WO2020156302A1

WO2020156302A1 - Detection system, detection method and apparatus, and computer-readable storage medium

Info

Publication number: WO2020156302A1
Application number: PCT/CN2020/073120
Authority: WO
Inventors: 苏阳; 马汉彬; 张研; 刘婉琛
Original assignee: 苏州奥素液芯电子科技有限公司
Priority date: 2019-02-02
Filing date: 2020-01-20
Publication date: 2020-08-06
Also published as: CN109868215B; CN109868215A

Abstract

A detection system, a detection method and apparatus, and a computer-readable storage medium. The detection system comprises: a lid with a sample inlet and a waste liquid outlet, an electrode array, a temperature control apparatus and a driving circuit, wherein the electrode array comprises a hydrophobic layer at the side close to the lid, and an electrode layer at the side away from the lid; a region composed of electrodes of the electrode layer comprises: a sample injection area, an amplification area, a mixing area and a waste liquid area; the areas are connected by means of a path formed by the electrodes; the driving circuit is configured to control the temperature control apparatus, and control the electrode array, so as to change the states of electrodes in the electrode array, thereby driving droplets between the lid and the electrode array; and the temperature control apparatus is configured to change the temperature of the electrode array based on the control of the driving circuit.

Description

Detection system, detection method and device, and computer readable storage medium

Technical field

The embodiments of the present application relate to, but are not limited to, a detection system, a detection method and device, and a computer-readable storage medium.

Background technique

Molecular biology methods play an extremely important role in rapid disease diagnosis, and nucleic acid amplification detection methods are widely used. However, because the detection equipment is expensive and the operation is complicated, and professional technical personnel are required to operate, the popularization and popularization of nucleic acid amplification detection methods are restricted. Polymerase chain reaction ((Polymerase Chain Reaction, PCR) is a very widely used nucleic acid amplification technology. In a typical PCR reaction, two oligonucleotide primers are used to hybridize with the target sequence, and deoxynucleoside triphosphates are added. Deoxyribonucleic acid (Deoxyribo Nucleic Acid, DNA) polymerase is used to produce double-stranded products, and a large number of target DNA copies are realized through continuous temperature cycling. PCR reaction has extremely high temperature precision requirements, so there are higher requirements for experimental equipment. This makes it more expensive to use. In contrast, Loop-mediated isothermal Amplification (LAMP) technology is a nucleic acid amplification technology that performs DNA amplification under constant temperature conditions. It uses 4-6 A designed primer and DNA polymerase perform a specific and rapid DNA copy under constant temperature conditions. The amplified product can be quickly detected qualitatively or quantitatively by observing the white precipitate of pyrophosphatase, gel electrophoresis turbidity detection, and real-time fluorescence detection. Compared with PCR technology, LAMP technology has the advantages of high sensitivity, high specificity, short time, less sample consumption, simple experimental process, etc. Because LAMP does not require precise temperature cycling process, it simplifies the complexity of the equipment and reduces the equipment Cost, more suitable for low-cost high-throughput rapid detection.

The current nucleic acid amplification technology is mainly completed in a test tube or on a 96- or 384-well plate platform with a sophisticated temperature control system. This method requires a lot of manual operation and the experimental equipment is relatively expensive, which is not conducive to the popularization and promotion of the technology .

With the rapid development of microfluidic technology and Lab-on-a-chip (LoC), many microfluidic chips for nucleic acid amplification detection have also emerged. Many microfluidic chips are also used in the rapid detection of DNA or ribonucleic acid (RNA). The function of the traditional microfluidic chip greatly depends on the design and processing technology of the microfluidic channel. The manufacturing process is complex and costly, and the structure and function are closely related, so the scalability is poor.

Summary of the invention

The following is an overview of the topics detailed in this article. This summary is not intended to limit the scope of protection of the claims.

On the one hand, an embodiment of the present application provides a detection system, including:

A cover with a sample inlet and a waste liquid outlet, an electrode array, a temperature control device and a drive circuit, the electrode array includes a hydrophobic layer on the side close to the cover, and an electrode layer on the side far from the cover, the A dielectric layer is included between the electrode layer and the hydrophobic layer, the electrode layer includes a plurality of electrodes, the driving circuit is respectively connected to the electrode array and the temperature control device, and the cover and the electrode array There is a space for droplets to move between, the sample inlet is set to allow droplets to enter the space between the cover and the electrode array, and the waste outlet is set to provide droplets from the cover and the electrode. The space between the arrays is moved out, the side of the cover close to the electrode array contains a hydrophobic layer, and the electrode layer of the electrode layer constitutes the area including: the sampling area, the amplification area, the mixing area and the waste liquid area. Are connected by a path formed by electrodes, each sample injection area has a sample inlet at a corresponding position on the lid, and each waste liquid area has a waste liquid outlet at a corresponding position on the lid, The amplification zone is an area where the sample to be detected and the auxiliary reactant react, and the mixing area is an area where one or more auxiliary reactants other than the sample to be detected are mixed, wherein:

The temperature control device is configured to change the temperature of the electrode array based on the control of the drive circuit;

The driving circuit is configured to control the temperature control device and control the electrode array to change the state of the electrodes in the electrode array so as to drive all the electrodes located between the cover and the electrode array. The droplets.

In another aspect, an embodiment of the present application provides a detection method, which is applied to the detection system described in any embodiment, including:

Controlling the electrode array to stir the separately injected auxiliary reactants;

Controlling the electrode array to mix and stir the auxiliary reactants to obtain an amplification reaction solution;

Controlling the electrode array to mix and stir the injected reaction solution containing the sample and at least part of the amplification reaction solution;

The temperature control device is activated so that the temperature of the electrode array meets the reaction demand.

In another aspect, an embodiment of the present application provides a detection device, including a memory and a processor, the memory stores a program, and when the program is read and executed by the processor, the detection device described in any of the embodiments is implemented. method.

In another aspect, an embodiment of the present application provides a computer-readable storage medium that stores one or more programs, and the one or more programs can be executed by one or more processors to Implement the detection method described in any embodiment.

After reading and understanding the drawings and detailed description, other aspects can be understood.

Figure overview

The accompanying drawings are used to provide a further understanding of the technical solutions of the embodiments of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions and do not constitute a limitation on the technical solutions.

Figure 1a is a schematic diagram of a detection system provided by an embodiment of the application;

Figure 1b is a schematic diagram of a detection system provided by an embodiment of the application;

2 is a schematic diagram of a detection system including a control terminal provided by an embodiment of the application;

FIG. 3 is a schematic diagram of electrode array connection provided by an embodiment of the application;

4 is a schematic diagram of the structure of an electrode array provided by an embodiment of the application;

FIG. 5 is a schematic diagram of functional partitions of an electrode array provided by an embodiment of the application;

Fig. 6 is a schematic diagram of an electrode array provided by an embodiment of the application;

Figure 7 is a schematic diagram of droplet transfer and fusion provided by an embodiment of the application;

Figure 8 is a schematic diagram of liquid drop stirring provided by an embodiment of the application;

Figure 9 is a schematic diagram of droplet separation provided by an embodiment of the application;

FIG. 10 is a flowchart of a detection method provided by an embodiment of the application;

Fig. 11 is a flowchart of a method for detecting Salmonella provided by an embodiment of the application;

FIG. 12 is a schematic diagram of injection and stirring of primers and the like in the Salmonella detection embodiment in the embodiment of the application;

13-15 are schematic diagrams of the separation of pure water in the Salmonella detection embodiment according to the embodiment of the application;

16 to 18 are schematic diagrams of the separation of the mixture of DNA polymerase and buffer in the Salmonella detection embodiment of the application;

Figures 19-21 are schematic diagrams of primer separation in the Salmonella detection embodiment in the embodiment of the application;

22 is a schematic diagram of agitation of the amplification reaction solution in the mixing zone in the Salmonella detection embodiment in the embodiment of the application;

FIG. 23 is a schematic diagram of the transfer of the amplification reaction solution in the Salmonella detection embodiment in the embodiment of the application;

FIG. 24 is a schematic diagram of agitating the amplification reaction solution in the amplification zone in the Salmonella detection embodiment in the embodiment of the application;

FIG. 25 is a schematic diagram of an amplification reaction solution in each amplification zone in the Salmonella detection example of the embodiment of the application;

FIG. 26 is a schematic diagram of injection of the sample and fluorescent probe mixture in the Salmonella detection embodiment in the embodiment of the application;

FIG. 27 is a schematic diagram of the transfer of the sample and the fluorescent probe mixture in the Salmonella detection embodiment in the embodiment of the application;

FIG. 28 is a schematic diagram of fluorescence detection in the Salmonella detection embodiment in the embodiment of the application;

FIG. 29 is a schematic diagram of electrode short-circuiting in an embodiment of the application;

Figure 30 is a schematic diagram of a detection device provided by an embodiment of the application;

FIG. 31 is a block diagram of a computer-readable storage medium provided by an embodiment of this application.

Detail

Hereinafter, the embodiments of the present application will be described in detail with reference to the drawings. In the case of no conflict, the embodiments of the application and the features in the embodiments can be combined with each other arbitrarily.

The steps shown in the flowchart of the drawings may be executed in a computer system such as a set of computer-executable instructions. Also, although the logical sequence is shown in the flowchart, in some cases, the steps shown or described may be performed in a different order than here.

Electrowetting dielectric (Electrowetting on dielectric, EWOD) technology is an emerging digital microfluidic technology, which can use microelectrodes and electrical signals to achieve precise control of a single droplet, and the accuracy can be as accurate as the skin level. The component processing technology is mature and the production cost is low. The integration of Thin Film Transistor (TFT) technology applied in the display field and EWOD technology can greatly reduce the number of physical connections, thereby greatly improving the scalability and throughput of the system. High-throughput rapid detection. Moreover, the system can realize automatic control and online programming, reducing time and labor costs. By integrating LAMP technology with EWOD technology, automatic low-cost high-throughput rapid detection can be realized, thereby promoting nucleic acid expansion. Increase the promotion and popularization of detection technology.

As shown in FIG. 1a, an embodiment of the present application provides a detection system, including: a cover 4 including a sample inlet 2 and a waste liquid outlet 3, an electrode array 5, a temperature control device 6 and a driving circuit 7. The driving circuit 7 is connected to the electrode array 5 and the temperature control device 6. The electrode array 5 includes a hydrophobic layer (not shown in FIG. 1a) on the side close to the cover 4, and an electrode layer on the side far away from the cover 4, between the electrode layer and the hydrophobic layer A dielectric layer (not shown in FIG. 1a), the electrode layer includes a plurality of electrodes, the driving circuit 7 is respectively connected to the electrode array 5 and the temperature control device 6, the cover 4 and the electrode There is a space between the arrays 5 for the droplets to move, and the droplets located between the cover 4 and the electrode array 5 can be manipulated by controlling the state of the electrodes in the electrode array 5. The sample inlet 2 is set to supply droplets Entering the space between the cover 4 and the electrode array 5, the waste liquid outlet 3 is set to allow liquid droplets to move out of the space between the cover 4 and the electrode array 5, and the cover 4 is close to the One side of the electrode array contains a hydrophobic layer (not shown in Figure 1a), where:

The temperature control device 6 is configured to change the temperature of the electrode array 5 based on the control of the drive circuit 7;

The driving circuit 7 is configured to control the temperature control device 6 and control the electrode array 5 to change the state of the electrodes in the electrode array 5 to drive the cover 4 and the The droplets between the electrode array 5.

In an exemplary embodiment, the cover 4 may be transparent and conductive, for example, realized by indium tin oxide (Indium Tin Oxide, ITO) glass.

In an exemplary embodiment, the dielectric layer helps isolate the droplet from the underlying electrode. The dielectric layer may include inorganic dielectric materials such as silicon dioxide, silicon nitride, aluminum oxide or high-k dielectric materials such as hafnium oxide, zirconium oxide or tantalum oxide.

In an exemplary embodiment, the dielectric layer may include an organic dielectric material.

In an exemplary embodiment, the thickness of the dielectric layer is thick enough to avoid breakdown, for example, 20 nm to 5 μm. The hydrophobic layer may include a suitable material, such as polytetrafluoroethylene (PTFE) or Cytop (RTM).

In an exemplary embodiment, the droplet 8 may contain an aqueous or non-aqueous liquid.

In an exemplary embodiment, the droplet 8 may contain a polar or non-polar liquid. For example, saline buffer, biological sample (e.g. DNA or protein) or body fluid (e.g. blood or urine). The droplet volume is, for example, sub-microliter or micro-level.

In an exemplary embodiment, the temperature control device 6 includes a heating resistor, a temperature sensor, an aluminum block, a temperature sensor, etc. The temperature control device 6 is controlled by a driving circuit 7 during operation, and the heating resistor is closely related to the aluminum block. For heating, the aluminum block disperses the heat evenly, the temperature sensor collects the temperature, and feeds the temperature signal back to the drive circuit 7. The drive circuit 7 will switch the heating resistor after reaching the target temperature to ensure that the temperature remains at the target temperature.

In an exemplary embodiment, the driving circuit 7 mainly includes a control unit (such as a single-chip microcomputer) and a control circuit. The control unit communicates with the control terminal to receive instructions, and applies control signals to the control circuit according to the instructions to control the electrode array 5 and Control of the temperature control device 6.

As shown in Fig. 1b, an embodiment of the present application provides a detection system. Based on the detection system shown in Fig. 1a, it further includes a detection device 1 arranged on the surface of the cover 4 away from the electrode array 5, so The detection device 1 is configured to detect liquid droplets in the space between the electrode array 5 and the cover 4 and output the detection result.

In an exemplary embodiment, the detection device 1 is, for example, a fluorescence detection device.

In an exemplary embodiment, the fluorescence detection device is configured to detect the fluorescence signal of the droplet located in the space between the electrode array and the cover and output the detection result.

In an exemplary embodiment, the fluorescence detection device is usually set at the position of the droplet after the reaction is finally completed, such as the position corresponding to the amplification zone described below. Wherein, the corresponding position refers to the position where the orthographic projection of the amplified region on the cover 4 is located.

In an exemplary embodiment, each amplification zone corresponds to a fluorescence detection device.

In an exemplary embodiment, the detection device 1 may also be other devices, such as an electron microscope.

As shown in Fig. 2, the detection system is connected to the control terminal 9, and the operator performs real-time control, detection and other operations on the detection system through the control terminal 9. Of course, it can also be set in advance to realize automatic detection. The control terminal 9 sends a control instruction to the drive circuit 7, and according to the control instruction, the drive circuit 7 controls the electrode array 5, turns off the electrodes or turns on (or activates) the electrodes, and drives the droplets. Wherein, closing the electrode is achieved, for example, by suspending the electrode or grounding, and opening the electrode is achieved, for example, by applying a bias voltage of a certain amplitude and frequency to the electrode. In addition, the drive circuit 7 controls the temperature control device 6 according to the control command to realize the control of the temperature of the electrode array 5. In addition, the control terminal 9 also controls the fluorescence detection device 1, and the control terminal 9 sends instructions to the fluorescence detection device 1 to activate the fluorescence detection device 1 to detect the fluorescence signal of the droplet, and receive the detection result of the fluorescence detection device 1.

In an exemplary embodiment, the control terminal 9 may be a computer, a tablet, a smart terminal or other types of terminals.

In an exemplary embodiment, the control terminal 9 may be a control terminal dedicated to the detection system.

In an exemplary embodiment, the above-mentioned detection system may further include a fluid processing system, for example, a tube (not shown), a valve (not shown), and a pump (not shown), which are configured to control the electrode array 5 and The supply and removal of liquid droplets in the spaces between the covers 4.

In an exemplary embodiment, the fluid processing system may be provided with an interface for connecting with other instruments, such as a flow cytometer, a next-generation sequencer, a mass spectrometer, an electrochemical workstation, etc.

In an exemplary embodiment, the electrode array 5 includes one or more electrodes 51, and the electrodes 51 may be connected to the driving circuit 7 using a passive connection or an active connection.

As shown in FIG. 3, in the passive connection mode, each electrode 51 is connected to the driving circuit 7 through a connecting wire 31.

In the active connection mode, the electrode layer further includes a control device corresponding to the electrode 51 one-to-one, the control device is connected to the corresponding electrode, and the control device includes a switch element configured to control the application to the electrode 51. The bias voltage of the electrode corresponding to the control device; the driving circuit 7 is connected to the control device in the electrode layer through a control line array, and the control line array includes a set of primary control lines and a set of secondary control lines, It is configured so that each control device can be individually addressed by a given primary control line and a given secondary control line. The primary control line is, for example, a row address line, and the secondary control line is, for example, a column address line. As shown in FIG. 3, each electrode 51 includes a pixel circuit 32 (that is, a control device), and each electrode 51 uses a row and column code connection mode. The counter electrode 51 can be realized by controlling the row address line 33 and the column address line 34. Opening and closing. The pixel circuit 32 includes, for example, a thin film transistor and a capacitor (1T1C), and the pixel circuit 32 may be other types of switching elements.

Among them, the active connection method can effectively reduce the number of connection lines and reduce the hardware complexity than the passive connection method.

In an exemplary embodiment, the shape of the electrode 51 may be a geometric shape such as a square, a hexagon, or other polygons.

In an exemplary embodiment, the edge of the electrode 51 has a sawtooth or wave shape (as shown in FIG. 4) to assist the droplet to move between the electrodes.

In an exemplary embodiment, there is a very narrow gap 41 (usually tens to hundreds of microns) between the electrodes 51 to form a physical barrier between the electrodes to prevent short circuits of the electrodes.

In an exemplary embodiment, the droplet volume that can be controlled by each electrode 51 is determined by the size of the electrode plane and the separation distance between the electrode array 5 and the upper cover 4, and the accuracy of the liquid volume that can be processed can be accurate to a scale.

The electrode array 5 can be designed as a functional structure capable of realizing a multi-channel LAMP reaction. The regions formed by the electrodes of the electrode layer include: sample injection region, amplification region, mixing region, and waste liquid region. The regions are connected by a path formed by electrodes, and each sample injection region is located on the cover. There is a sample inlet at the corresponding position, each waste liquid area has a waste liquid outlet at a corresponding position on the lid, the amplification area is the area where the sample to be tested reacts with the auxiliary reactant, and the mixing area The area where one or more auxiliary reactants other than the sample to be tested are mixed. Wherein, the corresponding position of each sample injection area on the cover refers to the position of the orthographic projection of the sample injection area on the cover, and the corresponding position of each waste liquid area on the cover refers to The position of the orthographic projection of the waste liquid area on the cover. One said injection zone and one said amplification zone constitute one channel, and said electrode array 5 includes a plurality of said channels. The connection between the regions by a path formed by electrodes includes: the sample injection area and the amplification area in each channel are connected by a path formed by electrodes, and the mixing area and the amplification area are connected by a path formed by electrodes, so The mixing area and the sample injection area located outside the channel are connected by a path formed by electrodes, and the amplification area is connected with at least one waste liquid area by a path formed by electrodes. The sample injection area includes a plurality of samples and auxiliary reactants respectively. Generally, each channel includes a sample injection zone, and the sample injection zone provided outside the channel is related to the number of auxiliary reactants, and each auxiliary reactant corresponds to one sample injection zone. The waste liquid after the reaction is transferred to the waste liquid area and then removed through the waste liquid outlet. In addition, the electrode layer also includes a ground electrode. The positional relationship between the ground electrode and each of the above-mentioned regions is mosaic, that is, between each of the above-mentioned regions is the ground electrode, and the bias voltage of the ground electrode is ground, which can prevent droplets from entering the ground electrode. The area only moves in the area other than the ground electrode, that is, in the injection area, mixing area, amplification area and waste liquid area mentioned above.

In an exemplary embodiment, the structure of each of the channels is the same, and there are at least two channels among the plurality of channels, and the electrodes at the same position of the at least two channels are short-circuited. For example, the electrodes in the same position of all channels are shorted.

In an exemplary embodiment, the electrodes at the same position in each channel may not be short-circuited. The structure of different channels can also be different.

As shown in FIG. 5, it is an electrode array that can realize 3 groups of LAMP in parallel. The area formed by the electrodes of the electrode layer of the electrode array 5 includes: sample injection area 52 (52 ₁ to 52 ₆ as shown in the figure), mixing In the area 53, the amplification area 54 (54 ₁ to 54 ₃ in Fig. 5) and the waste liquid area 55, the different areas are connected by a path formed by electrodes. In addition, the electrode layer also includes a ground electrode 56. The positional relationship between the ground electrode 56 and each of the above-mentioned regions is mosaic, that is, between each region is the ground electrode 56, and the bias voltage of the ground electrode 56 is ground, which can prevent droplets from entering The area where the ground electrode 56 is located only moves in the area other than the ground electrode 56, that is, moves in the above-mentioned sample injection area 52, mixing area 53, amplification area 54 and waste liquid area 55. The lid 4 corresponding to each sample injection area 52 has a sample inlet 2 for sample injection (including auxiliary reactants), and the lid 4 corresponding to each waste liquid area 55 has a waste liquid outlet 3 for drawing off the waste liquid. . Injection zone ₅₂₄ and expansion region ₅₄₁ forms a channel region ₅₂₅ and the sample amplified region ₅₄₂ forms a passage, the injection zone ₅₂₆ and expansion region ₅₄₃ forms a channel, from the injection zone 52 ₁ to 52 ₃ were injected into the auxiliary reactants, mixed in the mixing zone 53, respectively injected samples from the sample injection zone 52 ₄ to 52 ₆ , the samples were transferred to the corresponding amplification zone, the mixed auxiliary The reactants are transferred to the amplification zone, where the sample and auxiliary reactants react in the amplification zone. Take 3 sets of LAMP and real-time fluorescence detection as an example. Using this detection system, the user only needs to inject the original sample 6 times (3 injections of samples, 3 injections of auxiliary reactants), and the system can be fully automated 3 groups of LAMP and real-time fluorescence detection. Compared with the prior art, for each group of LAMP, it is necessary to inject 3 auxiliary reactants, 1 sample, a total of 4 times, and 3 groups of 12 injections in total. Using the detection system provided in this embodiment greatly reduces the number of operations. . If there are hundreds or more groups of LAMP, the reduction of the number of operations is more significant, and the detection efficiency is greatly improved. For example, for one hundred groups, 4*100=400 times are required in the related technology. Using the solution provided in this embodiment, 100 +3=103. The electrode array shown in FIG. 5 is only an example, and this application is not limited to this. For example, it may include more sample injection areas, more amplification areas, and more waste liquid areas. The waste liquid area is arranged in the peripheral area, which may include more Multiple electrodes, changing the shape of each area, etc.

The electrode array 5 can be expanded to increase the reaction throughput. For example, the electrode array shown in Figure 6 can perform 5 groups of LAMP at the same time. The electrode array 5 shown in FIG. 6 includes 5 channels (channel 61 to channel 65), and samples can be injected into each channel at the same time, thereby performing 5 sets of LAMP at the same time.

The electrode array 5 can also be cascaded and combined to increase the reaction flux. Among them, cascade is equivalent to using multiple identical electrode array chips, connecting the control pins one by one in parallel, and using the same drive circuit for control. The combination can use different electrode array chips and driving circuits, assembled together for use. The above 3 groups of LAMP and 5 groups of LAMP are just examples. The reaction flux is determined by the size of the electrode array. For example, hundreds of groups of LAMP or more groups of LAMP can be performed.

The basic operations of the droplet 8 on the electrode array 5 include transfer, fusion, stirring and separation. The above-mentioned operations on the droplets can be realized by controlling the electrodes on the electrode array 5.

As shown in Figure 7, the transfer operation of small droplets and large droplets can be realized by opening the corresponding electrodes, and droplets can be merged by the transfer operation.

For small droplets, as shown in Figure 7(a), a bias voltage of a certain frequency and amplitude is applied to the electrode 71 to open the electrode 71, and the other electrodes are closed, so that the droplet remains on the electrode 71. A bias voltage of a certain frequency and amplitude is applied to the electrode 72 to open the electrode 72, and the other electrodes are closed, and the droplets are transferred to the electrode 72.

For large droplets, as shown in Figure 7(b), a bias voltage of a certain frequency and amplitude is applied to the electrodes 71 to 77 to turn on the electrodes 71 to 77, and the remaining electrodes are closed to keep the drop on the electrode 71 ~ On the area formed by the electrode 77, then, apply a bias voltage of a certain frequency and amplitude to the electrodes 71~73, and the electrodes 77~80 to open the electrodes 71~73, the electrodes 77~80, and the other electrodes are closed, The droplets are transferred to the area constituted by the electrodes 71 to 73 and the electrodes 77 to 80.

As shown in Figure 8, the droplet stirring operation can be realized by turning on the corresponding electrodes in a cycle. Specifically:

Open the electrode 84, the electrode 85, the electrode 88 to the electrode 810, the electrode 813 and the electrode 814, and the other electrodes are closed, so that the liquid droplet is kept in the area formed by the electrode 84, the electrode 85, the electrode 88 to the electrode 810, the electrode 813, and the electrode 814;

Open electrode 89, electrode 810, electrode 813 to electrode 815, electrode 817, and electrode 818, and close the other electrodes, so that the droplet moves to the area composed of electrode 89, electrode 810, electrode 813 to electrode 815, electrode 817, and electrode 818;

Open electrode 810, electrode 811, electrode 814 to electrode 816, electrode 818, electrode 819, and close the other electrodes, so that the droplet moves to the area formed by electrode 810, electrode 811, electrode 814 to electrode 816, electrode 818, and electrode 819;

Open electrode 86, electrode 87, electrode 810 to electrode 812, electrode 815, electrode 816, and close the other electrodes, so that the droplet moves to the area constituted by electrode 86, electrode 87, electrode 810 to electrode 812, electrode 815, and electrode 816;

Open the electrode 82, the electrode 83, the electrode 85 to the electrode 87, the electrode 810, and the electrode 811, and the other electrodes are closed, so that the droplet moves to the area composed of the electrode 82, the electrode 83, the electrode 85 to the electrode 87, the electrode 810, and the electrode 811;

The electrode 81, the electrode 82, the electrode 84 to the electrode 86, the electrode 89, and the electrode 810 are opened, and the remaining electrodes are closed, so that the droplet moves to the area formed by the electrode 81, the electrode 82, the electrode 84 to the electrode 86, the electrode 89, and the electrode 810.

Through the above process, the stirring of the droplets is realized.

As shown in Figure 9, the separation operation of droplets can be realized by means of a functional electrode structure, and the separated volume can be precisely controlled by the number of opened electrodes. The specific implementation is as follows:

Open the electrodes 91 to 97, and close the other electrodes, so that the droplets remain in the area formed by the electrodes 91 to 97;

Open the electrode 91, the electrode 93, the electrode 94, the electrode 96, the electrode 97, the electrode 98, the electrode 99, the electrode 910, the other electrodes are closed, the droplet moves to the electrode 91, the electrode 93, the electrode 94, the electrode 96, the electrode 97, the electrode 98 , The area formed by the electrode 99 and the electrode 910.

Open electrode 98, electrode 99, electrode 910, as well as electrode 91, electrode 92, electrode 94, electrode 95, electrode 96, electrode 97, the rest of the electrodes are closed, the droplet is divided into two parts, one part is composed of electrode 98, electrode 99, electrode 910 The other part is in the area composed of electrode 91, electrode 92, electrode 94, electrode 95, electrode 96, and electrode 97, so as to realize the separation of droplets.

As shown in FIG. 10, an embodiment of the present application provides a detection method, which is applied to the detection system described in any embodiment, including:

Step 1001, controlling the electrode array 5 to stir the separately injected auxiliary reactants;

Among them, the auxiliary reactant refers to the reactant required for detection in addition to the sample. When there are multiple auxiliary reactants, they are injected separately.

Step 1002, controlling the electrode array 5 to mix and stir the auxiliary reactants to obtain an amplification reaction solution;

Wherein, the auxiliary reactant is moved to the same area to realize the mixing of the auxiliary reactant, and the auxiliary reactant is mixed uniformly by stirring.

Step 1003, controlling the electrode array 5 to mix and stir the injected reaction solution containing the sample and at least part of the amplification reaction solution;

In addition to the sample, the reaction solution containing the sample may also include a fluorescent probe for subsequent detection of the fluorescent signal.

Step 1004: Start the temperature control device 6 so that the temperature of the electrode array meets the reaction demand.

Taking LAMP as an example, turn on the temperature control device 6 to keep the temperature of the electrode array 5 at 65°C for 60 minutes;

Taking PCR as an example, 4 temperatures are involved, and the amplification process needs to cycle 30-40 times between 3 temperatures, and the temperature control device is controlled to make the electrode array 5 cycle 30-40 times between 3 temperatures.

In an embodiment, the controlling the electrode array 5 to mix and stir the auxiliary reactants to obtain the amplification reaction solution includes:

Control the electrode array 5 to transfer the auxiliary reactant from the sample injection area to the mixing area and stir to obtain an amplification reaction solution;

The controlling the electrode array 5 to mix and stir the injected reaction solution containing the sample and at least part of the amplification reaction solution includes:

Controlling the electrode array 5 to transfer the amplification reaction solution in the mixing zone to the amplification zone;

The electrode array 5 is controlled to transfer the injected reaction solution containing the sample to the amplification zone, and the electrode array 5 is controlled to stir the reaction solution containing the sample in the amplification zone and the amplification reaction solution.

In one embodiment, the electrode array 5 is controlled to transfer the auxiliary reactant from the sample injection area to the mixing area and stirred to obtain the amplification reaction solution, and the electrode array 5 is controlled to amplify the mixing area. The transfer of the reaction solution to the amplification area includes:

Step 10. Control the electrode array 5 to transfer part of the auxiliary reactant in the sample injection zone to the mixing zone, and control the electrode array 5 to stir the auxiliary reactant in the mixing zone to obtain an amplification reaction solution; The electrode array 5 is controlled to transfer the amplification reaction solution in the mixing zone to an amplification zone; this step 10 is repeated until the amplification zone required for detection contains the amplification reaction solution. For example, N groups of detection are required, so that the N amplification regions all contain the amplification reaction solution. In the case of multiple channels, inject the auxiliary reactants required by each channel at a time, and then separate part of the auxiliary reactants each time and move to the mixing zone, and move to the amplification zone after mixing with other auxiliary reactants to avoid The auxiliary reactants required for each channel are injected separately, reducing the number of manual operations and improving efficiency.

In an exemplary embodiment, the controlling the electrode array 5 to transfer the injected reaction solution containing the sample to the amplification zone includes: controlling the electrode array 5 to transfer the injected reaction solution containing the sample to the amplification zone in parallel. The amplified region. Through the parallel transfer, the detection of multiple channels can be realized together, and the detection efficiency is improved.

In an exemplary embodiment, the transfer may not be performed in parallel as needed.

In an exemplary embodiment, activating the temperature control device 6 so that the temperature of the electrode array 5 meets the reaction requirement includes:

The temperature control device 6 is activated so that the temperature of the electrode array 5 is the target temperature and maintained for a predetermined period of time. The predetermined duration may be determined according to the required duration. For example, keep the temperature at 65°C for 60 minutes.

In an exemplary embodiment, after the temperature control device 6 is activated so that the temperature of the electrode array meets the reaction demand, the method further includes: detecting the liquid droplets in the space between the electrode array 5 and the cover 4 The fluorescent signal is detected and the detection result is output.

In an exemplary embodiment, a combination of loop-mediated isothermal amplification and electrowetting dielectric technology is used to establish a method for rapid molecular detection of Salmonella. Salmonella is the main intestinal pathogen, which mainly causes clinical manifestations such as fever, gastroenteritis, diarrhea and sepsis. In China, the number of Salmonella infections is as high as 3 million each year. Therefore, the detection of Salmonella is of great significance.

According to the Salmonella invasion protein A (invasion protein A, invA) gene nucleotide sequence, primers were designed, and the invA gene fragment was amplified by strand displacement DNA polymerase under constant temperature conditions.

In this embodiment, the electrode array shown in FIG. 5 is used to complete the Salmonella invA gene detection of 3 sets of samples. The three groups of samples are: Salmonella genomic DNA (positive control), pure water (negative control), and samples to be tested.

Each reaction requires the use of 8uL pure water, 12.5uL DNA polymerase and buffer mixture, 2.5uL primers and 2uL DNA sample and fluorescent probe mixture. As shown in Figure 11, the specific process is as follows:

Step 1101, inject 24uL pure water, 37.5uL DNA polymerase and buffer mixture and 7.5uL primer from the sample inlet of pure water, DNA polymerase and buffer mixture, and primers respectively, and place them in the corresponding injection area Turn on the corresponding electrode and continue to stir the sample for a certain length of time (for example, 5 minutes) to make the sample fully uniform; the stirring direction is, for example, clockwise (as shown in Figure 12). This embodiment does not limit this, and the stirring direction can be other directions, such as counterclockwise, left and right, up and down stirring, and so on.

12, the pure water 24uL sample from the sample inlet zone ₅₂₁ is injected, the mixture 37.5uL DNA polymerase and a buffer zone from the injector ₅₂₂ injecting the sample inlet, the feed from the primer 7.5uL the sample inlet zone ₅₂₃ of sample injection, the respective open sample electrode stirring continued certain length of time.

For the stirring method, reference may be made to the description of the embodiment shown in FIG. 8, which will not be repeated here.

Step 1102: Turn on the corresponding electrode to separate 8uL pure water from 24uL pure water and transfer it to a position above the center of the mixing zone, during which other samples are still in a stirring state.

As shown in FIG. 13 to FIG, 14 will be separated from the water 8uL 24uL water injection zone ₅₂₁ out; FIG 15, the separated water 8uL transferred to a central position 53 on partial mixing zone .

For the separation method, refer to the description of the embodiment shown in FIG. 9, and for the transfer method refer to the description of the embodiment shown in FIG. 7, which will not be repeated here.

Step 1103: Turn on the corresponding electrode to separate the mixture of 12.5uL DNA polymerase and buffer from the mixture of 37.5uL DNA polymerase and buffer and transfer to the upper center of the mixing zone to fuse with 8uL pure water. During this period, other samples were still in agitation state.

As shown in Figure 16-17, the mixture of 12.5uL DNA polymerase and buffer is separated from the mixture of 37.5uL DNA polymerase and buffer in the sampling area 52 ₂ ;

As shown in Figure 18, transfer the separated 12.5uL DNA polymerase and buffer solution to a position above the center of the mixing zone 53, and merge with 8uL pure water.

For the realization of the separation and fusion of the droplets, reference may be made to the description of the embodiments shown in FIG. 7 and FIG. 9, which will not be repeated here.

Step 1104, open the corresponding electrode to separate the 2.5uL primer from the 7.5uL primer and transfer it to the upper center of the mixing zone to fuse with a mixture of 20.5uL pure water, DNA polymerase and buffer, and mix to form an amplification reaction solution . During this period, the other samples were still in a stirring state.

19 to FIG., The primer 2.5uL separated from the injection zone ₅₂₃ primer 7.5uL 20;

As shown in Figure 21, the separated 2.5uL primer is transferred to a position above the center of the mixing zone 53, and fused with the mixture of 20.5uL pure water, DNA polymerase and buffer.

In step 1105, the corresponding electrodes are cyclically opened, and the 23 uL amplification reaction solution in the mixing zone is fully stirred for 5 minutes to make it completely uniform (as shown in Figure 22).

Step 1106: Turn on the corresponding electrode to transfer the stirred 23uL amplification reaction solution to an amplification area. As shown in Figure 23, 23 uL of the amplification reaction solution was transferred to the amplification zone 54 ₁ .

Step 1107, the open loop amplification of the corresponding region of the liquid mixture electrode ₅₄₁ (i.e., amplification reactions) continue agitation operation (Figure 24).

Step 1108, repeat steps 1102-1107 so that all three amplification zones contain 23uL of amplification reaction solution, and the amplification reaction solution is in a stirring state.

As shown in FIG. 25, the amplified region ₅₄₂ and the amplified region ₅₄₃ contains the amplification reaction solution 23uL.

Step 1109: Inject three 2uL sample and fluorescent probe mixtures (1uL each of the sample and the fluorescent probe) from the three sample inlets. The three samples are: positive control, negative control and sample to be tested.

26, the DNA sample is injected and a fluorescent probe mixture from the sample inlet ₅₂₄ corresponding to the sample area, injection of DNA samples from a mixture of 2 and the fluorescent probe corresponding to the sample inlet ₅₂₅ into the region of the sample, from the feed the sample inlet zone ₅₂₆ corresponding to the sample injection and a fluorescent probe DNA samples 3 mixture.

Step 1110: Transfer the three samples and the fluorescent probe mixture to the corresponding amplification area in parallel, and fuse with the 23 uL amplification reaction mixture and continue stirring.

As shown in Figure 27, the DNA sample 1 and the fluorescent probe mixture in the sample injection area 52 ₄ are transferred to the amplification area 54 ₁ in parallel, and the DNA sample 2 and the fluorescent probe mixture in the sample injection area 52 ₅ are transferred to the amplification area. increasing region _542, the injector region ₅₂₆ DNA samples 3 and fluorescent probe amplification mixture was transferred to the region _543.

Step 1111: Turn on the temperature control device 6 to keep the temperature of the electrode array 5 at 65° C. for 60 minutes, and turn on the fluorescence detection device 1 to detect the fluorescence signal of the mixed solution in real time, as shown in FIG. 28.

Step 1112, after the reaction is completed, the mixed liquid is transferred to the waste liquid area 60 and extracted from the waste liquid outlet 3.

Among them, the three groups of electrodes in the sample injection area where the DNA sample and the fluorescent probe mixture are injected and the corresponding amplification area can be short-circuited to reduce the number of wires and perform parallel operations.

For example, as shown in Fig. 29, the electrode A1, the electrode B1, and the electrode C1 are the electrodes at the same position in the three channels and are short-circuited.

In an exemplary embodiment, the electrodes at the same position among multiple channels are short-circuited.

The above embodiments take LAMP as an example to illustrate the embodiments of the application, but the embodiments of the application are not limited to this, and can be applied to a variety of nucleic acid amplification detection methods including PCR, LAMP, and other biochemical reactions with similar reaction processes, such as SDA (Strand Displacement Amplification), HAD (Helix dependent Amplification), NEAR (Nicking enzyme amplification), MDA (Multiple Displacement Amplification, multiple replacement amplification), EMA (Enzymes Mediated Amplification, room temperature nucleic acid amplification) etc.

The solution provided in this embodiment is a fully automatic system. It only needs to inject the original sample to automatically complete all reactions and detections, which saves reaction time and reduces manual operations. The electrode is used to separate the droplets, which has high accuracy in droplet processing, saving samples and reducing Small experimental error; and it is a high-throughput processing platform, which saves experimental time and reduces experimental costs; has good scalability and can be used for various application scenarios and needs, including PCR, LAMP and other nucleic acid amplification detection methods and other similar reaction processes Biochemical reaction. In addition, the integration of thin film transistor technology can reduce the number of connection lines, reduce equipment complexity and equipment cost.

As shown in FIG. 30, an embodiment of the present application provides a detection device 300, which includes a memory 3010 and a processor 3020. The memory 3010 stores a program. When the program is read and executed by the processor 3020, any The detection method described in an embodiment.

An embodiment of the present application provides a control terminal, including the detection device 300 described above.

An embodiment of the present application also provides a system, including the detection system described in any embodiment and the aforementioned control terminal.

As shown in FIG. 31, an embodiment of the present application provides a computer-readable storage medium 310. The computer-readable storage medium 310 stores one or more programs 3110, and the one or more programs 3110 can be stored by one or more programs. Executed by two processors to implement the detection method described in any embodiment.

The drawings of the embodiments of the present application only relate to the structures related to the embodiments of the present application, and other structures can refer to the usual design. For the sake of clarity, in the drawings used to describe the embodiments of the present application, the thickness of layers or regions is enlarged or reduced, that is, these drawings are not drawn according to actual scale. It can be understood that when an element such as a layer, film, region or substrate is referred to as being "on" or "under" another element, the element can be "directly" on or "under" the other element. Or there may be intermediate elements.

A person of ordinary skill in the art can understand that all or some of the steps, functional modules/units in the system, and apparatus in the methods disclosed above can be implemented as software, firmware, hardware, and appropriate combinations thereof. In the hardware implementation, the division between functional modules/units mentioned in the above description does not necessarily correspond to the division of physical components; for example, a physical component may have multiple functions, or a function or step may consist of one or Multiple physical components cooperate to execute. Some or all of the components may be implemented as software executed by a processor, such as a digital signal processor or a microprocessor, or as hardware, or as an integrated circuit, such as an application specific integrated circuit. Such software may be distributed on a computer-readable medium, and the computer-readable medium may include a computer storage medium (or non-transitory medium) and a communication medium (or transitory medium). As is well known by those of ordinary skill in the art, the term computer storage medium includes volatile and nonvolatile implementations in any method or technology for storing information (such as computer readable instructions, data structures, program modules, or other data). Flexible, removable and non-removable media. Computer storage media include but are not limited to RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tapes, magnetic disk storage or other magnetic storage devices, or Any other medium used to store desired information and that can be accessed by a computer. In addition, as is well known to those of ordinary skill in the art, communication media usually contain computer readable instructions, data structures, program modules, or other data in a modulated data signal such as carrier waves or other transmission mechanisms, and may include any information delivery media .

Claims

A detection system includes: a lid with a sample inlet and a waste liquid outlet, an electrode array, a temperature control device, and a drive circuit. The electrode array includes a hydrophobic layer on the side close to the lid and a side far from the lid. The side includes an electrode layer, a dielectric layer is included between the electrode layer and the hydrophobic layer, the electrode layer includes a plurality of electrodes, the driving circuit is respectively connected to the electrode array and the temperature control device, the There is a space for liquid droplets to move between the cover and the electrode array, the sample inlet is arranged for liquid droplets to enter the space between the cover and the electrode array, and the waste liquid outlet is arranged for liquid droplets from The space between the cover and the electrode array is moved out, the side of the cover close to the electrode array includes a hydrophobic layer, and the area formed by the electrodes of the electrode layer includes: a sample injection area, an amplification area, and a mixing area The area is connected to the waste liquid area by a path formed by electrodes, and each sample injection area has a sample inlet at a corresponding position on the lid, and each waste liquid area corresponds to the lid. There is a waste liquid outlet at the location, the amplification zone is the area where the sample to be tested and the auxiliary reactant react, and the mixing zone is the area where one or more auxiliary reactants other than the sample to be tested are mixed, wherein:

The temperature control device is configured to change the temperature of the electrode array based on the control of the drive circuit;

The driving circuit is configured to control the temperature control device and control the electrode array to change the state of the electrodes in the electrode array so as to drive all the electrodes located between the cover and the electrode array. The droplets.
The detection system according to claim 1, further comprising: a detection device disposed on a side of the cover surface away from the electrode array, the detection device being configured to align the space between the electrode array and the cover The droplets are detected and the detection result is output.
The detection system according to claim 2, wherein the detection device is a fluorescence detection device, and the fluorescence detection device is configured to detect fluorescence signals of liquid droplets located in the space between the electrode array and the cover And output the test results.
The detection system according to claim 1, wherein:

The electrode layer further includes a control device corresponding to the electrode one-to-one, the control device is connected to the corresponding electrode, and the control device includes a switch element configured to control the bias applied to the electrode corresponding to the control device Voltage;

The driving circuit and the control device in the electrode layer are connected by a control line array. The control line array includes a set of primary control lines and a set of secondary control lines, configured so that each control device can be controlled by a given The primary control line and the given secondary control line are addressed separately.
The detection system according to claim 1, wherein one of the sample injection area and one of the amplification areas constitute a channel, and the electrode array includes a plurality of the channels.
The detection system according to claim 5, wherein the structure of each of the channels is the same, and in the plurality of channels, there are at least two channels, and the electrodes at the same position of the at least two channels are short-circuited.
The detection system according to claim 5, wherein the connection between the areas by a path formed by electrodes comprises: the sample injection area and the amplification area in each channel are connected by a path formed by the electrodes, and the mixing area is connected to the The amplification area is connected by a path formed by electrodes, the mixing area is connected with the sample injection area outside the channel by a path formed by electrodes, and the amplification area is connected with at least one waste liquid area by a path formed by electrodes.
A detection method, applied to the detection system according to any one of claims 1 to 7, comprising:

Controlling the electrode array to stir the separately injected auxiliary reactants;

Controlling the electrode array to mix and stir the auxiliary reactants to obtain an amplification reaction solution;

Controlling the electrode array to mix and stir the injected reaction solution containing the sample and at least part of the amplification reaction solution;

The temperature control device is activated so that the temperature of the electrode array meets the reaction demand.
8. The detection method according to claim 8, wherein the controlling the electrode array to mix and stir the auxiliary reactants to obtain an amplification reaction solution comprises:

Controlling the electrode array to transfer the auxiliary reactant from the sampling area to the mixing area and stirring to obtain an amplification reaction solution;

The controlling the electrode array to mix and stir the injected reaction solution containing the sample and at least part of the amplification reaction solution includes:

Controlling the electrode array to transfer the amplification reaction solution in the mixing zone to the amplification zone;

The electrode array is controlled to transfer the injected reaction solution containing the sample to the amplification zone, and the electrode array is controlled to stir the reaction solution containing the sample in the amplification zone and the amplification reaction solution.
The detection method according to claim 9, wherein the control controls the electrode array to transfer the auxiliary reactant from the sample injection area to the mixing area and stirs to obtain the amplification reaction solution, and controls the electrode array to transfer the The transfer of the amplification reaction solution from the mixing zone to the amplification zone includes:

Step 10. Control the electrode array to transfer part of the auxiliary reactant in the sample injection zone to the mixing zone, control the electrode array to stir the auxiliary reactant in the mixing zone to obtain an amplification reaction solution; The electrode array transfers the amplification reaction solution in the mixing zone to an amplification zone;

Repeat step 10 until the amplification area required for detection contains the amplification reaction solution.
The detection method according to claim 9, wherein the controlling the electrode array to transfer the injected reaction solution containing the sample to the amplification zone comprises: controlling the electrode array to concurrently transfer the injected reaction solution containing the sample Transfer to the amplification zone.
The detection method according to claim 8, wherein activating the temperature control device so that the temperature of the electrode array meets the reaction requirement comprises:

The temperature control device is activated so that the temperature of the electrode array is the target temperature and maintained for a predetermined period of time.
The detection method according to any one of claims 8 to 12, wherein after activating the temperature control device so that the temperature of the electrode array meets the reaction requirement, it further comprises: correcting the temperature between the electrode array and the cover The fluorescent signal of the droplet in the space is detected and the detection result is output.
A detection device, comprising a memory and a processor, the memory stores a program, and when the program is read and executed by the processor, the detection method according to any one of claims 8 to 13 is implemented.