Small-molecule Raman probe, bar code probe and coding method and application of small-molecule Raman probe and bar code probe

Abstract

The invention relates to the technical field of spectrum detection probes, in particular to a small molecule Raman probe, a bar code probe and a coding method and application thereof. The coding method comprises the following steps: sequentially coding all micromolecular probes in a micromolecular probe combination, if the micromolecular probes are not embedded into a carrier, recording the micromolecular probes as a first code, if the micromolecular probes are embedded into the carrier, recording the micromolecular probes as an Nth code according to different embedding amounts, and obtaining the bar code probe after all the micromolecular probes are coded. The invention provides a bar code probe obtained on the basis of coding of a plurality of small molecule probes, binary, ternary or higher coding can be realized according to whether the bar code probe is embedded into a carrier or not and according to different embedding amounts, and a bar code probe combination composed of a plurality of bar code probes is further obtained. According to the bar code probe combination provided by the invention, thousands of specific targets can be detected and analyzed through single imaging, the reliability is high, and the bar code probe combination has important significance in the field of biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of spectral detection probes, and in particular to a small molecule Raman probe, a barcode probe, and a coding method and application thereof. Background Art

[0002] Molecular probe labeling is a specific target detection method commonly used in the prior art. It uses specially designed probe molecules to label the substances and structures to be tested in biological systems, and realizes qualitative and quantitative detection of the molecules and structures to be tested through the specific signals of the probe molecules. It is widely used in basic biological research and clinical testing.

[0003] Fluorescence labeling is one of the most commonly used labeling methods, which mainly uses fluorescent dyes or proteins as probes, such as fluorescein, EGFP, piperazine dyes, etc. After the target substance is labeled with a fluorescent dye, the presence of the target substance can be confirmed by detecting the fluorescence information as needed in the subsequent experimental process to achieve qualitative detection. However, because the fluorescent probe has a small number of independent channels and has quenching problems, when the number of targets to be labeled is too large, the flux is insufficient, multiple staining is required, and the reliability is not high. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a small molecule Raman probe, a barcode probe and a coding method and application thereof.

[0005] The present invention is based on small molecule probes of aromatic compounds, and realizes binary, ternary or higher encoding to obtain barcode probes based on whether the small molecule probes are embedded in a carrier and the different embedding amounts. These barcode probes show different specific waveforms during Raman spectroscopy detection, and can accurately complete the detection of specific targets.

[0006] In a first aspect, the present invention provides a small molecule Raman probe, comprising a plurality of small molecule probes or isotope molecules thereof; the small molecule probes are aromatic compounds carrying cyano or alkynyl groups, or heterocyclic aromatic compounds carrying cyano or alkynyl groups.

[0007] The aromatic compounds of the present invention also include compounds derived from aromatic compounds by group substitution, such as substitution with any one or more of alkyl groups such as methyl, tert-butyl, hydroxyl, trifluoromethyl, amino, fluorine, chlorine or bromine; and substitution with one or more of nitrogen, oxygen or sulfur in a heterocyclic ring. The isotope molecule is obtained by isotope substitution on the basis of a small molecule probe, such as substitution of the same atom with an isotope of nitrogen, hydrogen or carbon.

[0008] Heterocyclic aromatic compounds carrying cyano or alkynyl groups have the following advantages:

[0009] (1) It has a narrow triple bond Raman linewidth;

[0010] (2) The resonance frequency can be easily adjusted by substituents, heterocycles, and isotopes, thereby achieving more distinguishable specific channels within a limited spectral bandwidth.

[0011] The following are some examples of small molecule probes:

[0012] 1-Ethynylpyrene, 2-Ethynyl-naphthalene, 9-Ethynylphenanthrene, 3-Ethynyl-1,1'-biphenyl, 4-Ethynylbiphenyl, 4,4'-diethynylbiphenyl, 4-amino-1-naphthocarbonitrile, 1-amino-2-cyanonaphthalene, 7-cyanoindole, indole-6-carbonitrile, 5-cyanoindole, 9-cyanophenanthrene, 5-amino-1-naphthocarbonitrile, 1-naphthocarbonitrile, 4-amino-4'-cyanobiphenyl, 3-hydroxy-1-naphthocarbonitrile, 4-cyano-4'-hydroxybiphenyl, (9ci)-1 H-benzimidazole-4-carbonitrile, 1H-benzimidazole-5-carbonitrile, 5-cyanoisoquinoline, 4-furan-2-benzonitrile, isoquinoline-8-carbonitrile, 1-amino-6-cyanonaphthalene, 2-naphthocarbonitrile, 4,4'-biphenyl dinitrile, benzofuran-5-carbonitrile, 4-(pyridin-3-yl)benzonitrile, 4-cyanoisoquinoline, 4-(pyridin-4-yl)benzonitrile, [1,1'-biphenyl]-3-carbonitrile, 3-cyanoisoquinoline, 6-cyanoquinoline, quinoline-4-carbonitrile, 3-cyanoisoquinoline Quinoline, 6-cyanoisoquinoline, 3-(pyridin-4-yl)benzonitrile, 1-cyanoisoquinoline, benzofuran-2-carbonitrile, 4-cyanoindole, 5-phenyl-2-pyridinecarbonitrile, 4-ethynylaniline, 4-tert-butylphenylacetylene, 1-ethynyl-4-nitrobenzene, 4-fluorophenylacetylene, phenylacetylene, 1-chloro-4-ethynylbenzene, 4-ethynyl-α,α,α-trifluorotoluene, 4-(trifluoromethoxy)phenylacetylene, 1-ethynyl-3,5-difluorobenzene, 5-ethynyl-1 ,2,3-trifluorobenzene, 3,5-bis(trifluoromethyl)phenylacetylene, 1-phenyl-2-(trimethylsilyl)acetylene, 3,4-diaminobenzonitrile, 4-aminobenzonitrile, 1,4-diphenylbutadiyne, 3,5-dimethyl-4-hydroxybenzonitrile, 4-phenylbenzonitrile, 4-tert-butylbenzonitrile, benzonitrile, p-chlorobenzonitrile, p-fluorobenzonitrile, terephthalonitrile, 4-(trifluoromethyl)benzonitrile, 2,4-difluorobenzonitrile, 2,4,5-trifluorobenzonitrile, tetrafluorophthalonitrile, tetrafluoroterephthalonitrile.

[0013] Preferably, the present invention provides a small molecule Raman probe comprising any one or more of the following:

[0014]

[0015] These 11 small molecule probes have the following advantages:

[0016] (1) Eight cyano-substituted molecular probe molecules can achieve equal spacing (about 12 cm -1 ) The triple bond Raman peak arrangement; the three alkynyl-substituted probe molecules can also be distinguished at equal intervals. And there is no interference from other peaks near these Raman peaks, which can achieve excellent 11-color Raman labeling.

[0017] (2) The triple bond Raman peak line widths of these molecules are all less than 10 cm -1 , which can realize high-density multi-color multiplexing marking.

[0018] (3) These molecules have stable structures, are transparent in the visible light band, have negligible electronic energy level absorption when excited by visible light and near-infrared light, are not easily photolyzed, and do not produce fluorescent background, thus enabling high signal-to-noise ratio Raman spectroscopy detection.

[0019] In a second aspect, the present invention provides a method for encoding a barcode probe, comprising:

[0020] All small molecule probes in the small molecule probe combination are encoded in sequence. If they are not embedded in the carrier, they are counted as the first encoding. If they are embedded in the carrier, they are counted as the Nth encoding according to the different embedding amounts. After all the encodings are completed, the barcode probes are obtained.

[0021] The first code described in the present invention is only a reference, which can be any coding method in the prior art, for example, "0" or "a". Correspondingly, if it is an embedded carrier, different codes are referred to according to the different embedding amounts. For example, if the first code is referred to as "0" and the embedding amount is a constant value, then N has only one choice (the second code), and the second code can only be referred to as "1"; but if the embedding amount uses two different values, then N includes two types (the second code and the third code), the second code can be referred to as "1", and the third code can be referred to as "2". Therefore, according to this method, "binary", "ternary" or higher-order coding can be achieved.

[0022] Furthermore, for all the small molecule probes in the small molecule probe combination, the characteristic peak spacing between each pair is greater than 10 wave numbers.

[0023] Furthermore, the carrier is a microsphere.

[0024] Furthermore, the microspheres are polymer microspheres or inorganic porous microspheres.

[0025] Furthermore, the surface of the microsphere is functionally modified; the functional modification includes functionalization of any one or more groups of amino, hydroxyl, carboxyl or thiol.

[0026] In a third aspect, the present invention provides a barcode probe combination, comprising: a plurality of barcode probes encoded by the encoding method.

[0027] Taking binary as an example (encoded as 0 or 1. 0 refers to no embedding in the carrier, and 1 refers to embedding in the carrier), assuming that three small molecule probes are used, then eight combinations of barcode probes such as "100", "010", "001", "110", "101", "011", "111" and "000" (that is, eight barcodes) can be obtained, which can be applied to the simultaneous detection of eight target substances. In actual applications, the number of small molecule probes can be increased, or a higher base can be used to achieve simultaneous detection of more target substances.

[0028] In situations where specific target substances need to be detected, the sample labeled with the barcode probe is sampled or imaged by Raman spectroscopy (e.g., T-SRS), that is, the specific barcode corresponding to the sample is obtained, and the detection and analysis of the specific target substance in the sample can be completed by comparing it with the barcode table. It can be seen that the barcode probe combination provided by the present invention can realize the detection and analysis of thousands of specific target substances through one imaging. In addition, it will not have the problem of probe bleaching, and has high reliability.

[0029] In a fourth aspect, the present invention provides the use of the barcode probe combination in Raman spectroscopy imaging or detection.

[0030] Furthermore, the Raman spectrum includes:

[0031] One or more of T-SRS, conventional frequency-domain stimulated Raman scattering, spontaneous Raman scattering, surface plasmon enhanced Raman scattering, resonance Raman scattering or tip enhanced Raman scattering.

[0032] T-SRS, also known as transient stimulated Raman scattering, is mainly used in the embodiments of the present invention. It is an ultrafast time-domain spectroscopy technology that can achieve high-spectral Raman data acquisition covering hundreds of wave numbers with only a single excitation and has the characteristics of high spectral resolution with a natural linewidth limit.

[0033] Furthermore, when the T-SRS method is used, any group of the substance to be tested, such as an alkynyl group or a cyano group, can be detected as long as each characteristic peak in the Raman spectrum obtained can be distinguished (for example, the spacing between different characteristic peaks is greater than 10 cm -1 ).

[0034] Furthermore, the application includes: one or more of super-multicolor microscopic imaging, spatial transcriptome imaging, spatial protein composition imaging or pathological imaging.

[0035] The application may also be an application in preparing a kit for Raman spectroscopy imaging or detection.

[0036] The present invention has the following beneficial effects:

[0037] The present invention provides a barcode probe encoding method and the encoded barcode probe based on aromatic compounds as small molecule probes. The distribution of peaks in the Raman spectrum can be adjusted by combining multiple small molecule probes and whether they are embedded in a carrier, and then the detection of specific signals can be achieved based on the difference in peak distribution. The barcode probe encoding method provided by the present invention can simultaneously achieve the labeling and detection of tens of thousands of specific targets, has high reliability, and has high application value in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 It is a schematic diagram of the encoding and application process of the barcode probe provided in Example 1 of the present invention.

[0040] Figure 2 It is a schematic diagram of the binary encoding scheme provided in Example 1 of the present invention.

[0041] Figure 3 This is a schematic diagram of the characteristic peak positions of 7 aromatic compounds provided in Example 2 of the present invention.

[0042] Figure 4 It is a schematic diagram of signals collected by 7 barcode probes provided in Example 2 of the present invention; the left figure is a schematic diagram of the position imaging of polymer microspheres bound by the barcode probes, and the right figure is a T-SRS spectrum diagram of the barcode probes.

[0043] Figure 5 This is a T-SRS spectrum diagram of the four barcode probes provided in Example 2 of the present invention.

[0044] Figure 6 It is a schematic diagram of the structures of 11 aromatic compounds used for encoding provided in Example 2 of the present invention.

[0045] Figure 7 These are the probe Raman spectra of 11 compounds provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] This embodiment provides a barcode probe design method, which specifically includes:

[0049] 1. Design of barcode probe

[0050] The design process is as follows Figure 1 As shown, specifically including:

[0051] (1) First, select small molecule probes with stable structures and strong signals (such as aromatic compounds carrying cyano or alkynyl groups) and introduce various substitution patterns such as group substitution, heterocyclic substitution, and isotope substitution. Group substitution includes methyl, hydroxyl, tert-butyl, trifluoromethyl, amino, fluorine, chlorine, bromine and other substitutions; heterocyclic substitution includes but is not limited to substitutions of nitrogen, oxygen or sulfur in five- and six-membered rings; isotope substitution includes but is not limited to substitutions of the same atom by related isotopes of nitrogen, hydrogen, and carbon. The Raman peak of a probe molecule containing one or more substitutions will shift by several to dozens of wave numbers relative to the original position. Therefore, a large number of small molecule probe combinations with different colors and relatively uniform intervals can be obtained through substitution and screening, which is convenient for subsequent encoding.

[0052] The following are examples of small molecule probes with stable structures and strong signals:

[0053] 1-ethynylpyrene, 2-ethynyl-naphthalene, 9-ethynylphenanthrene, 3-ethynyl-1,1'-biphenyl, 4-ethynylbiphenyl, 4,4'-diethynylbiphenyl, 4-amino-1-naphthocarbonitrile, 1-amino-2-cyanonaphthalene, 7-cyanoindole, indole-6-carbonitrile, 5-cyanoindole, 9-cyanophenanthrene, 5-amino-1-naphthocarbonitrile, 1-naphthocarbonitrile, 4-amino-4'-cyanobiphenyl, 3-hydroxy-1-naphthocarbonitrile, 4-cyano-4'-hydroxybiphenyl, (9 ci)-1H-benzimidazole-4-carbonitrile, 1H-benzimidazole-5-carbonitrile, 5-cyanoisoquinoline, 4-furan-2-benzonitrile, isoquinoline-8-carbonitrile, 1-amino-6-cyanonaphthalene, 2-naphthonitrile, 4,4'-biphenyl dinitrile, benzofuran-5-carbonitrile, 4-(pyridin-3-yl)benzonitrile, 4-cyanoisoquinoline, 4-(pyridin-4-yl)benzonitrile, [1,1'-biphenyl]-3-carbonitrile, 3-cyanoisoquinoline, 6-cyanoquinoline, quinoline -4-carbonitrile, 3-cyanoquinoline, 6-cyanoisoquinoline, 3-(pyridin-4-yl)benzonitrile, 1-cyanoisoquinoline, benzofuran-2-carbonitrile, 4-cyanoindole, 5-phenyl-2-pyridinecarbonitrile, 4-ethynylaniline, 4-tert-butylphenylacetylene, 1-ethynyl-4-nitrobenzene, 4-fluorophenylacetylene, phenylacetylene, 1-chloro-4-ethynylbenzene, 4-ethynyl-α,α,α-trifluorotoluene, 4-(trifluoromethoxy)phenylacetylene, 1-ethynyl -3,5-difluorobenzene, 5-ethynyl-1,2,3-trifluorobenzene, 3,5-bis(trifluoromethyl)phenylacetylene, 1-phenyl-2-(trimethylsilyl)acetylene, 3,4-diaminobenzonitrile, 4-aminobenzonitrile, 1,4-diphenylbutadiyne, 3,5-dimethyl-4-hydroxybenzonitrile, 4-phenylbenzonitrile, 4-tert-butylbenzonitrile, benzonitrile, p-chlorobenzonitrile, p-fluorobenzonitrile, terephthalonitrile, 4-(trifluoromethyl)benzonitrile, 2,4-difluorobenzonitrile, 2,4,5-Trifluorobenzonitrile, tetrafluorophthalonitrile, tetrafluoroterephthalonitrile (CAS No. 34993-56-1, 2949-26-0, 32870-98-7, 58650-11-6, 29079-00-3, 38215-38-2, 58728-64-6, 3100-67-2, 96631-87-7, 15861-36-6, 15861-24-2, 2510-55-6, 72016-73-0, 86-53-3, 4854- 84-6, 91059-46-0, 19812-93-2, 64574-21-6, 6287-83-8, 27655-41-0, 64468-77-5, 362606-11-9, 73399-92-5, 613-46-7, 1591-30-6, 79002-39-4, 294648-03-6, 34846-65-6, 144397-70-6, 24973-50-0, 26947-41-1, 23 395-72-4, 2973-27-5, 34846-64-5, 106778-42-1, 4350-55-4, 1198-30-7, 41717-32-2, 16136-52-0, 39065-45-7, 14235-81-5, 772-38-3, 937-31-5, 766-98-3, 536-74-3, 873-73-4, 705-31-7, 160542-02-9, 151361-87 4, 158816-55-8, 88444-81-9, 2170-06-1, 17626-40-3, 873-74-5, 886-66-8, 4198-90-7, 2920-38-9, 4210-32-6, 100-47-0, 623-03-0, 1194-02-1, 623-26-7, 455-18-5, 3939-09-1, 98349-22-5, 1835-65-0, 1835-49-0). It also includes the isotopic molecules of the above compounds.

[0054] (2) Taking a barcode probe as an example, its preparation method is as follows:

[0055] Select multiple aromatic compounds and collect Raman spectral signals to obtain corresponding characteristic peaks. According to the characteristic peak positions of these aromatic compound probes, they are sorted from small to large according to the wavenumber position and then encoded in sequence. The encoding method can be binary, ternary or higher. Taking the binary encoding scheme as an example, the aromatic compounds not embedded in the microspheres are encoded as "0", and the aromatic compounds embedded in the microspheres are encoded as "1" (the encoding diagram is shown in the figure). Figure 2As shown). Taking the ternary coding scheme as an example, the aromatic compounds not embedded in the microspheres are coded as "0", and the aromatic compounds embedded in the microspheres are coded as "1" or "2". At this time, "1" and "2" are coded according to the different embedding amounts. For example, the height of the characteristic peak coded as "1" is roughly half of the characteristic peak coded as "2". The height of the characteristic peak can be controlled by the embedding amount (concentration) of the corresponding aromatic compound. The same is true for higher-base coding schemes. These coding sequences are then defined as barcodes corresponding to unique barcode probes. Taking Table 1 as an example, different barcodes correspond to different detection substances:

[0056] Table 1 Barcode table corresponding to barcode probes

[0057] Compound A Compound B Compounds Compound W Compound X Barcode Matching 1 1 ··· 1 1 11···11 Sample1 1 1 ··· 1 0 11···10 Sample2 1 1 ··· 0 1 11···01 Sample3 ··· ··· ··· ··· ··· ··· Sample··· ··· ··· ··· ··· ··· ··· Sample··· 0 0 ··· 1 0 00···10 Samplem-2 0 0 ··· 0 1 00···01 Samplem-1 0 0 ··· 0 0 00···00 Samplem

[0058] Example 2

[0059] This embodiment provides a method for preparing multiple barcode probe combinations, which are as follows:

[0060] 1. Specific example of T-SRS spectrum of barcode probe combination (based on 7 aromatic compounds)

[0061] (1) First, select 7 aromatic compounds (commercial):

[0062] 4-ethynylaniline, 3,5-bis(trifluoromethyl)phenylacetylene, 1-phenyl-2-(trimethylsilyl)acetylene, 3,4-diaminobenzonitrile, 4-cyanobiphenyl, 2,4,5-trifluorobenzonitrile, 2,3,5,6-tetrafluoroterephthalonitrile (CAS numbers are 14235-81-5, 88444-81-9, 2170-06-1, 17626-40-3, 2920-38-9, 98349-22-5, 1835-49-0 respectively).

[0063] Among them, the T-SRS technology was used to collect alkynyl (CH≡C-) signals for the first three aromatic compound probes, and cyano (-C≡N) signals for the last four aromatic compound probes. The characteristic peaks collected by these seven aromatic compound probes were sorted from small to large according to the wave number position, namely:

[0064] 4-Ethynylaniline (2100cm-1), 3,5-bis(trifluoromethyl)phenylacetylene (2121cm-1), 1-phenyl-2-(trimethylsilyl)acetylene (2164cm-1), 3,4-diaminobenzonitrile (2220cm-1), 4-cyanobiphenyl (2233cm-1), 2,4,5-trifluorobenzonitrile (2248cm-1), 2,3,5,6-tetrafluoroterephthalonitrile (2260cm-1).

[0065] The results are as follows Figure 3 As shown in the figure, it can be seen that the distance between the characteristic peaks of each aromatic compound probe is greater than 10 wave numbers, and each characteristic peak position corresponds to only one small molecule probe, which meets the requirements of probe design.

[0066] (2) Binary coding is performed using the presence or absence of the T-SRS spectra of the seven aromatic compound probes. The characteristic peak of each probe is detected and recorded as "1", and the absence is recorded as "0". The order of 4-ethynylaniline, 3,5-bis(trifluoromethyl)phenylacetylene, 1-phenyl-2-(trimethylsilyl)acetylene, 3,4-diaminobenzonitrile, 4-cyanobiphenyl, 2,4,5-trifluorobenzonitrile, and 2,3,5,6-tetrafluoroterephthalonitrile is used as the coding channel, and each channel is recorded as "ABCDEFG" in sequence. Seven barcode IDs (i.e., seven barcode probes) are designed, namely:

[0067] ID:1(0110000), ID:2(0001010), ID:3(0100100), ID:4(1100100), ID:5(0001011), ID:6(1010100), ID:7(0001111).

[0068] Then, according to the specific code of the designed barcode, the corresponding aromatic compound and polymer microspheres are selected for mixing (the polymer microspheres are PMMA microspheres), and then the T-SRS signal is collected. The 7 barcode probes collect signals such as Figure 4 As shown, Figure 4 The middle left image shows the position of the polymer microspheres to which the barcode probe is bound. Figure 4 The middle right image shows the T-SRS spectrum of the barcode probe. Figure 4 It can be seen that the characteristic peak positions of the seven different spectra can be clearly identified, and then after decoding, the specific corresponding ID can be found from the barcode table (Table 2).

[0069] Table 2 Eight barcodes formed by T-SRS spectral labeling of seven barcode probes

[0070]

[0071]

[0072] (3) Ternary coding based on the presence and intensity of the T-SRS spectrum of aromatic compounds

[0073] Aromatic compounds with no characteristic peaks detected are coded as "0", and aromatic compounds with characteristic peaks detected are coded as "1" or "2" according to the height of the characteristic peaks. Figure 4Four different ternary barcode IDs were designed for the same coding channel, namely: ID: 1 (0100200), ID: 2 (1020200), ID: 3 (0121020), ID: 4 (2121022). These four different barcode IDs correspond to four barcode probes. The aromatic compounds corresponding to each barcode probe were mixed into polymer microspheres (polymer microspheres are polystyrene microspheres) for T-SRS signal collection. The obtained T-SRS spectrum is shown in the figure below. Figure 5 shown.

[0074] from Figure 5 It can be seen that the characteristic peak positions of different spectra can be clearly identified, and then after decoding, the specific corresponding ID can be found in the barcode book.

[0075] 2. Specific examples of barcode probes (including 11 aromatic compounds)

[0076] The aromatic compounds used are Figure 6 As shown in the figure, there are 11 kinds of aromatic compound probes, which are obtained by isotope substitution method using 4-ethynylbiphenyl, 1-cyanonaphthalene and 5-phenyl-2-pyridinecarbonitrile (CAS No. 29079-00-3, 86-53-3, 39065-45-7) as basic aromatic compound probes. These aromatic compound probes have significant spectroscopic advantages, as shown in the figure. Figure 7 As shown:

[0077] (1) These 11 molecules can achieve an equidistant (about 12 cm-1) triple-bond Raman peak arrangement, and there is no interference from other peaks near these Raman peaks, which can achieve excellent 11-color Raman labeling.

[0078] (2) The triple bond Raman peak line widths of these molecules are all less than 10 cm -1 , which can realize high-density multi-color multiplexing marking.

[0079] (3) These molecules have stable structures, are transparent in the visible light band, have negligible electronic energy level absorption when excited by visible light and near-infrared light, are not easily photolyzed, and do not produce fluorescent background, thus enabling high signal-to-noise ratio Raman spectroscopy detection.

[0080] In this case, the same binary or ternary encoding method as in step 1 above is used for barcode design and signal acquisition, which can achieve high-throughput rapid detection of 2048 or 177147 different targets at the same time.

[0081] The barcode probes encoded as above in the present application can be actually used in medical retrieval. By modifying the carboxyl groups on the surface of these encoded microsphere probes and coupling them with domain antibodies, they can be used for labeling and imaging of various cancer target proteins and further for medical detection.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A small molecule Raman probe, It is characterized in that include: Multiple small molecule probes or their isotope molecules; The small molecule probe is an aromatic compound carrying a cyano group or an alkynyl group, or a heterocyclic aromatic compound carrying a cyano group or an alkynyl group.

2. The small molecule Raman probe according to claim 1, It is characterized in that The small molecule probes include one or more of the following: 1-ethynylpyrene, 2-ethynyl-naphthalene, 9-ethynylphenanthrene, 3-ethynyl-1,1'-biphenyl, 4-ethynylbiphenyl, 4,4'-diethynylbiphenyl, 4-amino-1-naphthocarbonitrile, 1-amino-2-cyanonaphthalene, 7-cyanoindole, indole-6-carbonitrile, 5-cyanoindole, 9-cyanophenanthrene, 5-amino-1-naphthocarbonitrile, 1-naphthocarbonitrile, 4-amino-4'-cyanobiphenyl, 3-hydroxy-1-naphthocarbonitrile, 4-cyano-4 '-Hydroxybiphenyl, (9ci)-1H-benzimidazole-4-carbonitrile, 1H-benzimidazole-5-carbonitrile, 5-cyanoisoquinoline, 4-furan-2-benzonitrile, isoquinoline-8-carbonitrile, 1-amino-6-cyanonaphthalene, 2-naphthonitrile, 4,4'-biphenyl dinitrile, benzofuran-5-carbonitrile, 4-(pyridin-3-yl)benzonitrile, 4-cyanoisoquinoline, 4-(pyridin-4-yl)benzonitrile, [1,1'-biphenyl]-3-carbonitrile, 3-cyanoisoquinoline, 6-cyanoquinoline, quinoline- 4-carbonitrile, 3-cyanoquinoline, 6-cyanoisoquinoline, 3-(pyridin-4-yl)benzonitrile, 1-cyanoisoquinoline, benzofuran-2-carbonitrile, 4-cyanoindole, 5-phenyl-2-pyridinecarbonitrile, 4-ethynylaniline, 4-tert-butylphenylacetylene, 1-ethynyl-4-nitrobenzene, 4-fluorophenylacetylene, phenylacetylene, 1-chloro-4-ethynylbenzene, 4-ethynyl-α,α,α-trifluorotoluene, 4-(trifluoromethoxy)phenylacetylene, 1-ethynyl-3,5-difluorobenzene, 5- Ethylene-1,2,3-trifluorobenzene, 3,5-bis(trifluoromethyl)phenylacetylene, 1-phenyl-2-(trimethylsilyl)acetylene, 3,4-diaminobenzonitrile, 4-aminobenzonitrile, 1,4-diphenylbutadiyne, 3,5-dimethyl-4-hydroxybenzonitrile, 4-phenylbenzonitrile, 4-tert-butylbenzonitrile, benzonitrile, p-chlorobenzonitrile, p-fluorobenzonitrile, terephthalonitrile, 4-(trifluoromethyl)benzonitrile, 2,4-difluorobenzonitrile, 2,4,5-trifluorobenzonitrile, tetrafluorophthalonitrile, tetrafluoroterephthalonitrile.

3. A small molecule Raman probe, It is characterized in that Includes any one or more of the following:

4. A method for encoding a barcode probe, It is characterized in that include: All small molecule probes in the small molecule Raman probe described in any one of claims 1-3 are encoded in sequence. If they are not embedded in a carrier, they are counted as the first encoding. If they are embedded in a carrier, they are counted as the Nth encoding according to the different embedding amounts. After all the encoding is completed, a barcode probe is obtained.

5. The encoding method according to claim 4, It is characterized in that For all the small molecule Raman probes, the characteristic peak spacing between each other is greater than 10 wave numbers.

6. The encoding method according to claim 4 or 5, It is characterized in that The carrier is a microsphere; preferably, it is a polymer microsphere or an inorganic porous microsphere; the polymer microsphere is further preferably PMMA or polystyrene polymer microsphere.

7. The encoding method according to claim 6, It is characterized in that The surface of the microsphere is functionally modified; the functional modification includes the functionalization of any one or more groups of amino, hydroxyl, carboxyl or thiol.

8. A barcode probe combination, It is characterized in that include: A plurality of barcode probes encoded by the encoding method according to any one of claims 3 to 7.

9. Use of the barcode probe combination according to claim 8 in Raman spectroscopy imaging or detection.

10. The use according to claim 9, It is characterized in that The Raman spectrum includes: One or more of T-SRS, conventional frequency-domain stimulated Raman scattering, spontaneous Raman scattering, surface plasmon enhanced Raman scattering, resonance Raman scattering or tip enhanced Raman scattering; and / or, The applications include: one or more of super-multicolor microscopic imaging, spatial transcriptome imaging, spatial protein composition imaging or pathological imaging.

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