Multicolor biological imaging reagent based on triple-bond Raman as well as preparation method and application of multicolor biological imaging reagent

Abstract

The invention belongs to the field of biological imaging reagents, and relates to a multicolor biological imaging reagent based on triple bond Raman as well as a preparation method and application of the multicolor biological imaging reagent. The monomers 1-7 are respectively subjected to copolymerization reaction with (i) acrylic acid or (ii) styrene and acrylic acid in an aqueous solvent in the presence of an emulsifier and an initiator to form nanospheres with the particle size of 10-100nm; the preparation method comprises the following steps: activating carboxyl groups on the surfaces of nanospheres by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and then connecting the carboxyl groups on the surfaces of the nanospheres with amino groups of targeting molecules by using N-hydroxysuccinimide ester to obtain the Raman probe. A plurality of Raman probes with different Raman characteristic peaks are mixed together to obtain the multicolor biological imaging reagent. Compared with the existing Raman bead multicolor imaging reagent, the multicolor biological imaging reagent provided by the invention is easier to permeate tissues and be taken by cells, and has a higher signal-to-background ratio, so that the high definition of biological imaging is ensured. The preparation method disclosed by the invention is simple, mild in condition, environment-friendly and easy for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of biological imaging reagents, and relates to a multicolor biological imaging reagent based on triple bond Raman, and a preparation method and application thereof. Background Art

[0002] Bioimaging technology has important applications in biomedical research and clinical diagnosis, providing structural and functional information at the tissue, cellular, and molecular levels within the body. However, existing bioimaging reagents have several limitations. For one thing, many reagents have low signal-to-background noise ratios, resulting in unclear imaging results and difficulty accurately distinguishing target structures. Furthermore, single-color imaging reagents cannot simultaneously label and image multiple biomolecules or cell types, limiting comprehensive analysis of complex biological processes.

[0003] Existing multicolor imaging technologies such as fluorescence and mass spectrometry face major challenges. The repeated antigen repair process is cumbersome and faces the risk of antigen destruction. The subsequent data processing processes such as spectral splitting are complex. In particular, existing technologies face a monopoly on core technologies of imported equipment, and the instruments are expensive and have high costs of use and maintenance. Therefore, the development of new multicolor imaging technologies is of great significance.

[0004] In recent years, Raman spectroscopy, as a label-free, highly specific imaging method, has garnered widespread attention due to its ability to provide chemical information at the molecular level. However, traditional Raman imaging suffers from issues such as weak signal and strong background interference, limiting its widespread application in the biomedical field. Existing Raman bead multicolor imaging techniques utilize relatively large Raman beads, which makes tissue penetration and cellular uptake difficult, thus limiting their bioimaging capabilities. Furthermore, the limited number of distinguishable spectral channels further restricts the widespread application of this technology in the field of bioimaging. Summary of the Invention

[0005] The present invention provides a multicolor bioimaging reagent based on triple-bond Raman technology, as well as its preparation method and application. This multicolor bioimaging reagent comprises Raman beads with a particle size of less than 100 nm. These beads easily penetrate tissue and are taken up by cells, significantly enhancing the effectiveness of bioimaging. Furthermore, the multicolor bioimaging reagent of the present invention enables more precise Raman shift control and possesses stronger spectral orthogonality, effectively reducing signal crosstalk during multicolor imaging. Furthermore, the reagent also exhibits a high signal-to-background ratio, ensuring high-definition bioimaging.

[0006] The first aspect of the present invention provides a method for preparing a multicolor biological imaging reagent based on triple bond Raman, comprising:

[0007] Monomers 1 to 7 are copolymerized with (i) acrylic acid or (ii) styrene and acrylic acid in an aqueous solvent in the presence of an emulsifier and an initiator to form nanospheres with a particle size of 10 to 100 nm. The structural formulas of monomers 1 to 7 are as follows:

[0008] ;

[0009] The carboxyl groups on the surface of the nanospheres were activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and then N-hydroxysuccinimide (NHS) was used to link the carboxyl groups on the surface of the nanospheres with the amino groups of the target molecules to obtain Raman probes.

[0010] Multiple Raman probes with different Raman characteristic peaks are mixed together to obtain a multi-color biological imaging reagent.

[0011] In combination with the first aspect of the present invention, in some embodiments, the mass ratio of each monomer to acrylic acid is 2~5:1; and / or, the mass ratio of each monomer to styrene and acrylic acid is 2~5:1.3~2:1; and / or, the mass ratio of each monomer, emulsifier and initiator is 10~30:1:1~4; and / or, the concentration of each monomer in the aqueous solvent is 10~50g / L.

[0012] In conjunction with the first aspect of the present invention, in some embodiments, the average particle size of the nanospheres is 40-60 nm; and / or the multi-color bioimaging reagent has a 2160 cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 The Raman characteristic peak at .

[0013] In combination with the first aspect of the present invention, in some embodiments, the emulsifier is sodium dodecyl sulfate or sodium dodecylbenzene sulfonate; and / or the initiator is potassium persulfate or azobisisobutyronitrile; and / or the temperature of the copolymerization reaction is 70±5°C; and / or the copolymerization reaction is carried out under anaerobic conditions.

[0014] In combination with the first aspect of the present invention, in some embodiments, after the copolymerization reaction, the further steps include: removing unreacted monomers and emulsifiers by dialysis to obtain an aqueous dispersion of nanospheres; activating the carboxyl groups on the surface of the nanospheres using EDC, and then linking the carboxyl groups on the surface of the nanospheres to the amino groups of the targeting molecule using NHS, including: adding EDC to the aqueous dispersion of nanospheres at room temperature to activate the carboxyl groups on the surface of the nanospheres; adding NHS to the aqueous dispersion of nanospheres and stirring for at least 30 minutes, and then adding the targeting molecule to react for at least 2 hours.

[0015] In conjunction with the first aspect of the present invention, in some embodiments, dialysis uses a dialysis membrane with a pore size of 8-14 kD.

[0016] In combination with the first aspect of the present invention, in some embodiments, the targeting molecule is one or more of α-smooth muscle actin (α-SMA) antibody, cytokeratin 18 (CK-18) antibody, vimentin antibody, cluster of differentiation 163 (CD163) antibody, and cluster of differentiation 34 (CD34) antibody.

[0017] The second aspect of the present invention provides a triple bond Raman-based multicolor biological imaging reagent prepared by the above preparation method.

[0018] The third aspect of the present invention provides the application of the above-mentioned triple bond Raman-based multicolor biological imaging reagent in the field of biological imaging.

[0019] A fourth aspect of the present invention provides a biological imaging method comprising the following steps:

[0020] The triple-bond Raman-based multicolor bioimaging reagent is incubated with the biological sample for at least 2 hours, and then washed to remove unbound Raman probes;

[0021] The labeled biological samples were imaged using a Raman spectrometer at 2160 cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 The Raman signal at is the characteristic spectral band.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] 1. Compared with existing Raman bead multicolor imaging reagents, the multicolor bioimaging reagent provided by the present invention is easier to penetrate tissues and be taken up by cells, thereby significantly improving the effect of bioimaging; at the same time, the multicolor bioimaging reagent also has a high signal-to-background ratio, ensuring high-definition bioimaging.

[0024] 2. The preparation method of the present invention is simple, mild in conditions, environmentally friendly, and easy to scale up for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 Raman spectra of monomers 1 to 7.

[0027] Figure 2 These are transmission electron micrographs of the nanospheres prepared in Example 2 of the present invention, where a is a nanosphere formed by polymerization of monomer 2; b is a nanosphere formed by polymerization of monomer 3; c is a nanosphere formed by polymerization of monomer 5; d is a nanosphere formed by polymerization of monomer 6; and e is a nanosphere formed by polymerization of monomer 7.

[0028] Figure 3 These are the Raman spectra of each monomer before and after polymerization in Example 2 of the present invention, where M1 is the Raman spectrum of monomer 2; P1 is the Raman spectrum of the nanospheres of monomer 2; M2 is the Raman spectrum of monomer 3; P2 is the Raman spectrum of the nanospheres of monomer 3; M3 is the Raman spectrum of monomer 5; P3 is the Raman spectrum of the nanospheres of monomer 5; M4 is the Raman spectrum of monomer 6; P4 is the Raman spectrum of the nanospheres of monomer 6; M5 is the Raman spectrum of monomer 7; and P5 is the Raman spectrum of the nanospheres of monomer 7.

[0029] Figure 4 These are infrared spectra of the monomers before and after polymerization in Example 2 of the present invention, where M1 is the infrared spectrum of monomer 2; P1 is the infrared spectrum of the nanospheres of monomer 2; M2 is the infrared spectrum of monomer 3; P2 is the infrared spectrum of the nanospheres of monomer 3; M3 is the infrared spectrum of monomer 5; P3 is the infrared spectrum of the nanospheres of monomer 5; M4 is the infrared spectrum of monomer 6; P4 is the infrared spectrum of the nanospheres of monomer 6; M5 is the infrared spectrum of monomer 7; and P5 is the infrared spectrum of the nanospheres of monomer 7.

[0030] Figure 5a1 is the particle size diagram of a monomer 2 nanosphere;

[0031] Figure 5a2 This is the particle size diagram of the antibody modified with monomeric 2 nanospheres;

[0032] Figure 5b1 is the particle size diagram of a monomer 3 nanosphere;

[0033] Figure 5b2 This is the particle size diagram of the antibody modified with monomeric 3 nanospheres;

[0034] Figure 5c1 is the particle size diagram of a single 5-nanometer sphere;

[0035] Figure 5c2 This is the particle size diagram of the monomeric 5-nanometer sphere modified with antibody;

[0036] Figure 5d1 is the particle size diagram of a single 6-nanometer sphere;

[0037] Figure 5d2 This is the particle size diagram of the antibody modified with monomeric 6-nanometer spheres;

[0038] Figure 5e1 is the particle size diagram of a single 7-nanometer sphere;

[0039] Figure 5e2 This is the particle size diagram of the monomeric 7-nanometer sphere modified antibody.

[0040] Figure 6 This is the zeta potential diagram before and after the antibody is modified with 3 nanospheres in Example 3 of the present invention, where P1 is the zeta potential of a monomer 2 nanosphere, and α-SMA-P1 is the zeta potential after the monomer 2 nanospheres are modified with the α-SMA antibody; P2 is the zeta potential of a monomer 3 nanosphere, and CK-18-P2 is the zeta potential after the monomer 3 nanospheres are modified with the CK-18 antibody; P3 is the zeta potential of a monomer 5 nanosphere, and Vimentin-P3 is the zeta potential after the monomer 5 nanospheres are modified with the vimentin antibody; P4 is the zeta potential of a monomer 6 nanosphere, and CD163-P4 is the zeta potential after the monomer 6 nanospheres are modified with the CD163 antibody; P5 is the zeta potential of a monomer 7 nanosphere, and CD34-P5 is the zeta potential after the monomer 7 nanospheres are modified with the CD34 antibody.

[0041] Figure 7 This is a partial monochromatic imaging image of the Raman probe fluorescence and Raman contrast prepared in Example 3 of the present invention; wherein:

[0042] a1-a5 are a set of images of smooth muscle labeled with α-SMA antibody and compared with fluorescence labeling of monomeric 2-nanosphere modified smooth muscle: a1 is the overall fluorescence image, a2 is the overall Raman image, a3 is the local Raman image, a4 is the local fluorescence image, and a5 is the local Raman fluorescence superposition image;

[0043] b1 to b5 are a set of images of epithelial tumor cells labeled with CK-18 antibodies modified with monomeric 3-nanospheres and compared with fluorescence-labeled ones: b1 is the overall fluorescence image, b2 is the overall Raman image, b3 is the local Raman image, b4 is the local fluorescence image, and b5 is the local Raman-fluorescence superposition image;

[0044] C1 to C5 are a set of images of fibroblasts in the stroma labeled with monomeric 5-nanometer sphere-modified vimentin antibody and compared with fluorescently labeled images: C1 is the overall fluorescence image, C2 is the overall Raman image, C3 is the local Raman image, C4 is the local fluorescence image, and C5 is the local Raman-fluorescence superposition image.

[0045] d1 to d5 are a set of images of single cells labeled with 6-nanosphere-modified CD163 antibodies and compared with fluorescently labeled cells: d1 is the overall fluorescence image, d2 is the overall Raman image, d3 is the local Raman image, d4 is the local fluorescence image, and d5 is the local Raman-fluorescence superposition image;

[0046] e1~e5 are a set of pictures of vascular endothelium labeled with monomeric 7-nanometer balls modified with CD34 antibodies and contrast fluorescent labels: e1 is the overall fluorescence image, e2 is the overall Raman image, e3 is the local Raman image, e4 is the local fluorescence image, and e5 is the local Raman fluorescence superposition image.

[0047] Figure 8 These are two-color and three-color imaging images of the Raman probe fluorescence and Raman contrast prepared in Example 3 of the present invention, where a1 is a two-color fluorescence image; a2 is a two-color Raman image; b1 is a three-color fluorescence image; and b2 is a three-color Raman image.

[0048] Figure 9a1 2160cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman imaging of specific Raman signals;

[0049] Figure 9a2 The corresponding 2160 cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman spectra;

[0050] Figure 9b1 2186 cm is selected for the five-color Raman imaging in Example 5 of the present invention. -1 Raman imaging of specific Raman signals;

[0051] Figure 9b2 The corresponding 2186 cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman spectra;

[0052] Figure 9c1 2227 cm is selected for the five-color Raman imaging in Example 5 of the present invention. -1 Raman imaging of specific Raman signals;

[0053] Figure 9c2 In the five-color Raman imaging of Example 5 of the present invention, the corresponding 2227 cm -1 Raman spectra;

[0054] Figure 9d1 2241 cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman imaging of specific Raman signals;

[0055] Figure 9d2 The corresponding 2241 cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman spectra;

[0056] Figure 9e1 2260cm is selected for the five-color Raman imaging in Example 5 of the present invention. -1 Raman imaging of specific Raman signals;

[0057] Figure 9e2 The corresponding 2260 cm is selected in the five-color Raman imaging of Example 5 of the present invention. -1 Raman spectrum. DETAILED DESCRIPTION

[0058] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0059] For simplicity, the present invention only explicitly discloses certain numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range. Similarly, any upper limit can be combined with any other upper limit to form an unspecified range. In addition, although not explicitly stated, each point or individual value between the endpoints of a range is included in the range. Thus, each point or individual value can serve as its own lower limit or upper limit and be combined with any other point or individual value, or with other lower limits or upper limits, to form an unspecified range.

[0060] It should be noted that, in the description of the present invention, unless otherwise specified, “above” and “below” are inclusive, and the meaning of “multiple” in “one or more” is two or more. Relational terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms “include”, “comprising” or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence “comprising one…” do not exclude the presence of other identical elements in the process, method, article or device comprising the elements.

[0061] In the description of the present invention, the description with reference to the terms "any embodiment / method", "one embodiment / method", "some embodiments / methods", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in an appropriate manner in any one or more embodiments / methods or examples. In addition, those skilled in the art may combine and combine different embodiments / methods or examples and features of different embodiments / methods or examples described in this specification, unless they are contradictory.

[0062] In the description of the present invention, the term "high signal-to-background ratio" refers to a high ratio of signal to background, which means that the signal intensity is much greater than the background signal intensity, which helps to improve the sensitivity and accuracy of detection and is one of the key indicators for measuring the performance of imaging technology or detection methods.

[0063] In the description of the present invention, the term "biological silent region" refers to the 1800-2800 cm -1 In this region, the Raman signal of biomolecules is very weak and there is almost no background interference. Selecting probe molecules with characteristic signals in the biological silent zone can significantly reduce background interference and improve the signal-to-background ratio of detection, thereby achieving more sensitive and specific biological imaging or analysis.

[0064] In the description of the present invention, the term "Raman characteristic peak" refers to the specific wave number (cm2) generated by the molecular vibration or rotation energy level transition of a substance in the Raman spectrum. -1 The position, shape, and intensity of these peaks reflect the vibrational modes of chemical bonds or functional groups in the molecule and serve as the "fingerprint" of the substance's chemical structure.

[0065] The above summary of the invention is not intended to describe every disclosed embodiment or every implementation of the present invention. The following description more specifically illustrates exemplary embodiments. These embodiments can be used in various combinations. In each example, the examples are listed only as representative groups and should not be construed as exhaustive.

[0066] The present invention provides a method for preparing a multicolor biological imaging reagent based on triple bond Raman, comprising:

[0067] Monomers 1 to 7 are copolymerized with (i) acrylic acid or (ii) styrene and acrylic acid in an aqueous solvent in the presence of an emulsifier and an initiator to form nanospheres with a particle size of 10 to 100 nm. The structural formulas of monomers 1 to 7 are as follows:

[0068] ;

[0069] The carboxyl groups on the surface of the nanospheres were activated by EDC, and then NHS was used to connect the carboxyl groups on the surface of the nanospheres with the amino groups of the targeting molecules to obtain Raman probes.

[0070] Multiple Raman probes with different Raman characteristic peaks are mixed together to obtain a multi-color biological imaging reagent.

[0071] As shown in the structural formulas of monomers 1-7, all monomers contain ethylene bonds. During the copolymerization process, the ethylene bonds in the monomers participate in the reaction, allowing the monomers to self-polymerize into nanospheres. Alternatively, they can polymerize with (i) the ethylene bonds in acrylic acid or (ii) the ethylene bonds in acrylic acid and styrene, forming nanospheres copolymerized with monomer + acrylic acid or monomer + acrylic acid + styrene. These ethylene bonds polymerize to form the polymer backbone, while the triple bonds in the monomers, the carboxyl groups in acrylic acid, and the phenyl groups in styrene serve as side chains. The resulting nanospheres are enriched with numerous carboxyl groups, imparting excellent hydrophilicity, ensuring uniform dispersion in aqueous solvents and providing reliable anchor points for subsequent targeted molecular modification. The introduction of styrene ensures uniformity of the polymer backbone structure across the nanospheres, resulting in a uniform spherical shape and a uniformly enhanced Raman intensity.

[0072] like Figure 1 As shown, monomers 1 to 7 have different Raman characteristic peaks, although the Raman characteristic peak of monomer 1 is 2154 cm -1 The Raman characteristic peak of monomer 2 is 2160 cm -1 , the Raman characteristic peak of monomer 4 is 2214 cm -1 The Raman characteristic peak of monomer 5 is 2227 cm -1 There is a significant band overlap, but it can still be distinguished. In order to improve the recognition of characteristic peaks, the following five monomers are preferably used to prepare nanospheres: -1 Characteristic peaks of monomer 2, 2186 cm -1 Characteristic peaks of monomer 3, 2227cm -1 Characteristic peaks of monomer 5, 2241cm -1 Characteristic peaks of monomer 6 and 2260 cm -1 The monomers 7 with characteristic peaks were used to construct nanospheres with unique Raman fingerprint signals.

[0073] In some embodiments, the mass ratio of each monomer to acrylic acid is 2-5:1; and / or the mass ratio of each monomer to styrene and acrylic acid is 2-5:1.3-2:1; and / or the mass ratio of each monomer to emulsifier and initiator is 10-30:1:1-4; and / or the concentration of each monomer in the aqueous solvent is 10-50 g / L. By controlling the ratios and concentrations of monomers, initiator, emulsifier, and acrylic acid within the above ranges, the nanospheres can be regulated to have a particle size between 10 and 100 nm.

[0074] In some embodiments, the average particle size of the nanospheres is 40-60 nm; and / or the multi-color bio-imaging reagent has a 2160 cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 Nanospheres of this size are more easily taken up by cells, demonstrating excellent performance in bioimaging applications. Furthermore, the nanospheres disperse evenly in water and are less likely to aggregate, ensuring their stability and durability in vivo. The carboxyl groups on their surfaces also offer the potential for binding to biomolecules, enabling them to serve as multicolor bioimaging reagents, playing an important role in biomedical research and diagnosis.

[0075] In some embodiments, the emulsifier is sodium dodecyl sulfate or sodium dodecylbenzene sulfonate; and / or the initiator is potassium persulfate or azobisisobutyronitrile; and / or the copolymerization reaction temperature is 70±5°C; and / or the copolymerization reaction is carried out in the absence of oxygen. These specific emulsifier and initiator selections help control the polymerization process, thereby further tailoring the properties of the nanospheres. Sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, as emulsifiers, effectively reduce the surface tension of the system, promote the dispersion of the monomers in the aqueous phase, and facilitate the formation of nanospheres with uniform particle size and stable dispersion. Potassium persulfate and azobisisobutyronitrile, as initiators, effectively decompose at a set temperature to generate free radicals, initiating the polymerization reaction. Furthermore, controlling the copolymerization temperature within the range of 70±5°C optimizes the balance between reaction rate and nanosphere quality. Furthermore, conducting the copolymerization reaction in the absence of oxygen avoids the quenching of free radicals by oxygen, thereby improving polymerization efficiency and product purity. The combined effect of these conditions results in the prepared nanospheres having an ideal particle size distribution and excellent dispersion stability.

[0076] In some embodiments, after the copolymerization reaction, the following steps are further performed: removing unreacted monomers and emulsifiers by dialysis to obtain an aqueous dispersion of nanospheres; activating the carboxyl groups on the surface of the nanospheres using EDC, and then linking the carboxyl groups on the surface of the nanospheres to the amino groups of the targeting molecule using NHS, including: adding EDC to the aqueous dispersion of nanospheres at room temperature to activate the carboxyl groups on the surface of the nanospheres; adding NHS to the aqueous dispersion of nanospheres and stirring for at least 30 minutes, then adding the targeting molecule and reacting for at least 2 hours; and removing unattached targeting molecules and excess EDC and NHS by centrifugation to obtain a Raman probe. This surface modification not only improves the biocompatibility of the nanospheres but also imparts active targeting capabilities, enabling the nanospheres to more accurately reach the target location and improving the utilization efficiency of the Raman probe.

[0077] In some embodiments, dialysis uses a dialysis membrane with a pore size of 8-14 kD, which can remove small molecule unreacted products and retain the nanospheres, thereby obtaining a pure aqueous dispersion of nanospheres and preventing unreacted products from affecting the surface modification of the nanospheres.

[0078] In some embodiments, the targeting molecule is one or more of α-SMA antibody, CK-18 antibody, Vimentin antibody, CD163 antibody, and CD34 antibody. Each of these targeting molecules has a specific biological recognition ability and can specifically bind to the target cell or tissue. For example, α-SMA antibody is often used to mark smooth muscle cells, CK-18 antibody is often used to mark epithelial cells, Vimentin antibody can recognize a variety of mesenchymal cells, and CD163 and CD34 antibodies have high affinity for macrophages and vascular endothelial cells, respectively. By selecting specific targeting molecules, Raman probes can be designed for specific diseases or tissue types, thereby achieving more accurate disease diagnosis or treatment monitoring.

[0079] The triple-bond Raman-based multicolor biological imaging reagent is prepared by the above preparation method.

[0080] Application of the above multicolor bioimaging reagent in the field of bioimaging.

[0081] The biological imaging method provided by the present invention comprises the following steps: incubating the above-mentioned multi-color biological imaging reagent based on triple bond Raman with the biological sample for at least 2 hours, washing to remove the unbound Raman probe; imaging the labeled biological sample using a Raman spectrometer, selecting a 2160 cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 The Raman signal at is the characteristic spectral band.

[0082] Example

[0083] The following are examples of the present invention. The examples described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product instructions were used. Reagents or instruments used without manufacturer specified are all commercially available conventional products. Monomer 6 acrylonitrile was purchased from Aladdin Reagent Company with a purity of 99%; monomers 1 to 5 and 7 were synthesized in the laboratory.

[0084] Example 1: Synthesis of Monomers

[0085] 1. Synthesis of monomer 1

[0086]

[0087] To a solution of methyl 3-bromo-4-iodobenzoate (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL) were added triisopropylsilyl acetylene (218.4 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol), and bistriphenylphosphine palladium dichloride (14 mg, 0.2 mmol). The reaction system was cooled with liquid nitrogen and frozen, followed by the addition of 420 μL of dry triethylamine (TEA). N₂ protection was introduced, and the reaction was refluxed in an oil bath at 65°C for 24 h. After cooling to room temperature, the solvent was evaporated under reduced pressure, and a mixture of petroleum ether (PE) and ethyl acetate (EA) (10:1 by volume) was used as the eluent to afford 300 mg of the intermediate as a white solid in a 94.2% yield.

[0088] The above white solid intermediate (955 mg, 2.5 mmol) was dissolved in 20 mL of toluene, and tributyl vinyl tin (0.95 mL, 3 mmol) and tetrakistriphenylphosphine palladium (57.7 mg, 0.05 mmol) were added. The reaction system was cooled with liquid nitrogen and frozen. N2 was then introduced and refluxed in an oil bath at 115°C for 24 h. The solvent was then evaporated under reduced pressure, and the product was purified by column chromatography using a mixture of PE and EA (10:1 by volume) as the eluent to obtain a yellow liquid intermediate (0.7 g, 81.8%).

[0089] The yellow liquid intermediate (342 mg, 1 mmol) was dissolved in a mixture of methanol (MeOH, 5 mL) and water (1 mL). Lithium hydroxide (LiOH, 224 mg, 4 mmol) was added and stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and a mixture of dichloromethane (DCM) and MeOH (10:1 by volume) was used as the eluent to obtain 319 mg of monomer 1 as a white solid in a 97.2% yield. 1H NMR (400 MHz, CDCl3): δ 8.30 (d, J = 4.0 Hz, 1H), 7.91 (q, J = 8.0 Hz, 1H), 7.56 (d, J =8.0 Hz, 1H), 7.26 (s, 1H), 5.94 (d, J = 16.0 Hz, 1H), 5.54 (d, J = 12.0 Hz, 1H), 1.15 (s, 2H).

[0090] 2. Synthesis of monomer 4

[0091]

[0092] To a solution of methyl 3-bromo-4-iodobenzoate (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL) were added 4-pentyn-1-ol (100.94 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol), and bistriphenylphosphine palladium dichloride (14 mg, 0.2 mmol). The reaction system was cooled with liquid nitrogen and frozen, followed by the addition of 420 μL of dry TEA. N₂ protection was introduced, and the reaction was refluxed in an oil bath at 65°C for 24 h. After cooling to room temperature, the solvent was evaporated under reduced pressure. A mixture of DCM and MeOH (100:1 by volume) was used as the eluent to afford 300 mg of the intermediate as a pale yellow liquid in a 97.6% yield.

[0093] The above-mentioned pale yellow liquid intermediate (891 mg, 3 mmol) was dissolved in 20 mL of toluene, and tributyl vinyl tin (1.14 mL, 3.6 mmol) and tetrakistriphenylphosphine palladium (69 mg, 0.06 mmol) were added. The reaction system was cooled with liquid nitrogen and frozen. N2 was then introduced and refluxed in an oil bath at 115°C for 24 h. The solvent was then evaporated under reduced pressure, and the product was purified by column chromatography using a mixture of DCM and MeOH (100:1 by volume) as the eluent to obtain the yellow liquid intermediate (0.4 g, 54.6%).

[0094] The yellow liquid intermediate (244 mg, 1 mmol) was dissolved in a mixture of MeOH (5 mL) and water (1 mL). LiOH (96 mg, 4 mmol) was added and stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and a mixture of DCM and MeOH (10:1 by volume) was used as the eluent to obtain 83 mg of monomer 4 as a yellow solid in a 36.0% yield. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J = 4.0 Hz, 1H), 7.78 (q, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 20.0 Hz, 12 Hz, 1H), 5.98 (d, J = 20.0 Hz, 1H), 5.46 (d, J = 12.0 Hz, 1H), 3.53 (t, J =8.0 Hz, 2H), 2.55 (t, J = 8.0 Hz, 2H), 1.72 (m, 2H).

[0095] 3. Synthesis of Monomer 5

[0096]

[0097] 4-Bromobenzonitrile (1820.2 mg, 10 mmol) and tetrakis(triphenylphosphine)palladium (Pd(PPh³)⁴, 231 mg, 0.2 mmol) were added to a 250 mL three-necked flask. After removing all oxygen from the flask using a double-row pipette, tributyl vinyl tin (3804 mg, 12 mmol) was dissolved in 10 mL of toluene and injected into the flask via syringe. The mixture was cooled with liquid nitrogen and completely frozen. The oxygen was then removed three times, and the mixture was refluxed under condensation. The reaction mixture was moved to 120°C and 600 rpm for 24 h. After quenching with saturated potassium fluoride solution, the reaction solution was extracted with EA and deionized water, washed three times with deionized water, and then washed three times with saturated NaCl solution. The organic phase was dried over anhydrous NaSO⁴, concentrated by rotary evaporation, and purified by column chromatography using a mixture of PE and EA (20:1, by volume) as the eluent to obtain 435 mg of monomer 5 as a clear liquid in a 34.0% yield. 1H NMR (400 MHz, CDCl3): δ 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 6.73 (q, J = 16.0 Hz, 1H), 5.88 (d, J = 20.0 Hz, 1H), 5.45 (d, J = 12.0 Hz, 1H), 1.60 (s, 1H).

[0098] 4. Synthesis of Monomer 2: 2-Methacrylic acid and p-iodobenzyl alcohol undergo esterification to produce p-iodobenzyl 2-methacrylate, which then reacts with trimethylsilyl acetylene in the presence of cuprous iodide and bistriphenylphosphine palladium dichloride to produce Monomer 2.

[0099] 5. Synthesis of Monomer 3: 2-Methacrylic acid and trimethylsilylpropynyl alcohol undergo esterification reaction to produce Monomer 3.

[0100] 6. Synthesis of Monomer 7: 2-Methacrylic acid and trimethylsilylpropargyl alcohol undergo esterification to produce monomer 3; monomer 3 and propargyl alcohol undergo Sonogashira coupling under Pd / Cu catalysis to obtain monomer 7.

[0101] Example 2: Preparation of nanospheres

[0102] like Figure 1 As shown, the Raman characteristic peak of monomer 1 is 2154 cm -1 The Raman characteristic peak of monomer 2 is 2160 cm -1 , the Raman characteristic peak of monomer 4 is 2214 cm -1 The Raman characteristic peak of monomer 5 is 2227 cm -1 There is a significant band overlap phenomenon. In order to improve the recognition of characteristic peaks, this example specifically selects five monomers for nanosphere preparation: -1 Characteristic peaks of monomer 2, 2186 cm -1 Characteristic peaks of monomer 3, 2227cm -1 Characteristic peaks of monomer 5, 2241cm -1 Characteristic peaks of monomer 6 and 2260 cm -1 The monomer 7 with characteristic peaks was used to construct nanospheres with unique Raman fingerprint signals.

[0103] 1. Preparation of monomer 2 nanospheres: 0.06g of Raman characteristic peak at 2160cm -1 Monomer 2, 0.02g acrylic acid, 0.006g sodium dodecylbenzenesulfonate, and 5mL deionized water were added to the reaction vessel. After nitrogen bubbling to remove oxygen, the temperature was raised to 70°C and stirred continuously for 30 minutes. 0.008g potassium persulfate was added as an initiator and the reaction was fully reacted for 3 hours under constant pressure nitrogen protection. After the reaction was completed, the unreacted monomer and emulsifier were removed by dialysis (dialysis membrane pore size 8~14kD) to obtain pure Raman characteristic peak of 2160cm -1 Aqueous dispersion of monomeric 2 nanospheres.

[0104] 2. Preparation of monomer 3 nanospheres: 0.2g of Raman characteristic peak is 2186cm -1Monomer 3, 0.1g styrene, 0.05g acrylic acid, 0.008g sodium dodecylbenzenesulfonate, and 5mL deionized water were added to the reaction vessel. After nitrogen bubbling to remove oxygen, the temperature was raised to 70°C and stirred for 30 minutes. 0.03g potassium persulfate was added as an initiator and the reaction was fully reacted for 4 hours under constant pressure nitrogen protection. After the reaction was completed, the unreacted monomer and emulsifier were removed by dialysis (dialysis membrane pore size 8~14kD) to obtain pure Raman characteristic peak of 2186cm -1 Aqueous dispersion of monomeric 3 nanospheres.

[0105] 3. Preparation of monomer 5 nanospheres: 0.1g Raman characteristic peak is 2227cm -1 Monomer 5, 0.02g acrylic acid, 0.005g sodium dodecylbenzenesulfonate, and 5mL deionized water were added to the reaction vessel. After nitrogen bubbling to remove oxygen, the temperature was raised to 70°C and stirred continuously for 30 minutes. 0.015g potassium persulfate was added as an initiator and the reaction was carried out under constant pressure nitrogen protection for 3 hours. After the reaction, the unreacted monomer and emulsifier were removed by dialysis (dialysis membrane pore size 8~14kD), and the pure Raman characteristic peak was 2227cm -1 Aqueous dispersion of monomeric 5-nanospheres.

[0106] 4. Preparation of monomer 6 nanospheres: 0.2 g styrene, 0.15 g acrylic acid, 0.015 g sodium dodecylbenzene sulfonate, and 10 mL deionized water were added to a reaction vessel, nitrogen was introduced to deoxygenate, and the temperature was raised to 70 ° C. Stirring was continued for 30 min. 0.45 g of the Raman characteristic peak at 2241 cm was added. -1 Acrylonitrile (monomer 6) and 0.02g of azobisisobutyronitrile were fully reacted under constant pressure nitrogen protection for 2 hours. After the reaction, the unreacted monomer and emulsifier were removed by dialysis (dialysis membrane pore size 8~14kD) to obtain pure Raman characteristic peak of 2241cm -1 Aqueous dispersion of monomeric 6-nanospheres.

[0107] 5. Preparation of monomer 7 nanospheres: 0.05g of Raman characteristic peak is 2260cm -1 Monomer 7, 0.03g styrene, 0.02g acrylic acid, 0.003g sodium dodecylbenzenesulfonate, and 5mL deionized water were added to the reaction vessel. After nitrogen bubbling to remove oxygen, the temperature was raised to 70°C and stirred for 30 minutes. 0.01g potassium persulfate was added as an initiator and the reaction was fully reacted for 5 hours under constant pressure nitrogen protection. After the reaction was completed, the unreacted monomer and emulsifier were removed by dialysis (dialysis membrane pore size 8~14kD) to obtain pure Raman characteristic peak of 2260cm -1 Aqueous dispersion of monomeric 7-nanospheres.

[0108] Table 1 Nanospheres obtained in Example 2 and their preparation parameters

[0109]

[0110] Example 3: Synthesis of Raman Probes

[0111] In this embodiment, the targeting molecules (such as antibodies) are connected to the surface of the nanospheres by chemical coupling, thereby giving them specific recognition capabilities.

[0112] 1 mL of each of the five nanosphere aqueous dispersions prepared in Example 2 was treated separately: 5 μL of a 50 mg / mL LEDC aqueous solution was added and stirred for 30 minutes to activate the carboxyl groups on the surface of the nanospheres; 5 μL of a 50 mg / mL NHS aqueous solution was added as a coupling agent and stirred for another 30 minutes; and 5 μL of a targeting molecule (such as α-SMA antibody, CK-18 antibody, vimentin antibody, CD163 antibody, or CD34 antibody) was added and stirred at room temperature for 2 hours to covalently bind the targeting molecule to the carboxyl groups on the surface of the nanospheres through amide bonds to obtain a Raman probe.

[0113] In this embodiment, the surface of the monomer 2 nanosphere is modified with α-SMA antibody, the surface of the monomer 3 nanosphere is modified with CK-18 antibody, the surface of the monomer 5 nanosphere is modified with Vimentin antibody, the surface of the monomer 6 nanosphere is modified with CD163 antibody, and the surface of the monomer 7 nanosphere is modified with CD34 antibody. Figure 6 The changes in electrokinetic potential before and after nanosphere modification of antibodies are shown.

[0114] Example 4: Preparation of multicolor bioimaging reagents

[0115] The five Raman probes with different Raman characteristic peaks prepared in Example 3 were mixed at a Raman intensity ratio of 1:1:1:1:1 to obtain a multicolor bio-imaging reagent.

[0116] Each Raman probe in a multicolor bioimaging reagent generates a unique Raman signal at a specific wavelength. Commercially available Raman spectrometers can distinguish the signals of different Raman probes. Multicolor bioimaging reagents can be used to label different biomolecules or cellular structures, enabling simultaneous imaging of multiple targets.

[0117] Example 5: Application

[0118] In this example, the prepared multicolor bioimaging reagent is used for labeling and imaging of tumor cells.

[0119] The Raman probe prepared in Example 3 was co-incubated with tumor cells for 2 hours to allow the Raman probe to specifically bind to the surface of the tumor cells. Unbound Raman probes were removed by washing, and tumor cells were imaged using a commercially available Raman imaging system.

[0120] The results showed that Raman probes can clearly mark tumor cells and achieve single-color ( Figure 7 a2~e2 in Figure 7 a3~e3 in), two colors ( Figure 8 a2 in), three colors ( Figure 8 b2 in), five colors ( Figure 9a1 to Figure 9e1 ) and other multi-color imaging, with a signal-to-background ratio significantly higher than that of traditional fluorescent dyes Cy3, Cy5 and FTIC.

[0121] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a multicolor biological imaging reagent based on triple bond Raman, characterized in that: include: Monomers 1 to 7 are copolymerized with (i) acrylic acid or (ii) styrene and acrylic acid in an aqueous solvent in the presence of an emulsifier and an initiator to form nanospheres with a particle size of 10 to 100 nm. The structural formulas of monomers 1 to 7 are as follows: ； The carboxyl groups on the surface of the nanospheres were activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and then the carboxyl groups on the surface of the nanospheres were linked to the amino groups of the targeting molecules using N-hydroxysuccinimide ester to obtain a Raman probe. Multiple Raman probes with different Raman characteristic peaks are mixed together to obtain a multi-color biological imaging reagent.

2. The preparation method according to claim 1, wherein: The mass ratio of each monomer to acrylic acid is 2 to 5:1; and / or, The mass ratio of each monomer to styrene and acrylic acid is 2-5:1.3-2:1; and / or, The mass ratio of each monomer, emulsifier and initiator is 10-30:1:1-4; and / or, The concentration of each monomer in the aqueous solvent is 10-50 g / L.

3. The preparation method according to claim 1, wherein: The average particle size of the nanospheres is 40-60 nm; The multi-color biological imaging reagent has 2160cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 The Raman characteristic peak at .

4. The preparation method according to claim 1, wherein: The emulsifier is sodium lauryl sulfate or sodium dodecylbenzenesulfonate; and / or, The initiator is potassium persulfate or azobisisobutyronitrile; and / or, The temperature of the copolymerization reaction is 70±5°C; and / or, The copolymerization reaction is carried out under oxygen-free conditions.

5. The preparation method according to claim 1, wherein: After the copolymerization reaction, the method further comprises: removing unreacted monomers and emulsifiers by dialysis to obtain a nanosphere aqueous dispersion; The method utilizes 1-ethyl-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl groups on the surface of the nanospheres, and utilizes NHS to link the carboxyl groups on the surface of the nanospheres with the amino groups of the targeting molecules, comprising: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a nanosphere aqueous dispersion at room temperature to activate the carboxyl groups on the surface of the nanospheres; adding N-hydroxysuccinimide ester to the nanosphere aqueous dispersion, stirring for at least 30 minutes, and then adding the targeting molecules to react for at least 2 hours.

6. The preparation method according to claim 5, characterized in that: Dialysis was performed using a dialysis membrane with a pore size of 8~14kD.

7. The preparation method according to claim 1, wherein: The targeting molecule is one or more of α-smooth muscle actin antibody, cytokeratin 18 antibody, vimentin antibody, cluster of differentiation 163 antibody, and cluster of differentiation 34 antibody.

8. A multicolor bioimaging reagent based on triple bond Raman, characterized by: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the triple bond Raman-based multicolor bioimaging reagent according to claim 8 in the field of bioimaging.

10. A biological imaging method, characterized in that: The following steps are involved: Incubating the triple-bond Raman-based multicolor bioimaging reagent according to claim 8 with the biological sample for at least 2 hours, and washing to remove unbound Raman probes; The labeled biological samples were imaged using a Raman spectrometer at 2160 cm -1 、2186cm -1 , 2227cm -1 、2241cm -1 and 2260cm -1 The Raman signal at is the characteristic spectral band.

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