Multicolor bio-imaging reagent based on triple bond raman and preparation method and application thereof

Abstract

The application belongs to the field of biological imaging reagents, and relates to a multi-color biological imaging reagent based on a three-bond Raman and a preparation method and application thereof. Monomers 1-7 respectively undergo a 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 a particle size of 10-100 nm; carboxyl groups on the surface of the nanospheres are activated by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide to connect the carboxyl groups on the surface of the nanospheres with amino groups of a targeting molecule by using N-hydroxysuccinimide ester to obtain a Raman probe; and the Raman probes different in Raman characteristic peaks are mixed together to obtain the multi-color biological imaging reagent. Compared with existing Raman bead multi-color imaging reagents, the multi-color biological imaging reagent provided by the application is easier to penetrate tissues and be taken up by cells, has a higher signal-to-background ratio, and thus ensures high definition of biological imaging. The preparation method of the application is simple, mild in conditions, environmentally friendly, and easy to scale up.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bio-imaging reagents, and relates to a multi-color bio-imaging reagent based on three-bond Raman and a preparation method and application thereof. BACKGROUND

[0002] Bio-imaging technology has important applications in biomedical research and clinical diagnosis, as it can provide structural and functional information at the tissue, cell and molecular levels in living organisms. However, existing bio-imaging reagents have some limitations. On the one hand, many reagents have a low signal-to-background ratio, i.e., the ratio of signal to background noise is not high, resulting in unclear imaging results and difficulty in accurately distinguishing target structures. On the other hand, single-color imaging reagents cannot simultaneously label and image multiple biological molecules or cell types, limiting comprehensive analysis of complex biological processes.

[0003] Existing multi-color imaging technologies such as fluorescence and mass spectrometry face major challenges. The repeated antigen retrieval process is tedious and risks damaging the antigen, and the subsequent spectral separation data processing is complex. In particular, existing technologies face core technology monopoly of imported equipment, which is expensive and has high usage and maintenance costs. Therefore, it is of great significance to develop new multi-color imaging technologies.

[0004] In recent years, Raman spectroscopy technology has received widespread attention as a label-free and highly specific imaging method, as it can provide chemical information at the molecular level. However, traditional Raman imaging technology has weak signals and strong background interference, limiting its widespread application in the biomedical field. Existing Raman bead multi-color imaging technology uses Raman beads with a large particle size, which makes it difficult for them to penetrate tissues and be taken up by cells, thereby limiting their bio-imaging capabilities. In addition, the number of distinguishable spectral channels is limited, further restricting the widespread application of this technology in the field of bio-imaging. SUMMARY

[0005] The present application provides a multi-color bio-imaging reagent based on three-bond Raman technology and a preparation method and application thereof. The multi-color bio-imaging reagent comprises Raman beads with a particle size of less than 100 nm, which are easy to penetrate tissues and be taken up by cells, thereby significantly improving the effectiveness of bio-imaging. In addition, the multi-color bio-imaging reagent of the present application can achieve more precise Raman shift regulation, has stronger spectral orthogonality, and effectively reduces signal crosstalk in the multi-color imaging process. At the same time, the reagent also has a high signal-to-background ratio, ensuring the high definition of bio-imaging.

[0006] The first aspect of the present application provides a preparation method of a multi-color bio-imaging reagent based on three-bond Raman, comprising:

[0007] Monomers 1-7 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 10-100 nm; the structural formula of each of monomers 1-7 is as follows:

[0008] ;

[0009] The carboxyl groups on the surface of the nanospheres are activated by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and then the carboxyl groups on the surface of the nanospheres are connected to the amino groups of a targeting molecule by using N-hydroxysuccinimide ester (NHS) to obtain a Raman probe;

[0010] A multi-color bio-imaging reagent is obtained by mixing a plurality of Raman probes with different Raman characteristic peaks together.

[0011] In combination with the first aspect of the present application, 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, 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.

[0012] In combination with the first aspect of the present application, in some embodiments, the average particle size of the nanospheres is 40-60 nm; and / or, the multi-color bio-imaging reagent has Raman characteristic peaks at 2160 cm -1 , 2186 cm -1 , 2227 cm -1 , 2241 cm -1 and 2260 cm -1 .

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

[0014] In combination with the first aspect of the present application, in some embodiments, after the copolymerization reaction, the method further comprises: removing unreacted monomers and emulsifiers by dialysis to obtain a nanosphere aqueous dispersion; and activating the carboxyl groups on the surface of the nanospheres by using EDC, and then connecting the carboxyl groups on the surface of the nanospheres to the amino groups of a targeting molecule by using NHS, which comprises: adding EDC to the nanosphere aqueous dispersion at room temperature to activate the carboxyl groups on the surface of the nanospheres; and adding NHS to the nanosphere aqueous dispersion and stirring for at least 30 minutes, and then adding a targeting molecule and reacting for at least 2 hours.

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

[0016] In some embodiments of the first aspect of the application, the targeting molecule is one or more of an alpha-smooth muscle actin (a-SMA) antibody, a cytokeratin 18 (CK-18) antibody, a vimentin antibody, a cluster of differentiation 163 (CD163) antibody, and a cluster of differentiation 34 (CD34) antibody.

[0017] The second aspect of the application provides a triply Raman-based multicolor bioimaging reagent prepared by the above preparation method.

[0018] The third aspect of the application provides application of the above triply Raman-based multicolor bioimaging reagent in the field of bioimaging.

[0019] The fourth aspect of the application provides a bioimaging method, comprising the following steps:

[0020] The above triply Raman-based multicolor bioimaging reagent is co-incubated with a biological sample for at least 2 hours, and unbound Raman probes are removed by washing;

[0021] The labeled biological sample is imaged using a Raman spectrometer, and the Raman signals at 2160 cm -1 , 2186 cm -1 , 2227 cm -1 , 2241 cm -1 , and 2260 cm -1 are used as characteristic spectral bands for color development.

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

[0023] 1. Compared with existing Raman bead multicolor imaging reagents, the multicolor bioimaging reagent provided by the application is more easily penetrated into tissues and 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 the high definition of bioimaging.

[0024] 2. The preparation method of the application is simple, mild, environmentally friendly, and easy to scale up. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0026] Figure 1 Raman spectrum of monomer 1~7.

[0027] Figure 2 Transmission electron micrograph of nanospheres prepared in Example 2 of the present application, a is nanospheres polymerized from monomer 2; b is nanospheres polymerized from monomer 3; c is nanospheres polymerized from monomer 5; d is nanospheres polymerized from monomer 6; e is nanospheres polymerized from monomer 7.

[0028] Figure 3 Raman spectrum of monomer 1~7.

[0029] Figure 4 Raman spectrum of monomer 1~7.

[0030] Figure 5a1 Particle size distribution of monomer 2 nanospheres;

[0031] Figure 5a2 Particle size distribution of monomer 2 nanospheres after modification with antibody;

[0032] Figure 5b1 Particle size distribution of monomer 3 nanospheres;

[0033] Figure 5b2 Particle size distribution of monomer 3 nanospheres after modification with antibody;

[0034] Figure 5c1 Particle size distribution of monomer 5 nanospheres;

[0035] Figure 5c2 Particle size distribution of monomer 5 nanospheres after modification with antibody;

[0036] Figure 5d1 Particle size distribution of monomer 6 nanospheres;

[0037] Figure 5d2 Particle size distribution of monomer 6 nanospheres after modification with antibody;

[0038] Figure 5e1 Figure 7 is a particle size chart of monomer 7 nanospheres;

[0039] Figure 5e2 Figure 8 is a particle size chart of monomer 7 nanospheres after modification of antibodies.

[0040] Figure 6 Figure 9 is a zeta potential chart of the antibodies before and after modification of nanospheres in Example 3 of the present application, P1 is the zeta potential of monomer 2 nanospheres, a-SMA-P1 is the zeta potential of a-SMA antibodies modified by monomer 2 nanospheres; P2 is the zeta potential of monomer 3 nanospheres, CK-18-P2 is the zeta potential of CK-18 antibodies modified by monomer 3 nanospheres; P3 is the zeta potential of monomer 5 nanospheres, Vimentin-P3 is the zeta potential of vimentin antibodies modified by monomer 5 nanospheres; P4 is the zeta potential of monomer 6 nanospheres, CD163-P4 is the zeta potential of CD163 antibodies modified by monomer 6 nanospheres; P5 is the zeta potential of monomer 7 nanospheres, CD34-P5 is the zeta potential of CD34 antibodies modified by monomer 7 nanospheres.

[0041] Figure 7 Figure 10 is a monochrome imaging chart of the whole and local parts of the fluorescence and Raman contrast of the Raman probe prepared in Example 3 of the present application; wherein:

[0042] a1~a5 are a group of pictures of smooth muscle labeled by a-SMA antibodies modified by monomer 2 nanospheres and contrasted by fluorescent labels: a1 is the whole fluorescent image, a2 is the whole Raman image, a3 is the local Raman image, a4 is the local fluorescent image, and a5 is the local Raman fluorescent superimposed image;

[0043] b1~b5 are a group of pictures of tumor cells of epithelial origin labeled by CK-18 antibodies modified by monomer 3 nanospheres and contrasted by fluorescent labels: b1 is the whole fluorescent image, b2 is the whole Raman image, b3 is the local Raman image, b4 is the local fluorescent image, and b5 is the local Raman fluorescent superimposed image;

[0044] c1~c5 are a group of pictures of fibroblasts in the interstitium labeled by vimentin antibodies modified by monomer 5 nanospheres and contrasted by fluorescent labels: c1 is the whole fluorescent image, c2 is the whole Raman image, c3 is the local Raman image, c4 is the local fluorescent image, and c5 is the local Raman fluorescent superimposed image;

[0045] d1~d5 are a group of pictures of single cells labeled by CD163 antibodies modified by monomer 6 nanospheres and contrasted by fluorescent labels: d1 is the whole fluorescent image, d2 is the whole Raman image, d3 is the local Raman image, d4 is the local fluorescent image, and d5 is the local Raman fluorescent superimposed image;

[0046] E1-E5 are a set of images of vascular endothelium modified with CD34 antibody and contrast fluorescent labeling using monomeric 7 nanospheres: 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 superimposed image.

[0047] Figure 8 These are two-color and three-color imaging images of the Raman probe prepared in Example 3 of the present invention, showing fluorescence and Raman comparison. 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 In the five-color Raman imaging of Embodiment 5 of the present invention, a 2160cm chromatograph was selected. -1 Raman imaging of a specific Raman signal;

[0049] Figure 9a2 In the five-color Raman imaging of Embodiment 5 of the present invention, the corresponding 2160cm was selected. -1 Raman spectrum;

[0050] Figure 9b1 In the five-color Raman imaging of Embodiment 5 of the present invention, a 2186 cm⁻¹ was selected. -1 Raman imaging of a specific Raman signal;

[0051] Figure 9b2 In the five-color Raman imaging of Embodiment 5 of the present invention, the corresponding 2186 cm⁻¹ was selected. -1 Raman spectrum;

[0052] Figure 9c1 In the five-color Raman imaging of Embodiment 5 of the present invention, a 2227cm chromatograph was selected. -1 Raman imaging of a specific Raman signal;

[0053] Figure 9c2 In Embodiment 5 of this invention, the corresponding 2227cm was selected in the five-color Raman imaging. -1 Raman spectrum;

[0054] Figure 9d1 In the five-color Raman imaging of Embodiment 5 of the present invention, 2241 cm⁻¹ was selected. -1 Raman imaging of a specific Raman signal;

[0055] Figure 9d2 In the five-color Raman imaging of Embodiment 5 of the present invention, the corresponding 2241 cm⁻¹ was selected. -1 Raman spectrum;

[0056] Figure 9e1 In the five-color Raman imaging of Embodiment 5 of the present invention, a 2260cm chromatograph was selected. -1 Raman imaging of a specific Raman signal;

[0057] Figure 9e2 In the five-color Raman imaging of Embodiment 5 of the present invention, the corresponding 2260cm was selected. -1 Raman spectrum. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0059] For simplicity, this invention only explicitly discloses some 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. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value, or combined with other lower or upper limits, to form an unspecified range.

[0060] It should be noted that, in the description of this invention, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more. Relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0061] In the description of this invention, the terms "any embodiment / mode," "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment / mode or example, which are included in at least one embodiment / mode or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0062] In the description of this invention, the term "high signal-to-background ratio" refers to a high ratio of signal to background, meaning that the signal strength is much greater than the background signal strength. This 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 method.

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

[0064] In the description of this invention, the term "Raman characteristic peak" refers to a specific wavenumber (cm²) produced by a substance in a Raman spectrum due to molecular vibrational or rotational energy level transitions. -1 The position, shape, and intensity of these peaks reflect the vibrational modes of chemical bonds or functional groups in a molecule, serving as a "fingerprint" identifier of the chemical structure of a substance.

[0065] The above description of the invention is not intended to describe every disclosed embodiment or implementation of the invention. Instead, the following description provides more specific examples of exemplary embodiments. These embodiments can be used in various combinations. The examples listed are merely representative and should not be construed as exhaustive.

[0066] The present invention provides a method for preparing multicolor bioimaging reagents based on triple bond Raman spectroscopy, comprising:

[0067] Monomers 1-7 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 10-100 nm; the structural formulas of monomers 1-7 are as follows:

[0068] ;

[0069] The carboxyl groups on the surface of nanospheres were activated using EDC, and then the carboxyl groups on the surface of the nanospheres were linked to the amino groups of the target molecule using NHS to obtain a Raman probe.

[0070] By mixing multiple Raman probes with different Raman characteristic peaks together, a multicolor bioimaging reagent is obtained.

[0071] As shown in the structural formulas of monomers 1-7, all monomers 1-7 contain ethylene bonds. During the copolymerization process, the ethylene bonds in the monomers participate in the reaction, and the monomers can self-polymerize into nanospheres; they can also participate in the polymerization reaction together with the ethylene bonds in (i) acrylic acid or (ii) acrylic acid and styrene to form monomer + acrylic acid copolymer nanospheres, or monomer + acrylic acid + styrene copolymer nanospheres. After these ethylene bonds polymerize, they form the polymer backbone, while the triple bonds in the monomers, the carboxyl groups in acrylic acid, and the phenyl groups in styrene become side chains. The surface of the formed nanospheres is rich in carboxyl groups, giving them excellent hydrophilicity, ensuring that the nanospheres are uniformly dispersed in aqueous solvents, and providing reliable anchors for subsequent targeted molecular modification. The introduction of styrene makes the polymer backbone structure of each nanosphere more consistent, thereby making the spheres uniform in shape and the Raman intensity uniformly improved.

[0072] like Figure 1 As shown, monomers 1 to 7 have different Raman characteristic peaks, although the Raman characteristic peak of monomer 1 is at 2154 cm⁻¹. -1 Raman characteristic peak of monomer 2 at 2160 cm⁻¹ -1 Raman characteristic peak of monomer 4 at 2214 cm⁻¹ -1 Raman characteristic peak of monomer 5 at 2227 cm⁻¹ -1 Significant band overlap exists, but it is still distinguishable. To improve the identification of characteristic peaks, the following five monomers are preferred for nanosphere preparation: [The text then abruptly shifts to a seemingly unrelated topic about 2160 cm⁻¹ monomers and nanospheres.] -1 Characteristic peaks of monomers 2, 2186 cm⁻¹ -1 Characteristic peaks of monomers 3, 2227 cm⁻¹ -1 Characteristic peaks of monomers 5 and 2241 cm⁻¹ -1 Characteristic peaks of monomers 6 and 2260 cm⁻¹ -1 Monomer 7 with characteristic peaks was 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, 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 proportions and concentrations of monomers, initiators, emulsifiers, and acrylic acid within the above ranges, the particle size of the nanospheres can be controlled to be between 10 and 100 nm.

[0074] In some embodiments, the nanospheres have an average particle size of 40-60 nm; and / or, the multicolor bioimaging reagent has a diameter of 2160 cm⁻¹. -1 2186cm -1 2227cm -1 2241cm -1 and 2260cm -1 The Raman characteristic peaks at this location are observed. Nanospheres with this particle size are more easily taken up by cells, thus exhibiting excellent performance in bioimaging applications. Furthermore, the nanospheres are uniformly dispersed in water and do not easily aggregate, ensuring their stability and persistence in vivo. The carboxyl groups on their surface also provide the possibility of binding to biomolecules, enabling these nanospheres to play an important role in biomedical research and diagnosis as multicolor bioimaging reagents.

[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℃; and / or, the copolymerization reaction is carried out under anaerobic conditions. These specific choices of emulsifiers and initiators help control the polymerization process, thereby further regulating the properties of the nanospheres. Sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, as emulsifiers, can effectively reduce the surface tension of the system, promote monomer dispersion in the aqueous phase, and facilitate the formation of nanospheres with uniform particle size and stable dispersion. Potassium persulfate and azobisisobutyronitrile, as initiators, can effectively decompose to generate free radicals at the set temperature, initiating the polymerization reaction of the monomers. Furthermore, controlling the copolymerization reaction temperature within the range of 70±5℃ is beneficial for balancing the reaction rate and the mass of the nanospheres. Simultaneously, conducting the copolymerization reaction under anaerobic conditions can avoid the quenching effect of oxygen on free radicals, thereby improving polymerization efficiency and product purity. The combined effect of these conditions results in nanospheres with ideal particle size distribution and excellent dispersion stability.

[0076] In some embodiments, the copolymerization reaction is followed by: removing unreacted monomers and emulsifiers by dialysis to obtain an aqueous dispersion of nanospheres; activating the carboxyl groups on the surface of the nanospheres with EDC, and then linking the carboxyl groups on the surface of the nanospheres to the amino groups of the target molecule with 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 target molecule and reacting for at least 2 hours; removing unlinked target molecules and excess EDC and NHS by centrifugation to obtain the Raman probe. This surface modification not only improves the biocompatibility of the nanospheres but also endows them with active targeting capabilities, enabling the nanospheres to reach the target location more accurately and improving the utilization efficiency of the Raman probe.

[0077] In some embodiments, dialysis is performed using a dialysis membrane with a pore size of 8-14 kD. This dialysis membrane can remove unreacted small molecules while retaining the nanospheres, thereby obtaining an aqueous dispersion of pure nanospheres and avoiding the influence of unreacted substances on 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. These targeting molecules each possess specific biological recognition capabilities, enabling them to specifically bind to target cells or tissues. For example, α-SMA antibody is commonly used for labeling smooth muscle cells, CK-18 antibody is commonly used for labeling epithelial cells, Vimentin antibody can recognize various mesenchymal cells, while CD163 and CD34 antibodies have high affinity for macrophages and vascular endothelial cells, respectively. By selecting specific targeting molecules, Raman probes targeting specific diseases or tissue types can be designed, thereby achieving more precise disease diagnosis or treatment monitoring.

[0079] A multicolor bioimaging reagent based on triple bond Raman spectroscopy, prepared by the above method.

[0080] The above-mentioned multicolor bioimaging reagents are used in the field of bioimaging.

[0081] The bioimaging method provided by this invention includes the following steps: co-incubating the above-mentioned triple-bond Raman-based multicolor bioimaging reagent with a biological sample for at least 2 hours, washing away unbound Raman probes; and imaging the labeled biological sample using a Raman spectrometer, selecting a 2160 cm⁻¹ spectrometer. -1 2186cm -1 2227cm -1 2241cm -1 and 2260cm -1 The Raman signal at that location is characterized by a spectral band.

[0082] Example

[0083] The following are embodiments of the present invention. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Monomer 6-acrylonitrile was purchased from Aladdin Reagent Company, with a purity of 99%; monomers 1-5 and 7 were synthesized in the laboratory.

[0084] Example 1: Synthesis of Monomers

[0085] 1. Synthesis of monomer 1

[0086]

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

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

[0089] A yellow liquid intermediate (342 mg, 1 mmol) was dissolved in a mixed solution of methanol (MeOH, 5 mL) and water (1 mL), and lithium hydroxide (LiOH, 224 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, using a mixture of dichloromethane (DCM) and MeOH (volume ratio 10:1) as the eluent to give 319 mg of monomer 1 as a white solid, with a yield of 97.2%. 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), 4-pentyn-1-ol (100.94 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol), and palladium dichloride dichloride (14 mg, 0.2 mmol) were added. The mixture was cooled with liquid nitrogen to freeze the reaction system. 420 μL of dry TEA was added, and the system was refluxed in an oil bath at 65 °C for 24 h under N2 protection. After cooling to room temperature, the solvent was evaporated under reduced pressure, using a mixture of DCM and MeOH (100:1 v / v) as the eluent to give 300 mg of a pale yellow liquid intermediate, with a yield of 97.6%.

[0093] The above-mentioned pale yellow liquid intermediate (891 mg, 3 mmol) was dissolved in 20 mL of toluene, and tributylvinyltin (1.14 mL, 3.6 mmol) and tetrakis(triphenylphosphine)palladium (69 mg, 0.06 mmol) were added. The reaction system was cooled with liquid nitrogen to freeze it, then protected with N2, refluxed in an oil bath, and reacted at 115 °C for 24 h. The solvent was then evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of DCM and MeOH (100:1 v / v) 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 mixed solution of MeOH (5 mL) and water (1 mL), and LiOH (96 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, using a mixture of DCM and MeOH (volume ratio 10:1) as the eluent to give 83 mg of monomer 4 as a yellow solid, with a yield of 36.0%. 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. Synthetic 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 O2 from the flask using a double-row tube, tributylvinyltin (3804 mg, 12 mmol) was dissolved in 10 mL of toluene and injected into the flask using a syringe. The mixture was cooled with liquid nitrogen, completely frozen, and then O2 was removed three times. The mixture was refluxed and reacted at 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 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 v / v) as the eluent to obtain 435 mg of monomer 5, a clear liquid, with a yield of 34.0%. 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-methacrylic acid. The p-iodobenzyl 2-methacrylic acid then reacts with trimethylsilylacetylene under the catalysis of cuprous iodide and palladium dichloride to produce monomer 2.

[0099] 5. Synthesis of monomer 3: 2-Methacrylic acid and trimethylsilylpropynol undergo esterification to generate monomer 3.

[0100] 6. Synthesis of monomer 7: 2-Methylacrylic acid and trimethylsilylpropynol undergo esterification to generate monomer 3; monomer 3 and propynol are coupled via Sonogashira 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 at 2154 cm⁻¹. -1 Raman characteristic peak of monomer 2 at 2160 cm⁻¹ -1 Raman characteristic peak of monomer 4 at 2214 cm⁻¹ -1 Raman characteristic peak of monomer 5 at 2227 cm⁻¹ -1 Significant band overlap was observed. To improve the identification of characteristic peaks, this embodiment specifically selected five monomers for nanosphere preparation: using a 2160 cm⁻¹ nanosphere as an example. -1 Characteristic peaks of monomers 2, 2186 cm⁻¹ -1 Characteristic peaks of monomers 3, 2227 cm⁻¹ -1 Characteristic peaks of monomers 5 and 2241 cm⁻¹ -1 Characteristic peaks of monomers 6 and 2260 cm⁻¹ -1 Monomer 7 with characteristic peaks was used to construct nanospheres with unique Raman fingerprint signals.

[0103] 1. Preparation of monomeric 2 nanospheres: 0.06 g of the monomeric 2 nanospheres was prepared. -1 Monomer 2, 0.02 g acrylic acid, 0.006 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred continuously for 30 min. Then, 0.008 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was complete, unreacted monomers and emulsifiers were removed by dialysis (dialysis membrane pore size 8-14 kD), yielding a pure product with a Raman characteristic peak at 2160 cm⁻¹. -1 An aqueous dispersion of monomeric 2 nanospheres.

[0104] 2. Preparation of monomeric 3 nanospheres: 0.2 g of the Raman characteristic peak at 2186 cm⁻¹ was prepared. -1Monomer 3, 0.1 g styrene, 0.05 g acrylic acid, 0.008 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred for 30 min. Then, 0.03 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 4 h. After the reaction was complete, unreacted monomers and emulsifiers were removed by dialysis (dialysis membrane pore size 8-14 kD), yielding a pure product with a Raman characteristic peak at 2186 cm⁻¹. -1 An aqueous dispersion of monomeric 3 nanospheres.

[0105] 3. Preparation of monomeric 5 nanospheres: 0.1 g of the Raman characteristic peak at 2227 cm⁻¹ was prepared. -1 Monomer 5, 0.02 g acrylic acid, 0.005 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred continuously for 30 min. Then, 0.015 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was completed, unreacted monomers and emulsifiers were removed by dialysis (dialysis membrane with a pore size of 8-14 kD), yielding a pure product with a Raman characteristic peak at 2227 cm⁻¹. -1 An aqueous dispersion of monomeric 5-nanospheres.

[0106] 4. Preparation of monomeric 6 nanospheres: 0.2 g styrene, 0.15 g acrylic acid, 0.015 g sodium dodecylbenzenesulfonate, and 10 mL deionized water were added to a reaction vessel. After purging with nitrogen gas to remove oxygen, the temperature was raised to 70 °C and stirred continuously for 30 min. Then, 0.45 g of a Raman characteristic peak at 2241 cm⁻¹ was added. -1 Acrylonitrile (monomer 6) and 0.02 g of azobisisobutyronitrile were reacted under constant pressure nitrogen protection for 2 h. After the reaction, unreacted monomers and emulsifiers were removed by dialysis (dialysis membrane pore size 8~14 kD), yielding a pure product with a Raman characteristic peak at 2241 cm⁻¹. -1 An aqueous dispersion of monomeric 6 nanospheres.

[0107] 5. Preparation of monomeric 7 nanospheres: 0.05 g of the monomeric 7 nanospheres was prepared. -1 Monomer 7, 0.03 g styrene, 0.02 g acrylic acid, 0.003 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred for 30 min. Then, 0.01 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 5 h. After the reaction was complete, unreacted monomers and emulsifiers were removed by dialysis (dialysis membrane pore size 8-14 kD), yielding a pure product with a Raman characteristic peak at 2260 cm⁻¹. -1 An aqueous dispersion of 7-nanosphere monomers.

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

[0109]

[0110] Example 3: Synthesis of Raman Probe

[0111] In this embodiment, a target molecule (such as an antibody) is attached to the surface of the nanosphere by chemical coupling, giving it specific recognition ability.

[0112] Take 1 mL of each of the five nanosphere aqueous dispersions prepared in Example 2 and treat them separately: add 5 μL of 50 mg / mL LEDC aqueous solution and stir for 30 minutes to activate the carboxyl groups on the surface of the nanospheres; add 5 μL of 50 mg / mL NHS aqueous solution as a coupling agent and continue stirring for 30 minutes; add 5 μL of a targeting molecule (such as α-SMA antibody, CK-18 antibody, vimentin antibody, CD163 antibody, CD34 antibody), and continue stirring at room temperature for 2 hours to allow the targeting molecule to covalently bind to the carboxyl groups on the surface of the nanospheres through amide bonds, thus obtaining Raman probes.

[0113] In this embodiment, the surface of monomeric 2 nanospheres is modified with α-SMA antibody, the surface of monomeric 3 nanospheres is modified with CK-18 antibody, the surface of monomeric 5 nanospheres is modified with Vimentin antibody, the surface of monomeric 6 nanospheres is modified with CD163 antibody, and the surface of monomeric 7 nanospheres is modified with CD34 antibody. Figure 6 The changes in electrokinetic potential before and after the nanospheres were modified with the antibody were 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 in a Raman intensity ratio of 1:1:1:1:1 to obtain a multicolor bioimaging reagent.

[0116] Each Raman probe in a multicolor bioimaging reagent produces a unique Raman signal at a specific wavelength. These signals can be distinguished using a commercially available Raman spectrometer. By using multicolor bioimaging reagents to label different biomolecules or cellular structures, simultaneous imaging of multiple targets can be achieved.

[0117] Example 5: Application

[0118] In this embodiment, the prepared multicolor bioimaging reagent is used for the 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 the tumor cells were imaged using a commercially available ordinary Raman imaging system.

[0120] The results showed that the Raman probe could clearly label tumor cells and achieve monochromatic ( Figure 7 a2~e2, Figure 7 a3~e3), two-color ( Figure 8 a2), three colors ( Figure 8 (b2 in the middle), five colors ( Figures 9a1 to 9e1 Multicolor imaging, with a signal-to-background ratio significantly higher than that of traditional fluorescent dyes Cy3, Cy5 and FTIC.

[0121] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the 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 invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a multicolor bioimaging reagent based on triple bond Raman spectroscopy, characterized in that, include: Monomers 2, 3, 5, 6, and 7 undergo copolymerization reactions with (i) acrylic acid or (ii) styrene and acrylic acid in an aqueous solvent in the presence of an emulsifier and an initiator, respectively, to form nanospheres with a particle size of 40-60 nm; the structural formulas of monomers 2, 3, 5, 6, and 7 are as follows: ； The carboxyl groups on the surface of 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 target molecule using N-hydroxysuccinimide ester to obtain a Raman probe. The target molecule is one of α-smooth muscle actin antibody, cytokeratin 18 antibody, vimentin antibody, differentiation cluster 163 antibody, and differentiation cluster 34 antibody. Among them, the surface of monomeric nanosphere 2 was modified with α-smooth muscle actin antibody, the surface of monomeric nanosphere 3 was modified with cytokeratin 18 antibody, the surface of monomeric nanosphere 5 was modified with vimentin antibody, the surface of monomeric nanosphere 6 was modified with differentiation cluster 163 antibody, and the surface of monomeric nanosphere 7 was modified with differentiation cluster 34 antibody. Raman probes made from monomers 2, 3, 5, 6, and 7 were mixed together to obtain a multicolor bioimaging reagent; the multicolor bioimaging reagent has a 2160 cm⁻¹ diameter. -1 2186cm -1 2227cm -1 2241cm -1 and 2260cm -1 Raman characteristic peaks at the location.

2. The preparation method according to claim 1, characterized in that: 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 10~50 g / L.

3. The preparation method according to claim 1, characterized in that: The emulsifier is sodium dodecyl sulfate or sodium dodecylbenzene sulfonate; and / or... The initiator is potassium persulfate or azobisisobutyronitrile; and / or, The copolymerization reaction is carried out at a temperature of 70±5℃; and / or, The copolymerization reaction was carried out under anaerobic conditions.

4. The preparation method according to claim 1, characterized in that: The copolymerization process also includes: removing unreacted monomers and emulsifiers by dialysis to obtain an aqueous dispersion of nanospheres; The carboxyl groups on the surface of nanospheres were activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the carboxyl groups on the surface of the nanospheres were linked to the amino groups of the target molecule using NHS. The process included: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide to an aqueous dispersion of nanospheres at room temperature to activate the carboxyl groups on the surface of the nanospheres; adding N-hydroxysuccinimide ester to the aqueous dispersion of nanospheres, stirring for at least 30 minutes, and then adding the target molecule to react for at least 2 hours.

5. The preparation method according to claim 4, characterized in that: Dialysis uses dialysis membranes with a pore size of 8~14kD.

6. A multicolor bioimaging reagent based on triple bond Raman spectroscopy, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 5.

7. A bioimaging method, characterized in that, Includes the following steps: The multicolor bioimaging reagent based on triple bond Raman as described in claim 6 is co-incubated with biological samples for at least 2 hours, and unbound Raman probes are washed away. The labeled biological samples were imaged using a Raman spectrometer, with a 2160 cm⁻¹ spectroscopy setup. -1 2186cm -1 2227cm -1 2241cm -1 and 2260cm -1 The Raman signal at that location is characterized by a spectral band.

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