Via hole sensor and application thereof, and method for multiple analysis, multiple detection, multiple screening or multiple separation of to-be-analyzed object

Abstract

The invention belongs to the technical field of via hole sensors, and particularly relates to a via hole sensor, application of the via hole sensor and a method for multiple analysis, multiple detection, multiple screening or multiple separation of an object to be analyzed. The invention provides a via hole sensor which comprises a first electrode supporting plate, a second electrode supporting plate and a middle sensing layer arranged between the first electrode supporting plate and the second electrode supporting plate, a first liquid drop moving cavity is formed between the first electrode supporting plate and the middle sensing layer, and a second liquid drop moving cavity is formed between the second electrode supporting plate and the middle sensing layer. A second liquid drop moving cavity is formed between the second electrode supporting plate and the middle sensing layer, and a plurality of sensing micropores are formed in the middle sensing layer. The via hole sensor provided by the invention can be used for carrying out multiple repeated analysis, detection, screening and separation on the to-be-analyzed object with settable times, so that the analysis and detection accuracy of the to-be-analyzed object is effectively improved, and the purity and concentration of the to-be-analyzed object are improved; meanwhile, the cost and the timeliness can be considered on the premise that the detection accuracy and the purity are met.

Description

Technical Field

[0001] This invention belongs to the field of through-hole sensor technology, specifically relating to through-hole sensors and their applications, and methods for multiple analysis, multiple detection, multiple screening or multiple separation of analytes. Background Technology

[0002] A through-pore sensor is a sensor device that analyzes the random processes or heterogeneity of analyte molecules, such as composition, sequence, type, quantity, shape, size, and configuration, by driving the analyte through picometer-millimeter-sized pores. This analysis is achieved directly or indirectly through optical or electrical signals. For example, in a typical through-pore sensor, a single analyte is driven into and through a pore of a specific size by a force field (such as an electric field or magnetic field) or mechanical means. The perforation process alters the ion current passing through the pore (or changes the surface current, transverse current, or tunneling current on the membrane surface). By analyzing the changes in the amplitude, duration, and frequency of the analyzed current, the random processes or heterogeneity of the analyte molecules, such as composition, sequence, type, quantity, shape, size, and configuration, can be analyzed directly or indirectly.

[0003] Currently, when analyzing the stochastic processes or heterogeneity of analyte molecules, such as physical, chemical, and biological characteristics (e.g., the composition, sequence, type, quantity, shape, size, and configuration of the analyte), through-pore sensors of various sizes and types can be used to directly or indirectly analyze and detect the analyte through optical or electrical signals. Through-pore sensors offer several advantages: 1) They allow individual analytes in a population to be studied to pass through pores sequentially for counting, analysis, and detection, enabling the study of the entire population at the individual level; 2) They convert the physical, biological, and chemical characteristics of individual analytes into optical and electrical signals that can be directly or indirectly analyzed and detected; 3) They provide a controllable platform for designing and constructing biomimetic systems, which can be used to study complex interactions between biochemical molecules (e.g., the transfer characteristics of nuclear-pore complexes). However, due to various factors such as irregular motion, Brownian motion, fluctuations, and configuration changes of the analyte, as well as various noises (such as 1 / f noise from the detection device, white noise, parasitic capacitance noise, sampling circuit noise, etc.), the results of a single analysis and detection of the analyte through the through-hole sensor will produce various random errors, and the required accuracy cannot be achieved. Summary of the Invention

[0004] The purpose of this invention is to provide a through-hole sensor and its application, as well as a method for multiple analysis, detection, screening, or separation of analytes. The through-hole sensor provided by this invention can perform multiple repetitive analysis, detection, screening, and separation of analytes on a set number of times, effectively improving the accuracy of analysis and detection, as well as the purity and concentration of the analytes; at the same time, it can also balance cost and timeliness while meeting the requirements of detection accuracy and purity.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a through-hole sensor, including a first electrode support plate and a second electrode support plate, and an intermediate sensing layer disposed 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] The first electrode support plate is provided with a plurality of first electrode units, the second electrode support plate is provided with a plurality of second electrode units, and the intermediate sensing layer is provided with a plurality of sensing micropores that connect the first droplet moving chamber and the second droplet moving chamber.

[0008] Both the first and second droplet moving chambers are used to load a plurality of mutually separated drivable droplets. At least one of the drivable droplets contains the analyte. When the first droplet moving chamber is loaded with a plurality of first drivable droplets and the second droplet moving chamber is loaded with a plurality of second drivable droplets, any one of the first electrode units, any one of the first drivable droplets, any one of the sensing micropores, any one of the second drivable droplets, and any one of the second electrode units forms a closed loop with the external power supply.

[0009] Preferably, the driving method for the drivable droplet includes one or more of the following: capillary method, dielectric electrophoresis method, electrowetting method, dielectric wetting method, electrodewetting method, surfactant method, magnetic driving method, optical driving method, and thermal driving method.

[0010] Preferably, the sensing micropore is nanometer-sized or micrometer-sized, and the shape of the sensing micropore includes one or more of the following: slit, wire, tube, strip, field-effect transistor, and nanopore extended field-effect transistor.

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

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

[0013] Preferably, the number of sensing micropores is ≥2, and the intermediate sensing layer does not have droplet channels.

[0014] Preferably, the number of sensing micropores is one, and the number of droplet channels is one or two.

[0015] The present invention provides the application of the through-hole sensor described above in the analysis, detection, screening and separation of analytes, wherein the analytes include ions, atoms, molecules, polymers, microorganisms, cells or cell complexes.

[0016] This invention provides a method for multiple analyses or multiple detections of an analyte. When using the through-hole sensor described in the above technical solution, wherein the number of sensing micropores is ≥2, and the intermediate sensing layer does not have droplet channels, the method includes the following steps:

[0017] A first drivable droplet is loaded in a first droplet moving chamber, and a second drivable droplet is loaded in a second droplet moving chamber. The first drivable droplet and the second drivable droplet are respectively a droplet containing the analyte and a blank droplet. The first drivable droplet and the second drivable droplet are repeatedly driven to a number of sensing micropores. By applying a potential difference with interchangeable directions between the first electrode unit and the second electrode unit, multiple analyses or detections of the analyte are performed at the sensing micropores.

[0018] Alternatively, when using the via sensor described in the above technical solution, if the intermediate sensing layer of the via sensor is further provided with a plurality of droplet channels connecting the first droplet moving chamber and the second droplet moving chamber, the following steps are included:

[0019] A first drivable droplet is loaded into a first droplet moving chamber, and a second drivable droplet is loaded into a second droplet moving chamber. The first and second drivable droplets are a droplet containing the analyte and a blank droplet, respectively. The drivable droplets in the first and second droplet moving chambers are swapped through a droplet channel, and the drivable droplets in the first and second droplet moving chambers are driven to the sensing micropore. Multiple analyses or detections of the analyte are performed by applying a potential difference between the first and second electrode units.

[0020] This invention provides a method for multiple screening or separation of an analyte, employing the through-hole sensor described above, and includes the following steps:

[0021] One first drivable droplet is loaded in a first droplet moving chamber, and multiple second drivable droplets are loaded in a second droplet moving chamber. The first and second drivable droplets are respectively droplets containing the analyte and blank droplets. The first drivable droplet is driven to the sensing micropore, and the multiple second drivable droplets are driven to the sensing micropore in sequence. The analyte is screened or separated multiple times in the multiple blank droplets by applying a potential difference between the first and second electrode units.

[0022] This invention provides a via sensor. The invention presents the cis and trans chambers of a traditional via sensor in the form of flexibly driven droplets (specifically described as cis and trans droplets in this invention). That is, the relative positions of the two "chambers" and the sensing micropore are no longer fixed, but can be changed by flexibly driving the droplets that serve as the two "chambers". Simultaneously, the droplets serving as the two "chambers" can also establish relationships with other sensing micropores, either jointly or separately; that is, the droplets of the two "chambers" can become cis and / or trans chambers of other sensing micropores. Specifically, in this invention, a cis droplet carrying the analyte is driven to the cis side of the sensing micropore, and a trans droplet without the analyte is driven to the trans side of the sensing micropore, thereby temporarily forming a via sensor. When the analyte is driven from a cis-droplet through the sensing micropore and into a trans-droplet, the resulting optical or electrical signals are collected, completing the initial analysis and detection of the analyte. The trans-droplet containing the analyte is then driven back to the cis-side of the same or another sensing micropore, and another droplet is driven to the trans-side of the corresponding micropore, forming a new through-hole sensor. When the analyte is driven from the cis-droplet of this new through-hole sensor through the micropore and into a trans-droplet, the resulting optical or electrical signals are collected, completing one repeat analysis and detection of the analyte. This process can be repeated by continuously driving droplets to form through-hole sensors, allowing for a set number of repeated analyses and detections of the analyte, thereby improving the accuracy of analysis and detection. Furthermore, because the number of repeated analyses and detections can be customized, we can balance cost and timeliness while maintaining detection accuracy in different projects. Furthermore, the through-hole sensor provided by this invention can perform multiple screenings and separations of the analyte using the unique optical or electrical signal characteristics when the analyte passes through the sensing micropore. Multiple screenings and separations can significantly improve the purity and concentration of the analyte. The analyte after multiple screenings and separations can then be used again for further analysis and detection using the through-hole sensor provided by this invention, or for other subsequent applications.

[0023] In this invention, the specific implementation method of the through-hole sensor for multiple screening and separation is as follows: a droplet carrying a group of analytes is driven to the cis side of the micropore as a cis droplet. A droplet without analytes is driven to the inverse side of the micropore as an inverse droplet. Thus, a temporary through-hole sensor is formed. The group of analytes is driven one by one through the micropore and into the inverse droplet. Here, forward screening or reverse screening can be performed according to the specific situation of the group of analytes. The forward screening process is as follows: when one or more optical or electrical signals matching the target analyte are generated, it means that one or more target analytes have entered the inverse droplet. At this time, the inverse droplet is driven to the region of the target analyte for the next step. Then, a new droplet without analytes is driven to the inverse side of the micropore as an inverse droplet, and the process continues to wait for one or more optical or electrical signals matching the target analyte to be generated, and then the inverse droplet is driven to the region of the target analyte for the next step. In this way, each reverse droplet driven to the target analyte region contains one or more target analytes. Further processing can then proceed. The reverse screening process is as follows: when one or more optical or electrical signals that do not match the target analyte are generated, it indicates that one or more non-target analytes have entered the reverse droplet. At this time, the reverse droplet is driven to the non-target analyte region (such as a waste liquid region). Then, a new droplet without analyte is driven to the reverse side of the micropore as a reverse droplet, and the process continues to wait for one or more optical or electrical signals that do not match the target analyte to be generated, before driving the reverse droplet to the non-target analyte region (such as a waste liquid region). In this way, a relatively large number of target analytes are retained in the cis droplets, allowing further processing. Alternatively, forward and reverse screening can be performed simultaneously. The specific process is as follows: when one or more optical or electrical signals that match the target analyte are generated, it indicates that one or more target analytes have entered the reverse droplet. At this time, the reverse droplet is driven to the target analyte region for the next processing step. Next, a new droplet without the target analyte is driven onto the reverse side of the micropore as a reverse droplet. When one or more optical or electrical signals that do not match the target analyte are generated, it indicates that one or more non-target analytes have entered the reverse droplet. At this time, the reverse droplet is driven to the non-target analyte region (such as the waste liquid region). In this way, each reverse droplet driven to the target analyte region contains one or more target analytes. Further operations can then be performed on them. The actual forward screening process, reverse screening process, and simultaneous forward and reverse screening process can be interleaved. Alternatively, only the forward screening process, only the reverse screening process, or only the simultaneous forward and reverse screening process can be performed.

[0024] The through-hole sensor provided by this invention allows for repeated analysis, detection, screening, and separation of the analyte in a cyclical manner. For example, it can perform several analyses and detections followed by several screening and separations, and then repeat this process. Alternatively, it can perform several screening and separations followed by several analyses and detections, and then repeat this process. It can also perform only a few repeated analyses and detections or only a few screening and separations. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the basic structure of the through-hole sensor provided by the present invention;

[0026] Figure 2 This is a schematic diagram of the first analysis and detection process of the analyte in the through-hole sensor provided by the present invention.

[0027] Figure 3 This is a schematic diagram of the first analysis and detection process of the analyte in the through-hole sensor provided by the present invention.

[0028] Figure 4 This is a schematic diagram of the repeated analysis and detection process of the analyte in the through-hole sensor (three-dimensional) provided by the present invention;

[0029] Figure 5 This is a schematic diagram of the repeated analysis and detection process of the analyte in the through-hole sensor (three-dimensional) provided by the present invention;

[0030] Figure 6 This is a schematic diagram of the repeated analysis and detection process of the analyte in the through-hole sensor (three-dimensional) provided by the present invention;

[0031] Figure 7 This is a schematic diagram of the repeated analysis and detection process of the analyte in the two-dimensional through-hole sensor provided by the present invention;

[0032] Figure 8 This is a schematic diagram of the repeated analysis and detection process of the analyte in the two-dimensional through-hole sensor provided by the present invention;

[0033] Figure 9 This invention provides a method for single-molecule screening and separation of analytes in a porous sensor.

[0034] Figure 10 This invention provides a method for single-molecule screening and separation of analytes in a porous sensor.

[0035] Figure 11 This invention provides a batch successive approximation screening and separation method for analytes in a through-hole sensor;

[0036] Figure 12 This invention provides a batch successive approximation screening and separation method for analytes in a through-hole sensor;

[0037] Figure 13 These are the experimental results of Example 1 of the present invention;

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

[0039] Terminology Explanation:

[0040] The following explains the technical terms related to this invention:

[0041] Analyte: refers to various ions, atoms, molecules, polymers, viruses, bacteria and other microorganisms, as well as cells and cell complexes, whose physical, chemical and biological properties are to be detected and analyzed. Examples include amino acids, polypeptides, proteins, ribonucleic acid (RNA), deoxyribonucleic acid (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), glycolnucleic acid (GNA), threose nucleic acid (TNA), etc.), and nucleotide analogs (5-bromouracil, 2-aminopurine, 5-fluorouracil, hypoxanthine, adenosine-5'-monophosphate). 5'-monophosphate), 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl-5'-monophosphate

[0042] 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, etc. (including natural or synthetic nucleotide analogs such as 5'-monophosphate, inosine 5'-monophosphate, uridine-5'-diphospho-α-D-galactose disodium salt, etc.).

[0043] Through-pore sensors are sensor devices that analyze analytes by driving them through picometer-millimeter-sized pores, thereby directly or indirectly analyzing the random processes or heterogeneity of analyte molecules, such as composition, sequence, type, quantity, shape, size, and configuration, through optical or electrical signals. For example, in a typical through-pore sensor, a single analyte is driven into and through a pore of a specific size by a force field such as an electric field or magnetic field, or mechanically. The perforation process changes the ion current passing through the pore (or changes the surface current, transverse current, or tunneling current on the membrane surface, etc.). By analyzing the changes in the amplitude, duration, and frequency of the analyzed current, the random processes or heterogeneity of the analyte molecules, such as composition, sequence, type, quantity, shape, size, and configuration, can be analyzed directly or indirectly. The sensor micropores mentioned in this patent do not only refer to structures in the literal sense of "hole" in physics, but may also include structures such as slits, wires, tubes, and strips, such as nanogap, nanowire, nanotube, and nanoribbon. They may also be structures that constitute transistors such as field-effect transistors (FETs) or extended field-effect transistors (FETs). Broadly speaking, it refers to structures that allow analytes to pass through and cause changes in characteristic electrical or optical signals.

[0044] Correlation: In most cases, the correlation mentioned in this patent refers to the correlation between multiple analysis and testing data of a single analyte. Since multiple analysis and testing data are generated from the analysis and testing of the same individual analyte, these data are correlated and can be analyzed uniformly to improve the accuracy of the analysis and testing of that individual analyte.

[0045] Sequencing: In this patent, sequencing is a method for analyzing and detecting analytes. For example, sequencing nucleic acids refers to the process of determining the base composition and sequence of nucleic acid molecules; sequencing protein or polypeptide molecules refers to the process of determining the amino acid composition and sequence of protein or polypeptide molecules.

[0046] Screening: refers to the operation of separating each individual analyte from the mixture of analytes by passing it through a through-hole sensor one by one, based on the unique characteristics of the electrical or optical signals.

[0047] Ion current: In an electric field, positive and negative ions in an electrolyte solution move in opposite directions, forming an ion current. The ion current mentioned in this patent mostly refers to the current formed when an electric field is applied between the cis and anti-cis chambers of a through-hole sensor, causing positive and negative ions in the solution to move in opposite directions and pass through the sensor's micropores.

[0048] Lateral current: In this patent, the lateral current refers to the current relative to the ion current. First, the ion current is defined as the longitudinal current, which is the direction of the current formed by ions passing through the sensor micropore between the cis-chamber (top) and the anti-chamber. Therefore, the lateral current refers to the current at a 90-degree angle to the ion current direction. In the field of sensor micropore detection, it mostly refers to the current flowing through the metal solid film to be detected in the sensor micropore fabricated in a metal solid film (such as graphene film, gold film, platinum film, indium tin oxide (ITO) film, etc.). It is also sometimes called surface current. Broadly speaking, tunneling current can also be classified as lateral current.

[0049] Tunneling current: In through-hole sensors based on structures such as nanogap, nanowire, nanotube, and nanoribbon, or in through-hole sensors that form transistors such as field-effect transistors (FETs) or extended field-effect transistors (FETs), the difference in conductivity between the analyte and the electrolyte solution causes a change in the transverse current when the analyte passes through the hole. This transverse current when the analyte passes through the hole is called the tunneling current. In a broader sense, tunneling current can also be classified as transverse current.

[0050] MEMS (Micro-Electro Mechanical System): A micro-electro-mechanical system is a miniature intelligent system that integrates mechanical, electronic, optical and other functional components on a single chip or multiple chips to sense, identify, control and process natural information such as sound, light, heat, magnetic motion and so on.

[0051] Biomarkers are biochemical indicators that can mark changes or potential changes in the structure or function of systems, organs, tissues, cells, and subcellular structures. They have a very wide range of applications. Biomarkers can be used for disease diagnosis, disease staging, or to evaluate the safety and efficacy of new drugs or therapies in target populations. Detailed Implementation

[0052] This invention provides a through-hole sensor, including a first electrode support plate and a second electrode support plate, and an intermediate sensing layer disposed 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.

[0053] The first electrode support plate is provided with a plurality of first electrode units, the second electrode support plate is provided with a plurality of second electrode units, and the intermediate sensing layer is provided with a plurality of sensing micropores that connect the first droplet moving chamber and the second droplet moving chamber.

[0054] Both the first and second droplet moving chambers are used to load a plurality of mutually separated drivable droplets. At least one of the drivable droplets contains the analyte. When the first droplet moving chamber is loaded with a plurality of first drivable droplets and the second droplet moving chamber is loaded with a plurality of second drivable droplets, any one of the first electrode units, any one of the first drivable droplets, any one of the sensing micropores, any one of the second drivable droplets, and any one of the second electrode units forms a closed loop with the external power supply.

[0055] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0056] Figure 1 This is a schematic diagram of the through-hole sensor provided by the present invention. The following is in conjunction with... Figure 1 The via sensor provided by this invention will be described in detail.

[0057] The via sensor 100 provided by the present invention includes a first electrode support plate 102 and a second electrode support plate 103. A plurality of first electrode units 108 are disposed on the first electrode support plate, and a plurality of second electrode units 109 are disposed on the second electrode support plate. The first electrode units 108 and the second electrode units 109 are connected to an external power supply, and can form a positive or reverse potential difference from the first electrode unit 108 to the second electrode unit 109.

[0058] As one or more embodiments of the present invention, the first electrode unit 108 is embedded in the first electrode support plate 102.

[0059] As one or more embodiments of the present invention, the second electrode unit 109 is embedded in the second electrode support plate 103.

[0060] The through-hole sensor 100 provided by the present invention includes an intermediate sensing layer 101 disposed between a first electrode support plate 102 and a second electrode support plate 103. A first droplet movement chamber 106 is formed between the first electrode support plate 102 and the intermediate sensing layer 101, and a second droplet movement chamber 107 is formed between the second electrode support plate 103 and the intermediate sensing layer 101.

[0061] The via sensor 100 provided by this invention has a plurality of sensing micropores 104 on the intermediate sensing layer 101, connecting the first droplet moving chamber 106 and the second droplet moving chamber 107. When the analyte is driven through the sensing micropores 104 by the potential difference between the first electrode unit 108 and the second electrode unit 109 by an external power supply, the generated electrical or optical signals are collected for analysis and detection. In this invention, the sensing micropores 104 are preferably nanometer-sized or micrometer-sized. In this invention, the sensing micropores 104 generally refer to structures that allow the analyte to pass through and cause changes in characteristic electrical or optical signals. The sensing micropores 104 do not only represent structures in the literal sense of "hole" in physics, but may also be structures such as slits, wires, tubes, and strips. The shape of the sensing micropores 104 includes one or more of nanogap, nanowire, nanotube, nanoribbon, field-effect transistor (FET), and nanopore-extended field-effect transistor (FET). In this invention, the size of the sensing micropore 104 is set according to the size of the analyte. The size of the sensing micropore is related to the size of the analyte to be detected. The principle is that when the analyte passes through the pore, it can generate a specific blocking ion current pulse, transverse current, or tunneling current.

[0062] In specific embodiments of the invention, the through-hole sensor 100 may be configured with various sizes, types, and even modifications of sensing micropores 104, depending on the characteristics of different analytes and signal detection requirements. The sensing micropore 104 can utilize the drivable droplet-form cis- and trans-cavities described in this invention, as long as there is liquid on both sides and the analyte passes through the sensor from one side to the other for signal detection. In this invention, the sensing micropore 104, together with the cis- and trans-droplets loaded in the first droplet moving chamber 106 and the second droplet moving chamber 107, constitute the through-hole sensor unit structure.

[0063] The through-hole sensor 100 provided by the present invention has a first droplet moving chamber 106 and a second droplet moving chamber 107, both of which are used to contain a plurality of mutually separated drivable droplets, and at least one of the plurality of drivable droplets contains an analyte.

[0064] In this invention, 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 units 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.

[0065] In this invention, the driving method for the drivable droplet includes one or more of the following: capillary method, dielectric electrophoresis method, electrowetting method, dielectric wetting method, electrodewetting method, surfactant method, magnetic driving method, optical driving method, and thermal driving method.

[0066] In this invention, the capillary method is generally used in microfluidic droplet fabrication devices, where two phases of fluid are introduced at the intersection point: one phase is a continuous phase, and the other is a dispersed phase (droplet). By connecting submillimeter conduits to submillimeter T-junction and cross-junction devices, droplets can be generated as in microfluidic chip devices, which is a relatively simple method for droplet generation.

[0067] In this invention, the dielectric electrophoresis method is as follows: When a dielectric particle is placed in an electric field, due to the action of the applied electric field, the positive and negative charges in the particle will accumulate at both ends, resulting in an uneven charge distribution at both ends of the dielectric particle. Since the charges at both ends of the particle are opposite, this is called an electric dipole. The induced dipole moment is a vector, and its direction is from the end of the particle where the negative charge accumulates to the end where the positive charge accumulates. If the applied electric field is a uniform electric field, the dielectric particle is in equilibrium and will not move; if the applied electric field is a non-uniform electric field, the net force on the dielectric particle is not zero, and it 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 the direction of the electric field, exhibiting a positive dielectric electrophoresis 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 the direction of the electric field, exhibiting a negative dielectric electrophoresis phenomenon.

[0068] In this invention, the electrowetting method involves placing the droplet directly on an electrode array. When a voltage is applied, the surface tension between the liquid and solid interfaces changes, thereby causing a change in the wettability of the droplet.

[0069] In this invention, the dielectric wetting method is a type of electrically controlled surface tension-driven method based on the electrowetting of a dielectric film. It alters the wetting characteristics between the dielectric film and the surface liquid by applying a potential to a microelectrode array beneath the dielectric film. Specifically, it locally alters the three-phase contact angle between the microdroplet and the solid surface, causing asymmetrical deformation at both ends of the microdroplet and generating a pressure difference within the microdroplet, thereby enabling the manipulation and control of the microdroplet.

[0070] In this invention, the electrowetting method employs a mechanism opposite to electrowetting. The droplet-substrate interaction is not directly controlled by an electric field, but rather achieved through field-induced adhesion and separation of ionic surfactants on the substrate.

[0071] In this invention, the surfactant method is: using a functionalized surfactant, the droplets coated with this functionalized surfactant can be driven simultaneously by a magnetic field, optical field and electric field.

[0072] In this invention, the magnetic driving method is as follows: a micro magnet is added to the droplet, and the movement of the droplet is controlled by the force exerted by the external magnetic field on the droplet.

[0073] In this invention, the light-driven method is to convert light into a driving force for droplet motion through strategies such as photochemistry, photomechanics, photoinduced Marangoni effect, or photoinduced electric field.

[0074] In this invention, the thermal driving method is as follows: by creating a temperature difference at both ends of the droplet, the contact angles at both ends are inconsistent, forming a force directed towards the end with the smaller contact angle, thereby driving the droplet to move.

[0075] Considering the need for versatility in driving different droplets, this invention describes a droplet driving method based on magnetic force as an example in its embodiments. However, the droplet driving method of this invention is not limited to magnetic force driving.

[0076] As one or more embodiments of the present invention, the first droplet moving chamber 106 and / or the second droplet moving chamber 107 are filled with a liquid medium that is immiscible with the drivable droplet. The liquid medium helps the droplet move rapidly and reduces the driving voltage.

[0077] In this invention, the intermediate sensing layer 101 is further provided with a plurality of droplet channels 105 connecting the first droplet moving chamber and the second droplet moving chamber. The droplet channels 105 are used for droplet replacement chambers, thereby enabling repeated analysis and detection of the analyte, repeated screening of the analyte, or operations such as droplet loading, merging, separation, dilution, and concentration of the analyte. In this invention, the droplet channel 105 is a channel capable of driving droplets 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 a droplet is driven to the edge of the droplet channel 105, under the combined action of the tension generated by the droplet being squeezed by the first electrode support plate 102 or the second electrode support plate layer 103 and the driving force of the droplet, the droplet can be driven to pass through the droplet channel 105 and move to another chamber. In this invention, when the intermediate sensing layer 101 only has sensing micropores 104 and no droplet channels 105, it is called a through-hole sensor based on two-dimensional droplet movement. In this invention, when the intermediate sensing layer 101 has both sensing micropores 104 and droplet channels 105, it is called a through-hole sensor based on three-dimensional droplet movement.

[0078] In this invention, the number of sensing micro-holes 104 and droplet channels 105 is not limited to a 1:1 ratio. There can be one or more sensing micro-holes 104 forming a sensor array. Similarly, there can be one or more droplet channels 105 forming a droplet channel array. Figure 1 The sensing micropore 104 and the droplet channel 105 are only schematically described as two functional structures in this invention.

[0079] As one or more embodiments of the present invention, 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.

[0080] As one or more embodiments of the present invention, the number of the sensing micropores 104 is ≥2.

[0081] As one or more embodiments of the present invention, the number of the sensing micropores 104 is ≥2, and the intermediate sensing layer is not provided with droplet channels.

[0082] The present invention provides the application of the through-hole sensor described above in the analysis, detection, screening and separation of analytes, wherein the analytes include ions, atoms, molecules, polymers, microorganisms, cells or cell complexes.

[0083] This invention provides a method for multiple analyses or multiple detections of an analyte. When using the through-hole sensor described in the above technical solution, wherein the number of sensing micropores is ≥2, and the intermediate sensing layer does not have droplet channels, the method includes the following steps:

[0084] A first drivable droplet is loaded into a first droplet moving chamber, and a second drivable droplet is loaded into a second droplet moving chamber. The first drivable droplet and the second drivable droplet are respectively a droplet containing the analyte and a blank droplet. The first drivable droplet and the second drivable droplet are repeatedly driven to a number of sensing micropores. By applying a potential difference with interchangeable directions between the first electrode unit and the second electrode unit, multiple analyses or detections of the analyte are performed at the sensing micropores.

[0085] This invention provides a method for multiple analyses or multiple detections of an analyte. When using the through-hole sensor described in the above technical solution, and the intermediate sensing layer of the through-hole sensor is further provided with a plurality of droplet channels connecting the first droplet moving chamber and the second droplet moving chamber, the method includes the following steps:

[0086] A first drivable droplet is loaded into a first droplet moving chamber, and a second drivable droplet is loaded into a second droplet moving chamber. The first and second drivable droplets are a droplet containing the analyte and a blank droplet, respectively. The drivable droplets in the first and second droplet moving chambers are swapped through a droplet channel, and the swapped drivable droplets are driven to the sensing micropore. Multiple analyses or detections of the analyte are performed by applying a potential difference between the first and second electrode units.

[0087] This invention provides a method for multiple screening or separation of an analyte, employing the through-hole sensor described above, and includes the following steps:

[0088] One first drivable droplet is loaded in a first droplet moving chamber, and multiple second drivable droplets are loaded in a second droplet moving chamber. The first and second drivable droplets are respectively droplets containing the analyte and blank droplets. The first drivable droplet is driven to the sensing micropore, and the multiple second drivable droplets are driven to the sensing micropore in sequence. The analyte is screened or separated multiple times in the multiple blank droplets by applying a potential difference between the first and second electrode units.

[0089] The following combination Figures 2-3 The following describes the first analysis and detection of the analyte using the through-hole sensor provided by this invention:

[0090] Figure 2 and Figure 3This invention demonstrates the formation of the through-hole sensor and the initial analysis and detection process of the analyte. A cis-droplet 201 carrying the analyte 203 is driven to the cis-side of the sensing micro-hole 104 in the first droplet moving chamber 106. A trans-droplet 202 without the analyte is driven to the trans-side of the sensing micro-hole 104 in the second droplet moving chamber 107. At this point, the cis-droplet 201 and trans-droplet 202 are electrically connected only through the sensing micro-hole 104. This forms a through-hole sensor unit. Figure 2 When a potential difference is applied to the cis and trans droplets, the analyte sequentially passes through the sensing micro-orifice 104, completing the first analysis and detection of the analyte. The analyte enters the trans droplet 202. For example... Figure 3 In this invention, before analysis and detection, the droplets can be mixed, separated, concentrated, diluted, or otherwise processed as required.

[0091] The following combination Figures 4-8 The method for repeated analysis and detection of analytes using the through-hole sensor provided by this invention is described below:

[0092] This invention provides two methods for the repeated analysis and detection of analytes. Method 1: Repeated analysis and detection of analytes based on three-dimensional droplet movement; Method 2: Repeated analysis and detection of analytes based on two-dimensional droplet movement. The two methods can also be used in combination.

[0093] Repeated analysis and detection scheme for analytes based on three-dimensional droplet movement:

[0094] Figure 4 This paper exemplifies one of the methods for repeated analysis and detection of analytes based on three-dimensional droplet movement. Figure 3 After the first analysis and detection of the analyte is completed, the droplet 201 without analyte is driven away from the sensing micro-orifice 104 to the chip's waste liquid storage area, etc., and its position is occupied by the droplet 202 carrying the analyte passing through the droplet channel 105. The original position of the droplet 202 is occupied by a new droplet 300 without analyte. For example... Figure 4 The droplets 202 and 300, carrying the analyte, reconstitute the cis and trans droplets of the sensing micropore, forming a new through-hole sensor. When a potential difference is applied to the cis and trans droplets, the analyte passes through the sensing micropore 104 sequentially, completing the second analysis and detection of the analyte. This operation can be repeated multiple times, enabling repeatable analysis and detection with a set number of steps. Before each analysis and detection, the droplets can be mixed, separated, concentrated, or diluted as required.

[0095] Figure 5 This paper exemplifies a second method for the repeated analysis and detection of analytes based on three-dimensional droplet movement. Figure 3 After the first analysis and detection of the analyte, the droplet 201 without analyte is driven to a waiting area, where 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 droplets 201 and 202 are exchanged through the droplet channel 105, forming a new through-hole sensor. When a potential difference is applied to the cis- and trans-droplets, the analyte passes through the sensing micro-hole sequentially, completing the second analysis and detection of the analyte. This operation can be repeated multiple times, achieving repeatable analysis and detection with a set number of steps. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc., as required.

[0096] Figure 6 This paper exemplifies the third method for repeated analysis and detection of analytes based on three-dimensional droplet movement. Figure 6 In this design, there is a droplet channel 105 on each side of the sensing micropore 104. Figure 3 After the first analysis and detection of the analyte, the droplet 201, now free of analyte, is driven through one droplet channel to the reverse side of the sensing microorifice 104. Simultaneously, the droplet 202 carrying the analyte is driven through another droplet channel to the cis side of the sensing microorifice 104. This exchange of positions between droplets 201 and 202 is achieved through the two droplet channels, forming a new through-hole sensor. When a potential difference is applied to the cis and reverse droplets, the analyte sequentially passes through the sensing microorifice, completing the second analysis and detection. This operation can be repeated multiple times, enabling repeatable analysis and detection with a set number of steps. Before each analysis and detection, the droplets can be mixed, separated, concentrated, or diluted as required.

[0097] Repeated analysis and detection scheme for analytes based on two-dimensional droplet movement:

[0098] Figure 7 and Figure 8 This paper exemplifies a scheme for the repeated analysis and detection of analytes based on two-dimensional droplet movement. In this scheme, an analyte with n ( Figure 7 (Taking n=2 as an example) The operation is performed on a unit basis, with adjacent sensing micro-orifices 104. After the first analysis and detection of the analyte is completed in a sensing micro-orifice, both the droplet 201 without the analyte and the droplet 202 carrying the analyte move laterally, as shown in the example. Figure 7 The droplet 202, carrying the analyte, becomes a cis-droplet in the second sensing micro-orifice, while the droplet 201, without the analyte, becomes a trans-droplet. This forms a new through-hole sensor. Figure 8 When a potential difference opposite to that used in the first analysis and detection is applied to the cis and trans droplets, the analyte sequentially passes through the sensing micropores, completing the second analysis and detection. If two adjacent sensing micropores are used as a unit, after the second analysis and detection, the droplet 201 carrying the analyte and the droplet 202 without the analyte are driven back to the first sensing micropore to perform the same steps as the first analysis and detection, thus completing the third analysis and detection. In this way, analysis and detection can be performed back and forth between two sensing micropores, achieving repeatable analysis and detection with a set number of iterations. If n (n>2) adjacent sensing micropores are used as a unit, after the second analysis and detection, the droplet 201 carrying the analyte and the droplet 202 without the analyte will be driven to the third sensing micropore to perform the same steps as the first analysis and detection. That is, the same steps as the first analysis and detection are performed at the odd-numbered sensing micro-orifices, and the same steps as the second analysis and detection are performed at the even-numbered sensing micro-orifices. This continues until the nth analysis and detection is completed at the nth sensing micro-orifice, after which droplets 201 and 202 are driven to the (n-1)th sensing micro-orifice for the next analysis and detection. The droplet paths can also be arbitrarily set during this process to complete any number of analyses and detections at specific, configurable sensing micro-orifices. In this scheme, droplets 201 and 202 only move back and forth in a two-dimensional direction within their respective chambers, while the analyte shuttles between the top chamber and the second droplet moving chamber, performing a three-dimensional trajectory movement. Before each analysis and detection, the droplets can be mixed, separated, concentrated, diluted, etc., as required.

[0099] The following combination Figures 9-12 The method for repeated screening and separation of analytes using the through-hole sensor provided by this invention is described below:

[0100] This invention provides two exemplary schemes for screening and separating analytes. Scheme 1: Single-molecule screening and separation scheme; Scheme 2: Batch successive approximation screening and separation scheme. The two schemes can also be used in combination.

[0101] Single-molecule screening and separation schemes:

[0102] Figure 9 and Figure 10 The diagram illustrates a single-molecule screening and separation scheme for the analyte.

[0103] like Figure 9 A droplet 501 carrying the analyte is driven to the favorable side of the sensing microorifice, while a droplet 502 without the analyte is driven to the unfavorable side of the sensing microorifice. Additionally, the second droplet moving chamber has a perpendicular... Figure 9The second droplet moving chamber provides a droplet inlet channel 3 in the cross-sectional direction to provide new analyte-free droplets, and is always ready to provide new droplets to the second droplet moving chamber (reverse side) of the sensing micropore. At this time, droplets 501 and 502 and the sensing micropore 104 constitute a through-hole sensor. When a potential difference is applied to the cis and trans droplets, the analyte begins to pass through the sensing micropore. When the electrical or optical signal of a single analyte detected matches the characteristics of the target analyte, the droplet 502 containing the target analyte is moved along the first droplet outlet channel to a target analyte storage area, awaiting the next operation, or driven to other areas for parallel analysis, detection, etc. After this droplet is removed, a new analyte-free droplet moves from the droplet inlet channel 3 to the reverse side of the sensing micropore, continuing to drive the analyte in droplet 501 through the sensing micropore. If another target analyte molecule is detected, the above operation is repeated. If the electrical or optical signal of a single analyte does not match the characteristics of the target analyte, the droplet containing the non-target analyte is moved through the second liquid outlet channel 2 to a non-target analyte storage area to await the next step, or moved to a waste liquid storage area, such as... Figure 10 Before each screening and separation, the droplets can be mixed, separated, concentrated, diluted, or otherwise processed as required.

[0104] Batch successive approximation screening and separation scheme:

[0105] Figure 11 and Figure 12 This diagram illustrates a batch successive approximation screening and separation scheme. This scheme is suitable for the initial screening and separation of analytes at high concentrations, thereby improving the efficiency of screening and separation.

[0106] like Figure 11 A droplet 601 carrying a high concentration of analyte is driven into the first droplet moving chamber (the cis-side) of the sensing micropore, while a droplet 602 without analyte is driven into the second droplet moving chamber (the anti-cis-side). The second droplet moving chamber has a droplet inlet channel 3 perpendicular to the cross-sectional direction, providing new droplets without analyte, and is always ready to supply droplets to the anti-cis-side of the sensing micropore. At this point, droplets 601 and 602, along with the sensing micropore, constitute a through-hole sensor. When a potential difference is applied to the cis- and anti-cis-droplets, the analyte begins to pass through the sensing micropore. The number n of analytes separated in each anti-cis-droplet can be set. When the electrical or optical signals of the detected n analytes do not match the characteristics of the target analyte, the droplet 602 containing the target analyte is moved along the second liquid outlet channel 2 to a non-target analyte storage area, awaiting the next step, or moved to a waste liquid storage area, such as... Figure 12After the droplet is removed, a new droplet without analyte will be placed on the reverse side of the sensing micropore in the droplet inlet channel 3, continuing to drive the analyte in droplet 601 through the sensing micropore. If n non-target analyte molecules are detected, the above operation is repeated. If an electrical or optical signal matching the characteristics of the target analyte is detected in the next round of separation, the droplet containing one target analyte is moved to a target analyte storage area through the first droplet outlet channel 1, awaiting the next operation, or driven to other areas for single-molecule screening, separation, analysis, detection, etc. Before each screening and separation, the droplets can be mixed, separated, concentrated, diluted, etc., as required.

[0107] Repeated screening and separation of analytes:

[0108] The repeated screening and separation of analytes in this invention can refer to the droplet movement scheme of "repeated analysis and detection of analytes" described above.

[0109] To meet different detection needs, the solution in this invention can flexibly combine the repeatable screening and separation of analytes with the repeatable analysis and detection steps of analytes.

[0110] In summary, the via sensor provided by this invention has the following advantages:

[0111] The via sensor provided by this invention enables repetitive sequencing of nucleic acids, proteins, polypeptide chains, etc., by flexibly driving cis- and trans-droplets. To achieve sequencing accuracy requirements without wasting chip resources and saving time, the number of repetitive sequencing operations can be flexibly set. Before each sequencing run, the droplets can be mixed, separated, concentrated, diluted, etc., according to specific requirements.

[0112] The through-hole sensor provided by this invention enables repeatable detection of cells, molecules, and other analytes by flexibly driving cis- and trans-droplets. To achieve the required detection accuracy without wasting chip resources and saving time, the number of repeated detections can be flexibly set. Before each detection, the droplets can be mixed, separated, concentrated, or diluted according to specific requirements.

[0113] The through-hole sensor provided by this invention enables a single-cell and single-molecule counting method.

[0114] The through-hole sensor provided by this invention enables a repeatable single-cell and single-molecule counting method.

[0115] The through-hole sensor provided by this invention enables a method for simultaneously analyzing and detecting multiple analytes in the same sample.

[0116] The through-hole sensor provided by this invention enables a method for simultaneously and repeatedly analyzing and detecting multiple analytes in the same sample.

[0117] The through-hole sensor provided by this invention enables a method for screening and separating analytes. Before screening and separation, the droplets can be mixed, separated, concentrated, diluted, or otherwise processed according to specific requirements.

[0118] The through-hole sensor provided by this invention enables a method for repeatedly screening and separating analytes. To achieve the required purity during screening and separation while saving resources and time, the number of repeated screenings and separations can be flexibly set. Before each screening and separation, the droplets can be mixed, separated, concentrated, or diluted according to specific requirements.

[0119] The through-hole sensor provided by this invention can realize three schemes for repeated analysis and detection of analytes based on three-dimensional droplet movement.

[0120] The through-hole sensor provided by this invention enables a solution for repeated analysis and detection of analytes based on two-dimensional droplet movement.

[0121] The through-hole sensor provided by this invention enables two methods for screening and separating analytes: single-molecule screening and separation, and batch successive approximation screening and separation. The two methods can also be used in combination.

[0122] The through-hole sensor provided by this invention can realize repeated analysis, detection and repeated screening of analytes, separation and mixing, separation, concentration and dilution of droplets, and can be used in combination.

[0123] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0124] Example 1: Repeatable detection of single cells

[0125] The number of Staphylococcus aureus was detected in the solution.

[0126] One of the methods for repeated analysis and detection of analytes based on three-dimensional droplet movement is as follows: Figure 4Staphylococcus aureus was diluted to 1000 cells / µL using 5x TBE buffer. After adding ferric oxide magnetic particles, 1µL was added to the top chamber. This droplet was driven to the upper part of the sensing micropore using an electromagnet or permanent magnet as a cis-droplet. Before each detection, a trans-droplet without the analyte was driven to the bottom of the sensing micropore. The analyte was driven through the nanopore sensor, and ion current was detected. Five detections were performed. The average of the results from 2, 3, 4, and 5 detections was compared with the result of a single detection.

[0127] Results: Because each Staphylococcus aureus bacterium passing through the sensing microwell may not necessarily generate a sufficiently accurate discrimination signal due to conformational changes or detection noise, a single detection has a relatively large random error. As the number of detections increases, the occurrence of random errors is reduced or avoided.

[0128] Therefore, the closer the average value of the test results is to the actual value of 1000, the better. Figure 13 As shown.

[0129] Example 2: Single-cell screening and isolation

[0130] Staphylococcus aureus was screened and isolated from a mixture of Staphylococcus aureus and Bacillus subtilis.

[0131] The two bacteria differ significantly in cell size: Staphylococcus aureus is approximately 0.8 μm in diameter, while Bacillus subtilis is approximately 0.8 μm x (2 to 3 μm long). Therefore, the larger Bacillus subtilis generates a greater change in the ion current's resistance when passing through the perforated sensor. Thus, the bacterial species can be identified by observing the change in resistance current. The analyte screening and separation scheme one is used: the single-molecule screening and separation scheme. The principle is as follows... Figure 9 and Figure 10 Staphylococcus aureus was diluted to 80 cells / µL using 5x TBE buffer. Bacillus subtilis was also diluted to 80 cells / µL using 5x TBE buffer. Equal volumes of the two bacterial solutions were mixed, and ferric oxide magnetic particles were added. 1µL of this mixture was added to the top chamber, and the droplet was driven to the upper part of the sensing micropore using an electromagnet or permanent magnet as a cis-droplet. After each bacterial cell was detected and the trans-droplet had moved to a specific position, a new trans-droplet was driven to the bottom of the sensing micropore. The analyte was then driven through the nanopore sensor for ion current detection.

[0132] Results: 32 Staphylococcus aureus cells and 41 Bacillus subtilis cells were screened and isolated from 1 μL cis-droplets.

[0133] Example 3: Label-free, featureless DNA single-molecule repeatable sequencing

[0134] This embodiment primarily demonstrates the solution to the problem of label-free reproducible sequencing of multiple identical or similar nucleic acid molecules that are difficult to distinguish within the same cis-droplet. It employs a combination of single-molecule screening and separation schemes for the analyte and reproducible analysis and detection (using one of three schemes based on three-dimensional droplet movement, or a scheme based on two-dimensional droplet movement).

[0135] Synthetically synthesized oligonucleotides (C20G1A20) and (C20T1A20) were dissolved in 1M KCl (adjusted to pH 8.0 with 10mM Tris-HCl) solution, each diluted to 2000 copies / µL. 5µL of each was mixed to form 10µL. 1µL of this mixture was then added to the top chamber with iron oxide magnetic particles and driven to the upper part of the sensing micropore using an electromagnet or permanent magnet as a cis-droplet. Each trans-droplet accepted and transferred one DNA molecule. The next trans-droplet was driven to this position and waited to accept and transfer the next DNA molecule. The analyte was driven through the nanopore sensor, and ion current was detected. Once each DNA molecule was accepted and transferred by each trans-droplet, single-molecule screening and separation of DNA was completed. Next, each trans-droplet was driven to the cis-side of a new sensing micropore, and all DNA molecules were analyzed and detected in parallel (i.e., repeated sequencing). The number of repeated sequencing iterations was set to 15.

[0136] Results: A total of 462 DNA oligonucleotides were isolated through single-molecule screening and isolation. Statistical analysis was performed on the sequencing results of 15 sequencing runs for each DNA molecule. The final sequencing yielded 238 (C20G1A20) molecules and 224 (C20T1A20) molecules.

[0137] Example 4: Single-molecule reproducible sequencing of polypeptide chains

[0138] This embodiment primarily addresses the issue of sequencing accuracy in current porous sensor-based amino acid sequencing of proteins and polypeptide chains through reproducible sequencing.

[0139] A repeatable analysis and detection scheme for analytes based on two-dimensional droplet movement was employed. Bovine serum albumin (BSA) was prepared to a concentration of 200 pM using phosphate-buffered saline (PBS) containing 50 μM β-mercaptoethanol (BME), 250 mM NaCl, and 0.005% sodium dodecyl sulfate (SDS). After mixing, the solution was heated at 75°C for 60 minutes. After cooling, an equal volume of 250 mM NaCl was mixed. 1 μL of this solution was mixed with ferric oxide magnetic particles and added to the top chamber. This droplet was driven to the upper part of sensing micropore A using an electromagnet or permanent magnet as a cis-droplet. Separately, 1 μL of a protein-free solution was mixed with ferric oxide magnetic particles and added to the second droplet movement chamber, driven to the bottom of sensing micropore A as a trans-droplet. The analyte was then driven through the nanopore sensor, and ion current was detected. After all polypeptide molecules have passed through sensing micropore A and completed the first analysis and detection, the cis and trans droplets from sensing micropore A are driven to the top and bottom of sensing micropore B, respectively forming trans and cis droplets in the through-hole sensor B. A voltage opposite to that applied to through-hole sensor B is applied, driving the polypeptide molecules from the bottom droplet through the micropore B into the top droplet, thus completing the second analysis and detection. After the second analysis and detection, the cis and trans droplets from sensing micropore B are driven back to the bottom and top of sensing micropore A to begin the third analysis and detection. A total of 16 analyses and detections are performed between A and B.

[0140] Results: The first analysis and detection detected signals from 516 amino acid residues. The average of 16 analyses and detections yielded signals from 577 amino acid residues. Since bovine serum albumin (BSA) actually contains 583 amino acid residues, the average of the 16 detections is clearly more accurate. Due to the complexity of polypeptide sequencing, although this experiment has not yet achieved single-molecule polypeptide sequencing, the ability to more accurately detect the number of amino acid residues in polypeptide molecules lays a solid foundation for single-molecule polypeptide sequencing.

[0141] Example 5: Repeatable single-molecule counting of DNA

[0142] This embodiment primarily serves as an exemplary demonstration of the applicability of the technical method of this patent to repeatable single-molecule counting of DNA.

[0143] A repeatable analysis and detection scheme for analytes based on two-dimensional droplet movement was employed. λDNA was diluted to 6 pM with 1 M KCl solution. 1 μL of this solution was mixed with ferric oxide magnetic particles and added to the top chamber. An electromagnet or permanent magnet was used to drive this droplet to the top of sensing micropore A as a cis-droplet. Separately, 1 μL of DNA-free 1 M KCl solution was mixed with ferric oxide magnetic particles and added to the second droplet movement chamber, driving it to the bottom of sensing micropore A as a trans-droplet. The analyte was driven through the nanopore sensor, and ion current was detected. After all λDNA molecules had passed through sensing micropore A and the first count was completed, the cis- and trans-droplets from sensing micropore A were driven to the top and bottom of sensing micropore B, respectively forming trans- and cis-droplets in the through-pore sensor B. A voltage opposite to that applied to the through-pore sensor B was applied, driving polypeptide molecules from the bottom droplet through sensing micropore B into the top droplet. This completed the second count. After the second count is completed, the cis and trans droplets in sensing microorifice B are driven to the bottom and top of sensing microorifice C, respectively, to begin the third count. This process continues until the fifth count is completed in sensing microorifice E, at which point the droplet returns to sensing microorifice D for the sixth count. The droplet's path is ABCDEDCBA. After a total of nine counts, the droplet returns to sensing microorifice A.

[0144] Results: The first count was 3,219,680. The average count of 9 counts was 3,683,819. The average count of 9 counts is closer to the theoretical value. See Table 1.

[0145] Table 1. Statistical analysis of experimental results in Example 5

[0146]

[0147] As can be seen from the above embodiments, the perforated sensor provided by the present invention can perform repeated sequencing of nucleic acid, protein, and polypeptide chain molecules to correct random errors, thereby improving the accuracy of sequencing.

[0148] The perforated sensor provided by this invention allows for setting the number of repeat sequencing iterations for all analytes or for any portion of the target analytes, thus enabling the setting of sequencing accuracy for any molecule. This provides greater flexibility in sequencing applications. In practical research and clinical applications, it can balance other factors such as timeliness, cost, and throughput.

[0149] The porous sensor provided by this invention can sequence naked nucleic acid, protein, and polypeptide molecules. That is, the nucleic acid, protein, and polypeptide molecules to be sequenced do not require labeling, ligation, library construction, or other operations. It can most accurately reflect the sequence, abundance, and epigenetic information of the original molecules.

[0150] The perforated sensor provided by this invention can sequence extremely small amounts of nucleic acid, protein, and polypeptide molecules. For example, nucleic acid, protein, and polypeptide molecules in a single cell, or even a single nucleic acid, protein, and polypeptide molecule, can be sequenced.

[0151] The perforated sensor provided by this invention offers faster sequencing speeds for nucleic acids, proteins, and polypeptide chains. Taking nucleic acid sequencing as an example, Oxford Nanopore Technologies' bio-nanopore sequencing technology uses motor proteins to limit the rate of nucleic acid perforation, achieving a rate of approximately 100-500 nt / s. Pacific Biosciences of California's technology essentially employs a polymerase-based sequencing-while-synthesizing approach, achieving a rate of approximately 1-5 bp / s. In the method of this invention, the perforation rate for nucleic acids can reach 10 knt-10 Mnt / s.

[0152] The through-hole sensor provided by this invention can repeatedly detect analytes such as cells and molecules to correct random errors, thereby improving the accuracy of detection.

[0153] The through-pore sensor provided by this invention allows for setting the number of repeated detections for all analytes or for any portion of the target analytes, thus enabling the setting of detection accuracy for any molecule. This provides greater flexibility in detection applications. In practical research and clinical applications, it can balance other factors such as timeliness, cost, and throughput.

[0154] The through-hole sensor provided by this invention can detect naked cells, molecules, and other analytes. That is, the analytes do not require labeling or other processing. It can most accurately reflect the characteristic information of the analytes.

[0155] The through-hole sensor provided by this invention can detect trace amounts of analytes. For example, it can detect even a single analyte molecule.

[0156] The through-hole sensor provided by this invention can perform real-time screening and separation of a population of analytes on an individual basis.

[0157] The through-hole sensor provided by this invention can perform real-time screening and separation of a group of analytes on an individual basis.

[0158] The through-hole sensor provided by this invention can perform repeatable real-time screening and separation of analyte populations on an individual basis, thereby improving the concentration and purity of the target analyte.

[0159] The through-hole sensor provided by this invention allows for setting the number of repeated screening and separation cycles for the target analyte, thus enabling the setting of the screening and separation purity. This makes it more flexible in detection applications. In practical scientific research and clinical applications, it can balance other indicators such as timeliness, cost, and throughput.

[0160] The through-hole sensor provided by this invention can screen and separate trace amounts of analytes. For example, even a single target analyte molecule can be screened and separated.

[0161] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A via sensor, characterized in that, It includes a first electrode support plate and a second electrode support plate, and an intermediate sensing layer disposed 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. The first electrode support plate is provided with a plurality of first electrode units, the second electrode support plate is provided with a plurality of second electrode units, and the intermediate sensing layer is provided with a plurality of sensing micropores that connect the first droplet moving chamber and the second droplet moving chamber. Both the first and second droplet moving chambers are used to load a plurality of mutually separated drivable droplets. At least one of the drivable droplets contains the analyte. When the first droplet moving chamber is loaded with a plurality of first drivable droplets and the second droplet moving chamber is loaded with a plurality of second drivable droplets, any one of the first electrode units, any one of the first drivable droplets, any one of the sensing micropores, any one of the second drivable droplets, and any one of the second electrode units forms a closed loop with the external power supply.

2. The through-hole sensor according to claim 1, characterized in that, The driving method for the drivable droplet includes one or more of the following: capillary method, dielectric electrophoresis method, electrowetting method, dielectric wetting method, electrodewetting method, surfactant method, magnetic driving method, optical driving method, and thermal driving method.

3. The through-hole sensor according to claim 1, characterized in that, The sensing micropores are nanometer-sized or micrometer-sized, and the shape of the sensing micropores includes one or more of the following: slits, wires, tubes, strips, field-effect transistors, and nanopore extended field-effect transistors.

4. The through-hole sensor according to claim 1, characterized in that, The first droplet moving chamber and / or the second droplet moving chamber are filled with a liquid medium that is immiscible with the drivable droplet.

5. The through-hole sensor according to any one of claims 1 to 4, characterized in that, The intermediate sensing layer is also provided with several droplet channels that connect the first droplet moving chamber and the second droplet moving chamber.

6. The through-hole sensor according to any one of claims 1 to 4, characterized in that, The number of sensing micropores is ≥2, and the intermediate sensing layer does not have droplet channels.

7. The through-hole sensor according to claim 5, characterized in that, The number of the sensing micropores is 1, and the number of the droplet channels is 1 or 2.

8. The application of the through-hole sensor according to any one of claims 1 to 7 in the analysis, detection, screening and separation of analytes, wherein the analytes include ions, atoms, molecules, polymers, microorganisms, cells or cell complexes.

9. A method for multiple analyses or multiple detections of an analyte, characterized in that, When using the via sensor as described in claim 6, the following steps are included: A first drivable droplet is loaded in a first droplet moving chamber, and a second drivable droplet is loaded in a second droplet moving chamber. The first drivable droplet and the second drivable droplet are respectively a droplet containing the analyte and a blank droplet. The first drivable droplet and the second drivable droplet are repeatedly driven to a number of sensing micropores. By applying a potential difference with interchangeable directions between the first electrode unit and the second electrode unit, multiple analyses or detections of the analyte are performed at the sensing micropores. Alternatively, when using the through-hole sensor as described in claim 5, the following steps are included: A first drivable droplet is loaded into a first droplet moving chamber, and a second drivable droplet is loaded into a second droplet moving chamber. The first and second drivable droplets are a droplet containing the analyte and a blank droplet, respectively. The drivable droplets in the first and second droplet moving chambers are swapped through a droplet channel, and the drivable droplets in the first and second droplet moving chambers are driven to the sensing micropore. Multiple analyses or detections of the analyte are performed by applying a potential difference between the first and second electrode units.

10. A method for multiple screenings or separations of an analyte, characterized in that, The via sensor according to any one of claims 1 to 7 comprises the following steps: One first drivable droplet is loaded in a first droplet moving chamber, and multiple second drivable droplets are loaded in a second droplet moving chamber. The first and second drivable droplets are respectively droplets containing the analyte and blank droplets. The first drivable droplet is driven to the sensing micropore, and the multiple second drivable droplets are driven to the sensing micropore in sequence. The analyte is screened or separated multiple times in the multiple blank droplets by applying a potential difference between the first and second electrode units.

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