A nanocavity sensor for efficient excitation of fluorescence and methods of use thereof

Abstract

The application discloses a kind of nanocavity sensors for high-efficiency excitation fluorescence, including by lower to upper quartz substrate layer, microlens layer and metal film layer in turn;Microlens layer is located above quartz substrate layer, including several upward convex ball table type microlenses;Metal film layer is located above quartz substrate layer and microlens layer, and is provided with through nanometer hole corresponding to the center of ball table type microlens top plane one by one.When excitation light is incident, ball table type microlens can focus the light beam emitted in the lens cavity by bottom in nanometer hole bottom, form zero mode waveguide effect, and excite fluorescent substance in hole by evanescent field.Ball table type microlens carries out direction modulation to excitation light beam, and light beam is converted from parallel incidence to local enhanced excitation, realizes the modulation of excitation electromagnetic field distribution to nanometer hole bottom and nanocavity inside under solution environment, and uses focal line to describe electromagnetic field distribution.The device significantly improves the excitation efficiency of optical system to fluorescence.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, specifically to a nanocavity sensor for efficiently exciting fluorescence and its usage method. Background Technology

[0002] Fluorescence-based nanopore detection technology is an ultrasensitive method with the advantage of high spatiotemporal resolution. This method relies on the zero-mode waveguide (ZMW) effect to achieve localized optical field confinement through spatial isolation and background suppression, thereby enabling single-molecule-level detection. It has become an effective tool for exploring the mechanisms of reactions between biomolecules.

[0003] Zero-mode waveguides are a nanoscale detection technique that breaks the optical diffraction limit. By confining the propagation of the light field through subwavelength nanopores, they allow only evanescent waves to excite fluorescent molecules within a 30nm region at the bottom of the pore, compressing the observation volume to the order of 10⁻²¹ L, thus enabling single-molecule detection in high-concentration environments. Their cutoff wavelength characteristics (λ>1.7d) ensure that the light field cannot penetrate the pore, effectively suppressing background noise.

[0004] Traditional fluorescence-excited sensors typically involve depositing a 50nm-200nm thick metal film on a fused silica slide, followed by micro / nano fabrication processes to create nanopores (e.g., 50nm-250nm in diameter and depth greater than or equal to the metal film thickness) on the metal film surface. Figure 1 (As shown). However, for traditional zero-mode waveguide devices, since the unmodulated excitation light is incident parallel to the surface, it cannot be focused onto the nanopore test point. The number of excitation light photons that can effectively enter the nanopore is extremely low, resulting in low system excitation efficiency. Summary of the Invention

[0005] Therefore, this invention aims to solve the problems of excitation light not being able to be focused onto the nanopore test point and low system excitation efficiency in existing zero-mode waveguide devices, thereby providing a nanocavity sensor for efficient fluorescence excitation and its usage method.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] A nanocavity sensor for efficient fluorescence excitation includes:

[0008] Quartz base layer;

[0009] A microlens layer is disposed above the quartz substrate layer and includes a plurality of frustum-shaped microlenses arranged in an array and protruding in the direction away from the quartz substrate layer. The top of the protrusion of the frustum-shaped microlens is a circular top plane suitable for filling the solution to be tested. The bottom of the frustum-shaped microlens near the quartz substrate layer is a circular bottom plane. The diameter of the circular top plane is smaller than the diameter of the circular bottom plane.

[0010] A metal thin film layer is disposed above the quartz substrate layer and the microlens layer, and a coating layer is formed on the surface of the microlens layer and the quartz substrate layer. The metal thin film layer has a plurality of through-holes, and the plurality of nanopores are arranged one-to-one with the center of the circular top plane of the plurality of frustum-shaped microlenses.

[0011] Furthermore, the metal thin film layer covers the outer surface of the frustum-shaped microlens and forms a frustum-shaped focusing structure inside the metal thin film layer.

[0012] Furthermore, the microlens layer is integrally formed on top of the quartz substrate layer; the quartz substrate layer is made of fused silica, and the quartz substrate layer and the microlens layer are made of the same material.

[0013] Furthermore, the pore size of the nanopore is 50nm-250nm.

[0014] Furthermore, the thickness of the metal thin film layer is 50nm-200nm.

[0015] Furthermore, the thickness of the quartz substrate layer is 150μm-300μm.

[0016] A method of using the nanocavity sensor for efficient fluorescence excitation as described in any of the above claims includes the following steps:

[0017] S1: A parallel beam of light is emitted using a light source module, which penetrates from the quartz substrate into the frustum-shaped microlens. The parallel beam of light inside the frustum-shaped microlens converges into the nanopore within the frustum-shaped focusing structure inside the metal thin film.

[0018] S2: Using the causs line generation module, input the geometric parameters and material properties of the nanocavity sensor and the parameters of the parallel beam emitted by the light source module to generate causs lines corresponding to the propagation of the beam within the nanocavity.

[0019] S3: Using the caustic analysis module, the electromagnetic field distribution inside the nanocavity is analyzed based on the parameters generated by the caustic generation module.

[0020] Furthermore, in step S2, the incident angle of the parallel beam emitted by the light source module can be adjusted according to the caustic lines generated by the caustic line generation module, so that the beam within the spherical focusing structure can be precisely focused on the nanopore.

[0021] The technical solution of this invention has the following advantages:

[0022] 1. The nanocavity sensor for efficient fluorescence excitation provided by this invention achieves efficient focusing of the excitation beam by setting an upwardly convex frustum-shaped microlens in the microlens layer to efficiently modulate the excitation light field, thereby realizing efficient fluorescence excitation. Specifically, when the excitation light is incident, the frustum-shaped microlens can focus the beam emitted from the bottom into the lens cavity to the bottom of the nanopore based on the spherical curved surface structure, forming a zero-mode waveguide effect, and then excite the fluorescent material in the hole through the evanescent field. This structure modulates the direction of the excitation beam through the frustum-shaped microlens, changing the beam from parallel incident to localized enhanced excitation, realizing the modulation of the excitation photoelectric magnetic field distribution at the bottom of the nanopore and inside the nanocavity in a solution environment. This device retains the subwavelength optical field localization characteristics of zero-mode waveguide devices and improves the excitation efficiency of the optical path system for fluorescence.

[0023] 2. The nanocavity sensor for efficient fluorescence excitation provided by this invention features a planar top structure for the frustum-shaped microlens. This design avoids the destructive interference of the hemispherical and spherical cap structures' curved top surfaces with the principal optical axis excitation light, while also providing attachment points for sample molecules, thus improving detection convenience.

[0024] 3. The nanocavity sensor for efficient fluorescence excitation provided by this invention integrates the quartz substrate layer and the microlens layer into a single unit. This design avoids noise interference caused by heterogeneous structures and ensures detection sensitivity. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 A cross-sectional view of a conventional fluorescence excitation sensor;

[0027] Figure 2 A cross-sectional view of the nanocavity sensor for efficient fluorescence excitation provided by the present invention;

[0028] Figure 3Schematic diagram of the high-efficiency fluorescence excitation principle of this invention;

[0029] Figure 4 A comparison of the light field intensity distribution between the spherical microlens of this invention and the traditional hemispherical microlens structure;

[0030] Figure 5 A comparison diagram of aperture-field intensity points between the spherical microlens of this invention and the traditional hemispherical microlens;

[0031] Figure 6 Light field intensity distribution diagram of nanocavities with different aperture sizes in this invention;

[0032] Figure 7 A dotted line diagram of the aperture-field intensity of the nanocavity structure of this invention;

[0033] Figure 8 Light field intensity distribution diagrams at different excitation light incident angles of this invention;

[0034] Figure 9 Incident angle-field intensity dot plot of this invention;

[0035] Figure 10 Light field intensity distribution diagrams for different excitation wavelengths;

[0036] Figure 11 The excitation light wavelength-field intensity dotted line diagram of this invention;

[0037] Figure 12 Schematic diagram of the caustic lines and effective excitation region angle of the present invention;

[0038] Explanation of reference numerals in the attached figures: 1. Quartz substrate layer; 2. Metal thin film layer; 3. Microlens layer; 4. Nanopore; 5. Solution to be tested. Detailed Implementation

[0039] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0040] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0041] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0042] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] like Figures 2-10 The nanocavity sensor shown includes a quartz substrate 1, a microlens layer 3, and a metal thin film layer 2 for efficient fluorescence excitation. Specifically, the quartz substrate 1 is made of fused silica. The microlens layer 3 is disposed above the quartz substrate 1 and includes several arrayed and upwardly convex frustum-shaped microlenses, which are made of the same material as the quartz substrate 1. The metal thin film layer 2 covers the surface of the microlens layer 3. The top of the frustum-shaped microlenses has a plane, and a nanopore 4 with a diameter of 50nm-250nm and penetrating the metal thin film layer 2 is opened at the center of the plane. Several nanopores 4 correspond one-to-one with several microlenses.

[0044] This nanocavity sensor for efficient fluorescence excitation utilizes an upwardly convex frustum-shaped microlens in the microlens layer 3. When excitation light is incident, the frustum-shaped microlens, based on the curved surface of the frustum structure, focuses the light beam emitted from the bottom of the quartz substrate layer 1 into the lens cavity at the bottom of the nanopore 4, forming a zero-mode waveguide effect. This, in turn, excites the fluorescent material within the hole through an evanescent field. This structure modulates the direction of the excitation beam through the frustum-shaped microlens. The diameter of the circular top plane is smaller than the diameter of the circular bottom plane, achieving a focusing effect and transforming the beam from parallel incidence to localized enhanced excitation. This enables modulation of the photoelectric magnetic field distribution at the bottom of the nanopore 4 and inside the nanocavity in a solution environment. This device retains the subwavelength optical field localization characteristics of zero-mode waveguide devices while improving the excitation efficiency of the optical path system for fluorescence. This frustum-shaped microlens nanocavity sensor, used for efficient modulation of the excitation light field, effective focusing of the excitation beam, and thus efficient fluorescence excitation, can also be used for high-sensitivity detection of single molecules / weak fluorescence.

[0045] In this embodiment, the quartz substrate 1 is fused quartz, and the side of the metal thin film layer 2 away from the quartz substrate 1 and the microlens layer 3 is suitable for placing the test solution 5. The test solution 5 fills the area above the metal thin film layer 2 and inside the nanopore 4.

[0046] In this embodiment, the frustum-shaped microlens structure combines the features of a spherical surface and a planar surface. Specifically, the sides of the frustum are spherical band structures, while the bottom and top are circular planar structures. The spherical band structure has a certain radius of curvature; the top planar structure is a circular cross-section of a certain diameter (circular top plane), and it maintains a concentric mapping with the bottom planar structure (circular bottom plane). The spherical band sidewall structure formed by the middle portion of the circular top and bottom planes functions to converge the light beam. This design, with its circular top plane, avoids the destructive interference of the hemispherical and spherical cap structures' top curved surfaces with the principal optical axis excitation light, while also providing attachment points for sample molecules, improving detection convenience.

[0047] In this embodiment, by adjusting any one of the parameters of the spherical curvature, the radius of the upper and lower bases, and the spherical height of the spherical microlens, the distribution of the excitation light field of the spherical microlens can be effectively modulated. By designing the above parameters, the effective modulation of the excitation light field distribution can be achieved, satisfying the application of this sensor as a universal device.

[0048] In this embodiment, the quartz substrate 1 and the microlens layer 3 are integrally integrated. This arrangement avoids noise interference caused by heterogeneous structures and ensures detection sensitivity. Specifically, a metal thin film layer 2 is deposited and coated on the surfaces of the quartz substrate 1 and the microlens layer 3, and the nanopores 4 of the metal thin film layer 2 are connected to the corresponding frustum-shaped microlens nanocavities.

[0049] In this embodiment, the excitation efficiency of the excitation light field can be quantified by the numerical distribution of the light field intensity, and the focusing effect of the frustum-shaped microlens nanocavity on the excitation light and the electromagnetic field distribution inside the nanocavity and at the bottom of the aperture can be calculated using the finite element analysis method. Figure 4 and Figure 5 As shown in the figure, the superior structural characteristics of the frustum-shaped microlens in terms of optical field manipulation compared to existing traditional hemispherical microlenses are evident, and the superior excitation efficiency of the frustum-shaped microlens nanocavity compared to existing traditional hemispherical microlenses is also visible. An orthogonal simulation model was established based on the concept of controlled variables, such as... Figure 6 and Figure 7 As shown, the light field intensity distributions for both types of light sources were calculated under different aperture conditions; Figure 8 and Figure 9 As shown, the light field intensity distributions for both types of light were calculated under different excitation light incident angles; Figure 10 and Figure 11 As shown, the light field intensity distribution of both under different excitation wavelengths was calculated.

[0050] The simulation results of the above-described traditional microlenses and frustum-type microlens nanocavities show that the frustum-type microlens nanocavity in this application does not affect the local characteristics of the subwavelength optical field of the microlens and significantly improves the excitation efficiency. In this embodiment, the aperture of the nanopore 4 is 50nm-250nm. The thickness of the metal thin film layer 2 is 50nm-200nm, and the thickness of the metal thin film layer 2 remains consistent throughout its coating on the surfaces of the quartz substrate layer 1 and the microlens layer 3. The thickness of the quartz substrate layer 1 is 150um-300μm. Preferably, the aperture of the nanopore 4 is 150nm.

[0051] like Figure 10 , Figure 11 As shown, when the aperture of the nanopore 4 at the top of the convex spherical microlens is 150 nm, the light field intensity reaches its highest value. The corresponding optimal external excitation conditions are perpendicular incident excitation light (θ = 0°) and excitation wavelength λ = 532 nm. Under these conditions, compared to... Figure 1 Compared to conventional fluorescence excitation sensors, the excitation light field intensity at the bottom of the frustum-shaped microlens nanopore 4 in this application is increased by 201.71%, confirming that the frustum-shaped microlens nanocavity in this application has the ability to promote efficient fluorescence excitation. Figure 12 As shown, the caustic lines in the nanocavity of the ball-shaped microlens in this application are distributed in an "apple line" pattern, and the effective excitation region angle is -30° to 30°.

[0052] When using the nanocavity sensor of this application for efficient fluorescence excitation for single-molecule detection, the sample to be tested (the fluorescent sample solution to be detected) is added to the surface of the sensor chip, causing fluorescent single molecules to fill the nanopore 4 of the frustum-shaped microlens, and then observed using a fluorescence microscope. A laser with a wavelength matched to the sample wavelength is mounted externally to the fluorescence microscope to excite the fluorescent molecules. Finally, an EMCCD camera is used to capture, record, and quantify the fluorescence signal, achieving single-molecule fluorescence detection.

[0053] A method of using the above-mentioned nanocavity sensor for efficient fluorescence excitation includes the following steps: S1: A parallel beam of light is emitted using a light source module and passes through a quartz substrate 1 into a frustum-shaped microlens. The parallel beam of light in the frustum-shaped microlens is focused into a nanopore 4 within a frustum-shaped focusing structure inside a metal thin film.

[0054] S2: Using the causs line generation module, input the geometric parameters, material properties of the nanocavity sensor and the parameters of the parallel beam emitted by the light source module to generate causs lines corresponding to the propagation of the beam within the nanocavity.

[0055] S3: Using the caustic analysis module, the electromagnetic field distribution inside the nanocavity is analyzed based on the parameters generated by the caustic generation module.

[0056] This method of using a nanocavity sensor for efficient fluorescence excitation allows for the rapid analysis and description of the electromagnetic field distribution at the bottom of the nanopore 4 and inside the microlens nanocavity, based on the caustic generation and analysis modules. Conversely, the structural parameters for fabricating the frustum-shaped microlens can also be adjusted using the caustic data.

[0057] Specifically, in step S2, the incident angle of the parallel beam emitted by the light source module can be adjusted according to the caustic lines generated by the caustic line generation module, so that the beam inside the spherical focusing structure can be precisely focused on the nanopore 4.

[0058] Specifically, the beam of light eventually converges into the test solution 5 filled in the aperture. The test solution 5 contains fluorescent substances. After the beam of light converges into the test solution 5, it will excite the fluorescent substances in the test solution 5, and the beam of light will form an evanescent field in the aperture.

[0059] In summary, this nanocavity sensor for efficient fluorescence excitation utilizes an upwardly convex frustum-shaped microlens in the microlens layer 3. When excitation light is incident, the frustum-shaped microlens, based on its spherical curved surface structure, focuses the beam emitted from the bottom of the lens cavity onto the bottom of the nanopore 4, forming a zero-mode waveguide effect. This, in turn, excites the fluorescent material within the aperture through an evanescent field. This structure modulates the direction of the excitation beam through the frustum-shaped microlens, transforming the beam from parallel incidence to localized enhanced excitation. This achieves modulation of the photoelectric magnetic field distribution at the bottom of the nanopore 4 and inside the nanocavity in a solution environment, and the electromagnetic field distribution is described using caustics. This device retains the subwavelength optical field localization characteristics of zero-mode waveguide devices while improving the excitation efficiency of the optical path system for fluorescence.

[0060] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A nanocavity sensor for efficient fluorescence excitation, characterized in that, include: Quartz base layer (1); The microlens layer (3) is disposed above the quartz substrate layer (1) and includes a plurality of arrayed, protruding spherical microlenses facing away from the quartz substrate layer (1). The top of the spherical microlens is a circular top plane suitable for filling the solution to be tested (5). The bottom of the spherical microlens near the quartz substrate layer (1) is a circular bottom plane. The diameter of the circular top plane is smaller than the diameter of the circular bottom plane. The spherical microlens has a spherical curved sidewall. A metal thin film layer (2) is disposed above the quartz substrate layer (1) and the microlens layer (3), and forms a coating layer on the surface of the microlens layer (3) and the quartz substrate layer (1). The metal thin film layer (2) has a plurality of through-holes (4), and the plurality of nanoholes (4) are arranged one-to-one with the center of the circular top plane of the plurality of frustum-shaped microlenses. The metal thin film layer (2) covers the outer surface of the frustum-shaped microlens and forms a frustum-shaped focusing structure inside the metal thin film layer (2).

2. The nanocavity sensor for efficient fluorescence excitation according to claim 1, characterized in that, The microlens layer (3) is integrally formed on top of the quartz substrate layer (1); the quartz substrate layer (1) is made of fused silica, and the microlens layer (3) is made of the same material as the quartz substrate layer (1).

3. The nanocavity sensor for efficient fluorescence excitation according to claim 1, characterized in that, The pore size of the nanopore (4) is 50nm-250nm.

4. The nanocavity sensor for efficient fluorescence excitation according to claim 1, characterized in that, The thickness of the metal thin film layer (2) is 50nm-200nm.

5. The nanocavity sensor for efficient fluorescence excitation according to claim 1, characterized in that, The thickness of the quartz base layer (1) is 150μm-300μm.

6. A method of using the nanocavity sensor for efficient fluorescence excitation as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: A parallel beam of light is emitted using the light source module and passes through the quartz substrate (1) into the frustum-shaped microlens. The parallel beam of light in the frustum-shaped microlens converges into the nanopore (4) within the frustum-shaped focusing structure inside the metal thin film. S2: Using the causs line generation module, input the geometric parameters and material properties of the nanocavity sensor and the parameters of the parallel beam emitted by the light source module to generate causs lines corresponding to the propagation of the beam within the nanocavity. S3: Using the caustic analysis module, the electromagnetic field distribution inside the nanocavity is analyzed based on the parameters generated by the caustic generation module.

7. The method of using the nanocavity sensor for efficient fluorescence excitation according to claim 6, characterized in that, In step S2, the incident angle of the parallel beam emitted by the light source module is adjusted according to the caustic line generated by the caustic line generation module, and the beam in the spherical focusing structure is adjusted to be precisely focused on the nanopore (4).

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