Solid-state nanopore single molecule detection method and device based on GaN light-driven regulation and control

Abstract

The invention discloses a solid-state nanopore single molecule detection method and device based on GaN light-driven regulation and control, and belongs to the technical field of single molecule detection and micro-nano sensors. According to the invention, the solid nanopore is prepared on the insulating or semi-insulating film with the adjustable surface state, then the GaN light source is integrated in the adjacent area of the nanopore, and the surface state of the film material is injected or evacuated by using the photon-generated carrier generated by the GaN light source under the transfilm bias voltage. The effective surface charge density and the local potential field of the nanopore interface are reversibly changed, so that the light-operated modulation of the ion perforation current is caused, and the single-molecule identification of the target molecule can be realized by comparing the current event difference under the illumination condition and the non-illumination condition.

Description

Technical Field

[0001] This invention belongs to the field of single-molecule detection and micro / nano sensor technology, and particularly relates to a method and device for single-molecule detection in solid-state nanopores based on GaN light-driven modulation. Background Technology

[0002] Solid-state nanopores are a class of micro / nano sensing structures that utilize changes in ion current within nanoscale channels to achieve real-time detection of perforation and blockage events of individual molecules. Compared to traditional biological nanopores, solid-state nanopores offer significant advantages such as controllable pore size, strong chemical stability, high mechanical strength, and ease of large-scale integration, thus attracting widespread attention in fields such as nucleic acid analysis, protein detection, metal coordination complex identification, and environmental small molecule monitoring. Existing solid-state nanopores are typically fabricated using insulating or semi-insulating films such as Si3N4, Al2O3, SiO2, h-BN, and MoS2. Their surfaces possess a certain density of interface states, forming stable interface charges in the electrolyte, which significantly influence ion transport behavior. However, the surface states of traditional solid-state nanopores remain essentially fixed during detection, and the interface charge density is difficult to dynamically adjust. This results in the ion flux, local potential field distribution, and background noise characteristics of the nanopore being in a single, unadjustable state. This characteristic directly limits the controllability of solid-state nanopores, making it difficult to achieve reversible conductance switching or multi-level channel adjustment according to detection requirements, and also limiting their selectivity enhancement in complex molecular systems.

[0003] To overcome the limitations of traditional electro-driven methods, some studies have attempted to irradiate the nanopore interface with an external light source, hoping to modulate the ion current through light-induced surface reactions or changes in surface charge. However, external optical paths often struggle to effectively couple the light field to the nanopore opening region, resulting in significant energy loss during propagation and making it difficult to form a sufficiently strong and spatially confined local light field. Furthermore, this approach cannot control individual channels within a porous array, and the illumination range often covers the entire chip area, affecting controllability and failing to meet the independent adjustment capabilities required by array-level high-throughput detection platforms. Therefore, traditional external light control methods cannot provide a stable, reversible, and quantitative interface regulation mechanism for nanopores, and a usable single-molecule light-controlled detection scheme has not yet been developed.

[0004] Gallium nitride (GaN) is a typical wide-bandgap semiconductor material with excellent ultraviolet and blue light emission characteristics. It can be fabricated into micro-LEDs or optoelectronic control structures at the micrometer and submicrometer scale using mature processes. Under illumination, it can generate a large number of electron-hole pairs, forming a significant photogenerated carrier distribution at the interface. If a GaN light source is tightly coupled with a solid-state nanopore at the chip scale, the photogenerated carriers may exchange with the surface states of the nanopore film material, thereby changing the filling mode of the film material's surface states and enabling reversible changes in the effective surface charge density of the pore walls and the interfacial potential field with illumination. However, current technologies have not applied the photogenerated carrier control capability of GaN to the solid-state nanopore interface, nor have there been reports of integrated GaN micro-light sources and solid-state nanopore chips, and there is no mention of dynamically adjusting the nanopore's conduction state, single-molecule event amplitude, or entry barrier through light-driven processes. Therefore, how to construct a GaN-based optically driven solid-state nanopore control mechanism that can stably and controllably adjust the interfacial potential and ion transport behavior at the nanoscale, thereby improving the sensitivity, selectivity and scalability of single-molecule detection, has become an important problem that urgently needs to be solved in the current field. Summary of the Invention

[0005] To overcome the shortcomings of existing solid-state nanopore detection methods, such as unadjustable interfacial charge density, single control dimension, limited detection sensitivity and selectivity, and lack of local control over external light, this invention proposes a method for controlling the surface states of nanopore membrane materials using photogenerated carriers generated by a GaN light source. This method enables reversible, stable, and controllable dynamic adjustment of the interfacial potential field and ion transport behavior of the nanopore during detection, thereby improving the amplitude difference of single-molecule events, the contrast of entry barriers, and the event resolution capability. This gives solid-state nanopores the advantages of light-controlled switching, multi-level control, and independent addressability of multi-pore arrays.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation, comprising the following steps:

[0008] (1) Solid nanopores with a diameter of 1-10 nm are prepared on insulating or semi-insulating films with tunable surface states to obtain nanopore films;

[0009] (2) Integrating a GaN light source in the vicinity of the solid nanopore;

[0010] (3) Under transfilm bias, turn the GaN light source on or off, and record the current changes in the illuminated and unilluminated states.

[0011] (4) Identify the single-molecule blocking event of the target molecule based on the difference in current between the illuminated and unilluminated states.

[0012] This invention involves preparing solid-state nanopores on insulating or semi-insulating films with tunable surface states, and then integrating a GaN light source into the region adjacent to the nanopores. Under transmembrane bias, photogenerated carriers generated by the GaN light source are used to inject or evacuate the surface states of the film material, causing a reversible change in the effective surface charge density and local potential field at the nanopore interface. This induces photo-controlled modulation of the ion perforation current. By comparing the differences in current events under illuminated and unilluminated conditions, single-molecule recognition of target molecules can be achieved.

[0013] Further, in step (1), the insulating or semi-insulating film is a multilayer two-dimensional material; the insulating or semi-insulating film is selected from one or more of Si3N4, Al2O3, SiO2, h-BN, MoS2, WS2 and graphene film.

[0014] Furthermore, in step (1), the method for preparing the solid nanopore is selected from electron beam etching, helium ion beam etching, focused ion beam etching, or atomic layer etching.

[0015] Further, in step (2), the emission wavelength of the GaN light source is 250-450nm; the distance between the GaN light source and the solid nanopore is 100nm-5μm; and the working mode of the GaN light source is continuous or pulsed.

[0016] Furthermore, in step (2), the GaN light source is a micro-LED.

[0017] Furthermore, in step (3), the transmembrane bias voltage is 150-200mV.

[0018] Furthermore, in step (4), the amplitude of the monomolecular blocking event under illumination is ≥20% greater than that under non-illumination conditions.

[0019] Furthermore, step (1) also includes the step of modifying the surface of the solid nanopore with an aptamer.

[0020] This invention provides a GaN-based optically driven solid-state nanopore single-molecule detection device, comprising a solid-state nanopore chip, a GaN light source, a microfluidic cavity, a light source driving circuit, and a current acquisition module.

[0021] Furthermore, the GaN light source and the solid-state nanopore chip are integrated into an m×n nanopore array, where m and n represent the number of rows and columns, respectively.

[0022] Furthermore, the current acquisition module is a transimpedance amplifier array; the current acquisition module is connected to an external data analysis unit.

[0023] The present invention also provides an application of the method or device described in the above technical solutions in the single-molecule detection of biomarkers, metal coordination complexes or small molecule environmental pollutants.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] This invention is the first to introduce the GaN photogenerated carrier modulation mechanism into the interface of a solid-state nanopore. By changing the surface state filling of the nanopore membrane material, reversible regulation of the interface potential field and ion transport behavior is achieved, thus overcoming the limitations of traditional solid-state nanopores that rely on fixed surface charges and single bias voltage driving. This invention provides a new degree of optical control freedom, enabling nanopores to achieve ON / OFF switching, multi-level conduction, and adjustable event amplitude, improving the signal-to-noise ratio and recognition capability of single-molecule events. The light source and nanopore of this invention are integrated at the chip scale, enabling efficient optical coupling, localized modulation, and array expansion, avoiding energy loss and uncontrollability caused by external optical paths. The method provided by this invention is applicable to highly sensitive single-molecule detection of various targets, including biomarkers, metal coordination complexes, and environmentally relevant small molecules, and has broad application prospects. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0027] Figure 1 A schematic diagram illustrating the principle of the GaN-based optically driven single-molecule detection method for solid-state nanopores provided by this invention.

[0028] Figure 2 This is a schematic diagram illustrating the surface states and interfacial potential of the nanoporous membrane material controlled by GaN photogenerated carriers in this invention.

[0029] Figure 3 This is a schematic diagram illustrating the changes in nanopore ion current under illumination and non-illumination conditions according to the present invention.

[0030] Figure 4 This is a schematic diagram of the single-molecule event recognition based on light-controlled differences in this invention;

[0031] Figure 5 This is a schematic diagram of the GaN-nanopore array structure of the present invention;

[0032] Figure 6 This is a schematic diagram illustrating the differences in the photodynamic conformation and events of the aptamer of the present invention;

[0033] Figure 7 This is a schematic flowchart of the solid-state nanopore single-molecule detection method based on GaN optically driven regulation provided by the present invention.

[0034] Figure 8 The change in electrical conductivity of the nanopores in the MoS2 / h-BN two-dimensional material before and after illumination in Example 4;

[0035] Figure 9 This is a schematic diagram illustrating the differences in single-molecule events of PFOA molecules under light and non-light conditions in step (2) of Example 5;

[0036] Figure 10 This is a schematic diagram showing the difference in light-driven blocking events of AFP molecules in the aptamer-modified pores in step (2) of Example 6. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] This invention provides a GaN-based optically driven method for detecting single molecules in solid-state nanopores. By using a GaN light source, localized, pore-by-pore, pulsed, or continuous optical control operations are achieved on a single nanopore or nanopore array.

[0040] The schematic diagram of the solid-state nanopore single-molecule detection method based on GaN light-driven modulation provided by this invention is shown below. Figure 1 As can be seen, the method of the present invention uses a GaN micro-LED with an emission wavelength of 365nm as the light source to form a local field around a solid nanopore with a diameter of 1-10nm, which excites photogenerated carriers to regulate the surface states of the pore wall; ions in the electrolyte in the upper and lower cavities pass through the nanopore under the drive of the electric field, and the change in the surface state changes the ion transport resistance, generating a characteristic current difference, thereby realizing the recognition of single molecules.

[0041] The specific implementation method of the GaN-based light-driven single-molecule detection method for solid-state nanopores provided by this invention is as follows: First, solid-state nanopores with a diameter of 1-10 nm are prepared on an insulating or semi-insulating film with tunable surface states; then, a GaN light source is integrated in the vicinity of the solid-state nanopores, so that the illumination can form a local light field at the nanopore interface; when the light source is working, GaN generates a large number of photogenerated electrons and holes under illumination, and its charge carriers can inject or evacuate the surface states of the film material at the interface of GaN-nanopore film material, changing the surface state filling situation, thereby changing the effective surface charge density of the pore wall, resulting in the bending of the interface band and the redistribution of the local potential field, such as... Figure 2 As shown; under the action of transmembrane bias, the change in the interfacial potential field causes a change in ion perforation behavior, resulting in reversibly modulated nanopore conductivity that is related to the illumination state, such as... Figure 3 As shown; finally, by recording the differences in ion currents under illumination and non-illumination conditions, and analyzing them based on event amplitude, duration, or changes in the entry energy barrier, single-molecule recognition of target molecules can be achieved, such as... Figure 4 As shown.

[0042] In another embodiment, the present invention constructs an m×n nanopore-GaN light source array, where each nanopore unit is equipped with an independently driven GaN light source. Hole-by-hole addressing is achieved through row-column multiplexing circuitry, thereby enabling multi-channel parallel detection. The array structure is as follows: Figure 5 As shown, it can significantly improve detection throughput and realize independent optical control modes for different channels within the array, thereby improving the flexibility of the detection platform.

[0043] In another embodiment, the surface of the solid nanopore can be modified with specific aptamers. By photo-driven modulation of the aptamer conformation, differences in the entry energy barrier of target molecules under illumination can be created, further enhancing the difference in event amplitude and selectivity. Figure 6 As shown.

[0044] A flowchart illustrating the GaN-based light-driven solid-state nanopore single-molecule detection method provided by this invention is shown below. Figure 7 As can be seen, after the sample to be tested is input, the GaN light source is turned on or off to adjust the light control interface. At the same time, the ion current is detected and the current changes in the illuminated and unilluminated states are recorded. Then, the single-molecule blocking event of the target molecule is identified based on the current difference between the illuminated and unilluminated states.

[0045] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0046] Example 1

[0047] Fabrication of GaN-solid-state nanopore integrated chip:

[0048] (1) Using a commercially available low-stress Si3N4 / Si-based reinforcing film (structure Si3N4 / Si / Si3N4) as the substrate, the thickness of the Si3N4 film is about 20nm and the thickness of the single-sided polished silicon substrate is 200μm; after cleaning the Si3N4 / Si-based reinforcing film, photoresist is coated, and the window area is defined by back-side photolithography. The back-side Si3N4 film in the window area is removed by deep reactive ion etching to form a via on the silicon substrate. After the endpoint detection, the etching is stopped to obtain a Si3N4 suspension film structure with a central area of ​​50μm×50μm. After the suspension film is prepared, it is dried at a constant temperature and subjected to nitrogen blowing to obtain a clean and contamination-free film surface.

[0049] (2) The Si3N4 thin film on the front side of the suspended film area was etched by a helium ion microscope (HIM) with an accelerating voltage of 30kV. According to the beam current-aperture calibration curve, the beam current was set to 1.2-1.5pA and the residence time was 1-10ms. The aperture was controlled by single-point exposure, and a solid nanopore with a diameter of about 3.8nm was finally prepared. After etching, the aperture was confirmed by scanning electron microscopy, and the aperture morphology was further verified by low-dose electron beam under TEM to ensure that the channel was connected and that there was no significant damage at the edge.

[0050] (3) A GaN micro-LED with an emission wavelength of 365nm and a size of 20μm×20μm was selected as the light source chip. The GaN micro-LED was placed in a flip-chip manner with the light-emitting surface facing the solid nanopore at a position about 2.0μm away from the center of the solid nanopore. A precision positioning platform and an optical microscope were used to align the mounting position. Then, it was fixed to the chip substrate surface in the area adjacent to the solid nanopore by thin film welding. Subsequently, the positive and negative electrodes of the GaN micro-LED were led out to the external driving circuit by gold wire bonding. In order to ensure the stable operation of the light source in the electrolyte environment, a transparent insulating encapsulation film was further covered on the outside of the GaN micro-LED. The encapsulation thickness was about 3μm to ensure that the light path could pass through freely and to avoid electrochemical corrosion.

[0051] (4) The upper and lower cavities were prepared on the Si3N4 film by polydimethylsiloxane (PDMS) casting molding method to form a microfluidic cavity, and the solid nanopore was located in the center of the membrane between the two cavities. The electrolyte was a 1 mol / L KCl aqueous solution, and 10 mmol / L Tris buffer was added to maintain stable pH conditions to obtain the GaN-solid nanopore integrated chip. During the measurement, a 200 mV transmembrane bias voltage was applied through the Ag / AgCl electrode, the ion current was converted into a voltage signal by the transimpedance amplifier, and the current change was recorded by the high-speed data acquisition card at a sampling rate of 100 kHz.

[0052] Performance testing:

[0053] Testing Procedure: First, the steady-state conductivity and baseline noise of the solid-state nanopores were recorded under no-light conditions. The initial interface state was obtained by measuring the mean and standard deviation of the current over different time periods. Then, the GaN micro-LED was turned on and driven using a pulsed voltage mode, generating approximately 6mW / cm² with a 5V drive voltage. 2 The local illumination intensity was measured, and the current changes under illumination conditions were recorded simultaneously.

[0054] Results: The solid nanopore current was observed to increase significantly under illumination, with a steady-state conductance increase of about 12%. The current returned to the initial level after the light source was turned off, indicating that the interface potential field modulation has good reversibility.

[0055] A comparative analysis of transient current changes under illuminated and unilluminated conditions was conducted. The results show that the current step change induced by illumination exhibits a stable on / off boundary, with interface modulation speed on the order of milliseconds, demonstrating the rapid response capability of GaN photogenerated carriers. Analysis of steady-state conductance differences, noise amplitude, power spectral density, and step response confirms that the conductance change caused by illumination modulation originates from changes in surface state filling, rather than temperature effects, electrolyte evaporation, or external interference. In repeated experiments, no significant attenuation was observed after more than 100 illumination-off cycles, demonstrating the stability and reliability of interface modulation.

[0056] Example 1 presents a specific method for fabricating solid-state nanopores using Si3N4 thin films and integrating a GaN light source to achieve interfacial light-controlled modulation. It also verifies the core principle of this invention: through the tight integration of GaN micro-LEDs with solid-state nanopores, the surface states of the nanopore film material can be effectively modulated using photogenerated carriers, thereby enabling significant and reversible light-controlled characteristics in the interfacial potential field and ion transport behavior of the solid-state nanopores. This example not only demonstrates the feasibility of the method of this invention but also provides an experimental basis for subsequent differential enhancement and array-based extension of single-molecule events. The photogenerated carrier modulation mechanism proposed in this invention can significantly affect the interfacial potential field and ion transport behavior of solid-state nanopores, forming reversible and stable light-controlled conductivity characteristics.

[0057] Example 2

[0058] Single-molecule recognition experiments based on photo-driven aptamer conformational regulation:

[0059] (1) A Si3N4 film with a thickness of about 15 nm was selected as the nanoporous membrane material. Solid nanopores with a diameter of about 4 nm were prepared according to the method in Example 1. After the pore preparation and cleaning were completed, the nanopore opening and pore wall regions were surface activated to introduce hydroxyl groups. Subsequently, a stable aminated anchoring layer was formed on the pore wall surface by chemical modification with aminosilane 3-aminopropyltriethoxysilane. After silanization was completed and cured, the aptamer molecule with NHS active ester end group was placed in a primary amine-free buffer system to contact the aminated anchoring layer formed on the pore wall surface, so that the NHS end group and the pore wall amino group underwent an amidation reaction, thereby covalently fixing the aptamer to the nanopore opening and pore wall region. After the reaction was completed, the chip was thoroughly cleaned and the unreacted active sites were sealed to obtain aptamer-functionalized solid nanopores. The aptamer sequence was designed according to the charge characteristics, structural features and binding affinity of the target molecule. The concentration of the target molecule used was low, in the range of 1-10 nM, to ensure that the time distribution of single molecule events had sufficient sparsity for easy analysis.

[0060] (2) During the device assembly process, the nanopore chip containing aptamer modification obtained in step (1) is installed into the microfluidic cavity. The cavity structure is the same as in Example 1. The electrolyte is 1 mol / L KCl aqueous solution + 10 mmol / L Tris buffer system. The transmembrane bias is maintained at 180 mV to ensure that the target molecules have sufficient driving force to enter the pore area under the action of the electric field. Then, the GaN micro-LED is driven and adjusted to generate stable 365 nm local illumination in the voltage range of 5-6 V, so that the illumination covers an area of ​​about 3 μm around the nanopore.

[0061] (3) When baseline detection is performed under no light conditions, characteristic blocking events of aptamer-target molecule can be observed. The current amplitude is about 160-200 pA and the event duration is about 0.4-0.7 ms with a wide time distribution. Then, the GaN light source is turned on, and the local potential field is adjusted by photogenerated carriers on the hole wall and aptamer anchoring region. It is observed that the aptamer conformation under light conditions exhibits a higher tension state, which changes the binding energy barrier that the target molecule needs to overcome when entering the pore, resulting in a significant increase in event amplitude, with an average increase of about 35%, and some events even exceeding 50%. The event duration also shows a regular change, with most events showing a faster detachment process, indicating that light changes the local morphology of the aptamer binding potential energy surface.

[0062] (4) To eliminate non-specific factors, a light-on-off cycle test was conducted. The results showed that both light-on and light-off caused reversible changes in the amplitude and duration of the blocking event, and there was no significant decay after multiple cycles. This proved that the light-controlled regulation originated from the synergistic effect of the interface potential field and the conformational change of the aptamer, rather than the temperature rise or unstable light effect. In addition, by statistically analyzing the distribution of event amplitude and duration, the clustering boundary of the two types of events became more obvious under the illumination condition, which helps to improve the recognition accuracy of target molecules.

[0063] Example 2 provides a method for enhancing the recognition of target molecules by combining the GaN light-driven interface modulation mechanism with the functionalization of solid nanopore aptamers. The aim is to demonstrate that photogenerated carriers generated by GaN light sources can affect the conformation of aptamers or the local binding environment, thereby amplifying the differences between target molecules and background molecules in current events and improving the recognition of single-molecule events.

[0064] The results of Example 2 show that the signal difference of single-molecule events can be enhanced by the GaN photo-driven modulation mechanism without changing the aptamer concentration, pore size, electrolyte composition, etc. This method can be used in highly selective biological detection scenarios, especially suitable for molecular systems with similar structures but slight differences in binding potential.

[0065] Example 3

[0066] Construction of arrayed GaN nanoporous structures:

[0067] (1) A Si3N4 thin film chip containing a 3×3 suspended film array was prepared using the same method as in Example 1. Nine solid nanopores with diameters of 3-6 nm were formed by HIM point etching of each suspended film region. The spacing between the pores was kept at 50-80 μm to prevent coupling between the electric field and the diffusion field. Each solid nanopore was prepared using the method in Example 1 to ensure uniform pore size, clean film surface, and consistent structure between units.

[0068] (2) A 3×3 GaN micro-LED array is pre-fabricated in front of the array substrate so that the illumination area of ​​each GaN micro-LED exactly covers the position of a nanopore. The GaN light source array adopts a matrix driving method, with row lines and column lines connecting each row and column of micro-LEDs respectively. A logic controller selects a certain cross node to light up an LED individually, realizing the hole-by-hole addressing function. To reduce crosstalk, light-absorbing isolation trenches or surface matting coatings are set between GaN light sources to keep the illumination area highly localized.

[0069] (3) The array chip is assembled in a microfluidic structure containing upper and lower cavities, and parallel current acquisition of nine channels is achieved through multiple Ag / AgCl electrodes. The voltage source sequential excitation method is used to apply a transmembrane bias voltage of 150-200mV to each channel. The data acquisition system uses a multi-channel synchronous transimpedance amplifier to enable the current signals of the nine channels to be recorded simultaneously, and the synchronization error between different channels is less than 5μs.

[0070] The background current and noise distribution of all channels in the arrayed GaN nanopore structure constructed in Example 3 were recorded under no-light conditions, and it was confirmed that there was no current crosstalk within the array. Subsequently, by illuminating any GaN light source in the array through row and column addressing, it was observed that only the conductivity of the corresponding channel changed, while other channels remained static, proving that the light-controlled modulation of this embodiment has good locality. After illumination was turned on, the blocking event of the target molecule in the corresponding channel was significantly enhanced, while the unlit channels maintained the original event pattern, indicating that the array structure can achieve pore-by-pore differentiation and control.

[0071] The response characteristics of nine channels under different light intensities were tested. As the light source driving voltage increased from 3V to 7V, the increase in single-channel event rate increased from 12% to 45%, exhibiting a wide adjustable range and continuous response. The control curves of different channels showed good consistency, indicating that the fabrication process under the array structure has high repeatability and the light control effect is stable.

[0072] Example 3 demonstrates the scalability of this invention in the arraying direction. By integrating a GaN light source with solid-state nanopores in an m×n configuration and achieving per-pore addressing through a driving circuit, each pore can be independently controlled for light regulation, thus forming a high-throughput, multi-parameter, programmable single-molecule detection platform. This arrayed structure is suitable for future applications in simultaneous multi-parameter detection, complex environment sample analysis, and portable sensor systems.

[0073] Example 3 provides a system structure that integrates a GaN light source with a solid nanopore in an array form and realizes hole-by-hole addressing and synchronous detection. It is used to verify the scalability of the method of the present invention in a high-throughput platform, and demonstrates how to use an m×n array to extend solid nanopore detection from a single channel to a multi-channel, and realize hierarchical control and differentiated detection of each channel through independent optical control operation.

[0074] Example 4

[0075] Differences in the optical modulation of nanopores in MoS2 / h-BN two-dimensional materials:

[0076] (1) A MoS2 monolayer film with a thickness of about 1.2 nm and an h-BN monolayer film with a thickness of about 0.8 nm were prepared on a sapphire substrate by chemical vapor deposition. Then, the MoS2 monolayer film and the h-BN monolayer film were transferred to a windowed Si substrate by PMMA-assisted transfer method. The film was then annealed at 300 °C with argon to remove residual polymer and improve the bonding stability between the film and the substrate, forming a free-floating structure.

[0077] (2) Nanopores were etched on the surface of monolayer films using helium ion microscopy (HIM). Solid nanopores with a pore size of approximately 2.8 nm were obtained in the MoS2 monolayer film, and solid nanopores with a pore size of approximately 2.4 nm were obtained in the h-BN monolayer film. Due to the extreme thinness of the two-dimensional material, the pore boundaries were sharper, and the interface states mainly came from atomic vacancies and boundary defects. After etching, the chip was assembled in a PDMS-glass microfluidic cavity. A transmembrane bias of 200 mV was applied in 1 mol / L KCl aqueous solution + 10 mmol / L Tris buffer solution, and the ion current of the two types of channels was tested. Then, a GaN micro-LED was connected to provide approximately 6 mW / cm² to the pore area. 2 Under 365nm local illumination, the conductivity changes ΔG / G of the two types of materials before and after illumination were measured, where ΔG is the conductivity change caused by illumination and G is the conductivity change without illumination. The results are shown in […]. Figure 8 , Figure 8 The results show that both materials can generate blocking events under no-light conditions; under illumination, the event amplitude of MoS2 increases significantly, and the duration is also prolonged, with its two-dimensional distribution map showing greater migration; in contrast, although the light-controlled enhancement of h-BN is limited, the event distribution is more concentrated, providing a more stable recognition window. Repeated experiments confirmed the significant differences in material responses, demonstrating the significant depth of control that the method of this invention can achieve in multi-material systems.

[0078] This embodiment aims to verify the applicability of the GaN photogenerated carrier modulation mechanism proposed in this invention to nanopores of different two-dimensional materials and its differences in interface response, particularly its performance in two typical two-dimensional film materials: MoS2 and h-BN. Since two-dimensional materials have atomic-level thickness and significantly different band structures, their surface state densities, interfacial coupling strengths, and electrochemical stability also differ. Therefore, this embodiment can demonstrate material-dependent photo-controlled modulation and ion transport changes, supporting the material universality of this invention. The results show that the GaN photogenerated carrier modulation mechanism proposed in this invention is not only applicable to traditional insulating films such as Si3N4 and Al2O3, but also to typical two-dimensional material systems, exhibiting material-dependent interface modulation characteristics. MoS2 shows a stronger photo-controlled response and can be used in high-sensitivity scenarios; h-BN provides a more stable baseline and low noise advantages, suitable for high-precision background monitoring. This embodiment demonstrates that this invention has broad material compatibility, and different film materials can be selected according to actual application requirements to achieve optimized detection performance.

[0079] Example 5

[0080] Single-molecule detection of PFAS (taking PFOA as an example) based on GaN optically driven modulation:

[0081] (1) Solid nanopores with a diameter of about 3.5 nm were prepared on Si3N4 thin films (the method is the same as in Example 1); since PFOA (perfluorooctanoic acid) molecules are negatively charged, aptamer modification is not required, but the difference in entry energy barrier is enhanced by adjusting the surface states of the pore wall; the detection system uses a buffer solution of 1 mol / L KCl aqueous solution + 10 mmol / L Tris with a pH of 7.5.

[0082] (2) A transmembrane bias voltage of 200mV was applied under no-light conditions, and the results are shown in the figure. Figure 9 When PFOA molecules enter the pore, a blocking event occurs. However, due to the small molecular size and weak structural rigidity, the event amplitude is low (approximately 60-90 pA) and the duration is wide (0.05-0.2 ms), resulting in significant noise and making identification difficult. After the GaN micro-LED is turned on, the surface states at the pore opening are modulated by photogenerated carriers, and the interface potential field changes significantly. This causes the negatively charged PFOA to experience a stronger electrostatic repulsion or attraction effect in the pore opening region (depending on the modulation direction). The interface potential field under illumination increases the entry barrier for PFOA through the pore, significantly increasing the event amplitude to 100-140 pA and the duration to 0.1-0.3 ms, thereby improving the event signal-to-noise ratio by approximately 2 times. The illumination-off cycling experiment verifies that this modulation is reversible and reliable, and there is no long-term drift.

[0083] (3) To verify the applicability of environmental samples, PFOA standard was added to simulated surface water samples (containing Ca).2+ Mg 2+ Interfering components such as natural organic matter were included. The results showed that event categories were difficult to separate under no light conditions, while the event distribution of PFOA under light conditions was significantly shifted to the high amplitude region, forming clusters that were clearly distinguishable from background noise. This indicates that the method provided by the present invention can detect trace amounts of PFAS (perfluorinated and polyfluoroalkyl compounds) in complex environmental samples and is suitable for water monitoring and environmental law enforcement analysis.

[0084] Example 5 verifies the applicability of the method of the present invention in the detection of environmental pollutants. Using PFOA, a typical perfluorocarboxylic acid pollutant, as the target molecule, the GaN photo-enhancing mechanism is used to improve its event resolvability in solid nanopores, demonstrating the broad application prospects of the present invention in water quality detection and environmental monitoring.

[0085] Example 6

[0086] GaN-based photo-enhanced single-molecule detection of AFP biomarkers:

[0087] (1) Solid nanopores with a diameter of about 5 nm and a film thickness of 20 nm were prepared on Si3N4 film. The method of preparing the pores was the same as in Example 1. The nanopore opening and the pore wall region were subjected to surface activation treatment to introduce hydroxyl groups. Then, a stable aminated anchoring layer was formed on the pore wall surface by chemical modification with aminosilane 3-aminopropyltriethoxysilane, so that the surface could be covalently linked with the aptamer through NHS-EDC reaction. A DNA aptamer with high affinity for AFP (5′-AGC AGC ACAGAG GTC AGA TGA GGT TGG-3′) was used, and its dissociation constant was about 10-20 nM. After the aptamer modification was completed, the chip was assembled into a PDMS-glass microfluidic structure and a detection buffer of 1 mol / L KCl aqueous solution + 10 mmol / L Tris and pH 8.0 was added.

[0088] (2) When a transmembrane bias of 180 mV is applied under no-light conditions, the binding of AFP molecules at the aptamer-aperture interface will generate a typical blocking event with an amplitude of approximately 120-160 pA and a duration of 0.5-0.9 ms, and the event distribution density is low. Subsequently, the integrated and positioned GaN micro-LED is turned on to illuminate the aperture region at a wavelength of 365 nm, and the optical power density is maintained at 5-8 mW / cm². 2 Photogenerated carrier injection alters the surface state filling number near the aperture, enhancing the local interfacial potential and increasing the aptamer prestress. This changes the binding potential energy required for AFP molecules to enter the aperture, significantly increasing the amplitude of the blockage event. The results are shown in [Figure number missing]. Figure 10The results showed that the event amplitude increased by an average of 30-45%, with some events exceeding 60%, and the duration was shortened to 0.3-0.6ms, further enhancing the distinguishability of the events. The illumination-off cycle verification showed that the event parameters could be reversibly transformed, were highly consistent with the light control state, and did not decay after multiple cycles, indicating that the mechanism of the present invention is stable and reliable.

[0089] (3) To simulate real samples, AFP standard was added to human serum matrix diluted 10 times and the same experimental procedure was repeated. Due to the complex background of serum matrix, the event distribution overlapped under no light conditions; however, the amplitude and duration of AFP events shifted significantly under light conditions, forming separable two-dimensional feature clusters, thus achieving accurate identification.

[0090] Example 6 demonstrates the application of the method of the present invention in a biomedical detection scenario. Using serum-derived alpha-fetoprotein (AFP) as the detection target, highly sensitive single-molecule recognition is achieved through aptamer-modified solid nanopores combined with GaN photo-driven interface regulation mechanism. The results show that the method provided by the present invention can improve detection accuracy in complex biological backgrounds, has clinical translational potential, and has significant advantages in clinical biomarker detection. It can be used for early disease screening and trace analysis.

[0091] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation, characterized in that, Includes the following steps: (1) Solid nanopores with a diameter of 1-10 nm are prepared on insulating or semi-insulating films with tunable surface states to obtain nanopore films; (2) Integrating a GaN light source in the vicinity of the solid nanopore; (3) Under transfilm bias, turn the GaN light source on or off, and record the current changes in the illuminated and unilluminated states. (4) Identify the single-molecule blocking event of the target molecule based on the difference in current between the illuminated and unilluminated states.

2. The method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation according to claim 1, characterized in that, In step (1), the insulating or semi-insulating film is a multilayer two-dimensional material; the insulating or semi-insulating film is selected from one or more of Si3N4, Al2O3, SiO2, h-BN, MoS2, WS2 and graphene film.

3. The method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation according to claim 1, characterized in that, In step (1), the method for preparing the solid nanopore is selected from electron beam etching, helium ion beam etching, focused ion beam etching or atomic layer etching.

4. The method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation according to claim 1, characterized in that, In step (2), the emission wavelength of the GaN light source is 250-450nm; the distance between the GaN light source and the solid nanopore is 100nm-5μm; and the working mode of the GaN light source is continuous or pulsed.

5. The method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation according to claim 1, characterized in that, In step (4), the amplitude of the monomolecular blocking event under illumination is ≥20% greater than that under non-illumination conditions.

6. The method for detecting single molecules in solid-state nanopores based on GaN light-driven modulation according to claim 1, characterized in that, Step (1) also includes the step of modifying the surface of the solid nanopore with an aptamer.

7. A solid-state nanoporous single-molecule detection device based on GaN light-driven modulation, characterized in that, It includes a solid-state nanopore chip, a GaN light source, a microfluidic cavity, a light source driving circuit, and a current acquisition module.

8. The solid-state nanopore single-molecule detection device based on GaN light-driven modulation according to claim 7, characterized in that, The GaN light source and the solid-state nanopore chip are integrated into an m×n nanopore array.

9. The solid-state nanopore single-molecule detection device based on GaN light-driven modulation according to claim 7, characterized in that, The current acquisition module is a transimpedance amplifier array; the current acquisition module is connected to an external data analysis unit.

10. The application of the method according to any one of claims 1-6 or the device according to any one of claims 7-9 in the single-molecule detection of biomarkers, metal coordination complexes or small molecule environmental pollutants.

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