Nanopore single molecule capture translocation transmission optical inspection method and system

Abstract

The invention relates to a nanopore single molecule capture translocation transmission optical inspection method and system, and belongs to the technical field of nanopore single molecule detection. An optical detection method is combined with a solid-state nanopore electrical detection technology, a nanopore area and an opening area thereof are respectively excited by utilizing a dynamic focusing technology, and fluorescence intensity change and molecular Raman scattering fingerprint characteristics caused by target molecule via hole blockage are obtained, so that whether molecules really pass through nanopore channels or not is effectively judged, and the detection accuracy is improved. According to the invention, the verification of the via hole behavior of the target molecule is realized, the accuracy and reliability of detection and analysis are improved, the respective advantages of optical detection and electrical detection are integrated, and high-performance detection and analysis of the nanopore single molecule are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of nanopore single molecule detection, and relates to a nanopore single molecule capture translocation transmission optical inspection method and system. Background Art

[0002] Nanopore single-molecule detection technology, with its ultra-low detection limit analysis capability, has promoted the development of single-molecule sensing technology and has been widely used in the detection of DNA, RNA, proteins and their modifications and their interaction analysis. As a universal biological single-molecule sensing detection platform, nanopore single-molecule detection follows the principle of Coulter counter, analyzes the blocking current signal characteristics such as blocking current amplitude, duration and blocking frequency formed by target molecules such as DNA passing through nanopores, reflects the relevant molecular translocation information and reveals its basic physical properties, such as size, shape and charge.

[0003] At present, the traditional solid-state nanopore electrical detection technology has great uncertainty, which seriously limits the application research of solid-state nanopore in biomolecule detection. The mechanism of the transport behavior of the biomolecule to be detected through the nanopore channel driven by the transmembrane voltage is relatively complex, which is closely related to the nanopore detection system and the biomolecule system to be detected. At the same time, the nanopore electrical detection process involves the transient changes of weak ionic current caused by ions or molecules passing through the nanopore, which is then restricted by non-ideal noise from insulating films, electrolyte systems, and weak signal acquisition systems. Moreover, the characteristic signal of the ionic current caused by the analyte passing through the nanopore channel is easily affected by the ionic current interference signal caused by the collision of the analyte, blocking the nanopore or the translocation of non-analytes, which seriously hinders the accurate analysis of the biomolecule translocation event in the nanopore channel. In view of the challenges faced by single-molecule electrical detection of solid-state nanopores, integrating other detection methods to improve its single-molecule analysis capability and detection throughput is of great significance to the development of single-molecule detection technology based on solid-state nanopores. With the advantages of high bandwidth, polychromaticity and high-throughput measurement, optical detection methods have been introduced to analyze the transport and blockage behavior of biomolecules in nanopore channels, including fluorescence spectroscopy detection technology based on fluorescence luminescence mechanism and scattering spectroscopy optical detection technology based on molecular scattering mechanism.

[0004] Combining optical detection methods with solid-state nanopore electrical detection technology can give full play to the advantages of high throughput, high sensitivity and specificity of optical detection methods, and achieve the purpose of simultaneous and parallel detection and analysis through appropriate photoelectric detection structures and corresponding improvement strategies. However, fluorescently labeled biomolecules change the intrinsic morphological characteristics of biomolecules, and calcium ion fluorescence indication and nanopore localized molecular scattering strategies cannot fully monitor the perforation behavior of target molecules. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method and system for optical inspection of single-molecule capture and translocation transmission in nanopores, which integrates optoelectronic fingerprint feature information, solves the problem of inability to determine whether a molecule passes through the pore in conventional nanopore single-molecule optoelectronic detection, and realizes high-performance detection and analysis of nanopore single molecules.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for optical inspection of single-molecule capture and translocation transmission in nanopores, comprising the following steps:

[0008] S1: Prepare a plasmonic nanopore device: Use micro-nano processing, deposition or thin-film transfer methods to form a nanoscale thin film on a substrate, deposit a metal layer, and then prepare a plasmonic nanostructure array. Use electron beam lithography, focused ion beam or nanoimprint methods to form nanoscale holes in the nanostructure array;

[0009] S2: Construct an optical detection system: Place the plasmonic nanopore device in a sample cell, and integrate a Raman microspectroscopy system, a fluorescence microscope, a patch clamp system, and a synchronous detection control and data analysis system;

[0010] S3: Dynamic focusing and time-division excitation: Use a dynamic focusing mirror to focus the excitation light on the two open regions on both sides of the nanopore in a time-division manner, and respectively excite the fluorescence signal and Raman scattering signal of the biomolecule;

[0011] S4: Synchronous acquisition and analysis: Capture the target molecule under the drive of a transmembrane electric field, synchronously collect the ion current signal, the fluorescence intensity change signal and the Raman scattering fingerprint signal, and fuse and analyze the optoelectronic signals to determine the molecule's pore-passing behavior.

[0012] Furthermore, in S1, the nanoscale thin film is a suspended silicon nitride thin film with a thickness of 5-30 nm, including a two-dimensional material thin film of one or more of silicon nitride, aluminum oxide, graphene / molybdenum disulfide, and a thin film with an overlapping or sandwich structure of different materials; the thickness of the metal layer is 50-100 nm, and the material is selected from gold, silver, copper or their alloys.

[0013] Furthermore, the plasmonic nanostructure array unit includes metal nanostructures such as bowties, nanocolumns, nanoslits or nanobowls, and the size of the intermittent, slit or hole of the metal nanostructure is 10-20 nm.

[0014] Furthermore, in S2, the sample cell is provided with a double-sided glass observation window, and the excitation light sources of the Raman microspectroscopy system and the fluorescence microscope adopt a time-division multiplexing mode.

[0015] Furthermore, in S3, the focusing range of the dynamic focusing mirror is from the nanohole plasma nanostructure end to the other opening end, and the focusing accuracy is sub-micron level.

[0016] Furthermore, in S4, if the ion current blockage signal, the fluorescence intensity change signal and the target molecule Raman fingerprint feature are detected synchronously, it is determined to be a molecular through-hole behavior; if only the first two signals are detected, it is determined to be a molecular blockage or collision behavior.

[0017] Furthermore, the target molecule is DNA, RNA, protein or sugar, and the voltage range of the transmembrane electric field is 50mV to 500mV.

[0018] A system for implementing the method, comprising:

[0019] Plasmonic nanopore devices, which have nanoscale pores and plasmonic nanostructures;

[0020] The sample pool is equipped with a double-sided glass observation window and a solution chamber separated by a nanoporous film;

[0021] Dynamic focusing module, used to adjust the focusing position of the excitation light on both sides of the nanopore in a time-sharing manner;

[0022] Synchronous detection module, integrating Raman spectrometer, fluorescence detector and patch clamp electrical signal acquisition device;

[0023] The data analysis module is used to fuse the photoelectric signals and output the molecular via determination results.

[0024] Furthermore, the dynamic focusing module includes a programmable focusing mirror and a time-division multiplexing laser, and the focusing response time is less than 10ms.

[0025] Furthermore, the substrate of the plasma nanopore device is a low dielectric loss constant material such as silicon, silicon dioxide, aluminum oxide, zinc oxide or quartz glass.

[0026] The beneficial effects of the present invention are:

[0027] The optical detection method is combined with the solid-state nanopore electrical detection technology. Through the dynamic focusing technology, the excitation light is focused on the plasma nanopore area and the other opening area of ​​the nanopore, respectively. The fluorescence intensity change caused by the blockage of the target molecule through the hole and the molecular Raman scattering fingerprint characteristics obtained when the target molecule passes through the nanopore to the plasma micro-nanostructure area are obtained in a time-sharing manner. Then, the fused optoelectronic fingerprint feature information is used to analyze the single-molecule through-hole behavior of the solid-state nanopore, thereby realizing high-performance detection and analysis of single molecules in the nanopore.

[0028] The present invention combines the respective advantages of optical detection and electrical detection, integrates the fluorescence emission and the Raman scattering characteristics of target molecules enhanced by the plasma electromagnetic field, and verifies the translocation behavior of target molecules in the nanopore while performing nanopore optoelectronic detection and analysis on the target molecules. At the same time, the application of the dynamic focusing technology, combined with the time-sharing excitation of the excitation light, enables the target molecules to be excited by different lasers at both ends of the nanopore, and the corresponding optical characteristic information is obtained to achieve the verification of the target molecules passing through the nanopore. In addition, the irradiation of the laser on the nanopore can change the ion current conductance level of the nanopore, thereby improving the detection sensitivity of the nanopore ion current. It can be seen from this that the advantage of the present invention is that the change in the fluorescence signal caused by the blockage of biomolecules combined with the Raman fingerprint characteristics of the target molecules can capture the characteristics of the biomolecules passing through the pore at both ends of the nanopore, and then determine whether the target molecules truly pass through the nanopore, realizing the single-molecule detection and analysis of solid-state nanopores.

[0029] Other advantages, objects, and features of the present invention will be described to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. The objects and other advantages of the present invention can be realized and obtained through the following specification. Brief Description of the Drawings

[0030] In order to make the objects, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail with reference to the accompanying drawings. Among them:

[0031] Figure 1 is a process flow chart for the preparation of a plasma nanopore device;

[0032] Figure 2 is a schematic diagram of the capture and translocation of plasma nanopores;

[0033] Figure 3 is a nanopore single-molecule capture and translocation optical inspection system;

[0034] Figure 4 is a schematic diagram of laser dynamic focusing;

[0035] Figure 5 is a schematic diagram of the principle of optical inspection for the capture and translocation of plasma nanopore single molecules;

[0036] Figure 6 is a signal diagram of nanopore fluorescence and ion current. Detailed Description of the Embodiments

[0037] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the drawings provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0038] Among them, the drawings are only for illustrative purposes, showing only schematic diagrams, not physical diagrams, and should not be construed as a limitation to the present invention; for better illustrating the embodiments of the present invention, some components in the drawings will be omitted, enlarged or reduced, which does not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0039] In the drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "rear", etc. indicating the orientation or positional relationship, they are based on the orientation or positional relationship shown in the drawings. This is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the drawings are only for illustrative purposes and should not be construed as a limitation to the present invention. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0040] I. A preparation method of a plasma nanopore device applicable to photoelectric detection

[0041] As Figure 1 shown, the method includes the following steps:

[0042] (1) Prepare a nanoscale thin film with a thickness of 5 - 30 nm: Use micro-nano processing, deposition or thin film transfer methods to prepare a suspended silicon nitride thin film with a thickness of 5 - 30 nm on a silicon material substrate.

[0043] (2) Deposit a noble metal thin film, evaporate / deposit a 50 - 100 nm metal thin film on the above nanoscale thin film, and the metal can be gold, silver, copper or an alloy.

[0044] (3) Prepare a plasma nanostructure array: Use electron beam lithography method to prepare a plasma nanostructure array on the metal thin film. The unit structure of the nanostructure array is a bowtie structure, and the gap size of the metal bowtie nanostructure is 10 - 20 nm.

[0045] (4) Preparation of thin-film nanopores: Using a focused ion beam, nanoscale holes are prepared in the nanoscale silicon nitride thin film in the bow-tie plasmonic nanostructure, and the size range of the nanopores is 2 - 10 nm.

[0046] Figure 2 It is a schematic diagram of translocation transport captured by plasmonic nanopores.

[0047] II. Construction of an optical inspection system for single-molecule capture and translocation transport through nanopores

[0048] The specific steps are as follows:

[0049] (1) Load the prepared plasmonic nanopore device into a sample cell with double-sided glass observation windows designed and prepared independently.

[0050] (2) As Figure 3 shown, integrate a Raman microspectroscopy system, a fluorescence microscope, a patch clamp system, and a synchronous detection control and data analysis system to construct an optical inspection system for single-molecule capture and translocation transport through nanopores. Among them, the laser adopts a time-division multiplexing mode. As Figure 4 shown, it is used to excite the Raman scattering spectrum of biomolecules and the fluorescence of the calcium ion fluorescence indicator respectively. The dynamic focusing mirror is used to dynamically adjust the focal length of the excitation light, and focus on the plasmonic structure end and the fluorescence excitation end of the nanopore device respectively. The synchronous detection control and data analysis system mainly controls the selection of the laser and the focal length control of the dynamic focusing mirror, and synchronously collects optical and electrical signals. As Figure 6 shown, it realizes the synchronous acquisition and analysis of nanopore optoelectronic signals.

[0051] III. Optical inspection method for single-molecule capture and translocation transport through plasmonic nanopores

[0052] (1) Conduct routine cleaning treatments on the plasmonic nanopore device and the sample cell, including plasma treatment, soaking in ethanol and deionized water, etc.

[0053] (2) Inject the prepared buffer salt solution, calcium ions, and their calcium ion fluorescence indicator into both ends of the nanopore thin film in the sample cell respectively. Among them, calcium ions are added to the side of the plasmonic nanopore metal thin film, and the calcium ion fluorescence indicator and the DNA molecule to be detected are added to the other side.

[0054] (3) Load the sample cell after injection onto the stage, turn on the Raman or fluorescence excitation laser, adjust the dynamic focusing mirror, and focus on the plasmonic nanopore metal layer and the nanopore opening on the other side respectively, and mark the focusing range of the dynamic focusing mirror.

[0055] (4) Conduct DNA molecule capture translocation transmission experiments. Using a dynamic focusing mirror, set the dynamic focusing range. The fluorescence and Raman excitation lasers are alternately focused on both sides of the plasmonic nanopore. Driven by the transmembrane electric field, DNA molecules are captured by the plasmonic nanopore and translocate, while collecting DNA molecule translocation blocking ion current signals, blocking fluorescence signals, and plasmonic nanopore enhanced Raman scattering signals.

[0056] (5) Perform fusion analysis on the DNA molecule plasmonic nanopore capture translocation current signals, fluorescence, and Raman spectrum signals to determine whether the DNA molecule has passed through the pore, as Figure 5 shown. On the premise of obtaining the blocking ion current and fluorescence signals, if the Raman scattering fingerprint characteristics of the molecule to be measured are synchronously obtained, it indicates that the signal characteristics are pore passing behavior; if the Raman scattering fingerprint characteristics of the molecule to be measured are not obtained, it indicates that the signal characteristics are molecular blockage or collision behavior.

[0057] Example 1: Preparation of plasmonic nanopore device

[0058] Step 1: Prepare a suspended silicon nitride film

[0059] Deposit a 30-nm-thick low-stress silicon nitride film on the silicon substrate by low-pressure chemical vapor deposition (LPCVD).

[0060] Lithograph a window shape on one side of the silicon nitride film, etch the silicon nitride film on one side of the window by reactive ion etching (RIE), clean the surface photoresist with acetone, and finally remove the back region of the silicon substrate by wet etching to form a suspended silicon nitride film window (size: 10 μm × 10 μm).

[0061] Step 2: Deposit a noble metal film

[0062] Use an electron beam evaporator to evaporate a 100-nm-thick gold film on the surface of the silicon nitride film, and the evaporation rate is The vacuum is maintained at 5×10 -6 Torr.

[0063] Step 3: Prepare a bowtie plasmonic nanostructure array

[0064] Define a bowtie nanostructure pattern on the metal layer by electron beam lithography (EBL). The single structure size is 20 nm × 50 nm, and the gap is 10 nm.

[0065] Use ion beam etching (IBE) to remove the gold film not protected by the resist to form a periodic array (period: 100 nm).

[0066] Step 4: Prepare nanopores

[0067] Using a focused ion beam (FIB, Ga + ion beam, acceleration voltage 30 kV), a nanopore with a diameter of 5 nm was etched on the silicon nitride film in the central region of the bow-tie structure.

[0068] The morphology and size accuracy (error ±0.5 nm) of the nanopore were verified by transmission electron microscopy (TEM).

[0069] Example 2: Construction and calibration of a nanopore single-molecule detection system

[0070] System composition

[0071] Sample cell: Made of polytetrafluoroethylene, with quartz glass observation windows (thickness 0.17 mm) at both ends. The cavity is divided into two independent solution chambers by a nanopore film.

[0072] Optical module: Raman spectrometer: 785 nm laser (power 10 mW), equipped with a high-sensitivity CCD detector (spectral resolution 1 cm -1 ). Fluorescence microscope: 488 nm laser (power 5 mW), fluorescence filter set (bandwidth ±10 nm), EMCCD camera (quantum efficiency >90%).

[0073] Electrical module: Patch clamp amplifier (sampling rate 100 kHz, bandwidth 10 kHz), Ag / AgCl electrodes connected to both ends of the solution chamber.

[0074] Dynamic focusing module: Piezoelectric ceramic-driven adjustable focusing mirror (focusing range ±50 μm, response time <5 ms), synchronized with the laser control.

[0075] Calibration process

[0076] 1. Focus calibration: Dispersed fluorescent microspheres (diameter 100 nm) in the solution chamber, adjusted the laser focus to the metal end of the nanopore (error <200 nm) through the dynamic focusing mirror, and recorded the peak position of the fluorescence signal.

[0077] 2. Time-division excitation synchronization test: Set the laser switching period to 10 ms (Raman and fluorescence alternate excitation), and verified that there was no crosstalk in signal acquisition (cross-interference <1%).

[0078] 3. Electrical signal baseline calibration: Measured the baseline noise of the ion current (RMS noise <2 pA) under the condition of no molecule passing through the pore, and ensured that the signal-to-noise ratio >10.

[0079] Example 3: DNA molecule translocation detection experiment

[0080] Experimental conditions

[0081] Solution system: Metal end chamber: 1 M KCl buffer (pH 7.4) containing 0.5 M CaCl2 and 10 mM Tris-HCl. Non-metal end chamber: 1 M KCl buffer (pH 7.4) containing 10 mM Tris-HCl, 1 μM Fluo-8 (calcium ion fluorescence indicator) and 10 pM λ-DNA (48.5 kbp).

[0082] Transmembrane voltage: Apply a 200 mV DC voltage to drive the DNA molecules to migrate towards the metal end.

[0083] Operating procedure

[0084] 1. Dynamic focusing and signal acquisition:

[0085] Start the dynamic focusing lens program and switch the focus position (metal end → non-metal end) every 20 ms.

[0086] Synchronously trigger the Raman (785 nm) and fluorescence (488 nm) lasers, and collect the Raman spectrum (integration time 10 ms) and fluorescence intensity (frame rate 50 fps) respectively.

[0087] The patch clamp system records the ion current signal in real time (sampling frequency 50 kHz).

[0088] 2. Signal analysis:

[0089] Fluorescence signal processing: When the DNA molecules translocate through the nanopore, the voltage-driven calcium ion channels are blocked, and the binding of calcium ions to the fluorescence indicator is affected, resulting in a decrease in fluorescence intensity.

[0090] Raman fingerprint recognition: DNA characteristic peaks are detected (such as the phosphate backbone vibration peak at 1090 cm-1 and the deoxyribose peak at 780 cm-1), and the signal intensity is enhanced by 10 3 times (plasma enhancement effect).

[0091] Fusion determination: If the ion current blockage signal (amplitude > 50 pA, duration > 0.3 ms) appears synchronously with the decrease in fluorescence intensity and the Raman fingerprint (time deviation < 1 ms), it is determined as a DNA translocation event; if only the first two appear, it is a molecular blockage or pore mouth collision event.

[0092] Experimental results

[0093] Among 100 detections, 85 events met the translocation determination conditions, and the Raman fingerprint matching degree > 95%; 15 events only showed current blockage and fluorescence changes, and were determined as collisions.

[0094] Example 4: Optimization and verification of detection sensitivity

[0095] Parameter adjustment

[0096] Comparison of metal materials: Gold, silver, and copper alloys (Au 70 Ag 20 Cu 10 ) were used to fabricate nanopore devices, and the Raman enhancement factor (EF) was measured.

[0097] Results: The EF of gold nanopores was 10 5 , the EF of silver nanopores was 10 4 ×5, and the EF of copper alloy was 10 3 ×8 (due to poor oxidation stability).

[0098] Transmembrane voltage optimization: The DNA translocation speed and signal quality were measured in the range of 50 - 500 mV.

[0099] Optimal voltage: 200 mV (translocation time 1 - 3 ms, highest signal - to - noise ratio).

[0100] Sensitivity verification

[0101] Detection limit test: DNA was diluted to 10 fM, and clear translocation events could still be detected (signal - to - noise ratio > 5).

[0102] Through the dynamic focusing time - sharing excitation technology, combined with fluorescence, Raman, and ion current signals, the present invention significantly improves the accuracy and specificity of single - molecule translocation detection. Examples show that:

[0103] 1. Raman fingerprint features can effectively distinguish translocation and collision events (accuracy > 95%).

[0104] 2. The plasma nanostructure enhances the Raman signal intensity by 10 3 - 105 times, enabling the detection of low - concentration molecules.

[0105] 3. The system response time < 10 ms, suitable for high - throughput single - molecule analysis.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions, and they should all be covered within the scope of the claims of the present invention.

Claims

1. An optical inspection method for single-molecule capture, translocation and transmission through nanopores, characterized in that: It includes the following steps: S1: Prepare a plasmonic nanopore device: Use micro-nano processing, deposition, or thin-film transfer methods to form a nanoscale thin film on a substrate, deposit a metal layer, and then prepare a plasmonic nanostructure array. Use electron beam lithography, focused ion beam, or nanoimprinting methods to form nanoscale holes in the nanostructure array; S2: Construct an optical detection system: Place the plasmonic nanopore device in a sample cell, and integrate a Raman microspectroscopy system, a fluorescence microscope, a patch clamp system, and a synchronous detection control and data analysis system; S3: Dynamic focusing and time-division excitation: Use a dynamic focusing mirror to focus the excitation light on the two open regions on both sides of the nanopore in a time-division manner, and respectively excite the fluorescence signal and Raman scattering signal of biomolecules; S4: Synchronous acquisition and analysis: Capture target molecules under the drive of a transmembrane electric field, synchronously acquire ion current signal, fluorescence intensity change signal, and Raman scattering fingerprint signal, and fuse and analyze the optoelectronic signals to determine the molecule's behavior of passing through the pore.

2. The nanopore single-molecule capture translocation optical inspection method according to claim 1, wherein: In the above S1, the nanoscale thin film is a suspended silicon nitride thin film with a thickness of 5 - 30 nm, including a two-dimensional material thin film of one or more of silicon nitride, aluminum oxide, graphene / molybdenum disulfide, and a thin film with an overlapping or sandwich structure of different materials; the thickness of the metal layer is 50 - 100 nm, and the material is selected from gold, silver, copper, or their alloys.

3. The optical inspection method for nanopore single-molecule capture translocation transmission according to claim 1, characterized in that: The plasmonic nanostructure array unit includes metal nanostructures such as bowties, nanocolumns, nanoslits, or nanobowls, and the size of the gaps, slits, or holes in the metal nanostructures is 10 - 20 nm.

4. The nanopore single-molecule capture translocation transmission optical inspection method according to claim 1, wherein: In the above S2, the sample cell is provided with double-sided glass observation windows, and the excitation light sources of the Raman microspectroscopy system and the fluorescence microscope adopt a time-division multiplexing mode.

5. The optical inspection method for nanopore single-molecule capture and translocation transmission according to claim 1, characterized in that: In the above S3, the focusing range of the dynamic focusing mirror is from the plasmonic nanostructure end of the nanopore to the other open end, and the focusing accuracy is sub-micron level.

6. The nanopore single-molecule capture, translocation and transmission optical inspection method according to claim 1, wherein: In the above S4, if ion current blockage signal, fluorescence intensity change signal, and Raman fingerprint characteristics of the target molecule are synchronously detected, it is determined as the behavior of the molecule passing through the pore; if only the first two signals are detected, it is determined as the behavior of the molecule being blocked or colliding.

7. The optical inspection method for nanopore single-molecule capture and translocation transmission according to claim 1, characterized in that: The target molecule is DNA, RNA, protein, or sugar, and the voltage range of the transmembrane electric field is 50 mV - 500 mV.

8. A system for implementing the method according to any one of claims 1 to 7, characterized in that: It includes: A plasmonic nanopore device having nanoscale holes and plasmonic nanostructures; A sample cell configured with double-sided glass observation windows and a solution chamber separated by a nanopore thin film; A dynamic focusing module for adjusting the focusing position of the excitation light on both sides of the nanopore in a time-division manner; A synchronous detection module integrating a Raman spectrometer, a fluorescence detector, and a patch clamp electrical signal acquisition device; A data analysis module for fusing optoelectronic signals and outputting the determination result of the molecule passing through the pore.

9. The system according to claim 8, wherein: The dynamic focusing module includes a programmable focusing mirror and a time-division multiplexing laser, and the focusing response time is less than 10 ms.

10. The system according to claim 8, characterized in that: The substrate of the plasmonic nanopore device is a low dielectric loss constant material such as silicon, silicon dioxide, aluminum oxide, zinc oxide, or quartz glass.

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