Fully integrated molecular fluorescence detection platform
By designing a fully integrated molecular fluorescence detection platform containing fluorescent excitation, collection and detection layers in a biochip, the problems of complex processes and high cost are solved, and low-cost and efficient fluorescence signal detection is achieved.
Patent Information
- Application Number
- CN202510548293.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-22
AI Technical Summary
The existing biochips have complex processes and high costs, making it difficult to achieve low-cost fully integrated molecular fluorescence detection.
A fully integrated molecular fluorescence detection platform is designed, including periodically repeated subunits. The subunits are composed of a fluorescent excitation layer, a fluorescent collection layer and a fluorescent detection layer. The fluorescent collection layer includes a filter structure and a fluorescent crosstalk suppression structure. The crosstalk suppression structure includes a stop structure, an isolation structure and a distributed Bragg reflector layer, which are arranged at a specific position of the filter structure to suppress fluorescent crosstalk.
The process flow is simplified, the cost is reduced, and efficient fluorescence signal detection is achieved.
Smart Images
Figure CN120352399A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochips and relates to a fully integrated molecular fluorescence detection platform. Background Art
[0002] Biochip technology is a comprehensive high-tech technology that involves biology, chemistry, medicine, precision machining, optics, microelectronics, informatics and other fields. It is a hot topic with strong interdisciplinary nature. In recent years, the excitation and collection of biological fluorescence signals through optical integrated chips have gradually shown important value and potential in the field of fluorescent molecule detection, especially in the field of single-molecule fluorescence detection.
[0003] This technology uses semiconductor processes to achieve a monolithic integrated solution from fluorescence signal excitation, fluorescence signal collection, to fluorescence signal detection. By generating nanoscale sample restriction sites on a large scale on the chip surface and combining biochemical modification methods, spatial restriction at the single-molecule scale is achieved. In addition, compared to the traditional optical system method of exciting and collecting fluorescence signals, this technology uses an evanescent field to excite fluorescence through an optical waveguide, achieving simultaneous excitation of large-flux sites, and using the on-chip collection structure to make the sample restriction sites correspond to the detection pixels, achieving simultaneous collection of fluorescence signals at large-flux sites, and ultimately achieving fluorescence detection at large-flux sites.
[0004] Current biochips have problems with complex processes and high costs, which poses great challenges to chip processing. How to provide a fully integrated molecular fluorescence detection platform with low process difficulty and cost has become an important technical problem that technical personnel in this field need to solve urgently.
[0005] It should be noted that the above introduction to the technical background is only for the convenience of providing a clear and complete description of the technical solutions of the present application and for the convenience of understanding by those skilled in the art. It cannot be considered that the above technical solutions are well known to those skilled in the art simply because they are described in the background technology section of the present application. Summary of the invention
[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a fully integrated molecular fluorescence detection platform to solve the problems of high process difficulty and high cost of biochips in the prior art.
[0007] To achieve the above-mentioned and other related purposes, the present invention provides a fully integrated molecular fluorescence detection platform, comprising a plurality of periodically repeated subunits, wherein the subunits include a fluorescence excitation layer, a fluorescence collection layer and a fluorescence detection layer arranged in sequence from top to bottom, and the fluorescence collection layer includes:
[0008] A filtering structure for blocking laser light and allowing signals in the fluorescence band to pass through;
[0009] A fluorescence crosstalk suppression structure, including any one or combination of a diaphragm structure, an isolation structure, and a distributed Bragg reflector layer. The fluorescence crosstalk suppression structure is disposed at at least one of the following positions: above the filtering structure, below the filtering structure, on both sides of the filtering structure.
[0010] Optionally, the fluorescence collection layer further includes a microlens structure. The microlens structure adopts a multi-level Fresnel lens structure or a metasurface lens structure. The fluorescence crosstalk suppression structure is disposed at at least one of the following positions: above the filtering structure, below the filtering structure, on both sides of the filtering structure, on both sides of the microlens structure.
[0011] Optionally, the fluorescence crosstalk suppression structure includes a diaphragm structure disposed above and / or below the filtering structure. The diaphragm structure includes a light-shielding layer and a light-passing hole formed in the light-shielding layer. The size range of the light-passing hole of the diaphragm structure is 200 nanometers to 10 micrometers.
[0012] Optionally, the fluorescence crosstalk suppression structure includes a first diaphragm structure, a second diaphragm structure, and a third diaphragm structure arranged in sequence from top to bottom. The fluorescence collection layer includes a microlens structure disposed between the first diaphragm structure and the second diaphragm structure. The filtering structure is disposed between the second diaphragm structure and the third diaphragm structure.
[0013] Optionally, the fluorescence crosstalk suppression structure includes an isolation structure disposed on both sides of the microlens structure.
[0014] Optionally, the fluorescence crosstalk suppression structure includes a first diaphragm structure and a second diaphragm structure arranged in sequence from top to bottom. The filtering structure includes a resonant waveguide grating filter disposed between the first diaphragm structure and the second diaphragm structure. The resonant waveguide grating filter structure includes a planar waveguide layer and a periodic grating structure. The periodic grating structure is located above the planar waveguide layer and is connected to the planar waveguide layer. The fluorescence collection layer includes a microlens structure located below the planar waveguide layer and connected to the planar waveguide layer.
[0015] Optionally, the fluorescence collection layer includes a microlens structure located above the filtering structure. The fluorescence crosstalk suppression structure includes a distributed Bragg reflector layer located between the filtering structure and the microlens structure and an isolation structure located on both sides of the microlens structure.
[0016] Optionally, the fluorescence crosstalk suppression structure includes a distributed Bragg reflector layer disposed above and / or below the filtering structure. The distributed Bragg reflector layer includes alternately arranged first refractive index material layers and second refractive index material layers. The refractive index range of the first refractive index material layer is 1.6 - 3.5, and the refractive index range of the second refractive index material layer is 1.3 - 1.5. The distributed Bragg reflector layer blocks fluorescence signals with incident angles greater than a threshold angle θ and allows fluorescence signals with incident angles less than the threshold angle θ to pass through. The range of the threshold angle θ is 40° - 60°.
[0017] Optionally, the fluorescence crosstalk suppression structure includes isolation structures disposed on both sides of the filtering structure. The thickness of the isolation structure is not less than the thickness of the filtering structure, and the refractive index of the material of the isolation structure is less than the refractive index of the material of the filtering structure.
[0018] Optionally, the filtering structure includes a resonant waveguide grating filter or a distributed Bragg reflector filter.
[0019] Optionally, the resonant waveguide grating filter includes a planar waveguide layer and a periodic grating structure. The periodic grating structure is located above and / or below the planar waveguide layer and is connected to the planar waveguide layer. The periodic grating structure and the planar waveguide layer define a plurality of grooves, and the grooves are single-depth grooves or double-depth nested grooves.
[0020] Optionally, the fluorescence detection layer includes one of a CCD chip, a CMOS image sensing chip, a PD array, an SPAD array, a PMT array, and an SiPM array.
[0021] Optionally, the fluorescence excitation layer includes a cladding and a waveguide structure or a waveguide grating structure encapsulated in the cladding. A sample well is provided on the surface of the cladding above the waveguide structure or the waveguide grating structure. The sample well is used to load molecular substances that can excite fluorescence, and the distance between the bottom surface of the sample well and the top surface of the waveguide structure or the waveguide grating structure is less than the penetration distance of the waveguide evanescent wave.
[0022] As described above, the integrated molecular fluorescence detection platform of the present invention includes a plurality of periodically repeated subunits, and each subunit includes a fluorescence excitation layer, a fluorescence collection layer, and a fluorescence detection layer arranged in sequence from top to bottom. Among them, the fluorescence collection layer includes a filtering structure and a fluorescence crosstalk suppression structure. The filtering structure is used to block the laser and allow the signals in the fluorescence band to pass through. The fluorescence crosstalk suppression structure includes any one or a combination of a diaphragm structure, an isolation structure, and a distributed Bragg reflector layer, and the fluorescence crosstalk suppression structure is disposed on at least one of the two sides, above, and below the filtering structure, or the fluorescence collection layer further includes a microlens structure, and the fluorescence crosstalk suppression structure is disposed on at least one of the two sides of the microlens structure, the two sides, above, and below the filtering structure. The present invention provides a fully integrated fluorescence detection solution for the detection of fluorescence signals of biological samples, with a relatively simple and effective structure, which helps to simplify the process and optimize the cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 、 Figures 7 - 13 、 Figure 17 are respectively schematic structural diagrams of the subunits of the fully integrated molecular fluorescence detection platform of the present invention in different embodiments.
[0024] Figures 2 - 3 are respectively schematic structural diagrams of two fluorescence excitation layers.
[0025] Figures 4 - 6 are respectively schematic structural diagrams of three different resonant waveguide grating filters.
[0026] Figures 14 - 16 are respectively schematic structural diagrams of three multi-level Fresnel lens structures and the isolation structures on both sides thereof.
[0027] DESCRIPTION OF THE REFERENCE NUMERALS
[0028] 110, 210, 310, 410, 510, 610, 710, 810, 910 Fluorescence excitation layer
[0029] 1101 Cladding
[0030] 1102 Waveguide structure
[0031] 1103 Sample well
[0032] 1104 Waveguide grating structure
[0033] 120, 220, 320, 420, 520, 620, 720, 820, 920 Fluorescence collection layer
[0034] 121, 221, 321, 421, 521, 621, 721, 821, 921 Filtering structure
[0035] 1211 and 2211 flat waveguide layers
[0036] 1212 and 2212 periodic grating structures
[0037] 122, 222, 322, 422, 522, 622, 722, 822, 922 fluorescence crosstalk suppression structures
[0038] 1221, 2221, 3221, 4221, 8221 first aperture structures
[0039] 1222, 2222, 3222, 4222, 8222, 8223 second aperture structures
[0040] 1223 third aperture structure
[0041] 4223, 6222, 7221, 8224, 9222 isolation structures
[0042] 6221 and 9221 distributed Bragg reflector layers
[0043] 123, 223, 823, 923 microlens structures
[0044] 130, 230, 330, 430, 530, 630, 730, 830, 930 fluorescence detection layers Detailed implementation manners
[0045] The following describes the implementation manners of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.
[0046] It should be emphasized that the term "including / comprising" when used herein refers to the presence of features, whole pieces, steps or components, but does not exclude the presence or addition of one or more other features, whole pieces, steps or components.
[0047] Features described and / or illustrated for one implementation manner can be used in the same or similar manner in one or more other implementation manners, combined with features in other implementation manners, or replace features in other implementation manners.
[0048] When detailing the embodiments of the present invention, for ease of explanation, the schematic diagrams showing the device structure will be enlarged locally out of the general proportion, and the schematic diagrams are only examples and should not limit the scope of protection of the present invention herein. In addition, in actual production, three-dimensional spatial dimensions including length, width and depth should be included.
[0049] For convenience of description, spatial relationship terms such as "below", "beneath", "lower", "under", "above", "on" may be used herein to describe the relationship of one element or feature shown in the drawings to other elements or features. It will be understood that these spatial relationship terms are intended to encompass other orientations of the device in use or operation in addition to the orientation depicted in the drawings. Further, when a layer is referred to as being "between" two layers, it can be the only layer between the two layers, or there can be one or more intervening layers.
[0050] In the context of the present application, the structure in which the first feature is "above" the second feature as described may include embodiments in which the first and second features are in direct contact, and may also include embodiments in which additional features are formed between the first and second features, such that the first and second features may not be in direct contact.
[0051] It should be noted that the diagrams provided in this embodiment only illustrate the basic concept of the present invention schematically. Therefore, only the components related to the present invention are shown in the diagrams, rather than being drawn according to the number, shape and size of the components in actual implementation. The type, quantity and proportion of each component in actual implementation can be arbitrarily changed, and the layout type of its components may also be more complex.
[0052] The fully integrated molecular fluorescence detection platform of the present invention can realize the functions of exciting, collecting and detecting fluorescence signals. It includes a plurality of periodically repeated subunits, and each of the subunits includes a fluorescence excitation layer, a fluorescence collection layer and a fluorescence detection layer arranged in sequence from top to bottom. Among them, the fluorescence excitation layer realizes the confinement of fluorescent molecules and generates an excitation light field to excite fluorescent signals from the fluorescent molecules; the fluorescence collection layer realizes the collection, crosstalk suppression and filtering functions of the fluorescence signals; the fluorescence detection layer realizes the photoelectric signal conversion of the fluorescence signals for subsequent data processing.
[0053] In the fully integrated molecular fluorescence detection platform of the present invention, the fluorescence collection layer includes a filtering structure and a fluorescence crosstalk suppression structure. The filtering structure is used to block the laser and allow the signals in the fluorescence band to pass through. The fluorescence crosstalk suppression structure includes any one or combination of a diaphragm structure, an isolation structure and a distributed Bragg reflector layer, and the fluorescence crosstalk suppression structure is disposed at at least one of the following positions: above the filtering structure, below the filtering structure, and on both sides of the filtering structure.
[0054] In some embodiments, the fluorescence collection layer further includes a microlens structure for collimating the divergent fluorescence signal. The microlens structure adopts a multi-stage Fresnel lens structure or a metasurface lens structure. The fluorescence crosstalk suppression structure is disposed at at least one of the following positions: above the filtering structure, below the filtering structure, on both sides of the filtering structure, and on both sides of the microlens structure.
[0055] In some embodiments, the fluorescence crosstalk suppression structure includes a diaphragm structure disposed above and / or below the filtering structure. The diaphragm structure serves to reduce the fluorescence crosstalk between the sub-units.
[0056] Specifically, the diaphragm structure includes a light-shielding layer and a light-passing hole formed in the light-shielding layer. The material of the light-shielding layer can be an opaque metal or a metal composite. The light-passing hole can be a circular hole or a square hole, and the size (diameter or side length) of the light-passing hole ranges from 200 nanometers to 10 micrometers.
[0057] In some embodiments, the fluorescence crosstalk suppression structure includes a distributed Bragg reflector layer disposed above and / or below the filtering structure. The distributed Bragg reflector layer includes an alternating arrangement of a first refractive index material layer and a second refractive index material layer. The refractive index range of the first refractive index material layer is 1.6 - 3.5, and the refractive index range of the second refractive index material layer is 1.3 - 1.5. The distributed Bragg reflector layer blocks the fluorescence signal with an incident angle greater than the threshold angle θ and allows the fluorescence signal with an incident angle less than the threshold angle θ to pass through. The range of the threshold angle θ is 40° - 60°.
[0058] In some embodiments, the fluorescence crosstalk suppression structure includes an isolation structure disposed on both sides of the filtering structure. The thickness of the isolation structure is not less than the thickness of the filtering structure, and the refractive index of the material of the isolation structure is less than the refractive index of the material of the filtering structure.
[0059] In some embodiments, the fluorescence crosstalk suppression structure includes an isolation structure disposed on both sides of the microlens structure.
[0060] In some embodiments, the filtering structure includes a resonant waveguide grating (RWG) filter or a distributed Bragg reflector (DBR) filter.
[0061] The present invention proposes a variety of relatively concise and effective solutions from the perspectives of filters, crosstalk suppression, lenses, etc., which helps to simplify the process and optimize the cost.
[0062] For example, please refer to Figure 1 which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in an embodiment, including a fluorescence excitation layer 110, a fluorescence collection layer 120, and a fluorescence detection layer 130 arranged in sequence from top to bottom. Among them, the fluorescence collection layer 120 includes a filtering structure 121, a fluorescence crosstalk suppression structure 122, and a microlens structure 123. The fluorescence crosstalk suppression structure 122 includes a first aperture structure 1221, a second aperture structure 1222, and a third aperture structure 1223 arranged in sequence from top to bottom. The microlens structure 123 is arranged between the first aperture structure 1221 and the second aperture structure 1222, and the filtering structure 121 is arranged between the second aperture structure 1222 and the third aperture structure 1223.
[0063] Specifically, the function of the fluorescence excitation layer 110 is to restrict fluorescent molecules in physical space and generate an excitation light field through a waveguide device to excite the fluorescent molecules to generate a fluorescence signal. For example, please refer to Figure 2 which shows a schematic structural diagram of the fluorescence excitation layer 110. It includes a cladding layer 1101 and a waveguide structure 1102 coated in the cladding layer 1101. A sample well 1103 is provided on the surface of the cladding layer 1101 above the waveguide structure 1102. The sample well 1103 is used to load molecular substances that can excite fluorescence. The distance between the bottom surface of the sample well 1103 and the top surface of the waveguide structure 1102 is less than the penetration distance of the waveguide evanescent wave.
[0064] As an example, the waveguide structure 1102 adopts a straight waveguide. The sample well 1103 is located directly above the straight waveguide 1102. The sample well 1103 can be in the shape of a cylinder, a frustum of a cone, a cuboid, or other suitable shapes. The size of the sample well 1103 (such as diameter, side length, etc.) can be in the order of dozens to hundreds of nanometers. Fluorescent molecules can be fixed to the bottom of the sample well 1103 by chemical surface modification methods, so as to be excited by the waveguide evanescent light field and then generate a fluorescence signal.
[0065] For example, please refer to Figure 3 which shows another schematic structural diagram of the fluorescence excitation layer 110. It includes a cladding layer 1101 and a waveguide grating structure 1104 coated in the cladding layer 1101. A sample well 1103 is provided on the surface of the cladding layer 1101 above the waveguide grating structure 1104. The sample well 1103 is used to load molecular substances that can excite fluorescence. The distance between the bottom surface of the sample well 1103 and the top surface of the waveguide grating structure 1104 is less than the penetration distance of the waveguide evanescent wave, so as to be excited by the waveguide evanescent light field and then generate a fluorescence signal.
[0066] Specifically, the function of the fluorescence collection layer 120 is to filter and collect fluorescence signals. In Figure 1 the illustrated embodiment, the functions of the first aperture structure 1221, the second aperture structure 1222, and the third aperture structure 1223 are to reduce fluorescence crosstalk between sub-units. The function of the microlens structure 123 disposed between the first aperture structure 1221 and the second aperture structure 1222 is to collimate the divergent fluorescence signals. The function of the filtering structure 121 disposed between the second aperture structure 1222 and the third aperture structure 1223 is to block the laser wavelength.
[0067] As an example, the microlens structure 123 adopts a multi-level Fresnel lens structure or a metasurface lens structure. The multi-level Fresnel lens structure can be, but is not limited to, a 2-level, 3-level, or 4-level Fresnel lens structure.
[0068] As an example, the filtering structure 121 adopts a resonant waveguide grating filter, including a planar waveguide layer 1211 and a periodic grating structure 1212. The periodic grating structure 1212 is located above and / or below the planar waveguide layer 1211 and is connected to the planar waveguide layer 1211. The periodic grating structure 1212 and the planar waveguide layer 1211 define a plurality of grooves, and the grooves are single-depth grooves or double-depth nested grooves, which can be obtained by an etching process.
[0069] As an example, please refer to Figure 4 , which shows a schematic structural diagram of a resonant waveguide grating filter. Among them, the periodic grating structure 1212 is located above the planar waveguide layer 1211, and the groove defined by the periodic grating structure 1212 and the planar waveguide layer 1211 is a single-depth groove.
[0070] As an example, please refer to Figure 5 , which shows a schematic structural diagram of another resonant waveguide grating filter. Among them, the periodic grating structure 1212 is located above the planar waveguide layer 1211, and the groove defined by the periodic grating structure 1212 and the planar waveguide layer 1211 is a double-depth nested groove.
[0071] As an example, please refer to Figure 6 , which shows a schematic structural diagram of yet another resonant waveguide grating filter. Among them, the periodic grating structure 1212 is located above and below the planar waveguide layer 1211, and the grooves defined by the periodic grating structure 1212 and the planar waveguide layer 1211 are all single-depth grooves, and the grooves on both sides can have different depths.
[0072] As an example, the material of the resonant waveguide grating filter can be amorphous silicon (A-Si) or silicon nitride, and its filtering characteristic is to block the laser wavelength. By modulating the refractive index K of the material of the resonant waveguide grating, the filtering spectral tolerance of the RWG can be increased.
[0073] Since the laser in the waveguide is linearly polarized light, the scattered laser also has a high degree of linear polarization, which exactly conforms to the polarization filtering characteristic of the RWG. The blocking degree of the RWG for s-polarized light is much greater than that for p-polarized light. Therefore, ensuring that the input laser is consistent with the s-polarized light can maximize the utilization of the filtering characteristic of the RWG.
[0074] Specifically, the function of the fluorescence detection layer 130 is to detect the fluorescence signal and convert the photon signal into an electronic signal for subsequent signal processing. The fluorescence detection layer 130 can include one of a CCD chip, a CMOS image sensing chip, a PD array, a SPAD array, a PMT array, and a SiPM array.
[0075] As an example, please refer to Figure 7 , which shows a schematic structural diagram of a sub-unit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 210, a fluorescence collection layer 220, and a fluorescence detection layer 230 arranged in sequence from top to bottom. Among them, the fluorescence collection layer 220 includes a filtering structure 221, a fluorescence crosstalk suppression structure 222, and a microlens structure 223. The fluorescence crosstalk suppression structure 222 includes a first aperture structure 2221 and a second aperture structure 2222 arranged in sequence from top to bottom. The filtering structure 221 and the microlens structure 223 are both arranged between the first aperture structure 2221 and the second aperture structure 2222, and the microlens structure 223 is located below the filtering structure 221.
[0076] As an example, the filtering structure 221 adopts a resonant waveguide grating filter structure, which includes a planar waveguide layer 2211 and a periodic grating structure 2212. The periodic grating structure 2212 is located above the planar waveguide layer 2211 and is in close contact with the planar waveguide layer 2211.
[0077] As an example, the microlens structure 223 adopts a multi-stage Fresnel lens structure, which is located below the planar waveguide layer 2211 and is in close contact with the planar waveguide layer 2211.
[0078] As an example, please refer to Figure 8, which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 310, a fluorescence collection layer 320, and a fluorescence detection layer 330 arranged in sequence from top to bottom. Among them, the fluorescence collection layer 320 includes a filtering structure 321 and a fluorescence crosstalk suppression structure 322. The fluorescence crosstalk suppression structure 322 includes a first aperture structure 3221 and a second aperture structure 3222 arranged in sequence from top to bottom. The filtering structure 321 is arranged between the first aperture structure 3221 and the second aperture structure 3222.
[0079] As an example, the filtering structure 321 adopts a resonant waveguide grating filter structure or a distributed Bragg reflector.
[0080] As an example, please refer to Figure 9 , which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 410, a fluorescence collection layer 420, and a fluorescence detection layer 430 arranged in sequence from top to bottom. Among them, the fluorescence collection 420 includes a filtering structure 421 and a fluorescence crosstalk suppression structure 422. The fluorescence crosstalk suppression structure 422 includes a first aperture structure 4221 and a second aperture structure 4222 arranged in sequence from top to bottom, and includes an isolation structure 4223. The filtering structure 421 is arranged between the first aperture structure 4221 and the second aperture structure 4222. The isolation structure 4223 is arranged on both sides of the filtering structure 421. The thickness of the isolation structure 4223 is not less than the thickness of the filtering structure 421. The refractive index of the material of the isolation structure 4223 is less than the refractive index of the material of the filtering structure.
[0081] Specifically, the isolation structure 4223 can be obtained by etching an isolation groove in the material layer where the filtering structure 421 is located and filling the isolation groove with an isolation material.
[0082] Specifically, the first aperture structure 4221 and the second aperture structure 4222 are used to reduce the fluorescence crosstalk between each subunit, and the presence of the isolation structure 4223 can form a light field limitation for the filtering structure 421 in the horizontal direction, strengthening the crosstalk suppression performance.
[0083] As an example, the filtering structure 421 adopts a resonant waveguide grating filter structure or a distributed Bragg reflector. The isolation structure 4223 is located at the junction of adjacent periodic structures, and its filling material adopts one of silicon oxide, plastic, and su8.
[0084] As an example, please refer to Figure 10, which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 510, a fluorescence collection layer 520, and a fluorescence detection layer 530 sequentially arranged from top to bottom. Among them, the fluorescence collection 520 includes a filtering structure 521 and a fluorescence crosstalk suppression structure 522 located above the filtering structure 521. The filtering structure 521 can adopt a resonant waveguide grating filter or a distributed Bragg reflector filter, and the fluorescence crosstalk suppression structure 522 can adopt a distributed Bragg reflector layer.
[0085] As an example, the DBR structure adopted by the fluorescence crosstalk suppression structure 522 is composed of a combination of two or more layers of materials, and the constituent materials include at least two of amorphous silicon (A-Si), silicon nitride (SiN), and silicon oxide (SiOx). Its filtering characteristic is that for the fluorescence band signal, when the incident angle exceeds the threshold angle θ, it plays a blocking role, and when the incident angle is less than the threshold angle θ, it plays a passing role. The threshold angle θ is in the range of 40 to 60 degrees.
[0086] As an example, please refer to Figure 11 , which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 610, a fluorescence collection layer 620, and a fluorescence detection layer 630 sequentially arranged from top to bottom. Among them, the fluorescence collection 620 includes a filtering structure 621 and a fluorescence crosstalk suppression structure 622. The filtering structure 621 can adopt a resonant waveguide grating filter or a distributed Bragg reflector filter, and the fluorescence crosstalk suppression structure 622 includes a distributed Bragg reflector layer 6221 located below the filtering structure 621 and isolation structures 6222 located on both sides of the filtering structure 621.
[0087] As an example, please refer to Figure 12 , which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 710, a fluorescence collection layer 720, and a fluorescence detection layer 730 sequentially arranged from top to bottom. Among them, the fluorescence collection 720 includes a filtering structure 721 and a fluorescence crosstalk suppression structure 722. The filtering structure 721 can adopt a resonant waveguide grating filter or a distributed Bragg reflector filter, and the fluorescence crosstalk suppression structure 722 includes isolation structures 7221 located on both sides of the filtering structure 721.
[0088] As an example, please refer to Figure 13, which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 810, a fluorescence collection layer 820, and a fluorescence detection layer 830 arranged in sequence from top to bottom. Among them, the fluorescence collection layer 820 includes a filtering structure 821, a fluorescence crosstalk suppression structure 822, and a microlens structure 823. The fluorescence crosstalk suppression structure 822 includes a first aperture structure 8221, a second aperture structure 8222, and a third aperture structure 8223 arranged in sequence from top to bottom, and includes an isolation structure 8224. The microlens structure 823 is arranged between the first aperture structure 8221 and the second aperture structure 8222. The isolation structure 8224 is arranged on both sides of the microlens structure 823. The filtering structure 821 is arranged between the second aperture structure 8222 and the third aperture structure 8223.
[0089] Specifically, the isolation structure 8224 can be obtained by etching an isolation groove in the material layer where the microlens structure 823 is located and filling the isolation groove with an isolation material. The microlens structure 823 can adopt a multi-level Fresnel lens structure, for example, it can be, but is not limited to, a 2, 3, or 4-level Fresnel lens structure.
[0090] As an example, please refer to Figures 14 - 16 , in which, Figure 14 shows a schematic structural diagram of a two-level Fresnel lens structure and the isolation structure 8224 on both sides thereof, Figure 15 shows a schematic structural diagram of a three-level Fresnel lens structure and the isolation structure 8224 on both sides thereof, Figure 16 shows a schematic structural diagram of another multi-level Fresnel lens structure and the isolation structure 8224 on both sides thereof.
[0091] As an example, the filtering structure 121 adopts a resonant waveguide grating filter or a distributed Bragg reflector filter.
[0092] As an example, please refer to Figure 17, which shows a schematic structural diagram of a subunit of the fully integrated molecular fluorescence detection platform of the present invention in another embodiment, including a fluorescence excitation layer 910, a fluorescence collection layer 920, and a fluorescence detection layer 930 arranged in sequence from top to bottom. Among them, the fluorescence collection layer 920 includes a filtering structure 921, a fluorescence crosstalk suppression structure 922, and a microlens structure 923. The microlens structure 923 is located above the filtering structure 921. The filtering structure 921 can adopt a resonant waveguide grating filter or a distributed Bragg reflector filter. The microlens structure 923 can adopt a multi-level Fresnel lens structure, such as but not limited to a 2-level, 3-level, or 4-level Fresnel lens structure. The fluorescence crosstalk suppression structure 922 includes a distributed Bragg reflector layer 9221 located between the filtering structure 921 and the microlens structure 923 and isolation structures 9222 located on both sides of the microlens structure 923.
[0093] As an example, the distributed Bragg reflector layer 9221 blocks the fluorescence signal with an incident angle greater than the threshold angle θ and allows the fluorescence signal with an incident angle less than the threshold angle θ to pass through. The range of the threshold angle θ is 40° to 60°.
[0094] In summary, the integrated molecular fluorescence detection platform of the present invention includes a plurality of periodically repeated subunits. Each subunit includes a fluorescence excitation layer, a fluorescence collection layer, and a fluorescence detection layer arranged in sequence from top to bottom. Among them, the fluorescence collection layer includes a filtering structure and a fluorescence crosstalk suppression structure. The filtering structure is used to block the laser and allow the signal in the fluorescence band to pass through. The fluorescence crosstalk suppression structure includes any one or a combination of a diaphragm structure, an isolation structure, and a distributed Bragg reflector layer, and the fluorescence crosstalk suppression structure is arranged on at least one of the two sides, above, and below the filtering structure, or the fluorescence collection layer further includes a microlens structure, and the fluorescence crosstalk suppression structure is arranged on at least one of the two sides of the microlens structure, the two sides, above, and below the filtering structure. The present invention provides a fully integrated fluorescence detection solution for the detection of fluorescence signals of biological samples, with a relatively simple and effective structure, which helps to simplify the process and optimize the cost. Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.
[0095] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.
Claims
1. An all-integrated molecular fluorescence detection platform, characterized in that, It includes a plurality of periodically repeated subunits, and each subunit includes a fluorescence excitation layer, a fluorescence collection layer, and a fluorescence detection layer arranged in sequence from top to bottom. The fluorescence collection layer includes: A filtering structure for blocking the laser and allowing the signals in the fluorescence band to pass through; A fluorescence crosstalk suppression structure, including any one or a combination of a diaphragm structure, an isolation structure, and a distributed Bragg reflector layer. The fluorescence crosstalk suppression structure is disposed at least in one of the following positions: above the filtering structure, below the filtering structure, and on both sides of the filtering structure.
2. The fully integrated molecular fluorescence detection platform according to claim 1, wherein: The fluorescence collection layer further includes a microlens structure, and the microlens structure adopts a multi-level Fresnel lens structure or a metasurface lens structure. The fluorescence crosstalk suppression structure is disposed at least in one of the following positions: above the filtering structure, below the filtering structure, on both sides of the filtering structure, and on both sides of the microlens structure.
3. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence crosstalk suppression structure includes a diaphragm structure disposed above and / or below the filtering structure. The diaphragm structure includes a light-shielding layer and a light-passing hole formed in the light-shielding layer, and the size range of the light-passing hole of the diaphragm structure is 200 nanometers to 10 micrometers.
4. The fully integrated molecular fluorescence detection platform according to claim 3, wherein: The fluorescence crosstalk suppression structure includes a first diaphragm structure, a second diaphragm structure, and a third diaphragm structure arranged in sequence from top to bottom. The fluorescence collection layer includes a microlens structure disposed between the first diaphragm structure and the second diaphragm structure, and the filtering structure is disposed between the second diaphragm structure and the third diaphragm structure.
5. The fully integrated molecular fluorescence detection platform according to claim 4, wherein: The fluorescence crosstalk suppression structure includes an isolation structure disposed on both sides of the microlens structure.
6. The fully integrated molecular fluorescence detection platform according to claim 3, wherein: The fluorescence crosstalk suppression structure includes a first diaphragm structure and a second diaphragm structure arranged in sequence from top to bottom. The filtering structure includes a resonant waveguide grating filter disposed between the first diaphragm structure and the second diaphragm structure. The resonant waveguide grating filter structure includes a planar waveguide layer and a periodic grating structure. The periodic grating structure is located above the planar waveguide layer and is connected to the planar waveguide layer. The fluorescence collection layer includes a microlens structure located below the planar waveguide layer and connected to the planar waveguide layer.
7. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence collection layer includes a microlens structure located above the filtering structure. The fluorescence crosstalk suppression structure includes a distributed Bragg reflector layer located between the filtering structure and the microlens structure and an isolation structure located on both sides of the microlens structure.
8. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence crosstalk suppression structure includes a distributed Bragg reflector layer disposed above and / or below the filtering structure. The distributed Bragg reflector layer includes alternately arranged first refractive index material layers and second refractive index material layers. The refractive index range of the first refractive index material layer is 1.6 to 3.5, and the refractive index range of the second refractive index material layer is 1.3 to 1.
5. The distributed Bragg reflector layer blocks the fluorescence signals with incident angles greater than the threshold angle θ and allows the fluorescence signals with incident angles less than the threshold angle θ to pass through. The range of the threshold angle θ is 40° to 60°.
9. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence crosstalk suppression structure includes isolation structures disposed on both sides of the filtering structure. The thickness of the isolation structure is not less than that of the filtering structure, and the refractive index of the material of the isolation structure is less than that of the material of the filtering structure.
10. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The filtering structure includes a resonant waveguide grating filter or a distributed Bragg reflector filter.
11. The fully integrated molecular fluorescence detection platform according to claim 10, characterized in that: The resonant waveguide grating filter includes a planar waveguide layer and a periodic grating structure. The periodic grating structure is located above and / or below the planar waveguide layer and is connected to the planar waveguide layer. The periodic grating structure and the planar waveguide layer define a plurality of grooves, and the grooves are single-depth grooves or double-depth nested grooves.
12. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence detection layer includes one of a CCD chip, a CMOS image sensing chip, a PD array, a SPAD array, a PMT array, and a SiPM array.
13. The fully integrated molecular fluorescence detection platform according to claim 1 or 2, characterized in that: The fluorescence excitation layer includes a cladding and a waveguide structure or a waveguide grating structure encapsulated in the cladding. A sample well is provided on the surface of the cladding above the waveguide structure or the waveguide grating structure. The sample well is used to load a molecular substance that can excite fluorescence, and the distance between the bottom surface of the sample well and the top surface of the waveguide structure or the waveguide grating structure is less than the penetration distance of the waveguide evanescent wave.