Monomolecular observation and function research platform based on nanopore and detection method

Abstract

The invention discloses a nanopore-based single molecule observation and function research platform and a detection method, and belongs to the technical field of single molecule observation and detection. The invention discloses a nanopore-based single molecule observation and function research platform, which comprises an inverted microscope system, a single molecule observation and function research assembly, an openable electromagnetic shielding cover, a control system, an upper computer, a display system and a shockproof platform. Researchers can perform observation, detection and related function research work on a single molecule by using the platform, especially can realize observation, detection and related function research on DNA molecules and protein molecules, and the invention provides a novel detection platform for single molecule research, which is used in the fields of function research, sequencing and the like of the single molecule and has a wide application prospect. And position imaging, length detection and characteristic structure analysis of a single long-chain molecule are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of single molecule observation and detection, and specifically relates to a single molecule observation and function research platform and a detection method based on nanopores. Background Art

[0002] Many existing bio-measurement methods are based on collective measurements, that is, measuring and averaging a large number of molecules. Single molecule detection technology is a technology that can observe, detect and analyze individual biomolecules. Single molecule detection technology allows researchers to directly observe and analyze the characteristics and reaction processes of individual molecules, thereby providing more detailed and accurate information. Compared with existing collective measurement methods, single molecule detection technology has the following important significance.

[0003] First, single-molecule detection technology can provide nanoscale resolution and can analyze subtle changes and heterogeneity. Through single-molecule detection, we can have a comprehensive understanding of the structure, dynamic behavior and interaction of biological molecules. Secondly, single-molecule detection technology can analyze the heterogeneity of different members in a molecular population, help researchers better understand their functional mechanisms and phenotypic diversity, and reveal the heterogeneity of molecules. Thirdly, single-molecule detection technology can monitor and record the conversion process of individual molecules in real time, thereby providing key kinetic information, studying low-frequency biological processes and molecular events, and realizing the study of rare events and conversion processes. Single-molecule detection technology plays an important role in studying basic life processes such as DNA replication, protein folding, enzyme catalysis, and cell signaling. By observing single molecules, researchers can have a deeper understanding of the mechanisms and regulation methods of these basic principles; finally, single-molecule detection technology can provide more accurate diagnostic results and personalized treatment plans by detecting and analyzing the single-molecule behavior of specific biomarkers in patients.

[0004] In short, single-molecule detection technology provides a powerful tool for in-depth understanding of the structure, function and interaction of biological systems by observing and analyzing single molecules in real time and with high sensitivity. The development of single-molecule detection technology has promoted the research and development of single-molecule detection equipment. These devices include single-molecule manipulation tools such as optical tweezers, magnetic tweezers, and atomic force microscopes, combined with fluorescence microscopes, ion channel detection equipment, surface-enhanced Raman spectroscopy detection equipment, electrochemical systems and other technologies to achieve the detection and analysis of single molecules. However, most of these existing devices are based on the deconstruction of single-molecule mechanical information, and are analyzed and detected separately, and have certain limitations.

[0005] Therefore, it is necessary to develop an experimental platform for single molecule observation and functional research, which can provide a new system that can realize the detection of electrical information, spatial size and mechanical information measurement of single molecules in a centralized or integrated manner, realize the observation, detection and related functional research of single molecules in multiple dimensions, and facilitate researchers to carry out functional research, sequencing and other related work of single molecules, which will greatly promote the rapid development of life science related disciplines. Summary of the invention

[0006] Based on the technical problems existing in the prior art, the present invention provides a single molecule observation and function research platform and a detection method based on nanopores.

[0007] According to the first aspect of the technical solution of the present invention, a single molecule observation and functional research platform based on nanopores is provided, which includes an inverted microscope system, a single molecule observation and functional research component and an openable electromagnetic shielding cover, the inverted microscope system is used as a base for installing the single molecule observation and functional research component; the single molecule observation and functional research component is used for single molecule observation and functional research, and the single molecule observation and functional research component is placed inside the openable electromagnetic shielding cover, with the customized sample stage of the inverted microscope system as the installation plane; the openable electromagnetic shielding cover is the shell of the entire single molecule observation and functional research component, the single molecule observation and functional research component and the openable electromagnetic shielding cover constitute an instrument as a whole installed above the inverted microscope system, and the openable electromagnetic shielding cover shields interference signals and improves the sensitivity and accuracy of single molecule probe current detection. Further, the openable electromagnetic shielding cover is made of conductive material. Preferably, the openable electromagnetic shielding cover is made of aluminum alloy or stainless steel, and the entire openable electromagnetic shielding cover is physically connected to the inverted microscope system, the single molecule observation and functional research component, the control system, the host computer and the display system and the anti-vibration platform with the ground.

[0008] Furthermore, the single-molecule observation and functional research platform based on nanopores includes a control system, a host computer and display system, and an anti-vibration platform. The control system is a control system of the single-molecule observation and functional research platform based on nanopores. One end of the control system is connected to the various electrical output interfaces of the inverted microscope system and the single-molecule observation and functional research components, and the other end is connected to the host computer in the host computer and display system; the processing system in the host computer and display system is a control processing and display system designed according to the needs of single-molecule observation and functional research, which realizes the parameter setting, single-molecule detection and observation and functional analysis functions of the single-molecule observation and functional research platform based on nanopores; the anti-vibration platform is an optical anti-vibration platform, which is used to place the inverted microscope system, the control system, the host computer and the display system.

[0009] Preferably, the single-molecule observation and functional research components include a single-molecule probe, a detachable probe fixture, a nano-linear positioner, a micro-motion stage and a control motor, an XY-axis nano-positioner, a sample fixing fixture, a single-molecule sample, a miniature digital microscope, a low-noise high-speed current amplification circuit, and connecting fixtures.

[0010] Preferably, the control system includes a power module, an analog-to-digital acquisition card, a piezoelectric positioner controller, a stepper motor controller and a driver. The sample stage, which is the mounting surface of the single-molecule observation and functional research components, is integrally combined with the openable electromagnetic shielding cover to form a closed surface, and the closed surface and the openable electromagnetic shielding cover form a complete electromagnetic shielding body. Preferably, the power module supplies power to the analog-to-digital acquisition card, the piezoelectric positioner controller, the stepper motor controller and driver, the low-noise high-speed current amplification circuit, the inverted microscope system, the host computer and the display system.

[0011] More preferably, the openable electromagnetic shielding cover is in an openable mode, and the openable electromagnetic shielding cover is provided with a conductive glass observation window; the openable electromagnetic shielding cover in the openable mode facilitates the placement of samples and the replacement of probes; the conductive glass observation window is used to observe the operation of single-molecule observation and functional research components.

[0012] According to the second aspect of the technical solution of the present invention, the present invention provides a detection method using the above-mentioned nanopore-based single-molecule observation and functional research platform, which comprises the following steps: Step S1: material preparation and startup; Step S2: probe installation and sample placement; Step S3: inserting the probe and scanning and capturing the sample; Step S4: perform detection.

[0013] Compared with the prior art, the beneficial technical effects of the nanopore-based single-molecule observation and functional research platform of the present invention are as follows: (1) The single-molecule detection technology based on nanopores of the present invention can detect the length and characteristic structure of single-end fixed long-chain molecules. It can realize the position imaging, length detection and characteristic structure analysis of single long-chain molecules. It is a new type of single-molecule detection tool, providing a new method and tool for biomolecule detection, which will greatly promote the rapid development of single-molecule detection technology in the field of life sciences.

[0014] (2) The single-molecule probe detection based on nanomanipulation in the present invention can repeatedly detect the same sample to be tested, thereby improving the accuracy of detection.

[0015] (3) The single-molecule probe detection method based on ion current of the present invention can scan and image the sample surface. It can not only determine the sample surface morphology and molecular layout, but also perform longitudinal capture and single-molecule detection on characteristic points.

[0016] (4) The present invention realizes nanomanipulation technology combined with three single-molecule probe detection schemes, namely glass nanopore, biological nanopore and solid-state nanopore, on the same platform. Different sample detection requirements can be met by simply replacing the relevant single-molecule probes, which greatly expands the applicability of single-molecule detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the overall structure of the nanopore-based single-molecule observation and functional research platform according to the present invention; Figure 2 is a schematic diagram of the structure of a single molecule observation and functional research component according to the present invention; Figure 3 is a schematic diagram of a method for constructing a single-molecule probe according to the present invention; Figure 4 It is a control relationship diagram of the control system according to the present invention.

[0018] Explanation of the reference numerals in the accompanying drawings: 1. Inverted microscope system; 2. Single-molecule observation and functional research components; 3. Openable electromagnetic shielding cover; 4. Control system; 5. Host computer and display system; 6. Shockproof platform; 201. Single-molecule probe; 202. Removable probe clamp; 203. Nanolinear positioner; 204. Micro-motion stage and control motor; 205. XY-axis nanopositioner; 206. Sample fixing clamp; 207. Single-molecule sample; 208. Miniature digital microscope; 209. Low-noise high-speed current amplifier circuit; 210. Connecting fixtures. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0021] It should be noted that the concepts such as "first" and "second" mentioned in the present invention are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0022] It should be noted that the modifications of "one" and "plurality" mentioned in the present invention are illustrative rather than restrictive, and those skilled in the art should understand that, unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0023] In order to meet the needs of electrical information detection, spatial size and mechanical information measurement of single molecules, the present invention designs a single-molecule observation and functional research platform and observation method based on nanopores, which can be used to observe, detect and carry out related functional research on single molecules, especially for the observation, detection and related functional research of DNA molecules and protein molecules.

[0024] In response to the needs of electrical information detection, spatial size and mechanical information measurement of single molecules, the present invention designs a single molecule observation and function research platform and detection method based on nanopores, which can use the platform to observe, detect and conduct related functional research on single molecules, especially to observe, detect and conduct related functional research on DNA molecules and protein molecules. The single molecule observation and function research platform and detection method based on nanopores of the present invention belong to the field of single molecule observation and detection technology. The single molecule observation and function research platform based on nanopores is also a new detection platform for single molecule research, which is used in the fields of functional research and sequencing of single molecules, and realizes position imaging, length detection and characteristic structure analysis of single long-chain molecules.

[0025] like Figure 1 and Figure 2 As shown, a nanopore-based single-molecule observation and functional research platform of the present invention includes an inverted microscope system 1, a single-molecule observation and functional research component 2, an openable electromagnetic shielding cover 3, a control system 4, a host computer and display system 5 and an anti-vibration platform 6.

[0026] An inverted microscope system 1 is an inverted microscope system for observing single-molecule probes and sample positions. The inverted microscope system used in the present invention is very different from the existing upright microscope system; the inverted structure of the inverted microscope system can not only expand the size and load-bearing capacity of the sample stage carrying the sample, but also can perform position observation without interfering with the motion trajectory of the single-molecule probe, and realize the initial positioning of the single-molecule probe on the surface of the sample; for example, the inverted microscope system 1 is used as a base for installing the single-molecule observation and functional research component 2, and the sample stage of the inverted microscope system can be customized, with a table size of 300*250mm and a load-bearing capacity of more than 10kg. The sample stage has two-way adjustment functions such as XY, which is convenient for adjusting the sample to the focal length center of the objective lens. An observation window that can be opened and closed is provided in the center of the sample stage of the inverted microscope system. The positioning and observation of the sample and the single-molecule probe are performed by opening the observation window, and the detection of the single-molecule signal is realized by closing the observation window, so as to achieve the best shielding effect.

[0027] The single-molecule observation and functional research component 2 is a component used for single-molecule observation and functional research. It is a closed-loop system composed of a nano-manipulation system and an ion current detection system, and has the functions of ion current scanning and detection. For example, when the nano-manipulation single-molecule probe contacts the sample surface, the ion current of the single-molecule probe is detected by a low-noise high-speed current detection circuit, and is provided as a contact feedback signal to the nano-manipulation system to stop the needle; when the nano-manipulation scans the sample surface, the two-dimensional position signal of the nano-manipulation and the signal intensity of the ion current in the single-molecule probe constitute the morphology and position signal of the sample surface; when the nano-manipulation single-molecule probe detects a single-end fixed long-chain molecule, the current signal of the single-molecule probe feeds back the structure and electrical information of the long-chain molecule.

[0028] The electromagnetic shielding cover 3 can be opened to cover the single molecule observation and functional research component 2, so that the single molecule observation and functional research component is not interfered by external electromagnetic signals, thereby improving the accuracy and sensitivity of nano-manipulation and ion current detection.

[0029] The control system 4 is a control system for the nanopore-based single-molecule observation and functional research platform, which includes providing power for each module of the entire nanopore-based single-molecule observation and functional research platform, realizing signal conversion of low-noise high-speed current amplification circuits, and electrical control of piezoelectric positioners and stepper motors, and receiving parameter signals from the host computer and display system 5, so as to realize closed-loop control of the nanopore-based single-molecule observation and functional research platform.

[0030] The host computer and display system 5 is used to run the whole machine control system, realize the detection, scanning and functional data collection of single molecule samples, and realize the display, storage and analysis and processing of the collected data.

[0031] The anti-vibration platform 6 is used to place the inverted microscope system, control system, host computer and display system, and provide active vibration isolation for the system to prevent external vibration signals from interfering with the detection results.

[0032] Furthermore, the inverted microscope system 1 includes a microscope base, an eyepiece assembly, a CMOS camera, a multi-specification long working distance objective lens assembly, a light source, an optical path system and a customized sample stage. The microscope base is an optional cast heavy-weight microscope base, which is used to install other components of the microscope and provide sufficient load-bearing capacity and stability; the eyepiece assembly is an optical eyepiece used for human eye observation, which is installed at the top of the base optical path to facilitate eye observation; the CMOS camera is a high-resolution color CMOS camera, which is used to observe and record microscope pictures and videos, and the images observed are consistent with those observed by the eyepiece assembly; the multi-specification long working distance objective lens assembly is installed on the optical path system of the microscope base, which can focus the light source on the sample surface, and receive reflected and scattered optical signals, and transmit them to the eyepiece and CMOS camera. The multi-specification long working distance objective lens assembly is a turntable structure, through which The rotating turntable can switch between objective lenses of different specifications for observing samples with different magnification requirements; the light source is an LED light source, which is placed behind the microscope base, and the internal optical path of the base is connected to the objective lens to irradiate the sample surface; the optical path system is an optical structure connecting the light source, lens barrel, objective lens, semi-reflective and semi-transparent lens, and eyepiece, which is embedded in the microscope base to ensure the stability of the optical path; the customized sample stage forms a complete whole with the openable electromagnetic shielding cover, which is connected to the shielding cover by screws, and the load-bearing capacity meets the overall quality requirements of the entire single-molecule observation and functional research component 2. The sample stage is connected to the microscope base by screws, and the center position coincides with the focus of the optical system objective lens, which is convenient for sample placement and observation.

[0033] The customized sample stage serves as the mounting surface of the single-molecule observation and functional research component 2 and is a surface of the entire openable electromagnetic shielding cover 3, and forms a complete electromagnetic shielding body with the openable electromagnetic shielding cover 3. The light source of the inverted microscope system is electrically connected to the light source power supply, and the size of the light source can be adjusted in real time, which is convenient for observing the sample to be tested and debugging the auxiliary group. The CMOS camera is connected to the host computer and the display system 5 via a USB cable, and the communication control is performed by the host computer software, which can realize real-time observation, collection, storage and processing of sample information. Multi-specification long working distance objective lens assemblies are installed on the objective lens holder, and the lens is switched by rotating the objective lens holder. The objective lens holder is installed on the height adjustment table, and the focusing and observation of the sample are achieved by height adjustment.

[0034] The single-molecule observation and functional research component 2 is the core of the entire nanopore-based single-molecule observation and functional research platform, which is placed inside the openable electromagnetic shielding cover 3, with the customized sample stage of the inverted microscope system 1 as the installation plane. In a preferred embodiment, the sample stage is customized according to the inverted microscope system 1, and the sample stage of the inverted microscope system provides the load-bearing and size required by the single-molecule observation and functional research component.

[0035] like Figure 2As shown, the single molecule observation and functional research component 2 includes a single molecule probe 201, a probe detachable fixture 202, a piezoelectric linear positioner 203, a micro-motion stage and a control motor 204, an XY axis positioner 205, a sample fixing fixture 206, a single molecule sample 207, a miniature digital microscope 208, a low-noise high-speed current amplification circuit 209, and a connecting fixture 210. The piezoelectric linear positioner 203 is preferably a nano linear positioner; the XY axis positioner is preferably an XY axis nano positioner.

[0036] The functions and structures of the various components of the single-molecule observation and functional research component 2 are further described below.

[0037] The single-molecule probe 201 is used to construct an ion channel. Figure 3 The three structures described in . The ion channel is constructed by three types of nanopore devices. The upper and lower ends of the ion channel are two chambers, one inside the probe, injected with buffer solution and connected with electrodes; the other inside the sample pool, injected with solution and connected with electrodes, thus forming an electrode pair to detect the current at both ends of the ion channel in real time. When the single-molecule probe approaches the sample surface or captures a molecule, the current of the ion channel will change, thereby generating a characteristic signal to reflect the approach process and the characteristics of the captured molecule; refer to Figure 2 and Figure 3 .

[0038] The probe detachable fixture 202 is used to fix the single molecule probe 201. The fixed end of the single molecule probe 201 is a hollow structure probe with a diameter of 1 mm, which can be placed in the probe detachable fixture 202 for fixation and installed on the linear positioner 203. Figure 2 . The detachable clamp 202 is composed of a glass microelectrode probe holder and a detachable mechanical fastener, wherein the glass microelectrode probe holder is used to fix the single-molecule probe 201, and is usually made of transparent solid materials such as PMMA, which can be purchased directly; the detachable mechanical fastener is used to fix the glass electrode probe holder to the bottom of the nanolinear positioner 203, and is fixedly connected through the top screw interface of the nanolinear positioner. The detachable mechanical fastener can also be used to install the glass electrode probe holder, which is convenient for disassembly. One end of the glass microelectrode of the detachable clamp 202 is the clamped single-molecule probe 201, and the other end is the connection electrode output of the single-molecule probe 201. The single-molecule probe 201 is filled with an ion solution, preferably an ion solution of potassium chloride, sodium chloride, lithium chloride, etc., and an Ag / AgCl electrode is placed, and the electrode and the metal connector are connected to the electrode output end. The output end is connected to the low-noise high-speed current amplifier circuit 209 through an electrical connection.

[0039] refer to Figure 2, the nanolinear positioner 203 is used to locate the Z-axis position of the single-molecule probe 201. The nano-manipulation single-molecule probe moves flexibly in the Z-axis direction and records its position in real time. The nanolinear positioner 203 is a high-precision nanolinear positioner stacked with piezoelectric crystals. It has a cylindrical structure and a screw socket at the tip for installing detachable mechanical fasteners. The nanolinear positioner 203 has a nanopositioning accuracy of 0.2nm and a positioning stroke of 100um, corresponding to the spatial resolution accuracy and detection length range of detecting a single molecule. The nanolinear positioner 203 is installed on the micro-motion stage and the control motor 204 and is fixed by a bracket screw. The nanolinear positioner 203 is connected to the control system 4 via a coaxial cable.

[0040] refer to Figure 2 As shown, the micro-motion stage and control motor 204 is divided into two parts, one part is used to fix the nano linear positioner 203, control the linear positioner 203 and the single molecule probe 201 to move in a large range in the Z axis direction, and record the position in real time; the other part is used to fix the XY nano positioner 205, control the XY nano positioner and the sample fixing fixture 206 and the single molecule sample 207 to move in a large range in the XY plane, which is convenient for microscope observation and sample scanning. The micro-motion stage and control motor 204 is a linear displacement stage with an accuracy of 0.5um and a stroke greater than 25mm. The micro-motion stage and control motor are divided into two parts: the first part is used to adjust the vertical position of the single molecule probe perpendicular to the sample stage of the inverted microscope system 1, installed on the connecting fixture 210, and fixed by screws; the second part is used to adjust the horizontal position of the sample parallel to the sample stage, composed of two XY axes, and installed on the connecting fixture 210. The micro-motion stage and the control motor are connected to the control system 4 through a coaxial cable. The coaxial cable wiring includes a 5-phase stepper motor control line, a limit and origin connection line.

[0041] refer to Figure 2 The XY axis nanopositioner 205 is used to control the movement of the sample fixture 206 and the single molecule sample 207 in the XY plane, and to record the XY displacement value in real time, which together with the Z axis displacement provided by the nano linear positioner 203 constitutes the basic data of the 3D coordinate map. The XY axis nanopositioner 205 is a purchased 2D nanopositioner with a scanning accuracy higher than 1nm and a stroke of 100um. The upper end of the XY axis nanopositioner 205 is mounted on the second part of the micro-motion stage and the control motor 204, and the lower end is mounted with a sample fixture 206. The XY axis nanopositioner 205 is connected to the control system 4 via a coaxial cable.

[0042] refer to Figure 2The sample fixture 206 is used to fix the single-molecule sample 207 and the sample pool, and is controlled by the XY-axis nanopositioner 205, the micro-motion stage, and the control motor 204. The sample fixture 206 is a self-designed sample pool fixture, which is fixedly installed below the XY-axis nanopositioner 205 and is controlled by the micro-motion stage, the control motor 204, and the XY-axis nanopositioner 205 to perform precise movement in the XY direction; the sample fixture 206 can be placed on the sample fixture, such as a transparent cuvette with a diameter of 25 mm, and the center point is aligned with the objective lens optical axis and the probe center axis, ensuring that the sample in the sample pool can be viewed through the inverted microscope system 1 and the single-molecule probe 201 can detect the single-molecule sample 207 to be tested at various positions in the sample pool.

[0043] refer to Figure 2 , the single molecule sample 207 is the sample to be tested, which is placed in the sample pool and fixed on the sample fixing fixture 206. The single molecule sample 207 is a single-end fixed molecule to be tested or a single cell molecule adsorbed on a substrate; wherein, the single-end fixed molecule to be tested includes double-stranded DNA, single-stranded RNA, or double-stranded DNA or single-stranded RNA that is modified, methylated, or loaded with other molecules; the substrate is a commonly used glass cover glass or a substrate designed by the user according to the sample requirements, and the method of fixing the molecule to be tested on the substrate can be a conventional biotin modification link, or it can be other user-designed fixing methods. The single molecule sample 207 and the substrate are placed together in a sample pool fixed by the sample fixing fixture 206, such as a cuvette, and an ion solution is injected into the sample pool, and an Ag / AgCl electrode is placed in the ion solution. The electrode is connected to the center line of the coaxial cable, and the coaxial cable is connected to the analog signal output end of the analog-to-digital acquisition module of the control system 4.

[0044] The miniature digital microscope 208 is used to assist in observing the relative position between the single-molecule probe 201 and the solution in the sample pool and the substrate that fixes the single-molecule sample 207. First, it prevents the probe from colliding with the substrate and causing damage, and second, it observes and feedbacks the position of the probe and the surface of the solution and the surface of the substrate to improve the efficiency of probe approach and detection. The miniature digital microscope 208 is a miniature digital microscope that integrates a light source and a microscope, and the magnification is adjustable from 3X to 1000X. The miniature microscope 208 is fixed to the connecting fixture 210 and is installed on the horizontal plane of the sample pool, which is convenient for observing the position of the single-molecule probe 201 and the liquid surface and the sample surface; the miniature microscope is connected to the control system 4 via a USB cable.

[0045] The low-noise high-speed current amplifier circuit 209 is used to detect the tiny current at both ends of the ion channel constructed by the single-molecule probe 201. The low-noise high-speed current amplifier circuit 209 is a self-designed high-speed current acquisition circuit. The sampling speed of the low-noise high-speed current amplifier circuit 209 is greater than 100kHz, and the sampling noise is less than 2pA at a frequency of @5kHz. It has three levels of hardware Bessel filtering options of 1kHz, 5kHz and 10kHz, which can realize high-speed and low-noise single-molecule signal acquisition, support the application of the instrument in the detection of single molecules, and can realize the difference identification of the blocking current of different base molecules passing through the nanopore.

[0046] One end of the signal input end of the low-noise high-speed current amplifier circuit 209 is connected to the electrode interface of the single-molecule probe 201, and the other end is connected to the ground. The power input interface of the low-noise high-speed current amplifier circuit 209 is connected to the power module interface of the control system 4. The signal output interface of the low-noise high-speed current amplifier circuit 209 is connected to the analog signal input interface of the analog-to-digital acquisition circuit of the control system 4 through a coaxial cable.

[0047] The connection fixture 210 is used to mechanically connect the customized sample stage with the micro-motion stage and the control motor 204, and is an internal structural connection part of the single molecule observation and functional research component 2. The connection fixture 210 is a mechanical structure designed according to the connection relationship between each module, including the sample stage, the micro-motion stage and the control motor 204 mounting surface, the miniature digital microscope 208 fixing, and the low-noise high-speed current amplifier circuit 209 installation fixing.

[0048] like Figure 3 As shown, there are three methods for constructing the single molecule probe 201. The first method: the single molecule probe 201 is drawn using a laser drawing instrument, and in this embodiment, a Sutter-2000 is used to draw a glass nanopore probe with a length of 5 nm to 100 nm.

[0049] The second type: The single-molecule probe 201 uses a laser drawing instrument to draw a glass nanopore with a tip length of 1um, uses a hydrogen-oxygen flame to seal the tip, and then uses a grinder to grind the tip to form a thick-walled glass micropore with an opening size of 10um~100um. Finally, phospholipids and biological nanopores are embedded in the tip opening in a solution system to form a biological nanopore detection probe constructed by a probe structure.

[0050] The third type: The single-molecule probe 201 uses a laser drawing instrument to draw a glass nanopore with a 1um tip, fixes a solid film such as graphene, molybdenum disulfide or COF film on the tip of the glass nanopore in a solution, and then uses a local controllable dielectric breakdown method to process a solid nanopore with a specified aperture on the solid film. In this embodiment, a solid nanopore with a diameter of 1-30nm is preferred to form a solid nanopore detection probe. The existing technology of the local controllable dielectric breakdown method used in the present invention, such as the method described in patent CN201910670241.X.

[0051] The main function of the openable electromagnetic shielding cover 3 in the nanopore-based single-molecule observation and functional research platform is to shield interference signals and improve the sensitivity and accuracy of single-molecule probe current detection; the openable electromagnetic shielding cover 3 is the outer shell of the entire single-molecule observation and functional research component 2, thus forming an instrument as a whole installed above the inverted microscope system 1, so that the overall structure of the machine is reasonable and the functional division is reasonable.

[0052] The material for preparing the openable electromagnetic shielding cover can be a conductive material such as aluminum alloy, stainless steel, etc., and the openable electromagnetic shielding cover is made of conductive materials. Preferably, the openable electromagnetic shielding cover is made of aluminum alloy or stainless steel, and the entire openable electromagnetic shielding cover is physically connected to the inverted microscope system, single molecule observation and functional research components, control system, host computer and display system and shockproof platform with the ground. The openable electromagnetic shielding cover 3 is designed to be automatically openable and is provided with a conductive glass observation window; the openable mode facilitates the placement of samples and the replacement of probes; the conductive glass observation window is used to observe the operation of the instrument.

[0053] like Figure 4 As shown, the control system 4 in the nanopore-based single-molecule observation and functional research platform is the core control system of the nanopore-based single-molecule observation and functional research platform. The control system is correspondingly connected to each electrical output interface of the inverted microscope system 1 and the single-molecule observation and functional research component 2, and the other end is connected to the host computer and display system 5.

[0054] The control system 4 includes a power module, an analog-to-digital acquisition card, a piezoelectric positioner controller, a stepper motor controller and a driver. The power module supplies power to the analog-to-digital acquisition card, the piezoelectric positioner controller, the stepper motor controller and the driver, the low-noise high-speed current amplifier circuit 209, the inverted microscope system 1, and the host computer and display system 5.

[0055] The analog-to-digital acquisition card is an optional high-precision analog signal acquisition card with a single-channel sampling frequency higher than 200kHz, 16bit, and a ±10V sampling range. It provides a ±2.5V 12bit DAC output and provides an adjustable high-precision bias for system current detection. The analog-to-digital acquisition card is connected to the host computer and display system 5 via a USB cable and is controlled in real time by the host computer system software.

[0056] The piezoelectric positioner controller is a piezoelectric controller shared by the piezoelectric linear positioner and the XY axis piezoelectric positioner, providing high-precision control voltage for the piezoelectric linear positioner and the XY axis piezoelectric positioner, and detecting the capacitive sensor signals of the piezoelectric linear positioner and the XY axis piezoelectric potentiometer at the same time, and detecting the operating position of the piezoelectric positioner in real time, thereby achieving high-precision nano-drive positioning. The piezoelectric positioner controller is connected to the piezoelectric linear positioner and the XY axis piezoelectric positioner through three coaxial cables, each of which includes a piezoelectric drive voltage line and a capacitive sensor connection line. The piezoelectric positioner controller is connected to the host computer and the display system 5 through a USB interface, and is controlled in real time by the host computer system software.

[0057] The stepper motor controller and driver provide driving signals for the micro-motion stage and the control motor to achieve precise control of the stepper motor's operating position. The stepper motor controller and driver are connected to the stepper motor through three coaxial cables, each of which includes a five-phase stepper motor drive voltage line and three limit switch signal lines. The stepper motor controller and driver are connected to the host computer and display system 5 through a USB cable and are controlled in real time by the host computer system software.

[0058] The host computer and display system 5 is used to write the host computer system software to realize the relevant functions of the host computer and display system 5. The host computer system software is a control display software independently designed according to the needs of single molecule observation and functional research. The host computer system software can realize the functions of instrument parameter setting, single molecule detection, observation and functional analysis. When the user uses the instrument, he only needs to operate and control the instrument according to the user manual of the host computer system software to achieve the purpose of single molecule observation and functional research. In one embodiment, the host computer and display system 5 is a commonly used desktop computer.

[0059] The anti-vibration platform 6 is a commonly used optical anti-vibration platform on a desktop, which is used to place the mainframe of the single-molecule observation and functional research system, including the inverted microscope system 1 and the single-molecule observation and functional research component 2.

[0060] In another embodiment, the present invention provides a detection method based on a nanopore-based single molecule observation and functional research platform. In this embodiment, the length of a DNA molecule is tested using a glass nanopore. The specific steps are as follows: Step S1: Material preparation and startup. In this embodiment, a 10 nm glass nanopore drawn by laser is used as a single molecule probe, and the detection sample is a long DNA chain molecule fixed at one end on a cover glass substrate. It further includes the following steps: Step S11, preparing a glass nanopore and a single-end fixed DNA molecule sample; Step S12, turning on the power of the nanopore-based single-molecule observation and functional research platform, and opening the host computer system software in the host computer and display system.

[0061] Step S2: Probe installation and sample placement, which further includes the following steps: Step S21, installing the glass nanopore prepared in step S1 on the detachable fixture 202, installing the Ag / AgCl electrode and ensuring that it is connected to the signal input end of the low-noise current amplifier circuit 209; Step S22, prepare a new cuvette sample pool, inject 1M potassium chloride solution, so that the liquid level can completely immerse the DNA sample; preferably, the solution height is added to 3mm~8mm, more preferably, the solution height is added to 5mm in this embodiment. Place the DNA sample and substrate in the cuvette sample pool, connect the solution in the cuvette to the Ag / AgCl electrode, and ensure that it is connected to the analog signal coaxial cable output by the control system 4 analog-to-digital acquisition card to provide an adjustable bias voltage for the ion channel; Step S23, adjusting the sample stage of the inverted microscope system 1 so that the sample to be tested and the single-molecule probe are axially aligned and both can be observed through the inverted microscope system 1; Step S24: observe the relative position of the probe, the liquid surface of the solution in the sample pool, and the surface of the sample substrate through the miniature digital microscope 208, and adjust the probe to within the liquid surface to form an opening current I0.

[0062] Step S3: Probe insertion, sample scanning and capturing. The system software probe insertion function is turned on, and the probe is brought close to the sample surface and scanned and captured in three steps, which further includes the following steps: Step S31, needle insertion. Record the opening current I0, set the current blocking threshold I1 when the probe approaches the sample surface, or the percentage of current change during approach, such as 1%. That is, when the opening current of the glass nanopore single-molecule probe 201 changes by 1% during the probe approach process, it is determined that the probe has contacted the sample surface. Control the piezoelectric linear positioner 203 to make the probe approach the sample, and the low-noise current amplifier circuit 209 changes the opening current in real time. When a change exceeding the set threshold is detected, the needle insertion is stopped, and the surface probe has approached the sample surface; if the stroke of the piezoelectric linear positioner 203 reaches the maximum and still does not reach the approach threshold, it is necessary to retract the piezoelectric linear positioner 203 first, and then control the micro-motion stage and the control motor 204 to control the probe to approach the sample. The control distance is 95% of the stroke of the piezoelectric linear positioner 203, and then continue to control the piezoelectric linear positioner 203 to approach the sample surface, and repeat this process until the probe approaches the sample surface; Step S32, scanning. After the probe approaches the sample surface, the XY axis piezoelectric positioner 205 is controlled to scan the sample surface. The scanning point, accuracy and longitudinal accuracy can be set by adjusting the software setting parameters, such as the number of points, resolution and current detection accuracy of the XY axis scanning. Finally, a scanning morphology image is generated through the scanning coordinate points of the XY axis and the detection current value of the corresponding point of the single molecule probe. In this way, the distribution and surface morphology of the sample can be determined; Step S33, capture. When the probe scans the sample surface, the generated sample distribution and surface morphology indicate the sample distribution. The XY axis positioner 205 is adjusted to control the probe to the center of the characteristic sample, and the bias voltage between the single molecule probe electrode and the sample pool electrode is adjusted until a signal is detected that a single molecule sample enters the glass nanopore and is captured. This signal may be a signal of a single long-chain DNA molecule, or it may be a signal of two or more DNA molecules entering the nanopore. It can be judged based on the blocking current value, or the bias voltage can be turned off after the first molecule enters the nanopore, so that the nanopore only captures a single molecule.

[0063] Step S4: DNA molecule length detection: After capturing the DNA molecule, the piezoelectric linear positioner 203 is controlled to leave the sample surface until the DNA molecule exits the nanopore, and the length information of the DNA molecule can be obtained after repeated testing.

[0064] In another embodiment, the present invention provides a detection method based on a nanopore-based single molecule observation and functional research platform. This embodiment is a solid-state nanopore test protein molecule, and the specific steps are as follows: Step S1: material preparation and startup.

[0065] In this embodiment, the graphene nanopore on the glass microelectrode is used as a single molecule probe, and the detection sample is a long-chain DNA molecule modified with a protein molecule fixed at one end on a cover glass substrate. It further includes the following steps: Step S11, preparing a glass microelectrode, a graphene film transferred onto PMMA, and a single-end fixed DNA molecule sample modified with a protein molecule; Step S12, turning on the power of the entire system and opening the system software; Step S13, solid-state nanopore preparation.

[0066] Step S2: Probe installation and sample placement, which further includes the following steps: Step S21, installing the glass microelectrode of the graphene nanopore prepared in step S1 on the detachable fixture 202, installing the Ag / AgCl electrode and ensuring that it is connected to the signal input end of the low-noise current amplifier circuit 209; Step S22, prepare a new cuvette sample pool, inject 1M potassium chloride solution, so that the page height can completely immerse the protein molecule sample, such as the solution height is added to 5mm. Place the DNA sample and substrate in the cuvette sample pool, connect the solution in the cuvette to the Ag / AgCl electrode, and ensure that it is connected to the analog signal coaxial cable output by the control system 4 analog-to-digital acquisition card to provide an adjustable bias voltage for the ion channel; Step S23, adjusting the sample stage of the inverted microscope system 1 so that the sample to be tested and the single-molecule probe are axially aligned and both can be observed through the inverted microscope system 1; Step S24: observe the relative position of the probe, the liquid surface of the solution in the sample pool, and the surface of the sample substrate through the miniature digital microscope 208, and adjust the probe to within the liquid surface to form an opening current I0.

[0067] Step S3: Probe insertion, sample scanning and capturing. The system software probe insertion function is turned on, and the probe is brought close to the sample surface and scanned and captured in three steps, which further includes the following steps: Step S31, needle insertion. Record the opening current I0, set the current blocking threshold I1 for the probe to approach the sample surface, or the percentage of current change during approach, such as 1%. That is, when the opening current of the solid-state nanopore single-molecule probe 201 changes by 1% during the probe approach process, it is determined that the probe has contacted the sample surface. Control the piezoelectric linear positioner 203 to make the probe approach the sample, and the low-noise current amplifier circuit 209 changes the opening current in real time. When a change exceeding the set threshold is detected, the needle insertion is stopped, and the surface probe has approached the sample surface; if the piezoelectric linear positioner 203 reaches the maximum stroke and still does not reach the approach threshold, it is necessary to retract the piezoelectric linear positioner 203 first, and then control the micro-motion stage and the control motor 204 to control the probe to approach the sample. The control distance is 95% of the stroke of the piezoelectric linear positioner 203, and then continue to control the piezoelectric linear positioner 203 to approach the sample surface, and repeat this process until the probe approaches the sample surface; Step S32, scanning. After the probe approaches the sample surface, the XY axis piezoelectric positioner 205 is controlled to scan the sample surface. The scanning point, accuracy and longitudinal accuracy can be set by adjusting the software setting parameters, such as the number of points, resolution and current detection accuracy of the XY axis scanning. Finally, a scanning morphology image is generated through the scanning coordinate points of the XY axis and the detection current value of the corresponding point of the single molecule probe. In this way, the distribution and surface morphology of the sample can be determined; Step S33, capture. When the probe scans the sample surface, the generated sample distribution and surface morphology indicate the sample distribution. The XY axis positioner 205 is adjusted to control the probe to the center of the characteristic sample, and the bias voltage between the solid-state nanopore single-molecule probe electrode and the sample pool electrode is adjusted until a signal is detected that the single-molecule sample enters the solid-state nanopore and is captured.

[0068] Step S4: protein molecule detection. After capturing the long DNA chain modified with the protein molecule, the piezoelectric linear positioner 203 is controlled to leave the sample surface and return to the sample surface according to the length of the long DNA chain, and the blocking current signal of the protein molecule modified on the long DNA chain to the solid-state nanopore is repeatedly tested, so that the characteristic information of the protein molecule can be analyzed.

[0069] The present invention aims at the needs of single molecule electrical information detection, spatial size and mechanical information measurement, and invents and designs a single molecule observation and functional research platform based on nanopores, which can be used to observe, detect and conduct related functional research on single molecules, especially DNA molecules and protein molecules. The single molecule detection method proposed by the present invention can realize the position imaging, length detection and characteristic structure analysis of single long-chain molecules, is a new type of single molecule detection tool, provides a new method and tool for biomolecule detection, and will greatly promote the rapid development of single molecule detection technology in the field of life sciences.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanopore-based single-molecule observation and functional research platform, characterized in that: It includes an inverted microscope system (1), a single-molecule observation and functional research component (2), and an openable electromagnetic shielding cover (3); An inverted microscope system (1) serves as a base for mounting components for single-molecule observation and functional studies (2); The single-molecule observation and functional research component (2) is used for single-molecule observation and functional research. The single-molecule observation and functional research component (2) is placed inside the openable electromagnetic shielding cover (3) and uses the customized sample stage of the inverted microscope system (1) as the installation plane. The openable electromagnetic shielding cover (3) is the outer shell of the entire single-molecule observation and functional research component (2). The single-molecule observation and functional research component (2) and the openable electromagnetic shielding cover (3) constitute an instrument as a whole and are installed above the inverted microscope system (1). The openable electromagnetic shielding cover (3) shields interference signals and improves the sensitivity and accuracy of single-molecule probe current detection.

2. A nanopore-based single-molecule observation and functional research platform according to claim 1, characterized in that: It further comprises a control system (4), a host computer and display system (5) and a shockproof platform (6); the control system (4) is a control system of a single molecule observation and functional research platform based on nanopores, one end of the control system is connected to each electrical output interface of the inverted microscope system (1) and the single molecule observation and functional research component (2), and the other end is connected to the host computer in the host computer and display system; The processing system in the host computer and display system (5) is a control processing and display system designed according to the needs of single-molecule observation and functional research, which realizes the parameter setting, single-molecule detection and observation and functional analysis functions of the single-molecule observation and functional research platform based on nanopores; The anti-vibration platform (6) is an optical anti-vibration platform and is used to place the inverted microscope system (1), the control system (4), and the host computer and display system (5).

3. A nanopore-based single-molecule observation and functional research platform according to claim 2, characterized in that: The single-molecule observation and functional research component (2) includes a single-molecule probe (201), a detachable probe fixture (202), a nano-linear positioner (203), a micro-motion stage and a control motor (204), an XY-axis nano-positioner (205), a sample fixing fixture (206), a single-molecule sample (207), a miniature digital microscope (208), a low-noise high-speed current amplification circuit (209), and a connecting fixture (210).

4. A nanopore-based single-molecule observation and functional research platform according to claim 3, characterized in that: The inverted microscope system (1) includes a microscope base, an eyepiece assembly, a CMOS camera, a multi-specification long working distance objective lens assembly, a light source, an optical path system and a customized sample stage; the openable electromagnetic shielding cover (3) is made of conductive material.

5. A nanopore-based single-molecule observation and functional research platform according to claim 1, characterized in that: The openable electromagnetic shielding cover (3) is made of aluminum alloy or stainless steel. The entire openable electromagnetic shielding cover is physically connected to the ground with the inverted microscope system (1), the single molecule observation and functional research component (2), the control system (4), the host computer and display system (5) and the anti-vibration platform (6).

6. A nanopore-based single-molecule observation and functional research platform according to claim 1, characterized in that: The control system (4) includes a power module, an analog-to-digital acquisition card, a piezoelectric positioner controller, a stepper motor controller and a driver.

7. A nanopore-based single-molecule observation and functional research platform according to claim 2, characterized in that: The sample stage as the mounting surface of the single-molecule observation and functional research component (2) is integrally combined with the openable electromagnetic shielding cover (3) to form a closed surface, and the closed surface and the openable electromagnetic shielding cover (3) form a complete electromagnetic shielding body.

8. A nanopore-based single-molecule observation and functional research platform according to claim 1, characterized in that: The openable electromagnetic shielding cover (3) is in an openable mode, and the openable electromagnetic shielding cover (3) is provided with a conductive glass observation window; the openable electromagnetic shielding cover in the openable mode facilitates the placement of samples and the replacement of probes; the conductive glass observation window is used to observe the operation of the single molecule observation and functional research component (2).

9. A nanopore-based single-molecule observation and functional research platform according to claim 6, characterized in that: The power module supplies power to the analog-to-digital acquisition card, the piezoelectric positioner controller, the stepper motor controller and driver, the low-noise high-speed current amplifier circuit (209), the inverted microscope system (1), and the host computer and display system (5).

10. A detection method based on a nanopore single molecule observation and functional research platform, characterized in that: It includes the following steps: Step S1: material preparation and startup; Step S2: probe installation and sample placement; Step S3: inserting the probe and scanning and capturing the sample; Step S4: perform detection.

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