also lies in that the aerosol pollution problem caused by pipetting in the test tube reaction is reduced by automatic program control.

[0018] In summary, the application integrates the lysis technology, the magnetic bead nucleic acid extraction technology, the EWOD droplet driving technology and the RPA and CRISPR / Cas technology, the droplet is quickly moved in the RPA amplification area and the CRISPR detection area by the EWOD driving, the nucleic acid whole process automatic detection of multiple targets can be completed within 40 min, the sensitivity is high, and the specificity is strong compared with qPCR detection. Therefore, the EWOD-based whole process nucleic acid detection chip provided by the application has the advantages of small size, high detection sensitivity and reduction of aerosol pollution, and has a good application prospect in the nucleic acid detection field. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a full-process schematic diagram of a full-process multi-target nucleic acid detection chip according to a preferred embodiment of the present application;

[0020] Figure 2 is an exploded view of the full-process multi-target nucleic acid detection chip as shown in Figure 1

[0021] Figure 3 is a schematic diagram of contact angles of hydrophobic and hydrophilic regions;

[0022] Figure 4 is a control diagram before and after amplification; the left diagram is before amplification, and the right diagram is after amplification. DETAILED DESCRIPTION

[0023] The present application will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present application and are not used to limit the scope of the present application. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art, or follow the experimental methods suggested by the manufacturers of the kits and instrument equipment. The reagents and materials used in the embodiments can be obtained from commercial channels unless otherwise specified.

[0024] Example 1 Preparation of a multi-target full-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD

[0025] According to the present application, an ultra-sensitive nucleic acid detection chip for performing the full processes of lysis, elution, extraction, RPA amplification and CRISPR detection in a multi-target full-process nucleic acid detection chip is provided. The ultra-fast full-process detection is completed in 30 min, the RPA amplification and CRISPR detection processes are realized by electrode control of the rapid movement of microdroplets, thereby improving the sensitivity of detection, reducing aerosol pollution, and shortening the detection time.

[0026] According to Figure 1 , Figure 2 , a full-process nucleic acid detection chip based on EWOD according to a preferred embodiment of the present application is provided. The specific structure is described in detail as follows:

[0027] With reference to Figure 2 , the full-process nucleic acid detection chip comprises, from bottom to top, a PC substrate 1, an EWOD electrode layer 2, a PET dielectric layer 3, a first Teflon hydrophobic layer 4, a support layer 5, a second Teflon hydrophobic layer 6, and an ITO glass layer 7.

[0028] Referring to Figure 1 ​, the first Teflon hydrophobic layer 4 and the second Teflon hydrophobic layer 6 form a magnetic bead nucleic acid extraction area 10 and a nucleic acid isothermal amplification area 20 and a CRISPR detection area 30; the magnetic bead nucleic acid extraction area 10 includes: a lysis cavity 101, a first washing cavity 102, a second washing cavity 103 and an elution cavity 104.

[0029] In combination Figure 2 As shown, the EWOD electrode layer 2 includes an EWOD driving electrode 21 prepared by screen printing, which is aligned with the elution cavity 104, the nucleic acid isothermal amplification area 20 and the CRISPR detection area 30 below, realizes chemical binding and acoustic wave lysis of the sample in the lysis cavity 101, and the liquid droplets move in the RPA amplification area and the CRISPR detection area by controlling the EWOD driving electrode to be electrified to make the surface hydrophilic, realizing amplification and CRISPR detection.

[0030] Specifically, in combination Figure 1 、 Figure 2 As shown, the preparation process of the multi-target full-process nucleic acid detection chip is as follows:

[0031] First, PC material doped with 30-50% carbon is used as a PC substrate 1, and 0.4-0.5 mm thick thin SiO2 glass (not shown in the figure) is used above the PC substrate 1, and a specific shaped groove is engraved at the bottom of the PC substrate 1 to facilitate the movement of the magnetic bar and the installation of the heating block (37℃ constant temperature heating block);

[0032] Then, the EWOD electrode layer 2 is prepared by printing the EWOD driving electrode on the thin SiO2 glass by multi-layer screen printing process using conductive silver paste;

[0033] Next, a 2 μm thick PET dielectric layer 3 is sprayed on the EWOD electrode layer 2;

[0034] Then, Teflon is sprayed on the PET dielectric layer 3 as the first Teflon hydrophobic layer 4; 100-400 μm thick PET chamber is used as the support layer 5 above the first Teflon hydrophobic layer 4; the top layer uses 1.1 mm thick ITO glass layer 7 as the seal, and the side of the ITO glass layer 7 in contact with the support layer 5 is also sprayed with Teflon as the second Teflon hydrophobic layer 6;

[0035] The different glasses and support layers are bonded by high-temperature resistant and non-fluorescent UV glue. In addition, the spring needle is connected in a way that the spring needle is in contact with the external electrode to make the circuit conductive.

[0036] According to the application, in the magnetic bead nucleic acid extraction area and the nucleic acid amplification area, the Teflon hydrophobic layer and the PET dielectric layer are etched to expose the SiO2 with hydrophilicity. Therefore, the area where the droplet is located has hydrophilicity, and the outside has hydrophobicity, and the contact angle difference ensures that the sample to be detected, the eluent, and the extraction liquid are fixed in the hydrophilic area and cannot move. As shown in Figure 3 The contact angle of the hydrophilic area after hydrophilic treatment is 40.1°, and the contact angle of the hydrophobic area is 111.1°.

[0037] According to a preferred scheme of the application, PC material doped with 30-50% carbon is used as the substrate. By doping carbon elements, the thermal conductivity of the substrate is improved, which is beneficial to the rapid transmission and stability of temperature. In addition, the substrate can be prepared by injection molding process and hot pressing process, which can realize batch production and effectively reduce the substrate preparation cost of the chip.

[0038] According to a preferred scheme of the application, the hydrophilic droplet fixing area is prepared by etching the second Teflon hydrophobic layer and the ITO layer on the ITO glass plated with the second Teflon hydrophobic layer, which are respectively used as the lysis cavity 101, the first washing cavity 102, and the second washing cavity 103.

[0039] According to a preferred scheme of the application, each layer in the above structure is indispensable, and the two Teflon hydrophobic layers 4 and 6 are necessary conditions. The reason is that the interface of the EWOD droplet driving must be a hydrophobic interface, and the conventional EWOD chip preparation has two hydrophobic layers. The support layer 5 only uses PET material to provide a cavity with a thickness of 0.2-0.5 mm, and other materials such as PC can also be used to form the cavity required for the movement of silicone oil and reaction reagents.

[0040] Example 2 Application of the full-process nucleic acid detection chip in full-process nucleic acid detection

[0041] According to the embodiment, the application of the full-process nucleic acid detection chip based on EWOD as shown in Example 1 in full-process nucleic acid detection is provided, which includes the following steps:

[0042] 1) First, the detection sample (such as rice bacterial leaf streak bacteria, rice leaf juice, rice seed leachate, etc.) is added to the lysis cavity 101, and the entire lysis time is not more than 1 min.

[0043] 2) After the electric lysis is completed, the magnetic rod controls the magnetic beads to enter the lysis cavity 101, mixes by moving the magnetic rod, and then controls the magnetic rod to make the magnetic beads pass through the silicone oil and enter the first washing cavity 102. The above process is repeated to make the magnetic beads enter the second washing cavity 103.

[0044] 3) turn on the EWOD driving electrodes below the elution chamber 104, add the extraction solution in the elution chamber 104, control the magnetic bar to move the magnetic beads to the elution chamber 104 for elution, and then control the magnetic bar to move the magnetic beads back to the second washing chamber 103. Then control the EWOD driving electrodes to be powered on respectively, move 4 μL of reaction reagent in the elution chamber 104 to the nucleic acid constant temperature amplification area 20, and move the liquid droplets in the nucleic acid constant temperature amplification area 20 and the CRISPR detection area 30 by controlling the power-on and power-off of the EWOD driving electrodes, and the whole detection time is not more than 30 min. The moving time of the liquid droplets between adjacent electrodes is set to 500 ms-1000 ms to ensure that the liquid droplets can be moved stably, and the moving speed of the liquid droplets between the electrodes of the chip of the present application can reach 100 ms at the fastest.

[0045] 4) detect by means of fluorescence photographing (rice bacterial leaf spot bacteria are taken as an example, FAM channel), after 30 min, the fluorescence intensity of the positive and negative sample reactions before and after the reaction is controlled as shown in Figure 4

[0046] The above is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above embodiment of the present application can be variously changed. Any simple, equivalent changes and modifications made according to the content of the claims and the description of the present application fall within the scope of protection of the present application. The present application is not described in detail.​

Claims

1. A multi-target whole-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology, characterized in that the whole-process nucleic acid detection chip comprises, from bottom to top, a PC substrate doped with carbon, an EWOD electrode layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer. The first Teflon hydrophobic layer and the second Teflon hydrophobic layer form, in sequence, a magnetic bead nucleic acid extraction zone, a nucleic acid constant temperature amplification zone and a CRISPR detection zone that are connected in sequence. The magnetic bead nucleic acid extraction zone comprises, in sequence, a lysis cavity, a first washing cavity, a second washing cavity and an elution cavity. The magnetic bead nucleic acid extraction zone, the nucleic acid constant temperature amplification zone and the CRISPR detection zone are obtained by etching the second Teflon hydrophobic layer and the ITO film on the ITO glass layer by a dry etching process. The EWOD electrode layer comprises an EWOD driving electrode prepared by screen printing, and the EWOD driving electrode is aligned with the elution cavity, the nucleic acid constant temperature amplification zone and the CRISPR detection zone. By controlling the EWOD electrode to be powered on, the surface of the EWOD electrode is hydrophilic, and the droplets move in the magnetic bead nucleic acid extraction zone, the nucleic acid constant temperature amplification zone and the CRISPR detection zone, thereby realizing nucleic acid amplification and CRISPR detection. The PC substrate is made of PC material doped with 30-50% carbon elements.

2. The multi-target full-process nucleic acid detection chip according to claim 1, characterized in that, The PC substrate is formed by hot pressing or injection molding. 3.The multi-target full-process nucleic acid detection chip according to claim 1, characterized in that, The support layer has a thickness of 0.3-0.5 mm and provides a storage space for the droplets.

4. The multi-target full-process nucleic acid detection chip according to claim 1, characterized in that, By changing the structure of the screen printing plate, the manipulation of droplets in multiple paths and the parallel design of multiple channels are realized, thereby realizing the simultaneous detection of multiple targets.

5. The multi-target full-process multi-target nucleic acid detection chip according to claim 1, characterized in that, The method comprises the following steps:

6. The application of the multi-target whole-process nucleic acid detection chip based on RPA, CRISPR / Cas and EWOD technology according to any one of claims 1-5 in whole-process nucleic acid detection, characterized in that, S1. The detection sample is added to the lysis cavity, and the cell structure is destroyed by chemical and acoustic lysis to release the nucleic acid in the cell. S2. After lysis is completed, the magnetic beads are controlled to enter the lysis cavity by an external magnetic rod, mixed by moving the magnetic rod, and then moved through the first washing cavity and the second washing cavity by controlling the magnetic rod. S3. The EWOD driving electrode is connected to a power supply, nucleic acid extraction solution is added to the elution cavity, the magnetic beads are moved to the elution cavity by controlling the magnetic rod, and then the magnetic beads are moved back to the second washing cavity by controlling the magnetic rod. Then, the EWOD driving electrode is powered on, the reaction reagent in the elution cavity is moved to the nucleic acid constant temperature amplification zone, nucleic acid amplification is performed, and after completion, the EWOD driving electrode is powered on and off to move the droplets to the CRISPR detection zone, thereby realizing the detection of the fluorescence signal. S4. The nucleic acid detection is performed by means of fluorescence photography. The application includes the application in the detection of various rice pathogenic bacteria.

7. Use according to claim 6, characterized in that, ​

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