Via sensor and application thereof, and method for multiple analysis, multiple detection, multiple screening or multiple separation of analyte

Abstract

The present application relates to the technical field of via sensors, and in particular to a via sensor and an application thereof, and a method for multiple analysis, multiple detection, multiple screening or multiple separation of an analyte. The present application provides a via sensor, comprising a first electrode support plate, a second electrode support plate, and an intermediate sensing layer provided between the first electrode support plate and the second electrode support plate, wherein a first droplet movement chamber is formed between the first electrode support plate and the intermediate sensing layer, a second droplet movement chamber is formed between the second electrode support plate and the intermediate sensing layer, and a plurality of sensing micropores are provided on the intermediate sensing layer. The via sensor provided in the present application can perform multiple repeated analysis, detection, screening and separation of a set number of times on the analyte, thereby effectively improving the accuracy of analysis and detection of the analyte, and improving the purity and concentration of the analyte. Moreover, the costs and timeliness can also be taken into account while meeting the detection accuracy and the purity.

Description

Pore sensor and its application, method for multiple analysis, multiple detection, multiple screening or multiple separation of analyte TECHNICAL FIELD

[0001] The present application belongs to the technical field of pore sensor, and particularly relates to a pore sensor and its application, a method for multiple analysis, multiple detection, multiple screening or multiple separation of analyte. BACKGROUND

[0002] The pore sensor refers to a sensor device for directly or indirectly analyzing a random process or heterogeneity of an analyte molecule, such as a series of physical, chemical or biological characteristics including composition, sequence, species, quantity, shape, size, configuration, etc., by driving the analyte to pass through a pore with a size of picometer-millimeter, and then analyzing the analyte through optical or electrical signals. For example, in a typical pore sensor, a single analyte is driven into and passes through a pore with a specific size by a force field such as an electric field or a magnetic field or by mechanical driving, and the process of passing through the pore changes the ion current (or changes the surface current, transverse current or tunneling current, etc. on the membrane surface) passing through the pore. By analyzing the amplitude change, duration change and frequency change of the current, a random process or heterogeneity of an analyte molecule, such as a series of physical, chemical or biological characteristics including composition, sequence, species, quantity, shape, size, configuration, etc., can be directly or indirectly analyzed.

[0003] At present, when analyzing a random process or heterogeneity of an analyte molecule, such as physical, chemical, biological characteristics (such as a series of physical, chemical or biological characteristics including composition, sequence, species, quantity, shape, size, configuration, etc. of the analyte), various sizes and types of pore sensors can be used to directly or indirectly analyze and detect the analyte through optical or electrical signals. The pore sensor has many advantages as follows: 1) the pore sensor can make individual individuals in the analyte population to be studied pass through the pore one by one for counting, analysis and detection, that is, the population can be studied at the level of individual individuals; 2) the pore sensor can convert the physical, biological and chemical characteristics of the analyte individuals into optical and electrical signals which can be directly or indirectly analyzed and detected; 3) the pore sensor can provide a controllable platform for designing and constructing a biological simulation system, and can be used to study the complex interactions of biochemical molecules (such as the transfer characteristics of nuclear pore complex, etc.). However, due to various factors such as irregular motion, Brownian motion, fluctuation and configuration change of the analyte, and various noises (such as 1 / f noise, white noise, parasitic capacitance noise, sampling circuit noise, etc. from the detection device), the results of single analysis and detection of the analyte by the pore sensor produce various random errors, and the accuracy required cannot be achieved. SUMMARY

[0004] The application aims to provide a via-hole sensor and its application, a method for multiple analysis, multiple detection, multiple screening or multiple separation of an analyte, the via-hole sensor can repeatedly analyze, detect, screen and separate the analyte for a set number of times, effectively improving the accuracy of the analysis and detection of the analyte, and improving the purity and concentration of the analyte; meanwhile, the cost and timeliness can be considered under the premise of meeting the detection accuracy and purity.

[0005] To achieve the above-mentioned purpose, the application provides the following technical scheme:

[0006] The application provides a via-hole sensor, comprising a first electrode support plate and a second electrode support plate, and an intermediate sensing layer arranged between the first electrode support plate and the second electrode support plate, a first droplet moving chamber is formed between the first electrode support plate and the intermediate sensing layer, and a second droplet moving chamber is formed between the second electrode support plate and the intermediate sensing layer.

[0007] A plurality of first electrode units are arranged on the first electrode support plate, a plurality of second electrode units are arranged on the second electrode support plate, and a plurality of sensing micropores are arranged on the intermediate sensing layer and communicate the first droplet moving chamber and the second droplet moving chamber.

[0008] The first droplet moving chamber and the second droplet moving chamber are both used for loading a plurality of mutually separated driveable droplets, at least one of the plurality of driveable droplets contains an analyte, when the first droplet moving chamber is loaded with a plurality of first driveable droplets and the second droplet moving chamber is loaded with a plurality of second driveable droplets, any one of the first electrode units, any one of the first driveable droplets, any one of the sensing micropores, any one of the second driveable droplets and any one of the second electrode units form a closed loop with an external power supply.

[0009] Preferably, the driving method of the driveable droplet comprises one or more of a capillary method, a dielectrophoresis method, an electrowetting method, a dielectrowetting method, an electrowicking method, a surfactant method, a magnetic driving method, a light driving method and a thermal driving method.

[0010] Preferably, the sensing micropore is of nanoscale size or micrometer scale size, and the shape of the sensing micropore comprises one or more of a slit, a line, a tube, a strip, a field effect transistor and a nanopore extended field effect transistor.

[0011] Preferably, the first droplet moving chamber and / or the second droplet moving chamber is filled with a liquid medium that is immiscible with the driveable droplet.

[0012] Preferably, the intermediate sensing layer is further provided with a plurality of droplet channels that communicate the first droplet moving chamber and the second droplet moving chamber.

[0013] Preferably, the number of sensing microwells is ≥2, and the intermediate sensing layer is not provided with a droplet channel.

[0014] Preferably, the number of sensing microwells is 1, and the number of droplet channels is 1 or 2.

[0015] The application provides an application of the via sensor in the analysis, detection, screening and separation of an analyte, wherein the analyte includes ions, atoms, molecules, polymers, microorganisms, cells or cell complexes.

[0016] The application provides a method for multiple analysis or multiple detection of an analyte, when the via sensor is used, the number of sensing microwells is ≥2, and the intermediate sensing layer is not provided with a droplet channel, and the method comprises the following steps:

[0017] A first drivable droplet and a second drivable droplet are loaded in a first droplet moving chamber and a second droplet moving chamber, respectively, wherein the first drivable droplet and the second drivable droplet are an analyte-containing droplet and a blank droplet; the first drivable droplet and the second drivable droplet are repeatedly driven to the sensing microwells; a potential difference with a changeable direction is applied between the first electrode unit and the second electrode unit to perform multiple analysis or multiple detection of the analyte at the sensing microwells.

[0018] Or when the via sensor is used, the intermediate sensing layer of the via sensor is further provided with a plurality of droplet channels that communicate the first droplet moving chamber and the second droplet moving chamber, and the method comprises the following steps:

[0019] A first drivable droplet and a second drivable droplet are loaded in a first droplet moving chamber and a second droplet moving chamber, respectively, wherein the first drivable droplet and the second drivable droplet are an analyte-containing droplet and a blank droplet; the first drivable droplet and the second drivable droplet are repeatedly driven to the sensing microwells; a potential difference with a changeable direction is applied between the first electrode unit and the second electrode unit to perform multiple analysis or multiple detection of the analyte at the sensing microwells.

[0020] The application provides a method for multiple screening or multiple separation of an analyte, and the via sensor is used, and the method comprises the following steps:

[0021] loading 1 first driveable droplet in the first droplet moving chamber and loading multiple second driveable droplets in the second droplet moving chamber, the first driveable droplet and the second driveable droplets being analyte-containing droplets and blank droplets respectively; driving the first driveable droplet to the sensing microwell and driving the multiple second driveable droplets to the sensing microwell in sequence, and performing multiple screening or multiple separation of the analyte in the multiple blank droplets by applying a potential difference between the first electrode unit and the second electrode unit.

[0022] The application provides a through-hole sensor. The application presents the cis chamber and trans chamber of the traditional through-hole sensor in the form of flexibly driven droplets (which will be specifically described as cis droplets and trans droplets in the application). That is, the relative positions of the two "chambers" and the sensing microwell are no longer fixed, but can be changed by flexibly driving the droplets serving as the two "chambers". Meanwhile, the droplets serving as the two "chambers" can also establish a relationship with other sensing microwells together or separately, that is, the droplets serving as the two "chambers" can become the cis chamber and / or the trans chamber of other sensing microwells. Specifically, in the application, the cis droplet carrying the analyte is driven to the cis side of the sensing microwell, and the trans droplet not containing the analyte is driven to the trans side of the sensing microwell, thereby temporarily forming a through-hole sensor. After the analyte is driven from the cis droplet to pass through the sensing microwell and enter the trans droplet, the optical or electrical signal formed is collected, and the analysis and detection of the analyte are completed for the first time. The above-mentioned trans droplet containing the analyte is re-driven to the cis side of the sensing microwell or another sensing microwell, and another droplet is driven to the trans side of the corresponding microwell to form a trans droplet, thereby temporarily forming a new through-hole sensor. After the analyte is driven from the cis droplet of the new through-hole sensor to pass through the microwell and enter the trans droplet, the optical or electrical signal formed is collected, and the repeated analysis and detection of the analyte are completed. In this way, the through-hole sensor can be formed multiple times by continuously driving the droplets, so that the analyte can be repeatedly analyzed and detected for a set number of times, so as to improve the accuracy of the analysis and detection of the analyte. Moreover, the number of repeated analysis and detection can be personalized, so that we can consider the cost and timeliness in different projects under the premise of meeting the detection accuracy. In addition, the through-hole sensor provided by the application can perform multiple screening and separation of the analyte for a set number of times by the unique optical or electrical signal characteristics of the analyte passing through the sensing microwell. The multiple screening and separation can greatly improve the purity and concentration of the analyte. The analyte after multiple screening and separation can continue to be used for multiple analysis and detection in the through-hole sensor provided by the application, or for subsequent other applications.

[0023] In the present application, the specific implementation method of the via-hole sensor provided by the present application for multiple screening and separation is as follows: droplets carrying analyte groups are driven to the cis side of the micro-hole as cis droplets. Droplets without analytes are driven to the trans side of the micro-hole as trans droplets. Thus, a temporary via-hole sensor is formed. The analyte groups are driven through the micro-hole one by one in units of individuals and into the trans droplets. Here, positive screening or negative screening can be performed according to the specific conditions of the analyte groups. The process of positive screening is as follows: when one or more optical signals or electrical signals conforming to the target analyte are generated, it means that one or more target analytes have entered the trans droplets. At this time, the trans droplets are driven to the region of the target analyte for the next operation. Then, a new droplet without analytes is driven to the trans side of the micro-hole as a trans droplet, and one or more optical signals or electrical signals conforming to the target analyte are continuously generated, and the trans droplets are driven to the region of the target analyte for the next operation. In this way, each trans droplet driven to the region of the target analyte contains one or more target analytes. They can be subjected to the next operation. The process of negative screening is as follows: when one or more optical signals or electrical signals not conforming to the target analyte are generated, it means that one or more non-target analytes have entered the trans droplets. At this time, the trans droplets are driven to the region of the non-target analyte (such as a waste liquid region). Then, a new droplet without analytes is driven to the trans side of the micro-hole as a trans droplet, and one or more optical signals or electrical signals not conforming to the target analyte are continuously generated, and the trans droplets are driven to the region of the non-target analyte (such as a waste liquid region). In this way, more target analytes are retained in the cis droplets, and they can be subjected to the next operation. Positive and negative screening can also be performed simultaneously, and the specific process is as follows: when one or more optical signals or electrical signals conforming to the target analyte are generated, it means that one or more target analytes have entered the trans droplets. At this time, the trans droplets are driven to the region of the target analyte for the next operation. Then, a new droplet without analytes is driven to the trans side of the micro-hole as a trans droplet, and when one or more optical signals or electrical signals not conforming to the target analyte are generated, it means that one or more non-target analytes have entered the trans droplets. At this time, the trans droplets are driven to the region of the non-target analyte (such as a waste liquid region). In this way, each trans droplet driven to the region of the target analyte contains one or more target analytes. They can be subjected to the next operation. The actual process of positive screening, the process of negative screening, and the process of simultaneous positive and negative screening can be performed alternately. Only the process of positive screening, only the process of negative screening, or only the process of simultaneous positive and negative screening can be performed.

[0024] The through-hole sensor provided by the application can repeatedly analyze, detect and cross-screen and separate the analyte. For example, the analyte can be analyzed and detected for several times, then screened and separated for several times, and then analyzed and detected for several times, and so on. Alternatively, the analyte can be screened and separated for several times, then analyzed and detected for several times, and then screened and separated for several times, and so on. Alternatively, the analyte can be repeatedly analyzed and detected for several times or screened and separated for several times. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 is a schematic diagram of the basic structure of the through-hole sensor provided by the application;

[0026] Fig. 2 is a schematic diagram of the first analysis and detection of the analyte in the through-hole sensor provided by the application;

[0027] Fig. 3 is a schematic diagram of the first analysis and detection of the analyte in the through-hole sensor provided by the application;

[0028] Fig. 4 is a schematic diagram of the repeated analysis and detection of the analyte in the through-hole sensor (three-dimensional) provided by the application;

[0029] Fig. 5 is a schematic diagram of the repeated analysis and detection of the analyte in the through-hole sensor (three-dimensional) provided by the application;

[0030] Fig. 6 is a schematic diagram of the repeated analysis and detection of the analyte in the through-hole sensor (three-dimensional) provided by the application;

[0031] Fig. 7 is a schematic diagram of the repeated analysis and detection of the analyte in the through-hole sensor (two-dimensional) provided by the application;

[0032] Fig. 8 is a schematic diagram of the repeated analysis and detection of the analyte in the through-hole sensor (two-dimensional) provided by the application;

[0033] Fig. 9 is a method for single-molecule screening and separation of the analyte in the through-hole sensor provided by the application;

[0034] Fig. 10 is a method for single-molecule screening and separation of the analyte in the through-hole sensor provided by the application;

[0035] Fig. 11 is a method for batch-by-batch approximation screening and separation of the analyte in the through-hole sensor provided by the application;

[0036] Fig. 12 is a method for batch-by-batch approximation screening and separation of the analyte in the through-hole sensor provided by the application;

[0037] Fig. 13 is the experimental results of Example 1 of the application;

[0038] In the figure: 100 is a via hole sensor, 101 is an intermediate sensing layer, 102 is a first electrode support plate, 103 is a second electrode support plate, 104 is a sensing micro-hole, 105 is a droplet channel, 106 is a first droplet moving chamber, 107 is a second droplet moving chamber, 108 is a first electrode unit, 109 is a second electrode unit, 201 is a cis droplet, 202 is a first trans droplet, 203 is an analyte to be analyzed, 300 is a second trans droplet, 3 is a droplet inlet channel, 1 is a first droplet outlet channel, and 2 is a second droplet outlet channel.

[0039] Terminology:

[0040] The following technical terms related to the present application are explained:

[0041] Analyte: refers to various ions, atoms, molecules, polymers, viruses, bacteria, and other microorganisms, cells, and cell complexes, etc. whose physical, chemical, biological, and other characteristics are intended to be detected and analyzed. For example, amino acids, polypeptides, proteins, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), nucleotides (adenine, guanine, cytosine, thymine, uracil, dideoxyadenine, dideoxyguanine, dideoxycytosine, dideoxythymine, 5-methylcytosine, 5-hydroxymethylcytosine, etc.), nucleic acid analogs (peptide nucleic acid (PNA), locked nucleic acid (LNA), Morpholino (MNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), etc.), and nucleotide analogs (5-bromouracil, 2-aminopurine, 5-fluorouracil, hypoxanthine, adenosine 5'-monophosphate, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate, inosine 5'-monophosphate, uridine-5'-diphospho-α-D-galactose disodium salt, and other natural or artificially synthesized nucleotide analogs), nucleic acid-protein complexes, antigens, antibodies, antigen-antibody complexes, red blood cells, white blood cells, immune cells, platelets, stem cells, cell-antibody complexes, bacteria, fungi, viruses, viroids, metabolites, and the like.

[0042] Pore sensor: refers to a sensor device that directly or indirectly analyzes the random process or heterogeneity of the analyte molecules such as a series of physical, chemical or biological characteristics of composition, sequence, species, quantity, shape, size, configuration, etc. by driving the analyte to pass through the pore of picometer-millimeter level, and then analyzing the optical or electrical signal. For example, in a typical pore sensor, a single analyte is driven into and through a pore of a specific size by an electric field, a magnetic field, or other force field or mechanically, and the process of passing through the pore changes the ion current (or changes the surface current, transverse current or tunneling current, etc. on the membrane surface) passing through the pore. By analyzing the amplitude change, duration change and frequency change of the current, the random process or heterogeneity of the analyte molecules such as a series of physical, chemical or biological characteristics of composition, sequence, species, quantity, shape, size, configuration, etc. can be directly or indirectly analyzed. The sensor micropore mentioned in this patent not only represents the structure of the literal meaning of "pore" in physics, but also can be a structure such as a nanogap, a nanowire, a nanotube, a nanoribbon, etc. It can also be a structure that constitutes a transistor such as a field effect transistor (FET) or an extended field effect transistor (FET). It generally refers to a structure that can make the analyte pass through and cause characteristic changes in electrical or optical signals.

[0043] Correlation: In most cases, the correlation mentioned in this patent refers to the correlation of multiple analysis, detection information data of a single analyte. Since multiple analysis, detection information data are all generated by analyzing and detecting the same analyte individual, these information data have correlation and can be uniformly analyzed to improve the analysis and detection accuracy of the analyte individual.

[0044] Sequencing: In this patent, sequencing is a method for analyzing and detecting an analyte. For example, sequencing a nucleic acid refers to determining the base composition and sequence of a nucleic acid molecule; sequencing a protein or polypeptide molecule refers to determining the amino acid composition and sequence of a protein or polypeptide molecule.

[0045] Screening: refers to the operation of separating each analyte individual in a mixture of analytes from the mixture of analytes according to the unique characteristics of electrical or optical signals when each analyte individual passes through the pore sensor.

[0046] Ion current: In an electric field, positive and negative ions in an electrolyte solution move in opposite directions, forming an ion current. In this patent, the ion current mostly refers to the current formed by the movement of positive and negative ions in the solution in opposite directions through the sensor micropore when an electric field is applied between the cis-chamber and the trans-chamber of the pore sensor.

[0047] Transverse current: The transverse current mentioned in this patent is relative to the ion current. First, the ion current is defined as the longitudinal current, which is the direction of the current formed by the ions passing through the sensor micropore between the cis chamber (top) and the trans chamber. Then the transverse current refers to the current that is 90 degrees to the direction of the ion current. In the field of sensor micropore detection, it usually refers to the current flowing through the metal solid-state film in the sensor micropore made of metal solid-state film (such as graphene film, gold film, platinum-gold film, indium tin oxide (ITO) film, etc.). It is also called surface current. In a broad sense, the tunneling current can also belong to the transverse current.

[0048] Tunneling current: In the pore sensor based on the structure of nanogap, nanowire, nanotube, nanoribbon, etc. or the structure of transistor such as field effect transistor (FET) or extended field effect transistor (FET), due to the difference in conductivity of the analyte and the electrolyte solution, the transverse current will change when the analyte passes through the hole. The transverse current when the analyte passes through the hole is the tunneling current. In a broad sense, the tunneling current can also belong to the transverse current.

[0049] MEMS (Micro-Electro Mechanical System): Micro-electro-mechanical system, which is a micro intelligent system composed of mechanical, electronic, optical and other functional elements integrated on a single chip or multiple chips, which can sense, identify, control and process natural information such as sound, light, heat and magnetic movement.

[0050] Biomarker: It refers to a biochemical indicator that can mark the changes or possible changes of the system, organs, tissues, cells and subcellular structures or functions, and has very wide application. Biomarkers can be used for disease diagnosis, disease staging or to evaluate the safety and effectiveness of new drugs or new therapies in target populations. DETAILED DESCRIPTION

[0051] The application provides a pore sensor, which comprises a first electrode support plate and a second electrode support plate, and an intermediate sensing layer arranged between the first electrode support plate and the second electrode support plate, a first droplet moving chamber is formed between the first electrode support plate and the intermediate sensing layer, and a second droplet moving chamber is formed between the second electrode support plate and the intermediate sensing layer.

[0052] A plurality of first electrode units are arranged on the first electrode support plate, a plurality of second electrode units are arranged on the second electrode support plate, and a plurality of sensing micropores are arranged on the intermediate sensing layer and communicate the first droplet moving chamber and the second droplet moving chamber.

[0053] The first droplet moving chamber and the second droplet moving chamber are used for loading a plurality of mutually separated driveable droplets, at least one of the plurality of driveable droplets containing an analyte to be analyzed, when the first droplet moving chamber is loaded with a plurality of first driveable droplets and the second droplet moving chamber is loaded with a plurality of second driveable droplets, any one of the first electrode units, any one of the first driveable droplets, any one of the sensing microwells, any one of the second driveable droplets and any one of the second electrode units form a closed loop with an external power supply.

[0054] In the present application, all the preparation raw materials / components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0055] FIG. 1 is a structural schematic diagram of a via hole sensor provided by the present application. The via hole sensor provided by the present application will be described in detail below in combination with FIG. 1.

[0056] The via hole sensor 100 provided by the present application comprises a first electrode support plate 102 and a second electrode support plate 103. A plurality of first electrode units 108 are arranged on the first electrode support plate, and a plurality of second electrode units 109 are arranged on the second electrode support plate. The first electrode units 108 and the second electrode units 109 are connected with an external power supply, and can form a forward or reverse potential difference from the first electrode units 108 to the second electrode units 109.

[0057] As one or more embodiments of the present application, the first electrode units 108 are inlaid in the first electrode support plate 102.

[0058] As one or more embodiments of the present application, the second electrode units 109 are inlaid in the second electrode support plate 103.

[0059] The via hole sensor 100 provided by the present application comprises an intermediate sensing layer 101 arranged between the first electrode support plate 102 and the second electrode support plate 103. A first droplet moving chamber 106 is formed between the first electrode support plate 102 and the intermediate sensing layer 101, and a second droplet moving chamber 107 is formed between the second electrode support plate 103 and the intermediate sensing layer 101.

[0060] The through-hole sensor 100 provided in the present application is provided with a plurality of sensing micropores 104 on the intermediate sensing layer 101, which communicate the first droplet moving chamber 106 and the second droplet moving chamber 107. When the analyte to be analyzed is driven by the potential difference between the first electrode unit 108 and the second electrode unit 109 to pass through the sensing micropore 104, the generated electrical signal or optical signal is collected for analysis and detection. In the present application, the sensing micropore 104 is preferably of nanoscale or micrometer scale. In the present application, the sensing micropore 104 refers to a structure that can allow the analyte to pass through and cause characteristic changes in electrical or optical signals. The sensing micropore 104 not only represents the literal meaning of the structure of "hole" in the physical description, but also can be a structure such as a gap, a wire, a tube, a belt, etc. The shape of the sensing micropore 104 includes one or more of a nanogap, a nanowire, a nanotube, a nanoribbon, a field effect transistor (FET), and a nanohole extended field effect transistor (FET). In the present application, the sensing micropore 104 is sized according to the size of the analyte to be analyzed. In the present application, the size of the sensing micropore is related to the size of the analyte to be detected. The principle is that the analyte passing through the hole can generate specific blocked ion current pulses or transverse currents or tunneling currents.

[0061] In specific embodiments of the application, the through-hole sensor 100 can be provided with various sizes, types, and even various modified sensing micropores 104 according to the characteristics of different analytes to be analyzed and the detection requirements of signals. As long as the sensing micropore 104 has liquid on both sides and the analyte to be analyzed passes through the sensor from one side to the other side and the signal is detected, the structure can be applied to the cis-chamber and trans-chamber in the form of the driven droplet in the present application. In the present application, the sensing micropore 104 and the cis-droplet and trans-droplet loaded in the first droplet moving chamber 106 and the second droplet moving chamber 107 together constitute the through-hole sensor unit structure.

[0062] The through-hole sensor 100 provided in the present application is provided with a plurality of sensing micropores 104 on the intermediate sensing layer 101, which communicate the first droplet moving chamber 106 and the second droplet moving chamber 107. When the analyte to be analyzed is driven by the potential difference between the first electrode unit 108 and the second electrode unit 109 to pass through the sensing micropore 104, the generated electrical signal or optical signal is collected for analysis and detection. In the present application, the sensing micropore 104 is preferably of nanoscale or micrometer scale. In the present application, the sensing micropore 104 refers to a structure that can allow the analyte to pass through and cause characteristic changes in electrical or optical signals. The sensing micropore 104 not only represents the literal meaning of the structure of "hole" in the physical description, but also can be a structure such as a gap, a wire, a tube, a belt, etc. The shape of the sensing micropore 104 includes one or more of a nanogap, a nanowire, a nanotube, a nanoribbon, a field effect transistor (FET), and a nanohole extended field effect transistor (FET). In the present application, the sensing micropore 104 is sized according to the size of the analyte to be analyzed. In the present application, the size of the sensing micropore is related to the size of the analyte to be detected. The principle is that the analyte passing through the hole can generate specific blocked ion current pulses or transverse currents or tunneling currents.

[0063] In the present application, when the first droplet moving chamber 106 is loaded with a plurality of first drivable droplets, and the second droplet moving chamber 107 is loaded with a plurality of second drivable droplets, any one of the first electrode unit 108, any one of the first drivable droplets, any one of the sensing micropores 104, any one of the second drivable droplets, and any one of the second electrode units 109 form a closed loop with the external power supply.

[0064] In the present application, the driving method of the droplet includes one or more of a capillary method, a dielectrophoresis method, an electrowetting method, a dielectrowetting method, an electrowicking method, a surfactant method, a magnetic driving method, an optical driving method, and a thermal driving method.

[0065] In the present application, the capillary method generally introduces two-phase fluids at a cross point position in a microfluidic droplet preparation device, one phase being a continuous phase and the other phase being a dispersed phase (droplet). By connecting sub-millimeter conduits to sub-millimeter T-shaped and cross-shaped junction devices, droplets can be generated as in a microfluidic chip device, which is a relatively simple method for generating droplets.

[0066] In the present application, the dielectrophoresis method is that when a dielectric particle is in an electric field, positive and negative charges in the particle will gather to the two ends respectively due to the action of the applied electric field, causing uneven charge distribution at the two ends of the dielectric particle. Because the two ends of the particle have opposite electric properties, it is called an electric dipole. The induced dipole moment is a vector whose direction is from the negative charge gathering end to the positive charge gathering end of the particle. If the applied electric field is a uniform electric field, the dielectric particle is in an equilibrium state and does not move; if the applied electric field is a non-uniform electric field, the resultant force on the dielectric particle is not zero and the dielectric particle will move. When the dielectric constant of the dielectric particle is greater than the dielectric constant of the surrounding medium, the direction of the induced dipole moment is the same as that of the electric field, showing a positive dielectrophoresis phenomenon; when the dielectric constant of the dielectric particle is less than the dielectric constant of the surrounding medium, the direction of the induced dipole moment is opposite to that of the electric field, showing a negative dielectrophoresis phenomenon.

[0067] In the present application, the electrowetting method is that a droplet is directly located on an electrode array. When a voltage is applied, the surface tension between the liquid-solid interface changes, thereby causing a change in the wettability of the droplet.

[0068] In the present application, the dielectrowetting method is that the dielectrowetting method based on a medium is an electrically controlled surface tension driving method. It changes the wetting properties of the medium film and the surface liquid by applying an electric potential to the microelectrode array under the medium film, that is, by locally changing the three-phase contact angle of the microdroplet and the solid surface, causing the microdroplet to deform asymmetrically at the two ends, generating a pressure difference inside the microdroplet, thereby realizing the operation and control of the microdroplet.

[0069] In the present application, the electrowicking method is that a mechanism opposite to electrowetting is used. The droplet-substrate interaction is not directly controlled by the electric field, but is realized by the attachment and separation of field-induced ionic surfactants on the substrate.

[0070] In the present application, the surfactant method is that a functionalized surfactant is used. The droplet wrapped with such a functionalized surfactant can be driven by a magnetic field, optics, and an electric field at the same time.

[0071] In the present application, the magnetic force driving method is to add micro-magnets into the droplet, and to control the movement of the droplet by the action force of the external magnetic field on the droplet.

[0072] In the present application, the light driving method is to convert light into the driving force of the droplet movement by photochemistry, photomechanics, light-induced Marangoni effect, or light-induced electric field, etc.

[0073] In the present application, the thermal driving method is to make the contact angles of the two ends of the droplet inconsistent by creating a temperature difference between the two ends, so as to form an action force in the direction of the end with smaller contact angle, and to drive the droplet to move.

[0074] The present application takes into account the universality requirement of driving different droplets, and the droplet driving method based on magnetic force driving is taken as an example for description in the embodiments of the present application. However, the droplet driving method of the present application is not limited to the magnetic force driving method.

[0075] As one or more embodiments of the present application, the first droplet moving chamber 106 and / or the second droplet moving chamber 107 is filled with a liquid medium that is immiscible with the droplet. The liquid medium can help the droplet to move quickly and reduce the driving voltage.

[0076] In the present application, the intermediate sensing layer 101 is further provided with a plurality of droplet channels 105 that communicate the first droplet moving chamber and the second droplet moving chamber. The droplet channel 105 is used for the droplet replacement chamber, so as to perform repeated analysis and detection of the analyte, or repeated screening of the analyte, or to perform loading, merging, separation of the droplet, dilution and concentration of the analyte, etc. In the present application, the droplet channel 105 is a channel for the droplet to move from the first droplet moving chamber 106 to the second droplet moving chamber 107 or from the second droplet moving chamber 107 to the first droplet moving chamber 106. When the droplet is driven to the edge of the droplet channel 105, under the joint action of the tension generated by the droplet being extruded by the first electrode support plate 102 or the second electrode support plate layer 103 and the driving force of the droplet, the droplet will move to the other chamber through the droplet channel 105. In the present application, when the intermediate sensing layer 101 is only provided with the sensing microwell 104 and is not provided with the droplet channel 105, it is referred to as a via-hole sensor based on two-dimensional droplet movement. In the present application, when the intermediate sensing layer 101 is provided with both the sensing microwell 104 and the droplet channel 105, it is referred to as a via-hole sensor based on three-dimensional droplet movement.

[0077] In the present application, the number of sensing microwells 104 and droplet channels 105 is not limited to a 1:1 relationship. The sensing microwells 104 can have 1 to several in the form of a sensor array. The droplet channels 105 can also have 1 to several in the form of a droplet channel array. Figure 1 only schematically describes that the sensing microwells 104 and the droplet channels 105 are two functional structures in the present application.

[0078] As one or more embodiments of the present application, the first droplet moving chamber and / or the second droplet moving chamber are provided with a droplet inlet channel, a first droplet outlet channel and a second liquid outlet channel.

[0079] As one or more embodiments of the present application, the number of sensing microwells 104 is ≥2.

[0080] As one or more embodiments of the present application, the number of sensing microwells 104 is ≥2, and the intermediate sensing layer is not provided with a droplet channel.

[0081] The present application provides the application of the through-hole sensor described in the above technical solution in the analysis, detection, screening and separation of the analyte, which includes ions, atoms, molecules, polymers, microorganisms, cells or cell complexes.

[0082] The present application provides a method for multiple analysis or multiple detection of an analyte. When the through-hole sensor described in the above technical solution is used, the number of sensing microwells is ≥2, and the intermediate sensing layer is not provided with a droplet channel, the method comprises the following steps:

[0083] A first drivable droplet is loaded in the first droplet moving chamber, and a second drivable droplet is loaded in the second droplet moving chamber. The first drivable droplet and the second drivable droplet are respectively an analyte-containing droplet and a blank droplet. The first drivable droplet and the second drivable droplet are repeatedly driven to a plurality of sensing microwells. By applying an adjustable and reversible potential difference between the first electrode unit and the second electrode unit, multiple analysis or multiple detection of the analyte is carried out at the sensing microwells.

[0084] The present application provides a method for multiple analysis or multiple detection of an analyte. When the through-hole sensor described in the above technical solution is used, the intermediate sensing layer of the through-hole sensor is further provided with a plurality of droplet channels that communicate the first droplet moving chamber and the second droplet moving chamber, the method comprises the following steps:

[0085] loading one first droplet in the first droplet moving chamber and loading one second droplet in the second droplet moving chamber, the first droplet and the second droplet being analyte-containing droplet and blank droplet respectively; exchanging the droplets in the first droplet moving chamber and the second droplet moving chamber through the droplet channel, and driving the droplets in the first droplet moving chamber and the second droplet moving chamber to the sensing microwell, and performing multiple analysis or multiple detection of the analyte by applying a potential difference between the first electrode unit and the second electrode unit.

[0086] The application provides a method for multiple screening or multiple separation of an analyte, and the method comprises the following steps:

[0087] loading one first droplet in the first droplet moving chamber and loading multiple second droplets in the second droplet moving chamber, the first droplet and the second droplets being analyte-containing droplet and blank droplet respectively; driving the first droplet to the sensing microwell, and driving the multiple second droplets to the sensing microwell in sequence, and performing multiple screening or multiple separation of the analyte in the multiple blank droplets by applying a potential difference between the first electrode unit and the second electrode unit.

[0088] The following describes the first analysis and detection of an analyte by the through-hole sensor provided by the application in combination with FIG. 2 and FIG. 3:

[0089] FIG. 2 and FIG. 3 show the formation of the through-hole sensor provided by the application and the first analysis and detection of an analyte. The cis droplet 201 loaded with the analyte 203 is driven to the cis side of the sensing microwell 104 in the first droplet moving chamber 106. The trans droplet 202 not loaded with the analyte is driven to the trans side of the sensing microwell 104 in the second droplet moving chamber 107. At this time, the cis droplet 201 and the trans droplet 202 are electrically connected only through the sensing microwell 104. At this time, a through-hole sensor unit is formed. As shown in FIG. 2. When a potential difference is applied to the cis droplet and the trans droplet, the analyte passes through the sensing microwell 104 in sequence, and the first analysis and detection of the analyte is completed. The analyte enters the trans droplet 202. As shown in FIG. 3. In the application, the droplets can be mixed, separated, concentrated, diluted and the like before analysis and detection.

[0090] The following describes the method for repeated analysis and detection of an analyte by the through-hole sensor provided by the application in combination with FIG. 4 to FIG. 8:

[0091] The application provides two schemes for repeated analysis and detection of the analyte. Scheme one: repeated analysis and detection of the analyte based on three-dimensional droplet movement; scheme two: repeated analysis and detection of the analyte based on two-dimensional droplet movement. The two schemes can also be used in combination.

[0092] Scheme for repeated analysis and detection of the analyte based on three-dimensional droplet movement

[0093] Figure 4 exemplarily shows one of the schemes for repeated analysis and detection of the analyte based on three-dimensional droplet movement. After the first analysis and detection of the analyte in Figure 3 is completed, the droplet 201 without the analyte is driven out of the sensing micro-well 104 to a waste liquid storage area of the chip, and its position is occupied by the droplet 202 carrying the analyte passing through the droplet channel 105. The position of the original droplet 202 is then occupied by a new droplet 300 without the analyte. As shown in Figure 4, the droplet 202 carrying the analyte and the droplet 300 reconstitute the cis droplet and the trans droplet of the sensing micro-well. That is, a new through-hole sensor is formed. When a potential difference is applied to the cis droplet and the trans droplet, the analyte passes through the sensing micro-well 104 in turn, and the second analysis and detection of the analyte is completed. This operation can be performed multiple times, i.e., a set number of repeated analysis and detection is completed. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc. as required.

[0094] Figure 5 exemplarily shows another scheme for repeated analysis and detection of the analyte based on three-dimensional droplet movement. After the first analysis and detection of the analyte in Figure 3 is completed, the droplet 201 without the analyte is driven to a waiting area, and its original position is occupied by the droplet 202 carrying the analyte passing through the droplet channel 105. Then, the position of the original droplet 202 is occupied by the droplet 201 in the waiting area passing through the droplet channel 105. In this way, the positions of the droplet 201 and the droplet 202 are exchanged through the droplet channel 105. That is, a new through-hole sensor is formed. When a potential difference is applied to the cis droplet and the trans droplet, the analyte passes through the sensing micro-well in turn, and the second analysis and detection of the analyte is completed. This operation can be performed multiple times, i.e., a set number of repeated analysis and detection is completed. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc. as required.

[0095] Figure 6 illustrates a third scheme for repeated analysis and detection of the analyte based on three-dimensional droplet movement. In the scheme of Figure 6, each side of the sensing micro-well 104 has one droplet channel 105. After the first analysis and detection of the analyte is completed in Figure 3, the droplet 201 containing the analyte is driven through one of the droplet channels to the anti- order side of the sensing micro-well 104. Meanwhile, the droplet 202 containing the analyte is driven through the other droplet channel to the syn-order side of the sensing micro-well 104. In this way, the positions of the droplet 201 and the droplet 202 are exchanged through the two droplet channels. That is, a new through-well sensor is formed. When a potential difference is applied to the syn-order droplet and the anti-order droplet, the analyte sequentially passes through the sensing micro-well, and the second analysis and detection of the analyte is completed. This operation can be repeated multiple times, i.e., a set number of repeated analysis and detection is completed. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc. as required.

[0096] Scheme for repeated analysis and detection of the analyte based on two-dimensional droplet movement:

[0097] Fig. 7 and Fig. 8 illustrate a scheme for repeated analysis and detection of the analyte based on two-dimensional movement of the droplets. In this scheme, n (Fig. 7 shows an example of n=2) adjacent sensing microwells are used as a unit. After the first analysis and detection of the analyte is completed in one sensing microwell, the droplet 201 without the analyte and the droplet 202 with the analyte are moved horizontally, as shown in Fig. 7. They are moved to the second adjacent sensing microwell. At this time, the droplet 202 with the analyte becomes the cis droplet of the second sensing microwell, and the droplet 201 without the analyte becomes the trans droplet of the second sensing microwell. That is, a new via-sensor is formed, as shown in Fig. 8. When the cis droplet and the trans droplet are applied with a potential difference opposite to that in the first analysis and detection, the analyte passes through the sensing microwells in turn, and the second analysis and detection of the analyte is completed. If two adjacent sensing microwells are used as a unit, after the second analysis and detection is completed, the droplet 201 with the analyte and the droplet 202 without the analyte are driven back to the first sensing microwell, and the same steps as in the first analysis and detection are performed, so that the third analysis and detection is completed. In this way, the analysis and detection can be performed back and forth between the two sensing microwells, that is, the repeated analysis and detection for a set number of times are completed. If n (n>2) adjacent sensing microwells are used as a unit, after the second analysis and detection is completed, the droplet 201 with the analyte and the droplet 202 without the analyte are driven to the third sensing microwell, and the same steps as in the first analysis and detection are performed. That is, the same steps as in the first analysis and detection are performed in the odd-numbered sensing microwells, and the same steps as in the second analysis and detection are performed in the even-numbered sensing microwells. After the nth analysis and detection is completed in the nth sensing microwell, the droplet 201 and the droplet 202 are driven to the (n-1)th sensing microwell, and the next analysis and detection is performed. The path of the droplets can also be set arbitrarily in this process, so that the analysis and detection for an arbitrary number of times can be completed in a set sensing microwell. In this scheme, the droplets 201 and 202 move back and forth in the two-dimensional direction in the respective chambers, and the analyte moves in a three-dimensional trajectory between the top chamber and the second droplet movement chamber. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc. according to requirements.

[0098] The method for repeated screening and separation of the analyte by the via-sensor provided in the present application is described below in connection with Fig. 9 to Fig. 12.

[0099] Two schemes for screening and separating the analyte are provided illustratively in the present application. Scheme one: single-molecule screening and separation scheme; and scheme two: batchwise approaching screening and separation scheme. The two schemes can also be used in combination.

[0100] Single-molecule screening and separation scheme:

[0101] Fig. 9 and Fig. 10 schematically show the single molecule screening, separation scheme of analyte.

[0102] As shown in Fig. 9, the droplet 501 containing analyte is driven to the cis side of the sensing micro-hole, the droplet 502 without analyte is driven to the trans side of the sensing micro-hole, in addition, the second droplet moving chamber has a droplet inlet channel 3 which can provide new droplet without analyte in the direction perpendicular to the cross section of the second droplet moving chamber in Fig. 9, and is ready to provide new droplet to the second droplet moving chamber (trans side) of the sensing micro-hole. At this time, the droplets 501 and 502 and the sensing micro-hole 104 constitute a through-hole sensor. When the potential difference is applied to the cis droplet and the trans droplet, the analyte begins to pass through the sensing micro-hole. When the detected electrical or optical signal of the single analyte meets the characteristics of the target screening object, the droplet 502 containing the target screening object is moved to a target screening object storage area according to the first droplet outlet channel, waiting for the next operation, or driven to other areas for parallel analysis, detection and other operations. After the droplet is removed, a new droplet without analyte will be moved to the trans side of the sensing micro-hole from the direction of the droplet inlet channel 3, and the driving of the analyte in the droplet 501 through the sensing micro-hole continues. If the target screening object molecule is detected again, the above operation is repeated. If the detected electrical or optical signal of the single analyte does not meet the characteristics of the target screening object, the droplet containing the non-target screening object is moved to a non-target screening object storage area according to the second droplet outlet channel 2, waiting for the next operation, or moved to a waste storage area, as shown in Fig. 10. Before each screening and separation, the droplet can be mixed, separated, concentrated, diluted and the like according to requirements.

[0103] Batchwise successive approximation screening, separation scheme:

[0104] Fig. 11 and Fig. 12 schematically show the batchwise successive approximation screening, separation scheme. The batchwise successive approximation screening, separation scheme is suitable for preliminary screening and separation of analyte with high concentration. In turn, the efficiency of screening and separation can be improved.

[0105] As shown in Fig. 11, the droplet 601 containing a higher concentration of analyte is driven to the first droplet moving chamber of the sensing micowell (cis side), the droplet 602 containing no analyte is driven to the second droplet moving chamber of the sensing micowell (trans side), and in addition, the second droplet moving chamber has a droplet inlet channel 3 providing a new droplet containing no analyte and ready to be driven to the trans side of the sensing micowell. At this time, the droplets 601 and 602 and the sensing micowell constitute a through-hole sensor. When a potential difference is applied to the cis droplet and the trans droplet, the analyte begins to pass through the sensing micowell. The number n of analytes separated in each trans droplet can be set. When the electrical or optical signals of the n analytes detected do not meet the characteristics of the target screening substance, the droplet 602 containing the target screening substance is moved to a non-target screening substance storage area according to the second droplet outlet channel 2, waits for the next operation, or is moved to a waste storage area, as shown in Fig. 12. After the droplet is removed, a new droplet containing no analyte is provided to the trans side of the sensing micowell in the droplet inlet channel 3, and the analyte in the droplet 601 is continuously driven to pass through the sensing micowell. If n non-target screening substance molecules are detected again, the above operation is repeated. If the electrical or optical signals of the analytes separated in the next round meet the characteristics of the target screening substance, the droplet containing the target screening substance is moved to a target screening substance storage area according to the first droplet outlet channel 1, waits for the next operation, or is driven to other areas for single molecule screening, separation operation, or analysis and detection. Before each screening and separation, the droplet can be mixed, separated, concentrated, diluted, and the like according to requirements.

[0106] Repeated screening and separation of analytes

[0107] The repeated screening and separation of analytes in the present application can refer to the droplet moving scheme described above for the repeated analysis and detection of analytes.

[0108] The scheme in the present application can be flexibly combined with the repeated screening and separation of analytes and the repeated analysis and detection of analytes according to different detection requirements.

[0109] In summary, the through-hole sensor provided by the present application has the following advantages:

[0110] The through-hole sensor provided by the present application can realize repeated sequencing of nucleic acids, proteins, polypeptide chains, and the like by flexibly driving the cis droplet and the trans droplet. In order to meet the accuracy requirements of sequencing without wasting chip resources and saving time, the number of repeated sequencing can be flexibly set. The droplet can be mixed, separated, concentrated, diluted, and the like according to specific requirements before each sequencing.

[0111] The via sensor provided by the application can realize the method of repeatedly detecting other analytes such as cells and molecules by flexibly driving the cis droplet and the trans droplet. In order to meet the accuracy requirement of detection, save chip resources and save time, the number of repeated detection can be flexibly set. Before each detection, the droplet can be mixed, separated, concentrated, diluted and the like according to specific requirements.

[0112] The via sensor provided by the application can realize a single-cell, single-molecule counting method.

[0113] The via sensor provided by the application can realize a repeatable single-cell, single-molecule counting method.

[0114] The via sensor provided by the application can realize the method of simultaneously analyzing and detecting multiple analytes in the same sample.

[0115] The via sensor provided by the application can realize the method of repeatedly analyzing and detecting multiple analytes in the same sample.

[0116] The via sensor provided by the application can realize the method of screening and separating analytes. Before screening and separating, the droplet can be mixed, separated, concentrated, diluted and the like according to specific requirements.

[0117] The via sensor provided by the application can realize the method of repeatedly screening and separating analytes. In order to meet the purity requirement of screening and separating, save resources and save time, the number of repeated screening and separation can be flexibly set. Before each screening and separation, the droplet can be mixed, separated, concentrated, diluted and the like according to specific requirements.

[0118] The via sensor provided by the application can realize three schemes of repeatedly analyzing and detecting analytes based on three-dimensional droplet movement.

[0119] The via sensor provided by the application can realize the scheme of repeatedly analyzing and detecting analytes based on two-dimensional droplet movement.

[0120] The via sensor provided by the application can realize two schemes of screening and separating analytes: a single-molecule screening and separating scheme and a batch successive approximation screening and separating scheme. The two schemes can also be combined.

[0121] The via sensor provided by the application can realize the repeated analysis and detection of analytes, and the repeated screening and separation of analytes and the mixing, separation, concentration, dilution and the like of droplets can be combined.

[0122] In order to further illustrate the application, the technical schemes provided by the application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the application.

[0123] Example 1: Single cell repeatable detection

[0124] Detecting the number of Staphylococcus aureus from solution.

[0125] One of the repeatable analysis, detection schemes based on three dimensional droplet movement of the analyte to be analyzed, the principle as shown in Figure 4. Staphylococcus aureus is diluted to 1000 cells / uL using 5x TBE buffer. After adding iron oxide magnetic particles, 1 uL is added to the top chamber, and the droplet is driven to the top of the sensing microwell using an electromagnet or permanent magnet as a cis droplet. Before each detection, the trans droplet without analyte is driven to the bottom of the sensing microwell. Drive the analyte through the nanopore sensor and conduct ion current detection. Set the detection times to 5 times. The results of 2, 3, 4, 5 times of detection are averaged and compared with the results of 1 time of detection.

[0126] Results: Since each Staphylococcus aureus passes through the sensing microwell, it may not always produce a sufficiently accurate discrimination signal due to factors such as conformational changes or detection noise, that is, single detection has a relatively large random error. With the increase of detection times, the occurrence of random error is reduced or avoided,

[0127] Therefore, the average of the detection results is more and more close to the actual value 1000. As shown in Figure 13.

[0128] Example 2: Single cell screening separation

[0129] Screening and separating Staphylococcus aureus from a mixture of Staphylococcus aureus and Bacillus subtilis.

[0130] The two bacteria cell sizes are quite different, Staphylococcus aureus is about 0.8um in diameter. Bacillus subtilis is about 0.8um x (2 to 3um long). Therefore, the larger volume Bacillus subtilis produces a larger change in the blocking current of the ion current when passing through the pore sensor. Therefore, the change in the blocking current can be used to determine the bacterial species. Screening and separation scheme for the analyte to be analyzed: single molecule screening and separation scheme. The principle is shown in Figures 9 and 10. Staphylococcus aureus is diluted to 80 cells / uL using 5x TBE buffer. Bacillus subtilis is diluted to 80 cells / uL using 5x TBE buffer. Equal volumes of the two bacterial solutions are mixed, then 1uL of the mixture is added to the top chamber after adding iron oxide magnetic particles. An electromagnet or permanent magnet is used to drive the droplet onto the top of the sensing micropore as a cis droplet. After detecting each bacterial cell and moving the trans droplet to a specific position, a new trans droplet is driven to the bottom of the sensing micropore. The analyte is driven through the nanopore sensor and the ion current is detected.

[0131] Results: 32 Staphylococcus aureus cells and 41 Bacillus subtilis cells were screened and separated from 1uL of cis droplet.

[0132] Example 3: Label-free and featureless DNA single molecule repeatable sequencing

[0133] This example mainly illustrates the problem of label-free repeatable sequencing of multiple identical or similar nucleic acid molecules that are not easy to distinguish in the same cis droplet. The combination of the single molecule screening and separation scheme for the analyte to be analyzed and the repeat analysis and detection of the analyte to be analyzed (one of the three schemes based on three-dimensional droplet movement or the scheme based on two-dimensional droplet movement can be used) is used.

[0134] Synthetic polynucleotide (C20G1A20) and (C20T1A20) are dissolved in 1M KCl (10mM Tris-HCl adjusted to pH 8.0) solution, each diluted to 2000 copies / uL. 5uL of each is mixed into 10uL, 1uL of which is mixed with iron oxide magnetic particles and added to the top chamber, using an electromagnet or permanent magnet to drive the droplet onto the top of the sensing nanopore as a cis droplet. Each trans droplet receives one DNA molecule, i.e. transfer. The next trans droplet is driven to the position and waits to receive the next DNA molecule for transfer. The analyte to be analyzed is driven through the nanopore sensor and ion current detection is performed. After each DNA molecule is received and transferred by each trans droplet, the single molecule screening and separation of DNA is completed. Next, each trans droplet is driven to a new sensing nanopore cis side, and all DNA molecules are subjected to parallel repeated analysis and detection (i.e. repeated sequencing). The number of repeated sequencing is set to 15 times.

[0135] Results: 462 molecules of DNA polynucleotide were separated by single molecule screening and separation. Statistical analysis was performed on the results of 15 times sequencing of each DNA molecule. A total of 238 (C20G1A20) molecules and 224 (C20T1A20) molecules were sequenced.

[0136] Example 4: Single molecule repeated sequencing of polypeptide chain

[0137] This example mainly improves the sequencing accuracy in the current protein and polypeptide chain amino acid sequence sequencing based on the nanopore sensor by repeated sequencing.

[0138] The detection scheme is based on the repeated analysis of the analyte using two-dimensional droplet movement. Bovine serum albumin (BSA) is prepared in phosphate buffered saline (PBS) at a concentration of 200 pM with 50 uM β-mercaptoethanol (BME), 250 mM NaCl, 0.005% sodium dodecyl sulfate (SDS), mixed well and heated at 75 °C for 60 minutes. After cooling, it is mixed with an equal volume of 250 mM NaCl. 1 uL is mixed with iron oxide magnetic particles and added to the top chamber. An electromagnet or permanent magnet is used to drive the droplet to the top of sensor well A as the cis droplet. Another 1 uL of solution without protein is mixed with iron oxide magnetic particles and added to the second droplet movement chamber, which is driven to the bottom of sensor well A as the trans droplet. The analyte is driven through the nanopore sensor and ion current detection is performed. After all the polypeptide chain molecules have passed through sensor well A and the first analysis / detection is complete, the cis and trans droplets of sensor well A are driven to the top and bottom of sensor well B, respectively, to form the trans and cis droplets of transwell sensor B. The opposite voltage is applied to transwell sensor B as compared to transwell sensor A, and the polypeptide chain molecules are driven from the bottom droplet through sensor well B into the top droplet. The second analysis / detection is then completed. After the second analysis / detection is complete, the cis and trans droplets of sensor well B are driven back to the bottom and top of sensor well A, respectively, to start the third analysis / detection. A total of 16 analyses / detections are performed between A and B.

[0139] Results: A total of 516 amino acid residues were detected in the first analysis / detection. The average of 16 analyses / detections statistically resulted in 577 amino acid residues. Since the actual number of amino acid residues of bovine serum albumin (BSA) is 583, it is clear that the average of 16 detections is more accurate. Due to the complexity of polypeptide chain sequencing, although this experiment has not yet achieved single molecule polypeptide chain sequencing, it can more accurately detect the number of amino acid residues of the polypeptide chain molecule, laying a solid foundation for single molecule polypeptide chain sequencing.

[0140] Example 5: DNA Repeated Single Molecule Counting

[0141] This example mainly exemplarily demonstrates the applicability of the technical method of the present patent to DNA repeated single molecule counting.

[0142] The repeat analysis and detection scheme based on two-dimensional droplet movement is adopted. The λDNA is diluted to 6 pM by 1 M KCl solution. 1 uL of the solution mixed with the oxidized iron magnetic particles is added to the top chamber, and the electromagnet or permanent magnet is used to drive the droplet to the top of the sensing micro-hole A as the cis droplet. Another 1 uL of 1 M KCl solution without DNA is mixed with the oxidized iron magnetic particles, and then added to the second droplet movement chamber, and driven to the bottom of the sensing micro-hole A as the trans droplet. The analyte is driven to pass through the nanopore sensor and ion current detection is performed. After all the λDNA molecules pass through the sensing micro-hole A and the first counting is completed, the cis droplet and the trans droplet of the sensing micro-hole A are driven to the top and the bottom of the sensing micro-hole B, respectively, to form the trans droplet and the cis droplet of the through-hole sensor B. The voltage opposite to that of the hole sensor A is applied to the through-hole sensor B, and the polypeptide chain molecules are driven to pass through the sensing micro-hole B from the bottom droplet to the top droplet. In this way, the second counting is completed. After the second counting is completed, the cis droplet and the trans droplet of the sensing micro-hole B are driven to the bottom and the top of the sensing micro-hole C, respectively, to start the third counting. In this way, when the fifth counting is completed in the sensing micro-hole E, the sixth counting is started in the sensing micro-hole D. That is, the route of the droplet is A-B-C-D-E-D-C-B-A. In this way, a total of 9 times of counting is completed, and the droplet returns to the sensing micro-hole A.

[0143] Results: The first counting result is 3219680. The average counting result of 9 times of counting is 3683819. The average counting result of 9 times of counting is closer to the theoretical value. As shown in Table 1.

[0144] Table 1: Statistics of experimental results of Example 5

[0145] From the above examples, it can be seen that the through-hole sensor provided by the present application can perform repeated sequencing on nucleic acids, proteins, and polypeptide chain molecules to correct random errors, thereby improving the accuracy of sequencing.

[0146] The through-hole sensor provided by the present application can set the number of repeated sequencing of all analytes, or the number of repeated sequencing of any part of the target analyte, that is, the sequencing accuracy of any molecule can be set, so the application of sequencing is more flexible. In actual scientific research and clinical application, time efficiency, cost, and throughput and other indicators can be considered.

[0147] The through-hole sensor provided by the present application can sequence naked nucleic acids, proteins, and polypeptide chain molecules. That is, the nucleic acids, proteins, and polypeptide chain molecules to be sequenced do not need to be labeled, connected, or library constructed. The sequence, abundance, and epigenetic information of the original molecule can be most truly reflected.

[0148] The via sensor provided in the present application can sequence trace amounts of nucleic acids, proteins, and polypeptide chains. For example, nucleic acids, proteins, and polypeptide chains in a cell or even a single nucleic acid, protein, or polypeptide chain can be sequenced.

[0149] The via sensor provided in the present application can provide a faster sequencing speed for nucleic acids, proteins, and polypeptide chains. Taking nucleic acid sequencing as an example, the biological nanopore sequencing technology of Oxford Nanopore Technologies in the United Kingdom uses a motor protein to limit the speed of nucleic acid via, with a rate of about 100-500 nt / s. The technology of Pacific Biosciences of California essentially uses a polymerase to sequence while synthesizing, with a rate of about 1-5 bp / s. In the method of the present application, the via rate of nucleic acids can reach 10 knt-10 Mnt / s.

[0150] The via sensor provided in the present application can repeatedly detect the analyte such as cells and molecules to correct random errors, thereby improving the accuracy of detection.

[0151] The via sensor provided in the present application can set the number of repeated detections of all analytes, or the number of repeated detections of any part of the target analyte, i.e., the detection accuracy of any molecule, so it is more flexible in the application of detection. In actual scientific research and clinical applications, timeliness, cost, and throughput and other indicators can be considered.

[0152] The via sensor provided in the present application can detect naked cells, molecules, and other analytes. That is, the analyte can not need to be labeled or operated. It can most truly reflect the characteristic information of the analyte.

[0153] The via sensor provided in the present application can detect trace amounts of analytes. For example, even a single analyte molecule can be detected.

[0154] The via sensor provided in the present application can real-time screen and separate the analyte population in individual units.

[0155] The via sensor provided in the present application can real-time screen and separate the analyte population in individual units.

[0156] The via sensor provided in the present application can repeatedly real-time screen and separate the analyte population in individual units, thereby improving the concentration and purity of the target analyte.

[0157] The via hole sensor provided in the application can set the repeated screening and separation times of the target analyte to be analyzed, that is, the screening and separation purity of the target analyte to be analyzed, and is more flexible in the application of detection. In actual scientific research and clinical application, timeliness, cost, throughput and other indicators can be considered.

[0158] The via hole sensor provided in the application can screen and separate trace amounts of analytes. For example, even a target analyte molecule can be screened and separated.

[0159] Although the above embodiment describes the application in detail, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity according to the embodiment, which all belong to the protection scope of the application.

Claims

1. A via sensor, comprising: The through-hole sensor comprises a first electrode support plate and a second electrode support plate, and an intermediate sensing layer arranged between the first electrode support plate and the second electrode support plate, a first droplet moving chamber is formed between the first electrode support plate and the intermediate sensing layer, and a second droplet moving chamber is formed between the second electrode support plate and the intermediate sensing layer; A plurality of first electrode units are arranged on the first electrode support plate, a plurality of second electrode units are arranged on the second electrode support plate, and a plurality of sensing micropores are arranged on the intermediate sensing layer, the sensing micropores being in communication with the first droplet moving chamber and the second droplet moving chamber; The first droplet moving chamber and the second droplet moving chamber are used for loading a plurality of mutually separated driveable droplets, at least one of the driveable droplets containing an analyte to be analyzed, when the first droplet moving chamber is loaded with a plurality of first driveable droplets and the second droplet moving chamber is loaded with a plurality of second driveable droplets, any one of the first electrode units, any one of the first driveable droplets, any one of the sensing micropores, any one of the second driveable droplets, and any one of the second electrode units form a closed loop with an external power supply.

2. The via sensor of claim 1, wherein, The driving method of the driveable droplets comprises one or more of a capillary method, a dielectrophoresis method, an electrowetting method, a dielectrowetting method, an electrowicking method, a surfactant method, a magnetic driving method, a light driving method, and a thermal driving method.

3. The via sensor of claim 1, wherein, The sensing micropores are of nanoscale or micrometer scale, and the shape of the sensing micropores comprises one or more of a slit, a line, a tube, a strip, a field effect transistor, and a nanopore field effect transistor.

4. The via sensor of claim 1, wherein, The first droplet moving chamber and / or the second droplet moving chamber is / are filled with a liquid medium that is immiscible with the driveable droplets.

5. The via sensor according to any one of claims 1 to 4, wherein The intermediate sensing layer is further provided with a plurality of droplet channels in communication with the first droplet moving chamber and the second droplet moving chamber.

6. The via sensor according to any one of claims 1 to 4, wherein The number of the sensing micropores is greater than or equal to 2, and the intermediate sensing layer is not provided with a droplet channel. Alternatively, the number of the sensing micropores is 1, and the number of the droplet channels is 1 or 2.

7. The via sensor of claim 1, wherein, The plurality of first electrode units are embedded in the first electrode support plate, and the plurality of second electrode units are embedded in the second electrode support plate.

8. The through-hole sensor according to any one of claims 1 to 7 is used in the analysis, detection, screening, and separation of an analyte, the analyte comprising ions, atoms, molecules, polymers, microorganisms, cells, or cell complexes.

9. A method of multiple analysis or multiple detection of an analyte to be analyzed, characterized in that, When the through-hole sensor according to claim 6 is used, the following steps are included: A first driveable droplet is loaded in the first droplet moving chamber, and a second driveable droplet is loaded in the second droplet moving chamber, the first driveable droplet and the second driveable droplet being an analyte-containing droplet and a blank droplet, respectively; the first driveable droplet and the second driveable droplet are repeatedly driven to the sensing micropores, the analyte is analyzed or detected multiple times at the sensing micropores by applying an adjustable and reversible potential difference between the first electrode units and the second electrode units; Alternatively, when the through-hole sensor according to claim 5 is used, the following steps are included: Loading one first droplet in the first droplet moving chamber and one second droplet in the second droplet moving chamber, the first droplet and the second droplet are analyte-containing droplet and blank droplet respectively; exchanging the droplets in the first droplet moving chamber and the second droplet moving chamber through the droplet channel, driving the droplets in the first droplet moving chamber and the second droplet moving chamber to the sensing microwell, and performing multiple analysis or multiple detection of the analyte by applying a potential difference between the first electrode unit and the second electrode unit.

10. A method of multiple screening or multiple isolation of an analyte to be analyzed, characterized in that, The via sensor of any one of claims 1-7, comprising the following steps: Loading one first droplet in the first droplet moving chamber and multiple second droplets in the second droplet moving chamber, the first droplet and the second droplets are analyte-containing droplet and blank droplet respectively; driving the first droplet to the sensing microwell, and sequentially driving the multiple second droplets to the sensing microwell, and performing multiple screening or multiple separation of the analyte in the multiple blank droplets by applying a potential difference between the first electrode unit and the second electrode unit.

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