Nano-cavity sensor for efficiently exciting fluorescence and use method of nano-cavity sensor

Abstract

The invention discloses a nano-cavity sensor for efficiently exciting fluorescence. The nano-cavity sensor comprises a quartz substrate layer, a micro-lens layer and a metal film layer which are sequentially arranged from bottom to top, the micro-lens layer is located above the quartz substrate layer and comprises a plurality of spherical-table-shaped micro-lenses protruding upwards; and the metal film layer is positioned on the quartz substrate layer and the micro lens layer, and is provided with through nano holes which are in one-to-one correspondence with the centers of the top planes of the table-shaped micro lenses. When exciting light enters, the spherical table type micro lens can focus a light beam emitted into the lens cavity from the bottom to the bottom of the nanopore to form a zero-mode waveguide effect, and fluorescent substances in the nanopore are excited through an evanescent field. The spherical table type micro lens modulates the direction of an excitation light beam, the light beam is converted into local enhanced excitation from parallel incidence, modulation of excitation photoelectric magnetic field distribution at the bottom of a nanopore and in a nanocavity in a solution environment is achieved, and distribution of an electromagnetic field is described by using a caustics line. According to the device, the excitation efficiency of a light path system on fluorescence is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence detection technology, and in particular to a nano-cavity sensor for efficient excitation of fluorescence and a method of using the same. BACKGROUND

[0002] The fluorescence-based nanopore detection technology has the advantage of high spatial and temporal resolution as an ultra-sensitive method. This method relies on the Zero-Mode Waveguide (ZMW) effect to achieve spatial isolation and background suppression, thereby realizing the confinement of localized light field and single-molecule level detection. It has become an effective tool for exploring the reaction mechanism between biomolecules.

[0003] Zero-Mode Waveguide is a nanoscale detection technology that breaks through the optical diffraction limit. It restricts the propagation of light field through subwavelength nanopores, allowing only evanescent waves to excite fluorescent molecules in the bottom of the hole within about 30 nm, thereby compressing the observation volume to the order of 10-21 L, and realizing single-molecule detection in a high-concentration environment. Its cutoff wavelength characteristic (λ>1.7d) ensures that the light field cannot penetrate the hole, effectively suppressing background noise.

[0004] Traditional fluorescence excitation sensors usually deposit a 50-200 nm thick metal film on a fused quartz glass substrate, and then form a nanopore with a diameter of 50-250 nm and a depth of ≥metal film thickness on the surface of the metal film through micro-nano processing technology (such as shown in FIG. 1). Figure 1 However, for traditional zero-mode waveguide devices, since the unmodulated excitation light is parallel incident, it cannot be focused on the nanopore test point, and the number of excitation light photons effectively entering the nanopore is extremely low, resulting in low system excitation efficiency. SUMMARY

[0005] Therefore, the present application aims to solve the problem of low system excitation efficiency caused by the inability of excitation light to focus on the nanopore test point in the prior art zero-mode waveguide device, and to provide a nano-cavity sensor for efficient excitation of fluorescence and a method of using the same.

[0006] To solve the above technical problems, the technical solutions of the present application are as follows:

[0007] A nano-cavity sensor for efficient excitation of fluorescence, comprising:

[0008] a quartz substrate layer;

[0009] a microlens layer disposed above the quartz substrate layer, comprising a plurality of arrayed and protruding spherical-tapered microlenses facing away from the quartz substrate layer, the spherical-tapered microlenses having a circular top plane at the top of the protrusion suitable for filling the solution to be tested, and a circular bottom plane at the bottom of the protrusion close to the quartz substrate layer, the diameter of the circular top plane being smaller than that of the circular bottom plane;

[0010] a metal thin film layer disposed above the quartz substrate layer and the microlens layer, and forming a cladding layer on the surface of the microlens layer and the quartz substrate layer, the metal thin film layer having a plurality of through nano-pores, and the plurality of nano-pores corresponding to the centers of the circular top planes of the plurality of spherical-tapered microlenses.

[0011] Further, the metal thin film layer covers the outer surface of the spherical-tapered microlenses and forms a spherical-tapered focusing structure inside the metal thin film layer.

[0012] Further, the microlens layer is integrally formed above the quartz substrate layer; the quartz substrate layer is made of fused quartz, and the quartz substrate layer and the microlens layer are made of the same material.

[0013] Further, the aperture of the nano-pore is 50-250 nm.

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

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

[0016] A use method of the nano-cavity sensor for efficiently exciting fluorescence, comprising the following steps:

[0017] S1: using a light source module to emit a parallel light beam, which penetrates into the spherical-tapered microlenses from the quartz substrate layer, and the parallel light beam in the spherical-tapered microlenses converges to the nano-pore in the spherical-tapered focusing structure inside the metal thin film;

[0018] S2: using a caustic line generation module to input the geometric parameters, material properties of the nano-cavity sensor, and the parameters of the parallel light beam emitted by the light source module, and generating a caustic line corresponding to the light beam propagation in the nano-cavity;

[0019] S3: using a caustic line analysis module to analyze the electromagnetic field distribution inside the nano-cavity according to the parameters generated by the caustic line generation module.

[0020] Further, in the S2 step, the incidence angle of the parallel light beam emitted by the light source module can be adjusted according to the generated caustic line, so that the light beam in the ball table type focusing structure can be accurately converged in the nanometer hole.

[0021] The technical scheme of the present application has the following advantages:

[0022] 1. The nanocavity sensor for efficient fluorescence excitation provided by the present application can efficiently modulate the excitation light field by arranging the upwardly protruding ball table type microlens on the microlens layer, so as to realize effective convergence of the excitation light beam and efficient excitation of fluorescence. Specifically, when the excitation light is incident, the ball table type microlens can focus the light beam emitted from the bottom into the lens cavity to the bottom of the nanometer hole based on the spherical band surface structure, form a zero-mode waveguide effect, and then excite the fluorescent substance in the hole through the evanescent field. The structure modulates the excitation electromagnetic field distribution at the bottom of the nanometer hole and inside the nanocavity in the solution environment by directionally modulating the excitation light beam through the ball table type microlens, which not only retains the subwavelength light field localization characteristics of the zero-mode waveguide device, but also improves the excitation efficiency of the optical system on the fluorescence.

[0023] 2. The nanocavity sensor for efficient fluorescence excitation provided by the present application, the top of the ball table type microlens is a planar structure. In this way, the top planar design of the ball table can avoid the interference cancellation of the top curved surface of the hemisphere and the spherical cap structure on the main optical axis excitation light, and at the same time can provide an attachment point for the sample molecules, improving the detection convenience.

[0024] 3. The nanocavity sensor for efficient fluorescence excitation provided by the present application, the quartz substrate layer and the microlens layer are integrally arranged. In this way, the noise caused by the heterostructure can be avoided, and the detection sensitivity can be ensured. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present application or the technical scheme in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor.

[0026] Figure 1 It is a sectional view of the existing conventional fluorescence excitation sensor;

[0027] Figure 2 It is a sectional view of the nanocavity sensor for efficient fluorescence excitation provided by the present application;

[0028] Figure 3The schematic diagram of the high-efficiency fluorescence excitation principle of the application;

[0029] Figure 4 The comparison chart of the light field intensity distribution of the ball table type microlens and the traditional hemispherical microlens structure;

[0030] Figure 5 The aperture-field intensity point line comparison chart of the ball table type microlens and the traditional hemispherical microlens structure;

[0031] Figure 6 The light field intensity distribution chart of different aperture nanocavities;

[0032] Figure 7 The aperture-field intensity point line chart of the nanocavity structure;

[0033] Figure 8 The light field intensity distribution chart of different excitation light incident angles;

[0034] Figure 9 The incident angle-field intensity point line chart;

[0035] Figure 10 The light field intensity distribution chart of different excitation light wavelengths;

[0036] Figure 11 The excitation light wavelength-field intensity point line chart;

[0037] Figure 12 The caustic line and effective excitation area angle schematic diagram;

[0038] The figure mark explanation: 1, quartz substrate layer; 2, metal thin film layer; 3, microlens layer; 4, nanohole; 5, solution to be measured. DETAILED DESCRIPTION

[0039] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0040] In the description of the present application, it should be noted that the orientations or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0041] In the description of the present application, it should be noted that unless specifically defined and limited otherwise, the terms "mounting", "connected", "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through intermediate medium, or it can be internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0042] In addition, the technical features involved in the different embodiments of the application described below can be combined with each other as long as there is no conflict.

[0043] As Figures 2-10 A nanocavity sensor for efficient excitation of fluorescence, as shown in the drawings, comprises a quartz substrate layer 1, a microlens layer 3 and a metal film layer 2; specifically, the quartz substrate layer 1 is made of fused quartz; the microlens layer 3 is arranged above the quartz substrate layer 1 and comprises a plurality of array-arranged and upwardly-protruding ball-typed microlenses, which are made of the same material as the quartz substrate layer 1; the metal film layer 2 is coated on the surface of the microlens layer 3, the top of the ball-typed microlens is provided with a flat surface, a nanohole 4 with a diameter of 50-250 nm and penetrating through the metal film layer 2 is arranged at the center of the flat surface, and the plurality of nanoholes 4 correspond to the plurality of microlenses one by one.

[0044] This nanocavity sensor for efficient excitation of fluorescence, by arranging the upwardly-protruding ball-typed microlenses on the microlens layer 3, when the excitation light is incident, the ball-typed microlens can focus the light beam emitted in the lens cavity at the bottom of the nanohole 4 based on the curved surface of the ball-typed structure, forming a zero-mode waveguide effect, and then exciting the fluorescent substance in the hole through the evanescent field. The structure modulates the direction of the excitation light beam through the ball-typed microlens, and the diameter of the circular top flat surface is smaller than that of the circular bottom flat surface, which can achieve the effect of light condensation, so that the light beam is converted from parallel incidence to local enhanced excitation, realizing the modulation of the excitation light electromagnetic field distribution at the bottom of the nanohole 4 and inside the nanocavity in the solution environment. This device not only retains the subwavelength light field localization characteristics of the zero-mode waveguide device, but also improves the excitation efficiency of the light path system to fluorescence; this ball-typed microlens nanocavity sensor for efficient modulation of excitation light field, effective convergence of excitation light beam and efficient excitation of fluorescence can also be used for high-sensitivity detection of single molecules / weak fluorescence.

[0045] In this embodiment, the quartz substrate layer 1 is fused quartz, and the side of the metal film layer 2 away from the quartz substrate layer 1 and the microlens layer 3 is suitable for placing the to-be-tested solution 5, and the to-be-tested solution 5 is filled in the metal film layer 2 and inside the nanohole 4.

[0046] In the embodiment, the ball table type microlens structure has the characteristics of combining spherical surface and plane. Specifically, the side of the ball table is a ball band structure, the bottom and top are circular plane structures. The ball band structure has a certain curvature radius; the top plane structure is a circular cross section (circular top plane) with a certain diameter, and is concentrically mapped with the bottom plane structure (circular bottom plane), and the ball band side wall structure formed by the middle part of the circular top plane and the circular bottom plane has the function of converging light beams. In this way, the design of the circular top plane of the ball table type microlens can avoid the interference cancellation of the top curved surface of the half-sphere and spherical cap structure to the excitation light of the main optical axis, and at the same time can provide an attachment point for the sample molecules, improving the detection convenience.

[0047] In the embodiment, by adjusting any one of the parameters of the spherical curvature of the ball table type microlens, the radius of the upper and lower bottoms, and the spherical height, the distribution of the ball table type microlens excitation light field can be effectively modulated. By designing the above parameters, the excitation light field distribution can be effectively modulated to meet the application of the sensor as a universal device.

[0048] In the embodiment, the quartz substrate layer 1 and the microlens layer 3 are integrally arranged. In this way, the noise caused by the heterostructure can be avoided, and the detection sensitivity can be ensured. Specifically, the metal thin film layer 2 is deposited on the surface of the quartz substrate layer 1 and the microlens layer 3, and the nanometer hole 4 of the metal thin film layer 2 penetrates the corresponding ball table type microlens nanometer cavity.

[0049] In the embodiment, the numerical distribution of the light field intensity can be used to quantify the excitation efficiency of the excitation light field, and the finite element analysis method can be used to calculate the convergence effect of the ball table type microlens nanometer cavity on the excitation light and the electromagnetic field distribution inside the nanometer cavity and the hole bottom. Figure 4 and Figure 5 As shown in the figures, the ball table type microlens has excellent structural characteristics in light field regulation compared with the existing traditional half-sphere type microlens, and the superiority of the ball table type microlens nanometer cavity in excitation efficiency compared with the existing traditional half-sphere type microlens can be seen; based on the control variable idea, an orthogonal simulation calculation model is set, as shown in Figure 6 and Figure 7 As shown in the figures, the light field intensity distribution of the two under different aperture conditions is calculated respectively; as shown in Figure 8 and Figure 9 As shown in the figures, the light field intensity distribution of the two under different excitation light incidence angles is calculated respectively; as shown in Figure 10 and Figure 11 As shown in the figures, the light field intensity distribution of the two under different excitation wavelengths is calculated respectively.

[0050] It can be seen from the simulation of the traditional microlens and the ball table type microlens nanocavity that the ball table type microlens nanocavity in the application does not affect the localization characteristics of the microlens subwavelength light field, and greatly improves the excitation efficiency. In the embodiment, the aperture of the nanohole 4 is 50 nm-250 nm. The thickness of the metal thin film layer 2 is 50 nm-200 nm, and the thickness of the metal thin film layer 2 coated on the surface of the quartz substrate layer 1 and the microlens layer 3 is uniform. The thickness of the quartz substrate layer 1 is 150 um-300 um. Preferably, the aperture of the nanohole 4 is 150 nm.

[0051] As shown in Figure 10 , Figure 11 , when the aperture of the convex ball table type microlens top nanohole 4 is 150 nm, the light field intensity can reach the highest value, and at this time the corresponding external optimal excitation condition is that the excitation light is vertically incident (θ=0°) and the excitation wavelength λ=532 nm. Under this condition, compared with the existing traditional fluorescence excitation sensor as shown in Figure 1 , the excitation light field intensity at the bottom of the ball table type microlens nanohole 4 in the application is increased by 201.71%, which proves that the ball table type microlens nanocavity in the application has the ability to promote high-efficiency fluorescence excitation. As shown in Figure 12 , the focal line in the nanocavity of the ball table type microlens in the application is in the form of an "apple line", and the effective excitation area angle is -30° to 30°.

[0052] When using the nanocavity sensor for high-efficiency fluorescence excitation in the application for single molecule detection, the sample to be detected (fluorescent sample solution to be detected) is added to the surface of the sensor chip, so that the fluorescent single molecule fills into the ball table type microlens nanohole 4, and a fluorescence microscope is used for observation. The fluorescence microscope is externally equipped with a laser matched with the wavelength of the sample to be detected to excite the fluorescent molecules. Finally, an EMCCD camera is used to record and quantify the fluorescence signal, realizing single molecule fluorescence detection.

[0053] A use method for the nanocavity sensor for high-efficiency fluorescence excitation described above, comprising the following steps: S1: using a light source module to emit a parallel light beam, which enters the ball table type microlens from the quartz substrate layer 1, and the parallel light beam in the ball table type microlens converges to the nanohole 4 in the ball table type focusing structure inside the metal thin film;

[0054] S2: using a focal line generation module, inputting the geometric parameters, material properties of the nanocavity sensor and the parameters of the parallel light beam emitted by the light source module, and generating a focal line corresponding to the light beam propagation in the nanocavity;

[0055] S3: through a focal line analysis module, analyzing the electromagnetic field distribution inside the nanocavity according to the parameters generated by the focal line generation module.

[0056] The method for using the nanocavity sensor for efficiently exciting fluorescence can quickly analyze the electromagnetic field distribution at the bottom of the nanopore 4 and inside the microlens nanocavity according to the caustic line generation module and the caustic line analysis module. Conversely, the structure parameters of the ball table type microlens can also be adjusted through the caustic line data.

[0057] Specifically, in the S2 step, the incident angle of the parallel light beam emitted by the light source module can be adjusted according to the caustic line generated by the caustic line generation module, and the light beam can be accurately converged in the nanopore 4 through the focusing structure of the ball table type.

[0058] Specifically, the light beam is finally converged in the to-be-measured solution 5 filled in the hole, the to-be-measured solution 5 contains fluorescent substances, and after the light beam is converged in the to-be-measured solution 5, the fluorescent substances in the to-be-measured solution 5 are excited, and the light beam forms an evanescent field in the hole.

[0059] In summary, the nanocavity sensor for efficiently exciting fluorescence is provided with the upwardly protruding ball table type microlens in the microlens layer 3, when the excitation light is incident, the ball table type microlens can focus the light beam emitted from the bottom into the lens cavity to the bottom of the nanopore 4 based on the spherical band surface structure, form a zero-mode waveguide effect, and then excite the fluorescent substances in the hole through the evanescent field. The structure modulates the direction of the excitation light beam through the ball table type microlens, converts the parallel incidence of the light beam into local enhanced excitation, realizes the modulation of the electromagnetic field distribution at the bottom of the nanopore 4 and inside the nanocavity in the solution environment, and uses the caustic line to describe the electromagnetic field distribution. The device not only retains the subwavelength light field localization characteristics of the zero-mode waveguide device, but also improves the excitation efficiency of the optical system on the fluorescence.

[0060] Obviously, the above embodiments are only examples for clearly illustrating, but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and cannot be exhausted. The changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A nanocavity sensor for efficient excitation of fluorescence, characterized in that, The application relates to a nano-cavity sensor, which comprises the following parts: a quartz substrate layer (1); a microlens layer (3) arranged above the quartz substrate layer (1) and comprising a plurality of array-arranged spherical-tapered microlenses protruding away from the quartz substrate layer (1), wherein the top of each spherical-tapered microlens is a circular top plane suitable for filling a solution (5) to be detected, the bottom of each spherical-tapered microlens close to the quartz substrate layer (1) is a circular bottom plane, and the diameter of the circular top plane is smaller than that of the circular bottom plane; a metal film layer (2) arranged above the quartz substrate layer (1) and the microlens layer (3) and forming a coating layer on the surface of the microlens layer (3) and the quartz substrate layer (1), wherein the metal film layer (2) is provided with a plurality of nano-pores (4) penetrating through the metal film layer (2), and the plurality of nano-pores (4) are arranged in one-to-one correspondence with the centers of the circular top planes of the plurality of spherical-tapered microlenses.

2. The nanocavity sensor for efficient excitation of fluorescence according to claim 1, wherein, The metal film layer (2) covers the outer surface of the spherical-tapered microlenses and forms a spherical-tapered focusing structure inside the metal film layer (2).

3. The nanocavity sensor for efficient excitation of fluorescence according to claim 1, wherein, The microlens layer (3) is integrally formed above the quartz substrate layer (1); the quartz substrate layer (1) is made of fused quartz, and the microlens layer (3) is made of the same material as the quartz substrate layer (1).

4. The nanocavity sensor for efficient excitation of fluorescence according to claim 1, wherein, The aperture of the nano-pore (4) is 50-250 nm.

5. The nanocavity sensor for efficient excitation of fluorescence according to claim 1, wherein, The thickness of the metal film layer (2) is 50-200 nm.

6. The nanocavity sensor for efficient excitation of fluorescence according to claim 1, wherein, The thickness of the quartz substrate layer (1) is 150-300 microns.

7. A method of use of the nanocavity sensor for efficient excitation of fluorescence according to any of the preceding claims 1-6, characterized in that, The application further discloses a nano-cavity sensor detection method, which comprises the following steps: S1: using a light source module to emit parallel light beams, which penetrate into the spherical-tapered microlenses from the quartz substrate layer (1), and the parallel light beams in the spherical-tapered microlenses converge to the nano-pores (4) in the spherical-tapered focusing structure inside the metal film; S2: using a caustic line generation module to input the geometric parameters and material properties of the nano-cavity sensor and the parameters of the parallel light beams emitted by the light source module, and generating caustic lines corresponding to the light beam propagation in the nano-cavity; S3: using a caustic line analysis module to analyze the electromagnetic field distribution inside the nano-cavity according to the parameters generated by the caustic line generation module.

8. The method of using a nanocavity sensor for efficient excitation of fluorescence according to claim 7, wherein, In the S2 step, the incident angle of the parallel light beams emitted by the light source module can be adjusted according to the caustic lines generated by the caustic line generation module, and the light beams can be accurately converged in the spherical-tapered focusing structure to the nano-pores (4).

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