Method for detecting a fluorescent marker and detection device

Abstract

The application provides a fluorescence marker detection method and a detection device, the detection method comprises encoding based on position difference and / or difference of Stokes shift of emission spectrum peaks of various fluorescent materials in a fluorescent material combination. The detection method can break the limitation of fluorescence encoding on emission spectrum overlap, utilize emission spectrum and Stokes shift co-encoding technology, realize super-multiple encoding of multiplexed immunohistochemistry / immunofluorescence, meanwhile, the detection method does not depend on iterative sequential antibody labeling and imaging technology, and does not need multispectral imaging technology to realize super-multiple detection of multiplexed immunohistochemistry / immunofluorescence, is suitable for conventional imaging instruments, is low in cost and fast in scanning speed.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, and in particular to a method and equipment for detecting fluorescent markers. Background Technology

[0002] Conventional immunohistochemistry / immunofluorescence (IHC / IF) is commonly used as a diagnostic technique in histopathology, but it has certain limitations. The most significant limitation is that this technique can only detect one marker on a tissue sample at a time. This can lead to the loss of important prognostic and diagnostic information in patient samples. For example, tumor-infiltrating CD8+ T cells can be identified by recognizing the expression of antigens such as CD8, CD3, FOXP3, and CD20. Identifying only one of these antigens may lead to false positives or false negatives. Similarly, the identification of a single antigen also leads to another drawback of IHC / IF-based biomarker assessment—high inter-observer variability. Different individuals may arrive at subjective and non-reproducible assessments of the same slide due to a lack of cross-validation among multiple interpretive criteria.

[0003] To address these challenges, researchers have recently developed multiplex immunohistochemistry / immunofluorescence (mIHC / IF) technology. This technique allows for the simultaneous detection of multiple markers on a single tissue section and has begun to be introduced into research and clinical settings. mIHC / IF technology can provide crucial information about the cancer microenvironment, prognosis, treatment, and recurrence; it allows for the simultaneous examination of different components of the tumor microenvironment using multiplex detection methods, providing insights into biological crosstalk at the tumor-host interface and offering information from the subcellular to the cell population level; and it enables the simultaneous assessment of the expression and localization of multiple biomarkers, as well as their co-expression or interactions between cells.

[0004] To achieve multiplex immunohistochemistry / immunofluorescence (mIHC / IF), there are currently the following main approaches: non-fluorescence-based multiplex imaging techniques and fluorescence-based multiplex imaging techniques.

[0005] Non-fluorescence-based multiplex imaging techniques include multiplex immunohistochemical serial staining and mass spectrometry imaging. Multiplex immunohistochemical serial staining is essentially a repeated experiment in traditional immunohistochemistry (it can be understood as creating a layer for a single marker with each slide preparation and imaging step; multiple layers are then superimposed to form an image containing repeatedly superimposed markers, thus achieving multivariate detection). Because it requires multiple image superpositions, the process is complex; it involves repeated substrate elution, antibody stripping, incubation, slide preparation, and sample preparation, resulting in a long processing time. Mass spectrometry-based imaging techniques visualize the elemental or molecular components of fixed cells or tissues using mass spectrometry. By distinguishing the molecular weight of different markers or different labels, multivariate detection is achieved. However, mass spectrometry-based imaging techniques are relatively immature, costly, and have slow scanning speeds, limiting their development.

[0006] Fluorescence-based multiplex imaging techniques include traditional multiplex immunofluorescence imaging based on the position of different fluorescence emission spectral peaks, multispectral imaging techniques based on spectral splitting and analysis, and iterative sequential antibody labeling and imaging techniques (MxIF and CycIF). Among these, multiplex immunofluorescence imaging based on the position of different fluorescence emission spectral peaks is fast, mature, and widely used. However, due to the limitation of spectral overlap, current multiplex immunofluorescence imaging systems can generally only detect a maximum of 4-6 fluorescently labeled biomarkers simultaneously, which cannot meet the needs of joint detection of multiple biomarkers in tumor tissue, adjacent normal tissue, and the tumor microenvironment. Multispectral imaging techniques based on spectral splitting and analysis (such as those developed by Akoya Biosciences) Automated quantitative pathological imaging systems (QPIs) can identify and quantify multiple overlapping biomarkers (up to eight) by recognizing and separating overlapping fluorescence signals, without being affected by autofluorescence interference due to signal non-mixing. However, due to the slow scanning and resolution speed of multispectral imaging technology, its imaging speed is very slow compared to conventional imaging techniques, making it difficult to improve throughput. Furthermore, multispectral imaging technology is expensive, requiring specialized instruments for imaging analysis, and is controlled by companies such as Akoya and PerkinElmer, resulting in limited versatility. Iterative sequential antibody labeling and imaging technology achieves multivariate detection through repeated staining-imaging-quenching (antigen retrieval) cycles. Theoretically, this approach can overcome the limitations of overlapping fluorescence emission spectra for more diverse detection, but it is very time-consuming. Moreover, the reactivity of antigens and antibodies may decrease due to repeated antigen retrieval, leading to biased results. Additionally, image overlap is required between multiple imaging sessions, and displacement can easily occur during this process, resulting in poor imaging quality. Therefore, there is an urgent need to develop a new, low-cost, and easy-to-operate ultramultivariate multicoding technology. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a method and device for detecting fluorescent biomarkers. Unlike traditional mIHC / IF technology, which encodes based on the positional differences of fluorescence emission peaks, this invention utilizes both the positional differences of the emission peaks and the differences in Stokes shifts of the fluorescent materials for co-encoding. This enables ultra-multivariate detection of multiplex immunohistochemistry / immunofluorescence without relying on iterative sequential antibody labeling and imaging techniques. It is suitable for conventional imaging instruments, low in cost, and offers fast scanning speed.

[0008] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0009] The first aspect of the present invention discloses a method for detecting fluorescent markers, the method comprising encoding based on the positional differences and / or Stokes shift differences of the emission spectral peaks of various fluorescent materials in a fluorescent material combination.

[0010] Furthermore, the detection method includes co-coding based on the positional differences of the emission spectral peaks and the differences in Stokes shifts of various fluorescent materials in the fluorescent material combination.

[0011] Furthermore, the fluorescent material combination includes a combination of inorganic fluorescent materials, a combination of organic fluorescent materials, or a combination of the inorganic fluorescent materials and the organic fluorescent materials.

[0012] Furthermore, in the fluorescent material combination, at least one fluorescent material has a Stokes shift difference of more than 50 nm from the Stokes shifts of the other fluorescent materials; and / or at least one fluorescent material has an anti-Stokes shift, which is different from the Stokes shifts of the other fluorescent materials.

[0013] Furthermore, the inorganic fluorescent material includes at least one or more of quantum dots and upconversion nanoparticles.

[0014] Further, the quantum dots include one or more of silicon quantum dots, germanium quantum dots, cadmium sulfide quantum dots, cadmium selenide quantum dots, cadmium telluride quantum dots, zinc selenide quantum dots, lead sulfide quantum dots, lead selenide quantum dots, indium phosphide quantum dots, indium arsenide quantum dots, and carbon quantum dots. Preferably, the quantum dots are selected from these categories. A series of nanocrystals.

[0015] Further, the upconversion nanoparticles are preferably materials composed of inorganic nanocrystals doped with rare earth ions. More preferably, the upconversion nanoparticles are materials composed of inorganic nanocrystals doped with yttrium ions. More preferably, the upconversion nanoparticles are materials containing NaYF4.

[0016] Furthermore, the organic fluorescent material includes at least one or more of the following: organic fluorescent dyes, organic small molecule luminescent materials, organic polymer luminescent materials, and fluorescent proteins.

[0017] Furthermore, the organic fluorescent dye includes Series of fluorescent dyes, Alexa Series of fluorescent dyes, eVolve TM One or more of the following: a series of fluorescent dyes, Opal series of fluorescent dyes, fluorescein isothiocyanate (FITC), rhodamine series of fluorescent dyes, cyanine fluorescent dyes, Texas Red, 4',6-diamidinyl-2-phenylindole (DAPI), propidium iodide (PI), and Hoechst 33342.

[0018] Furthermore, the organic small molecule luminescent material includes one or more of the following: diazoles and their derivatives, triazoles and their derivatives, coumarin derivatives, 1,8-naphthalimide derivatives, pyrazoline derivatives, triphenylamine derivatives, porphyrin compounds, carbazole derivatives, pyrazine derivatives, thiazole derivatives, and perylene derivatives.

[0019] Furthermore, the organic polymer luminescent material includes luminescent materials with conjugated polymer structures or side-chain polymer luminescent materials, preferably one or more of polyphenylene, polythiophene, polyfluorene, polytriphenylamine and their derivatives.

[0020] Furthermore, the fluorescent protein includes one or more of phycoerythrin (PE), allophycocyanin (APC), and green fluorescent protein (GFP).

[0021] Furthermore, the fluorescent material combination is any combination of two or three of the following: quantum dots, organic fluorescent dyes, and upconversion nanoparticles.

[0022] Furthermore, the Stokes shift of the organic fluorescent dye is <150 nm, preferably <50 nm; the Stokes shift of the quantum dot is >150 nm, preferably >200 nm; and the upconversion nanoparticle has an anti-Stokes shift, preferably <-100 nm.

[0023] Furthermore, the Stokes shift represents the difference between the position of the emission spectral peak and the position of the excitation spectral peak. When the difference is greater than 0, it is called the Stokes shift; when the difference is less than 0, it is called the anti-Stokes shift.

[0024] Furthermore, the encoding based on the positional differences of the emission spectral peaks of each fluorescent material in the fluorescent material combination includes performing a first re-encoding on the positions of different emission spectral peaks of different fluorescent materials. If the position of each distinguishable emission spectral peak corresponds to a specific target analyte, then the first re-encoding is performed; if the position of each distinguishable emission spectral peak corresponds to at least two specific target analytes, then the second re-encoding is performed.

[0025] Furthermore, the encoding based on the differences in Stokes shifts of various fluorescent materials in the fluorescent material combination includes a second encoding of fluorescent materials with the same or similar emission spectral peak positions using different Stokes shifts, different anti-Stokes shifts, or the difference between Stokes shifts and anti-Stokes shifts.

[0026] Furthermore, the first recoding of the positions of different emission spectral peaks of different fluorescent materials includes the following steps:

[0027] Step S1: Establish corresponding relationships between different fluorescent materials and different reactants;

[0028] Step S2: React reactants labeled with different fluorescent materials with the sample to be tested;

[0029] Step S3: Select the corresponding fluorescence channel based on the type of fluorescent material, and the detection equipment will automatically switch to the second filter corresponding to the fluorescence channel for filtering;

[0030] Step S4: Excite different fluorescent materials with various excitation lights. The emission light of fluorescent materials whose emission spectral peaks are indistinguishable passes through the same second filter corresponding to the same fluorescence channel; the emission light of fluorescent materials whose emission spectral peaks are distinguishable passes through different second filters corresponding to different fluorescence channels.

[0031] Step S5: Detect the emitted light after it has passed through different second filters using a detector;

[0032] Step S6: Based on the results detected by the detector after filtering through different second filters, determine one or more fluorescent materials in the sample to be tested that are compatible with the same second filter;

[0033] Step S7: When there is only one fluorescent material adapted to the same second filter, the type and intensity of the fluorescent material can be determined by the first recoding, and the type and content of the reactants and target detectants corresponding to the fluorescent material can be further determined.

[0034] Furthermore, the second encoding of fluorescent materials with emission spectral peaks at the same or similar positions using different Stokes shifts or different anti-Stokes shifts includes the following steps:

[0035] Step S8: Excite several fluorescent materials in the sample to be tested described in step S6 that are adapted to the same second filter with different excitation light; wherein, the several fluorescent materials adapted to the same second filter are fluorescent materials with the same or similar emission spectral peak positions;

[0036] Step S9: Use a detector to detect the emitted light after it has passed through the same second filter, and record the excitation light corresponding to these emitted lights;

[0037] Step S10: Based on different excitation light, emission light and / or second filter information, further determine the types of several fluorescent materials adapted to the same second filter, thereby further determining the types and contents of the reactants and target detectants corresponding to the fluorescent materials.

[0038] Furthermore, the target detection substance includes one or more of the following: protein, nucleic acid, lipid, small molecule compound, cell, extracellular vesicle, and exosome.

[0039] Furthermore, the reactants include one or more of antibodies, nucleic acids, and ligands that can specifically react with the target detectable.

[0040] Furthermore, establishing corresponding relationships between different fluorescent materials and different reactants includes cross-linking different fluorescent materials with corresponding reactants through chemical cross-linking, bio-coupling, or physical cross-linking methods.

[0041] Furthermore, the detection methods described above are applied in the fields of immunohistochemistry and / or immunofluorescence.

[0042] A second aspect of the present invention discloses a detection device for fluorescent markers, the detection device comprising detection using the detection method described above.

[0043] Furthermore, the detection device includes an excitation light source, which includes excitation light generated by multiple light sources with narrow wavelength ranges and / or excitation light generated by light sources with wide wavelength ranges; the excitation light source includes at least two or more of the ultraviolet light band, visible light band, and infrared light band.

[0044] Furthermore, the detection device also includes a filter, which includes a second filter group and / or a first filter group; the second filter group is used to filter the emitted light of the fluorescent material; the first filter group is used to filter the excitation light generated by the light source with a wide wavelength range to obtain excitation light of multiple specific wavelength ranges.

[0045] Furthermore, the detection device also includes a detector for detecting the emitted light of the fluorescent material after it has been excited by the excitation light and filtered by the second filter group.

[0046] The beneficial effects of this invention are as follows:

[0047] Using the detection method and equipment for fluorescent markers described in this invention, the theoretically maximum number of codes achievable through encoding by the positions of different emission spectral peaks and different Stokes shifts equals the number of different emission spectral peak positions multiplied by the number of different excitation lights. This co-encoding method overcomes the limitation of overlapping fluorescence emission spectra, achieving ultra-multivariate encoding for multiplex immunohistochemistry / immunofluorescence (mIHC / IF). This method does not rely on iterative sequential antibody labeling and imaging techniques, nor does it require multispectral imaging technology to achieve ultra-multivariate detection of multiplex immunohistochemistry / immunofluorescence. It is suitable for conventional imaging instruments, low in cost, and fast in scanning speed. Attached Figure Description

[0048] Figure 1 These are the emission spectra of different organic fluorescent dyes and quantum dots in embodiments of the present invention. Figure 1 A represents the emission spectrum of the organic fluorescent dye; Figure 1 B represents the emission spectrum of the quantum dot; Figure 1 C represents the overlap of the emission spectra of organic fluorescent dyes and quantum dots.

[0049] Figure 2These are the excitation spectra (dashed lines) and emission spectra (color blocks) of different organic fluorescent dyes and quantum dots in embodiments of the present invention. Figure 2 A represents the excitation spectrum (dashed line) and emission spectrum (color patch) of the organic fluorescent dye; Figure 2 B represents the excitation spectrum (dashed line) and emission spectrum (color block) of the quantum dot.

[0050] Figure 3 These are the excitation spectrum (dashed line) and emission spectrum (color block) of the upconversion nanoparticles in this embodiment of the invention.

[0051] Figure 4 The difference (dashed line) refers to the difference in excitation light (color patch) between quantum dots and organic fluorescent dyes with the same or similar emission spectral peak positions (color patches) in the embodiments of the present invention. Figure 4 A represents the difference in excitation light between Alexa 488 organic fluorescent dye and Qdot 525 quantum dot, whose emission spectral peaks are located at similar positions; where... Figure 4 B represents the difference in excitation light between Alexa 532 organic fluorescent dye and Qdot 565 quantum dot, whose emission spectral peaks are located at similar positions; where... Figure 4 C represents the difference in excitation light between Alexa 568 organic fluorescent dye and Qdot 605 quantum dot, whose emission spectral peaks are located at similar positions; where Figure 4 D represents the difference in excitation light between Alexa 635 organic fluorescent dye and Qdot 655 quantum dots with similar emission spectral peak positions; where... Figure 4 E represents the difference in excitation light between Alexa 680 organic fluorescent dye and Qdot 705 quantum dot, whose emission spectral peaks are located at similar positions.

[0052] Figure 5 This is a schematic diagram illustrating the principle of multiplex immunohistochemistry / immunofluorescence technology based on emission spectroscopy and Stokes shift co-encoding in an embodiment of the present invention.

[0053] Figure 6 This is a schematic diagram of the operation process of multiplex immunohistochemistry / immunofluorescence technology based on emission spectroscopy and Stokes shift co-encoding in an embodiment of the present invention.

[0054] Figure 7 This is a schematic diagram of the detection results of multiplex immunohistochemistry / immunofluorescence based on emission spectrum and Stokes shift co-encoding in an embodiment of the present invention. Detailed Implementation

[0055] To better understand the content of this invention, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] In this invention, the excitation light refers to light with a specific wavelength that matches the excitation spectrum of the fluorescent material. The excitation light can induce photoluminescence in the fluorescent material, producing emitted light of another wavelength. The excitation light source includes light sources with a wide wavelength range, such as mercury lamps, xenon lamps, and halogen lamps, as well as light sources with a narrow wavelength range, such as lasers of various colors.

[0057] In this invention, the Stokes shift and the anti-Stokes shift refer to the difference between the peaks of the emission spectrum and the absorption spectrum. When the difference is greater than 0, it is the Stokes shift, and when the difference is less than 0, it is the anti-Stokes shift.

[0058] Conventional detection methods and equipment use a specific excitation light to excite fluorescent materials with a specific emission peak position, establishing a one-to-one relationship between the emission peak position and the excitation light. However, in this invention, fluorescent materials with emission peak positions that are the same or similar have different Stokes shifts or anti-Stokes shifts, meaning that fluorescent materials with the same or similar emission peak positions require different excitation light to be excited. For example, among fluorescent materials, quantum dots, organic fluorescent dyes, and upconversion nanoparticles have the same or similar emission spectral peak positions. The quantum dots have a very large Stokes shift, and their emission spectral peaks are generally located in the visible light range, typically excited by excitation light in the ultraviolet range. In this invention, quantum dots are excited by excitation light in the ultraviolet or near-ultraviolet bands. The organic fluorescent dyes have a smaller Stokes shift, and their emission spectra are generally in the visible light range, with their excitation spectra near the emission spectrum, also generally in the visible light range. In this invention, organic fluorescent dyes are excited by excitation light in the visible light band. The upconversion nanoparticles have an anti-Stokes shift, and their emission spectra are generally in the visible light range, but their excitation light is in the near-infrared range. In this invention, upconversion nanoparticles are excited by excitation light in the near-infrared band.

[0059] All quantum dots used in this invention were purchased from Thermo Fisher Scientific. A series of nanocrystals.

[0060] All fluorescent dyes used in this invention were purchased from ThermoFisher's Alexa. A series of fluorescent dyes.

[0061] The upconversion nanoparticles used in this invention were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0062] The antibodies used in this invention were purchased from Biolegend.

[0063] The imaging instrument used in this invention is a modified Olympus SLIDEVIEW VS200.

[0064] The technical solution of this application and how the technical solution of this application solves the above-mentioned technical problems are described in detail below with specific embodiments. These specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments. The embodiments of this application will now be described with reference to the accompanying drawings.

[0065] Example 1: Preparation of Samples Using Multiplex Immunohistochemistry / Immunofluorescence Techniques

[0066] In this embodiment, the antibodies corresponding to proteins that are highly expressed in the tissue are selected as follows: anti-GAPDH antibody, anti-α-actin antibody, anti-β-actin antibody, anti-α-tubulin antibody, anti-β-tubulin antibody, anti-transferrin antibody, anti-cytokeratin antibody, anti-lamin B1 antibody, anti-PCNA antibody, anti-SDHA antibody, and anti-histone antibody.

[0067] In this preferred embodiment, quantum dots, organic fluorescent dyes, and upconversion nanoparticles are selected as a fluorescent material combination for the antibody.

[0068] In this embodiment, a corresponding relationship is established between different fluorescent materials and different reactants. Preferably, different fluorescent materials are cross-linked with corresponding reactants through chemical cross-linking, bio-coupling, or physical cross-linking methods.

[0069] Among them, anti-GAPDH antibody, anti-α-actin antibody, anti-β-actin antibody, anti-α-tubulin antibody, and anti-β-tubulin antibody were labeled with Qdot 525, Qdot 565, Qdot 605, Qdot 655, and Qdot 705, respectively, according to the instructions; anti-transferrin antibody, anti-cytokeratin antibody, anti-lamin B1 antibody, anti-PCNA antibody, and anti-SDHA antibody were labeled with Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 635, and Alexa Fluor 680, respectively, according to the instructions; and anti-histone antibody was labeled with upconversion nanoparticles.

[0070] In this preferred embodiment, the sample pretreatment method includes the following steps:

[0071] FFPE-prepared 4μm thick paraffin tissue sections were dewaxed with xylene and then rehydrated with a gradient of 100%, 95%, 85%, and 75% ethanol.

[0072] Antigen retrieval was performed using a preheated epitope retrieval reagent. The antigen was then subjected to microwave heat retrieval for 15 min, followed by inactivation of endogenous catalase by incubation at room temperature with 3% H2O2 for 10 min.

[0073] Nonspecific epitopes were blocked by incubation at room temperature for 20 minutes using 10% sheep serum;

[0074] The primary antibody labeled with fluorescent material was placed in a refrigerator at 4°C overnight for treatment;

[0075] Wash with TBST for 5 min × 3 times. Sample preparation is complete.

[0076] The primary antibodies labeled with fluorescent materials are, respectively, an anti-GAPDH antibody labeled with Qdot 525, an anti-α-actin antibody labeled with Qdot 565, an anti-β-actin antibody labeled with Qdot 605, an anti-α-tubulin antibody labeled with Qdot 655, an anti-β-tubulin antibody labeled with Qdot 705, an anti-transferrin antibody labeled with Alexa Fluor 488, an anti-cytokeratin antibody labeled with Alexa Fluor 532, an anti-lamin B1 antibody labeled with Alexa Fluor 568, an anti-PCNA antibody labeled with Alexa Fluor 635, an anti-SDHA antibody labeled with Alexa Fluor 680, and an anti-histone antibody labeled with upconversion nanoparticles.

[0077] It should be noted that there are various methods for sample preparation. As long as the prepared sample uses the position of the emission spectrum peak and the co-coding of the excitation light during the imaging process, it falls within the protection scope of this invention.

[0078] The samples and fluorescent materials selected in this embodiment are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0079] Example 2: Encoding of Emission Spectral Peak Positions in Multiplex Immunohistochemistry / Immunofluorescence Samples

[0080] The samples prepared in Example 1 were imaged using a SLIDEVIEW VS200 multi-element fluorescence microscope. The imaging results are as follows:

[0081] Imaging was performed on the anti-transferrin antibody labeled with Alexa Fluor 488, the anti-cytokeratin antibody labeled with Alexa Fluor 532, the anti-lamin B1 antibody labeled with Alexa Fluor 568, the anti-PCNA antibody labeled with Alexa Fluor 635, and the anti-SDHA antibody labeled with Alexa Fluor 680. The imaging results are as follows: Figure 1As shown in A, Figure 1 From left to right in Figure A are the fluorescence emission spectra of the organic fluorescent dyes Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 635, and Alexa Fluor 680. We can encode the corresponding markers by the position of different emission spectral peaks of the organic fluorescent dyes, with a maximum of 5 codes being performed simultaneously.

[0082] Similarly, we imaged the anti-GAPDH antibody labeled Qdot 525, the anti-α-actin antibody labeled Qdot 565, the anti-β-actin antibody labeled Qdot 605, the anti-α-tubulin antibody labeled Qdot 655, and the anti-β-tubulin antibody labeled Qdot 705. The imaging results are as follows: Figure 1 As shown in B, Figure 1 From left to right in B are the fluorescence emission spectra of quantum dots Qdot 525, Qdot 565, Qdot 605, Qdot 655, and Qdot 705. We can encode the corresponding markers by the position of different emission spectral peaks of the quantum dots, with a maximum of 5 codes at the same time.

[0083] However, due to the overlap in the emission spectra of quantum dots and organic fluorescent dyes, such as Figure 1 As shown in C, although we can encode both organic fluorescent dyes and quantum dots in five ways based on the positional differences of their emission spectral peaks, in reality, due to the overlapping of the emission spectra of quantum dots and organic fluorescent dyes, we can only encode a maximum of five ways.

[0084] Example 3: Feasibility Analysis of Stokes Shift-Based Encoding of Multiplex Immunohistochemistry / Immunofluorescence Samples

[0085] The spectra of the fluorescent material on the sample prepared in Example 1 were analyzed, and the results are as follows: Figure 2 , Figure 3 As shown:

[0086] Figure 2 From left to right, A shows the fluorescence emission spectra (color blocks) and excitation spectra (dashed lines) of the organic fluorescent dyes Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 568, Alexa Fluor 635, and Alexa Fluor 680. Figure 2 B, from left to right, shows the fluorescence emission spectra (color blocks) and excitation spectra (dashed lines) of quantum dots Qdot 525, Qdot 565, Qdot 605, Qdot 655, and Qdot 705, respectively. Figure 3 The images show the excitation spectrum (dashed line) and emission spectrum (color blocks) of the upconversion nanoparticles. (Summary) Figure 2 and Figure 3 We can see that the emission spectra of organic fluorescent dyes, quantum dots, and upconversion particles overlap, but their excitation spectra are significantly different. The excitation light of organic fluorescent dyes is generally in the visible light range (400-800 nm), while the excitation light of quantum dots with the same emission wavelength is generally in the ultraviolet region (<400 nm), and the excitation light of upconversion nanoparticles with the same emission wavelength is generally in the infrared region (>800 nm). This is due to the different Stokes shifts or anti-Stokes shifts of organic fluorescent dyes, quantum dots, and upconversion particles.

[0087] We take organic fluorescent dyes Alexa Fluor 488 and quantum dot Qdot 525, Alexa Fluor 532 and quantum dot Qdot 565, Alexa Fluor 568 and quantum dot Qdot 605, Alexa Fluor 635 and quantum dot Qdot 655, and Alexa Fluor 680 and quantum dot Qdot 705 with similar or identical emission spectra as examples, through... Figure 4 Comparing the excitation light of these combinations reveals that, although their emission spectra are similar or overlap, the excitation light is significantly different, especially compared to the excitation light of upconversion nanoparticles. Figure 3 Significant differences were observed in all samples. This further demonstrates the feasibility of encoding using different excitation light when the emission spectral peaks of different fluorescent materials are at the same or similar positions.

[0088] Example 4: Co-coding of samples based on emission spectra and Stokes shifts using multiplex immunohistochemistry / immunofluorescence techniques

[0089] The samples prepared in Example 1 were imaged using a modified SLIDEVIEW VS200 multi-element fluorescence microscope. The modified SLIDEVIEW VS200 was equipped with a light source emitting ultraviolet light, a light source emitting infrared light, and lasers in the visible light range (488nm, 532nm, 561nm, 637nm, and 680nm lasers), as well as corresponding second filters: EVOS Light Cube, GFP 2.0, EVOS Light Cube, YFP 2.0, EVOS Light Cube, RFP 2.0, a custom filter (allowing light from 640-670nm to pass through), and EVOS Light Cube, Cy5 2.0. The imaging principle is as follows: Figure 5As shown, select the corresponding fluorescence channel, excite it with different excitation light, filter the emitted light with the second filter corresponding to the fluorescence channel, and then detect it with a detector. Record the corresponding excitation light, filter and detected emitted light, and determine the type of fluorescent material based on the above information. Based on the correspondence between fluorescent material and antibody, determine the type of antibody and protein.

[0090] In this embodiment, the co-encoding based on the positional differences of the emission spectral peaks and the differences in Stokes shifts of the various fluorescent materials in the fluorescent material combination includes the following steps:

[0091] Step S1: Establish corresponding relationships between different fluorescent materials and different reactants;

[0092] Step S2: React reactants labeled with different fluorescent materials with the sample to be tested;

[0093] Step S3: Select the corresponding fluorescence channel according to the type of fluorescent material, and the detection device will automatically switch to the second filter corresponding to the fluorescence channel for filtering;

[0094] Step S4: Excite different fluorescent materials with various excitation lights. The emission light of fluorescent materials whose emission spectral peaks are indistinguishable passes through the same second filter corresponding to the same fluorescence channel; the emission light of fluorescent materials whose emission spectral peaks are distinguishable passes through different second filters corresponding to different fluorescence channels.

[0095] Step S5: Detect the emitted light after it has passed through different second filters using a detector;

[0096] Step S6: Based on the results detected by the detector after filtering through different second filters, determine one or more fluorescent materials in the sample to be tested that are compatible with the same second filter;

[0097] Step S7: When there is only one fluorescent material adapted to the same second filter, the type and intensity of the fluorescent material can be determined by the first recoding, and the type and content of the reactants and target detectants corresponding to the fluorescent material can be further determined.

[0098] Step S8: Excite the several fluorescent materials adapted to the same second filter as described in step S6 with different excitation light; wherein, the several fluorescent materials adapted to the same second filter are fluorescent materials with the same or similar emission spectral peak positions;

[0099] Step S9: Use a detector to detect the emitted light after it has passed through the same second filter, and record the excitation light corresponding to these emitted lights;

[0100] Step S10: Based on different excitation light, emission light and / or second filter information, further determine the types of several fluorescent materials adapted to the same second filter, thereby further determining the types and contents of the reactants corresponding to the fluorescent materials and the target detectables.

[0101] Specific implementation methods are as follows Figure 6 As shown, the implementation methods of steps S1 and S2 are the same as those described in Example 1, with different filters representing the first level of coding and different Stokes shifts (represented by different excitation light) representing the second level of coding.

[0102] For EVOS Light Cube, GFP 2.0 filter (first-order coding):

[0103] Step S3: Select the fluorescence channel corresponding to the EVOS Light Cube filter, GFP 2.0;

[0104] Step S4: Excite the fluorescence labeled on the sample with ultraviolet light, lasers of different wavelengths, and infrared light;

[0105] Step S5: Detect the results after passing through the EVOS Light Cube, GFP 2.0 filter using a detector;

[0106] Step S6: The detector detected fluorescence under two different excitation lights after passing through the EVOS Light Cube, GFP 2.0 filter. There are two fluorescent materials in the sample that are compatible with the EVOS Light Cube, GFP 2.0 filter, so it is determined that a second encoding is required.

[0107] Step S8: Re-excite the fluorescent material labeled on the sample using ultraviolet light, lasers of different wavelengths, and infrared light;

[0108] Step S9: Detect the emitted light after passing through the EVOS Light Cube and GFP 2.0 filter using a detector, and record the excitation light (ultraviolet light, 488nm laser) corresponding to these emitted lights (second re-encoding);

[0109] Step S10: The fluorescent material that can be excited by ultraviolet light and whose emitted light can be filtered by the EVOS Light Cube and GFP 2.0 filter is Qdot 525. According to Example 1, Qdot 525 corresponds to an anti-GAPDH antibody containing the target analyte GAPDH protein, and according to... Figure 7The relative fluorescence intensity of the target analyte GAPDH protein is 33%; the fluorescent material that can be excited by a 488nm laser and whose emitted light can be filtered by an EVOS Light Cube and a GFP 2.0 filter is Alexa 488. According to Example 1, Alexa 488 corresponds to an anti-transferrin antibody containing the target analyte transferrin, and according to... Figure 7 The relative fluorescence intensity of the target analyte, transferrin, was 35%.

[0110] For EVOS Light Cube, YFP 2.0 filter (first-order encoding):

[0111] Step S3: Select the fluorescence channel corresponding to the EVOS Light Cube, YFP 2.0 filter;

[0112] Step S4: Excite the fluorescence labeled on the sample with ultraviolet light, lasers of different wavelengths, and infrared light;

[0113] Step S5: Use a detector to detect the result after passing through the EVOS Light Cube, YFP 2.0 filter;

[0114] Step S6: The detector detected fluorescence under two different excitation lights after passing through the EVOS Light Cube, YFP 2.0 filter. There are two fluorescent materials in the sample that are compatible with the EVOS Light Cube, YFP 2.0 filter, so it is determined that a second encoding is required.

[0115] Step S8: Re-excite the fluorescent material labeled on the sample using ultraviolet light, lasers of different wavelengths, and infrared light;

[0116] Step S9: Use a detector to detect the emitted light after it has passed through the EVOS Light Cube and YFP 2.0 filter, and record the excitation light (ultraviolet light, 532nm laser) corresponding to these emitted lights (second re-encoding);

[0117] Step S10: The fluorescent material that can be excited by ultraviolet light and whose emitted light can be filtered by the EVOS Light Cube YFP 2.0 filter is Qdot 565. According to Example 1, Qdot 565 corresponds to an anti-α-actin antibody, containing the target analyte α-actin, and according to... Figure 7The relative fluorescence intensity of the target analyte α-actin was 54%; the fluorescent material that can be excited by a 532nm laser and whose emitted light can be filtered by the EVOS Light Cube, YFP 2.0 filter is Alexa532. According to Example 1, Alexa 532 corresponds to an anti-keratin antibody containing the target analyte keratin, and according to... Figure 7 The relative fluorescence intensity of the target molecule, keratin, was 35%.

[0118] For EVOS Light Cube, RFP 2.0 filter (first-order encoding):

[0119] Step S3: Select the fluorescence channel corresponding to the EVOS Light Cube, RFP 2.0 filter;

[0120] Step S4: Excite the fluorescence labeled on the sample with ultraviolet light, lasers of different wavelengths, and infrared light;

[0121] Step S5: Use a detector to detect the result after passing through the EVOS Light Cube, RFP 2.0 filter;

[0122] Step S6: The detector detected fluorescence under two different excitation lights after passing through the EVOS Light Cube, RFP 2.0 filter. There are two fluorescent materials in the sample that are compatible with the EVOS Light Cube, RFP 2.0 filter, so it is determined that a second encoding is required.

[0123] Step S8: Re-excite the fluorescent material labeled on the sample using ultraviolet light, lasers of different wavelengths, and infrared light;

[0124] Step S9: Use a detector to detect the emitted light after it has passed through the EVOS Light Cube and the RFP 2.0 filter, and record the excitation light (ultraviolet light, 561nm laser) corresponding to these emitted lights (second re-encoding);

[0125] Step S10: The fluorescent material that can be excited by ultraviolet light and whose emitted light can be filtered by the EVOS Light Cube RFP 2.0 filter is Qdot 605. According to Example 1, Qdot 605 corresponds to an anti-β-actin antibody, containing the target analyte β-actin, and according to... Figure 7The relative fluorescence intensity of the target analyte β-actin was 64%; the fluorescent material that can be excited by a 561nm laser and whose emitted light can be filtered by an EVOS Light Cube RFP 2.0 filter is Alexa568. According to Example 1, Alexa 568 corresponds to an anti-lamin B1 antibody, containing the target analyte laminin B1, and according to... Figure 7 The relative fluorescence intensity of the target substance, laminin B1, was 32%.

[0126] For custom-designed filters (allowing light to pass through 640-670nm) (first-level coding):

[0127] Step S3: Select the fluorescence channel corresponding to the customized filter (allowing light to pass through 640-670nm);

[0128] Step S4: Excite the fluorescence labeled on the sample with ultraviolet light, lasers of different wavelengths, and infrared light;

[0129] Step S5: Use a detector to detect the result after filtering with a custom filter (allowing light to pass through at 640-670nm);

[0130] Step S6: After the detector detects the light filtered by the custom filter (allowing light to pass through 640-670nm), fluorescence is detected under three different excitation lights. There are two fluorescent materials in the sample that are compatible with the custom filter (allowing light to pass through 640-670nm), so it is determined that a second encoding is required.

[0131] Step S8: Re-excite the fluorescent material labeled on the sample using ultraviolet light, lasers of different wavelengths, and infrared light;

[0132] Step S9: Use a detector to detect the emitted light after it has passed through a custom filter (allowing light from 640-670nm to pass through), and record the excitation light (ultraviolet light, 637nm laser, infrared light) corresponding to these emitted lights (secondary encoding).

[0133] Step S10: The fluorescent material that can be excited by ultraviolet light and whose emitted light can be filtered by a custom filter (allowing light of 640-670nm to pass through) is Qdot 655. According to Example 1, Qdot 655 corresponds to an anti-α-tubulin antibody containing the target analyte α-tubulin, and according to... Figure 7The relative fluorescence intensity of the target analyte α-tubulin was 27%; the fluorescent material that can be excited by a 637nm laser and whose emitted light can be filtered by a custom filter (allowing light from 640-670nm) is Alexa 635. According to Example 1, Alexa 635 corresponds to an anti-PCNA antibody containing the target analyte PCNA, and according to... Figure 7 The relative fluorescence intensity of the target analyte PCNA is 53%; the fluorescent material that can be excited by infrared light and whose emitted light can be filtered by a custom-designed filter (allowing light of 640-670nm) is upconversion nanoparticles. According to Example 1, the upconversion nanoparticles correspond to an anti-histone antibody containing the target analyte histone, and according to... Figure 7 The relative fluorescence intensity of the target histone was 57%.

[0134] For EVOS Light Cube, Cy5 2.0 filter (first-order encoding):

[0135] Step S3: Select the fluorescence channel corresponding to the EVOS Light Cube, Cy5 2.0 filter;

[0136] Step S4: Excite the fluorescence labeled on the sample with ultraviolet light, lasers of different wavelengths, and infrared light;

[0137] Step S5: Use a detector to detect the result after filtering with EVOS Light Cube, Cy5 2.0;

[0138] Step S6: The detector detected fluorescence under two different excitation lights after passing through the EVOS Light Cube, Cy5 2.0 filter. There are two fluorescent materials in the sample that are compatible with the EVOS Light Cube, Cy5 2.0 filter, so it is determined that a second encoding is required.

[0139] Step S8: Re-excite the fluorescent material labeled on the sample using ultraviolet light, lasers of different wavelengths, and infrared light;

[0140] Step S9: Use a detector to detect the emitted light after it has passed through the EVOS Light Cube and Cy5 2.0 filter, and record the excitation light (ultraviolet light, 680nm laser) corresponding to these emitted lights (second re-encoding);

[0141] Step S10: The fluorescent material that can be excited by ultraviolet light and whose emitted light can be filtered by the EVOS Light Cube Cy5 2.0 filter is Qdot 705. According to Example 1, Qdot 705 corresponds to an anti-β-tubulin antibody, containing the target analyte β-tubulin, and according to... Figure 7 The relative fluorescence intensity of the target analyte β-tubulin was 36%; the fluorescent material that can be excited by a 680nm laser and whose emitted light can be filtered by the EVOS Light Cube Cy5 2.0 filter is Alexa680. According to Example 1, Alexa 680 corresponds to an anti-SDHA antibody containing the target analyte SDHA, and according to... Figure 7 The relative fluorescence intensity of the target analyte SDHA was 19%.

[0142] This invention also relates to a detection device for fluorescent markers. The detection device includes an excitation light source, comprising excitation light generated by multiple light sources with narrow wavelength ranges and / or excitation light generated by light sources with broad wavelength ranges; the excitation light source includes at least two or more of the ultraviolet, visible, and infrared wavelength ranges. The detection device also includes filters, comprising a second filter group and / or a first filter group; the second filter group is used to filter the emission light of the fluorescent material; the first filter group is used to filter the excitation light generated by the broad wavelength range light source to obtain excitation light with multiple specific wavelength ranges. The detection device also includes a detector, which is used to detect the emission light of the fluorescent material after excitation by the excitation light and filtering by the second filter group.

[0143] The detection equipment can support the detection method shown in the above embodiments and achieve the corresponding technical effects.

[0144] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for detecting a fluorescent marker, characterized in that, The detection method includes co-coding based on the differences in the positional differences of the emission spectral peaks and the differences in the Stokes shifts of various fluorescent materials in the fluorescent material combination; The encoding based on the positional differences of the emission spectral peaks of each fluorescent material in the fluorescent material combination includes a first re-encoding of the positions of different emission spectral peaks of different fluorescent materials. If the position of each distinguishable emission spectral peak corresponds to a specific target analyte, then the first re-encoding is performed; if the position of each distinguishable emission spectral peak corresponds to at least two specific target analytes, then the second re-encoding is performed. The encoding based on the differences in Stokes shifts of various fluorescent materials in the fluorescent material combination includes a second encoding of fluorescent materials with the same or similar emission spectrum peak positions using different Stokes shifts, different anti-Stokes shifts, or the difference between Stokes shifts and anti-Stokes shifts. The first re-encoding of the positions of different emission spectral peaks of different fluorescent materials includes the following steps: Step S1: Establish corresponding relationships between different fluorescent materials and different reactants; Step S2: React reactants labeled with different fluorescent materials with the sample to be tested; Step S3: Select the corresponding fluorescence channel according to the type of fluorescent material, and the detection device will automatically switch to the second filter corresponding to the fluorescence channel for filtering; Step S4: Excite different fluorescent materials with various excitation lights. The emission light of fluorescent materials whose emission spectral peaks are indistinguishable passes through the same second filter corresponding to the same fluorescence channel; the emission light of fluorescent materials whose emission spectral peaks are distinguishable passes through different second filters corresponding to different fluorescence channels. Step S5: Detect the emitted light after it has passed through different second filters using a detector; Step S6: Based on the results detected by the detector after filtering through different second filters, determine one or more fluorescent materials in the sample to be tested that are compatible with the same second filter; Step S7: When there is only one fluorescent material adapted to the same second filter, the type and intensity of the fluorescent material can be determined by the first recoding, and the type and content of the reactants and target detectants corresponding to the fluorescent material can be further determined.

2. The detection method as described in claim 1, characterized in that, The fluorescent material combination includes combinations of inorganic fluorescent materials, combinations of organic fluorescent materials, or combinations of the inorganic fluorescent materials and the organic fluorescent materials.

3. The detection method as described in claim 2, characterized in that, In the fluorescent material combination, at least one fluorescent material has a Stokes shift difference of more than 50 nm from the Stokes shift of the other fluorescent materials; and / or at least one fluorescent material has an anti-Stokes shift, which is different from the Stokes shift of the other fluorescent materials.

4. The detection method as described in claim 2, characterized in that, The inorganic fluorescent material includes at least one or more of quantum dots and upconversion nanoparticles.

5. The detection method as described in claim 2, characterized in that, The organic fluorescent material includes at least one or more of the following: organic fluorescent dyes, organic small molecule luminescent materials, organic polymer luminescent materials, and fluorescent proteins.

6. The detection method as described in claim 2, characterized in that, The fluorescent material combination is any combination of two or three of the following: quantum dots, organic fluorescent dyes, and upconversion nanoparticles.

7. The detection method as described in claim 1, characterized in that, The second encoding of fluorescent materials with emission spectral peaks at positions that are the same or similar, using different Stokes shifts or different anti-Stokes shifts, includes the following steps: Step S8: Excite several fluorescent materials in the sample to be tested described in step S6 that are adapted to the same second filter with different excitation light; wherein, the several fluorescent materials adapted to the same second filter are fluorescent materials with the same or similar emission spectral peak positions; Step S9: Use a detector to detect the emitted light after it has passed through the same second filter, and record the excitation light corresponding to these emitted lights; Step S10: Based on different excitation light, emission light and / or second filter information, further determine the types of several fluorescent materials adapted to the same second filter, thereby further determining the types and contents of the reactants and target detectants corresponding to the fluorescent materials.

8. The detection method according to any one of claims 1 and 7, characterized in that, The target analytes include one or more of the following: proteins, nucleic acids, lipids, small molecule compounds, cells, and extracellular vesicles.

9. The detection method as described in claim 8, characterized in that, The reactants include one or more of antibodies, nucleic acids, and ligands that can specifically react with the target detectable.

10. The detection method as described in claim 1, characterized in that, Establishing corresponding relationships between different fluorescent materials and different reactants includes cross-linking different fluorescent materials with corresponding reactants through chemical cross-linking, bio-coupling, or physical cross-linking methods.

11. The detection method according to any one of claims 1 and 7, characterized in that, The detection method is applied in the fields of immunohistochemistry and / or immunofluorescence.

12. A detection device for fluorescent markers, characterized in that, The detection equipment includes detection using the detection method as described in any one of claims 1 to 11.

13. The detection device as described in claim 12, characterized in that, The detection device includes an excitation light source, which includes at least two or more of the ultraviolet light band, visible light band, and infrared light band.

14. The detection device as described in claim 13, characterized in that, The detection device further includes filters, which include a second filter group and / or a first filter group; the second filter group is used to filter the emitted light of the fluorescent material; the first filter group is used to filter the excitation light generated by the light source with a wide wavelength range to obtain excitation light with multiple specific wavelength ranges.

15. The detection device as described in claim 14, characterized in that, The detection device further includes a detector, which is used to detect the emitted light of the fluorescent material after it has been excited by the excitation light and filtered by the second filter group.

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