WO2019179386A1

WO2019179386A1 - Raman probe, and manufacturing method and application thereof

Info

Publication number: WO2019179386A1
Application number: PCT/CN2019/078450
Authority: WO
Inventors: 叶坚; 张雨晴; 顾雨清
Original assignee: 上海交通大学
Priority date: 2018-03-19
Filing date: 2019-03-18
Publication date: 2019-09-26
Also published as: CN110286112A; CN110286112B

Abstract

A Raman probe, having a nano-scale core (1), a first Raman signal layer (2), and a shell layer (3). The nano-scale core (1) is coated by the first Raman signal layer (2). Raman signal molecules are distributed within the first Raman signal layer (2). The shell layer (3) has a first layer (31) and a second layer (32). The first Raman signal layer (2) is coated by the first layer (31). The second layer (32) coats the outside of the first layer (31) and has a gap (4). The Raman probe has the advantages of having a high Raman signal strength and superior signal repeatability. The manufacturing method of a Raman probe is simple, and applicable to ultra-sensitive and ultra-fast Raman detection and imaging techniques.

Description

一种拉曼探针及其制备方法和应用Raman probe and preparation method and application thereof

技术领域Technical field

本发明属于纳米材料领域，涉及一种拉曼探针及其制备方法和应用。The invention belongs to the field of nano materials and relates to a Raman probe and a preparation method and application thereof.

背景技术Background technique

拉曼光谱是一种表征分子振动的指纹光谱。金属纳米颗粒在入射光的作用下产生等离激元共振现象，使得金属纳米颗粒表面吸附的分子的拉曼光谱得到极大增强，这称为表面增强拉曼散射效应(SERS)。近年来，结合了金属纳米颗粒(即SERS基底)和拉曼信号分子的新型拉曼探针受到越来越多的关注。在金属纳米颗粒上标记不同拉曼信号分子，可以得到具有不同信号的超灵敏拉曼探针，并有望实现多指标的分子检测和生物成像应用。Raman spectroscopy is a fingerprint spectrum that characterizes molecular vibration. The metal nanoparticles generate plasmon resonance under the action of incident light, which greatly enhances the Raman spectrum of the molecules adsorbed on the surface of the metal nanoparticles. This is called surface enhanced Raman scattering effect (SERS). In recent years, new Raman probes incorporating metal nanoparticles (ie, SERS substrates) and Raman signal molecules have received increasing attention. By labeling different Raman signal molecules on metal nanoparticles, ultrasensitive Raman probes with different signals can be obtained, and multi-index molecular detection and bioimaging applications are expected.

传统的探针是将拉曼信号分子吸附在金纳米颗粒表面，增强效果一般、拉曼信号重复性差、稳定性较差(Qian X M，Nie S M.Chem.Soc.Rev.，2008，37，912-920)。因此制备一种拉曼信号强、颗粒稳定性高、拉曼信号重复性好，特别是能够适用于不同拉曼信号分子标记的拉曼探针是一个迫切的科学和技术问题。Conventional probes adsorb Raman signal molecules on the surface of gold nanoparticles, and have a general enhancement effect, poor Raman signal repeatability, and poor stability (Qian X M, Nie S M. Chem. Soc. Rev., 2008, 37). , 912-920). Therefore, it is an urgent scientific and technical problem to prepare a Raman signal with high Raman signal, high particle stability and good Raman signal repeatability, especially for Raman probes with different Raman signal molecular markers.

经对现有技术的文献检索发现，Dong-Kwon Lim等人(Lim D K，Jeon K S，Hwang J H，et al.Nature nanotechnology，2011，6(7)：452-460)制备了具有1.2nm缝隙的核壳金纳米颗粒，通过将特制的DNA和拉曼信号分子包裹在缝隙结构中，可以得到具有较强拉曼信号的金纳米颗粒。A literature search of the prior art found that Dong-Kwon Lim et al. (Lim D K, Jeon K S, Hwang J H, et al. Nature nanotechnology, 2011, 6(7): 452-460) prepared 1.2. The core-shell gold nanoparticles in the nm gap can be obtained by encapsulating the special DNA and Raman signal molecules in the gap structure to obtain gold nanoparticles with strong Raman signals.

Srikanth Singamaneni和叶坚等人(Gandra N，Singamaneni S.Adv.Mater.2013，25，1022-1027；Lin L，Zapata M，Ye J，et al.Nano Lett.，2015，15(10)，6419-6428；Lin L，Gu H C，Ye J.Chem.Commun.，2015，51，17740-17743；Zhang Y Q，Xiao Z Y，Ye J，et al.ACS Appl.Mater.Interfaces 2017，9，3995-4005)制备了具有亚纳米缝隙的内嵌拉曼信号分子的核壳结构金纳米颗粒，内嵌的拉曼信号分子在金核和完整的金壳之间形成缝隙结构，使颗粒的拉曼信号得到增强。Srikanth Singamaneni and Ye Jian et al. (Gandra N, Singamaneni S. Adv. Mater. 2013, 25, 1022-1027; Lin L, Zapata M, Ye J, et al. Nano Lett., 2015, 15(10), 6419 -6428; Lin L, Gu H C, Ye J. Chem. Commun., 2015, 51, 17740-17743; Zhang Y Q, Xiao Z Y, Ye J, et al. ACS Appl. Mater. Interfaces 2017, 9, 3995-4005) Preparation of core-shell structure gold nanoparticles with embedded Raman signal molecules with sub-nano gaps. The embedded Raman signal molecules form a gap structure between the gold core and the intact gold shell, so that the particles are pulled. The MANN signal is enhanced.

然而，由于检测灵敏度和成像速度的需求，目前在近红外区拉曼探针的增强性能还需提高，以及在近红外区拉曼成像速度也还需提高。However, due to the need for detection sensitivity and imaging speed, the enhancement performance of the Raman probe in the near-infrared region needs to be improved, and the Raman imaging speed in the near-infrared region also needs to be improved.

发明内容Summary of the invention

针对现有技术的不足，本发明提供一种拉曼探针，其特征在于，具有纳米核、第一拉曼信号层和壳层；所述纳米核被所述第一拉曼信号层包被；所述第一拉曼信号层内分布有拉曼信号分子；所述壳层具有第一层和第二层；所述第一拉曼信号层被所述第一层包被；所述第二层包覆在所述第一层外，具有缝隙。In view of the deficiencies of the prior art, the present invention provides a Raman probe characterized by having a nano core, a first Raman signal layer and a shell layer; the nano core is coated by the first Raman signal layer a Raman signal molecule distributed in the first Raman signal layer; the shell layer has a first layer and a second layer; the first Raman signal layer is coated by the first layer; The second layer is wrapped outside the first layer and has a slit.

进一步地，所述缝隙为能够增强拉曼光谱信号强度的结构。Further, the slit is a structure capable of enhancing the intensity of the Raman spectral signal.

在一个具体实施方式中，所述纳米核、所述第一拉曼信号层和所述壳层组合成盛开的花朵状，花朵的相邻花瓣之间形成所述缝隙。In a specific embodiment, the nanocore, the first Raman signal layer, and the shell layer are combined into a flower shape in full bloom, and the gap is formed between adjacent petals of the flower.

在另一个具体实施方式中，在所述拉曼探针的截面上，所述壳层呈齿轮状，齿轮的相邻齿之间形成所述缝隙。In another embodiment, on the cross section of the Raman probe, the shell layer is gear-shaped, and the gap is formed between adjacent teeth of the gear.

进一步地，所述缝隙的数目为多个；所述缝隙的大小、形状不完全一致。Further, the number of the slits is plural; the size and shape of the slits are not completely identical.

进一步地，所述缝隙在所述第一拉曼信号层中的拉曼信号分子的作用下形成。Further, the slit is formed by the action of Raman signal molecules in the first Raman signal layer.

进一步地，所述第一拉曼信号层中的拉曼信号分子包括带有硝基的硫醇化合物。Further, the Raman signal molecule in the first Raman signal layer comprises a thiol compound bearing a nitro group.

进一步地，所述第一拉曼信号层中的拉曼信号分子选自同时含有巯基和硝基的化合物。Further, the Raman signal molecule in the first Raman signal layer is selected from the group consisting of a compound containing both a thiol group and a nitro group.

进一步地，所述第一拉曼信号层中的拉曼信号分子选自同时含有巯基、硝基和苯环的化合物。Further, the Raman signal molecule in the first Raman signal layer is selected from the group consisting of a compound containing a thiol group, a nitro group and a benzene ring.

进一步地，所述第一拉曼信号层中的拉曼信号分子选自4-硝基苯硫醇(4-NITROBENZENETHIOL，简称4-NBT)、3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑中的一种或多种；结构式如下：Further, the Raman signal molecule in the first Raman signal layer is selected from the group consisting of 4-nitrobenzenethiol (4-NITROBENZENETHIOL, abbreviated as 4-NBT), 3-nitrobenzylthiol, 2-amino One or more of 5-nitrobenzenethiol, o-nitrothiophenol, 2-mercapto-6-nitrobenzothiazole, and 2-mercapto-5-nitrobenzimidazole; the structural formula is as follows:

进一步地，所述第一层为封闭结构。Further, the first layer is a closed structure.

进一步地，所述缝隙内分布有拉曼信号分子。Further, a Raman signal molecule is distributed in the slit.

在一个具体实施方式中，所述第一拉曼信号层的拉曼信号分子与所述缝隙内的拉曼信号分子是相同的。这有利于进一步增强该拉曼探针的拉曼信号。In a specific embodiment, the Raman signal molecules of the first Raman signal layer are identical to the Raman signal molecules within the gap. This is advantageous to further enhance the Raman signal of the Raman probe.

在另一个具体实施方式中，所述第一拉曼信号层的拉曼信号分子与所述缝隙内的拉曼信号分子是不同的。这有利于该拉曼探针形成多指标检测的拉曼信号。In another embodiment, the Raman signal molecules of the first Raman signal layer are different from the Raman signal molecules within the gap. This facilitates the formation of a Raman signal for multi-indicator detection by the Raman probe.

进一步地，所述缝隙内的拉曼信号分子通过作用力吸附到所述壳层上。Further, the Raman signal molecules in the slit are adsorbed onto the shell layer by a force.

进一步地，所述壳层为金壳层、银壳层、铜壳层或者铂壳层。Further, the shell layer is a gold shell layer, a silver shell layer, a copper shell layer or a platinum shell layer.

进一步地，所述缝隙内的拉曼信号分子包括可与所述壳层产生静电吸附作用或化学共价结合的分子。优选地，所述缝隙内的拉曼信号分子选自对腈基苯硫醇、3-氟苯硫醇、2-苯硫酚、3、4-二氯苯硫酚、4-硝基苯硫醇、3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑中的一种或多种。Further, the Raman signal molecules in the gap comprise molecules that can electrostatically or chemically covalently bond to the shell layer. Preferably, the Raman signal molecule in the gap is selected from the group consisting of p-cyanobenzenethiol, 3-fluorobenzenethiol, 2-thiophenol, 3,4-dichlorothiophenol, 4-nitrobenzene sulfur Alcohol, 3-nitrobenzylthiol, 2-amino-5-nitrobenzenethiol, o-nitrothiophenol, 2-mercapto-6-nitrobenzothiazole and 2-mercapto-5-nitrate One or more of the benzimidazoles.

进一步地，所述纳米核为金纳米核、银纳米核、铜纳米核或铂纳米核。Further, the nano core is a gold nano core, a silver nano core, a copper nano core or a platinum nano core.

进一步地，还具有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；所述外层结构包被在所述第二层外。带氨基的高分子化合物，如多环芳烃(PAH)；带负电的高分子化合物，如聚乙二醇(PEG)、聚苯乙烯磺酸钠(PSS)等；巯基化合物，如巯基十一烷酸(MUA)、巯基十一烷醇等。Further, having an outer layer structure; the outer layer structure is mesoporous silica, a mercapto compound or a polymer compound capable of electrostatically adsorbing or chemically covalently bonding with the shell layer; Is outside the second layer. A polymer compound having an amino group such as a polycyclic aromatic hydrocarbon (PAH); a negatively charged polymer compound such as polyethylene glycol (PEG), sodium polystyrene sulfonate (PSS), or the like; a mercapto compound such as nonyldecane Acid (MUA), mercapto undecyl alcohol, and the like.

本发明还涉及一种如上所述的拉曼探针的制备方法，其特征在于，包括如下步骤：The invention also relates to a method for preparing a Raman probe as described above, characterized in that it comprises the following steps:

步骤一、将原料纳米核颗粒加入到表面活性剂的水溶液里，离心，重分散在表面活性剂的水溶液中，得到以表面活性剂为稳定剂的纳米核；Step 1: adding the raw material nano-nuclear particles to the aqueous solution of the surfactant, centrifuging, and redispersing in the aqueous solution of the surfactant to obtain a nano-nucleus with a surfactant as a stabilizer;

步骤二、在所述步骤一得到的以表面活性剂为稳定剂的纳米核中加入拉曼信号分子溶液，离心，重分散在表面活性剂的水溶液中，制备得到在纳米核的外表面包被有所述第一拉曼信号层的纳米颗粒，即在纳米核的外表面修饰有所述拉曼信号分子的纳米颗粒；Step 2: adding a Raman signal molecule solution to the nano-nuclear obtained by using the surfactant as a stabilizer in the first step, centrifuging, redispersing in an aqueous solution of the surfactant, and preparing the outer surface of the nano-nucleus to be coated. The nanoparticles of the first Raman signal layer, that is, the nanoparticles having the Raman signal molecule modified on the outer surface of the nano core;

步骤三、将所述步骤二得到的在纳米核的外表面包被有所述第一拉曼信号层的纳米颗粒加入到含有表面活性剂的水溶液、金属离子化合物溶液、还原剂混合的生长液中，得到具有所述壳层包被在所述第一拉曼信号层外的纳米颗粒，即得到所述拉曼探针；所述金属离子化合物溶液选自氯金酸溶液、硝酸银溶液、氯化铜溶液、硫酸铜溶液和氯铂酸溶液中的一种或多种。当壳层为金壳层时，与还原剂反应的所述金属离子化合物溶液一般是氯金酸溶液；当壳层为银壳层时，与还原剂反应的所述金属离子化合物溶液一般是硝酸银溶液；当壳层为铜壳层时，与还原剂反应的所述金属离子化合物溶液一般是氯化铜溶液和/或硫酸铜溶液；当壳层为铂壳层时，与还原剂反应的所述金属离子化合物溶液一般是氯铂酸溶液。Step 3: adding the nanoparticles coated with the first Raman signal layer on the outer surface of the nano core obtained in the step 2 to a growth liquid containing a surfactant, an aqueous solution of a metal ion compound, and a reducing agent. Obtaining a nanoparticle having the shell layer coated outside the first Raman signal layer, thereby obtaining the Raman probe; the metal ion compound solution is selected from the group consisting of a chloroauric acid solution, a silver nitrate solution, and a chlorine One or more of a copper solution, a copper sulfate solution, and a chloroplatinic acid solution. When the shell layer is a gold shell layer, the metal ion compound solution reacted with the reducing agent is generally a chloroauric acid solution; when the shell layer is a silver shell layer, the metal ion compound solution reacted with the reducing agent is generally nitric acid. a silver solution; when the shell layer is a copper shell layer, the metal ion compound solution reacted with the reducing agent is generally a copper chloride solution and/or a copper sulfate solution; and when the shell layer is a platinum shell layer, reacts with the reducing agent The metal ion compound solution is typically a chloroplatinic acid solution.

进一步地，步骤二在步骤一得到的以表面活性剂为稳定剂的纳米核中加入拉曼信号分子溶液后，混合震荡2-20分钟后，再进行后续操作。Further, in step 2, after adding the Raman signal molecule solution to the nano-nucleus with the surfactant as the stabilizer obtained in the first step, the mixture is shaken for 2-20 minutes, and then the subsequent operation is performed.

进一步地混合震荡时间为5-10分钟，优选为5分钟。The mixing time is further mixed for 5-10 minutes, preferably 5 minutes.

进一步地，在所述步骤三得到的具有所述壳层的纳米颗粒外包覆介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物。Further, the nanoparticle having the shell layer obtained in the step 3 is coated with mesoporous silica, a mercapto compound or a polymer compound which can electrostatically adsorb or chemically covalently bond with the shell layer.

进一步地，还包括步骤四，向所述步骤三得到的具有所述壳层的纳米颗粒中加入拉曼信号分子，离心，重分散在表面活性剂的水溶液中，得到所述第二层的缝隙中修饰有拉曼信号分子的纳米颗粒。Further, the method further includes a step 4, adding a Raman signal molecule to the nanoparticle having the shell layer obtained in the step 3, centrifuging, and redispersing in an aqueous solution of the surfactant to obtain a gap of the second layer. Nanoparticles modified with Raman signal molecules.

进一步地，在所述步骤四得到的所述第二层的缝隙中修饰有拉曼信号分子的纳米颗粒外包覆介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物。Further, the nanoparticle modified with the Raman signal molecule in the gap of the second layer obtained in the step 4 is coated with mesoporous silica, a mercapto compound or may be electrostatically adsorbed with the shell layer or Chemically covalently bonded polymeric compounds.

进一步地，所述步骤一中的原料纳米核颗粒的制备方法包括柠檬酸钠热还原法、种子生长法、聚乙烯吡咯烷酮保护还原法或紫外光引发还原法。Further, the preparation method of the raw material nano-nuclear particles in the first step comprises a sodium citrate thermal reduction method, a seed growth method, a polyvinylpyrrolidone protective reduction method or an ultraviolet light-induced reduction method.

进一步地，所述表面活性剂选自十六烷基氯化铵、十六烷基溴化铵、聚乙烯吡咯烷酮中的一种或者多种。Further, the surfactant is selected from one or more of cetyl ammonium chloride, cetyl ammonium bromide, and polyvinyl pyrrolidone.

进一步地，所述步骤三中的还原剂选自抗坏血酸、盐酸羟胺、甲醛中的一种或者多种。Further, the reducing agent in the third step is selected from one or more of ascorbic acid, hydroxylamine hydrochloride and formaldehyde.

本发明还涉及如上所述的拉曼探针的应用，比如，在单颗粒检测方面的应用。The invention also relates to the use of Raman probes as described above, for example in the application of single particle detection.

在一个具体实施方式中，所述拉曼探针的第二层外包被有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；包括以下步骤：In a specific embodiment, the second layer of the Raman probe is overcoated with an outer layer structure; the outer layer structure is mesoporous silica, a mercapto compound or can be electrostatically adsorbed with the shell layer or Chemically covalently bonded polymeric compounds; including the following steps:

A、将所述拉曼探针的溶液滴于硅片上，干燥后将硅片固定在原子力-显微共焦拉曼联用光谱仪上进行单颗粒测试；A, the solution of the Raman probe is dropped on a silicon wafer, and after drying, the silicon wafer is fixed on an atomic force-microscopic confocal Raman spectrometer for single particle test;

B、首先对有拉曼探针的硅片进行原子力显微成像，找到并确认硅片上的多个单颗粒；然后对单颗粒依次进行拉曼光谱采集，并对结果进行分析。B. Firstly, atomic force microscopy imaging of the silicon wafer with Raman probe is performed to find and confirm a plurality of single particles on the silicon wafer; then, the single particles are sequentially subjected to Raman spectroscopy, and the results are analyzed.

在另一个具体实施方式中，所述应用基于拉曼流式的单颗粒标记液相芯片；包括以下步骤：In another embodiment, the application is based on a Raman flow single particle labeling liquid phase chip; comprising the steps of:

a、基于拉曼流式的单颗粒标记液相芯片，单颗粒拉曼探针作为生物标记物，内嵌不同的拉曼信号分子实现编码，得到编码后的单颗粒拉曼探针；然后将每种所述编码后的单颗粒拉曼探针共价交联上针对特定检测物，即靶分子的捕获分子，得到针对不同检测物的编码单颗粒拉曼探针；所述捕获分子包括抗原、抗体和/或核酸探针；a Raman flow-based single-particle labeling liquid phase chip, single-particle Raman probe as a biomarker, embedded with different Raman signal molecules to achieve encoding, to obtain a coded single-particle Raman probe; Each of the encoded single-particle Raman probes is covalently cross-linked to a specific detector, ie, a capture molecule of a target molecule, to obtain a single-particle Raman probe encoding a different analyte; the capture molecule includes an antigen , antibodies and/or nucleic acid probes;

b、先把多种所述针对不同检测物的编码单颗粒拉曼探针混合，再加入微量待检样本，形成悬液；在所述悬液中，所述待检样本中的靶分子与所述针对不同检测物的编码单颗粒拉曼探针表面交联的捕获分子发生特异性结合；最后使用显微共焦拉曼光谱仪识别所述针对不同检测物的编码单颗粒拉曼探针的编码及与之特异性结合的靶分子，从而识别待检样本。b. first mixing a plurality of the encoded single-particle Raman probes for different detection substances, and then adding a trace amount of the sample to be tested to form a suspension; in the suspension, the target molecules in the sample to be tested are The specific molecules of the single-particle Raman probe surface cross-linked capture molecules for different detections are specifically bound; finally, the micro-confocal Raman spectrometer is used to identify the single-particle Raman probes for different detections. The target molecule is encoded and specifically bound to identify the sample to be examined.

又如，在细胞水平成像中的应用。Another example is the application in cell level imaging.

进一步地，所述拉曼探针的第二层外包被有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；包括以下步骤：Further, the second layer of the Raman probe is overcoated with an outer layer structure; the outer layer structure is mesoporous silica, a mercapto compound or may be electrostatically adsorbed or chemically covalently bonded to the shell layer. High molecular compound; includes the following steps:

1)、将细胞与所述拉曼探针的溶液共孵育，使所述拉曼探针进入细胞内部；1) co-incubating the cells with the solution of the Raman probe to allow the Raman probe to enter the interior of the cell;

2)、用拉曼光谱仪对经所述步骤1)处理过的细胞进行拉曼成像，对成像结果进行分析。2) Raman imaging of the cells treated in the step 1) by Raman spectroscopy, and analyzing the imaging results.

进一步地，所述步骤1)中将细胞与0.001-100nmol/L的所述拉曼探针的溶液共同置于细胞培养箱中，以37℃孵育0.5-24h，使所述拉曼探针进入细胞内部。Further, in the step 1), the cells are placed together with a solution of 0.001-100 nmol/L of the Raman probe in a cell incubator, and incubated at 37 ° C for 0.5-24 h to allow the Raman probe to enter. Inside the cell.

进一步地，所述步骤2)中每个像素点的积分时间为0.7-10ms，激光功率为1％-10％，高分辨率的细胞成像可在3-20s内完成；所述高分辨率的细胞成像指细胞成像的像素点大于等于50×50像素点。Further, the integration time of each pixel in the step 2) is 0.7-10 ms, the laser power is 1%-10%, and the high-resolution cell imaging can be completed within 3-20 s; Cell imaging refers to the pixel point of cell imaging greater than or equal to 50 x 50 pixels.

再如，在医学成像中的应用。Another example is the application in medical imaging.

进一步地，包括以下步骤：Further, the following steps are included:

I、将所述拉曼探针均匀分散于生理盐水或pH＝7.4的PBS溶液中制成0.01-50nmol/L的拉曼探针溶液；I, the Raman probe is uniformly dispersed in physiological saline or PBS solution of pH=7.4 to prepare a Raman probe solution of 0.01-50 nmol/L;

II、向试验动物体内局部注射经超声分散的所述步骤I制得的0.01-50nmol/L的拉曼探针溶液；II. Injecting 0.01-50 nmol/L of Raman probe solution prepared by the step I obtained by ultrasonication into the test animal;

III、注射0.5-24h后，使用拉曼光谱仪对经所述步骤II处理的试验动物的感兴趣部位进行拉曼成像，并对成像结果进行分析。III. After 0.5-24 hours of injection, the region of interest of the test animal treated in the step II was subjected to Raman imaging using a Raman spectrometer, and the imaging results were analyzed.

进一步地，所述步骤III中每个像素点的积分时间为0.7-100ms。优选地，所述步骤III中每个像素点的积分时间为0.7-10ms。Further, the integration time of each pixel in the step III is 0.7-100 ms. Preferably, the integration time of each pixel in the step III is 0.7-10 ms.

再如，在离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用。As another example, the application in the imaging of isolated tissues, isolated organs, dead human bodies or dead animals.

进一步地，包括以下步骤：Further, the following steps are included:

II、将离体组织、离体器官与经超声分散的所述步骤I制得的0.01-50nmol/L的拉曼探针溶液共孵育10-60分钟，或者向已经死亡的人体或已经死亡的动物体内局部注射经超声分散的所述步骤I制得的0.01-50nmol/L的拉曼探针溶液；II. Incubating the ex vivo tissue and the isolated organ with the 0.01-50 nmol/L Raman probe solution prepared by the step I of the ultrasonic dispersion for 10 to 60 minutes, or to the dead human body or having died. a 0.01-50 nmol/L Raman probe solution prepared by the step I obtained by ultrasonic dispersion is locally injected into the animal;

III、使用拉曼光谱仪对经所述步骤II处理的离体组织、离体器官、已经死亡的人体或已经死亡的动物体的感兴趣部位进行拉曼成像，并对成像结果进行分析。III. Raman imaging was performed on the isolated tissue treated by the step II, the isolated organ, the dead human body or the dead animal body using a Raman spectrometer, and the imaging results were analyzed.

进一步地，所述步骤III为在孵育10-60分钟后，使用拉曼光谱仪对经所述步骤II处理的离体组织、离体器官、已经死亡的人体或已经死亡的动物体的感兴趣部位进行拉曼成像，并对成像结果进行分析。Further, the step III is a portion of interest of the isolated tissue, the isolated organ, the dead human body or the dead animal body treated by the step II after Rab spectroscopy for 10 to 60 minutes after incubation. Raman imaging was performed and the imaging results were analyzed.

本发明还涉及如上所述的拉曼探针在生物医学检测领域中的应用，在DNA检测、RNA检测、外泌体检测和/或抗原抗体检测中的应用，在制备肿瘤检测试剂盒、肿瘤治疗试剂盒、肿瘤检测治疗一体化试剂盒或者肿瘤药物中的应用，以及在防伪领域中的应用。The invention also relates to the application of the Raman probe as described above in the field of biomedical detection, in the application of DNA detection, RNA detection, exosome detection and/or antigen antibody detection, in preparing a tumor detection kit, tumor Therapeutic kits, tumor detection and treatment integration kits or applications in oncology drugs, and applications in the field of anti-counterfeiting.

SERS的增强原理：目前学术界普遍认同的SERS机理主要有电磁场增强机理和化学增强机理两类。电磁场增强机理：表面等离子体共振引起的局域电磁场增强被认为是最主要的贡献。化学增强机理：金属与吸附分子在入射光的作用下发生电荷转移而产生电子共振。一般认为化学增强的作用较电磁场增强的作用弱。The principle of SERS enhancement: At present, the SERS mechanism generally recognized by the academic community mainly includes electromagnetic field enhancement mechanism and chemical enhancement mechanism. Electromagnetic field enhancement mechanism: Local electromagnetic field enhancement caused by surface plasmon resonance is considered to be the most important contribution. Chemical strengthening mechanism: metal and adsorbed molecules undergo charge transfer under the action of incident light to generate electron resonance. It is generally believed that the effect of chemical enhancement is weaker than that of electromagnetic field enhancement.

缝隙增强拉曼探针的增强机理：首先，与传统的二聚体相比，纳米核与壳层(简称核壳)之间的亚纳米缝隙提供了大量的电磁场增强和化学增强热点。其次，核壳之间的分子连接处的电荷转移效应，会产生强的化学增强和适当的电磁增强。强的化学增强主要是由于电子由分子的高能轨道转移到金属层(包括纳米核和壳层)，再由金属层转移到分子的低能轨道。The enhancement mechanism of the slit-enhanced Raman probe: First, the sub-nano gap between the nano-nucleus and the shell layer (referred to as the core shell) provides a large number of electromagnetic field enhancement and chemical enhancement hotspots compared with the conventional dimer. Second, the charge transfer effect at the molecular junction between the core shells produces strong chemical enhancement and proper electromagnetic enhancement. Strong chemical enhancement is mainly due to the transfer of electrons from the high energy orbitals of the molecules to the metal layers (including the nanocores and shells), which are then transferred from the metal layers to the low energy orbitals of the molecules.

本发明的拉曼探针的第一拉曼信号层位于核壳之间，其相对于由金、银、铜或铂构成的核壳结构(纳米核与壳层)来说，相当于核壳之间的缝隙层，当其大小属于亚纳米级别时，即等同于上一段所述的核壳之间的亚纳米缝隙。因此本发明的拉曼探针能够具有缝隙增强拉曼探针的增强机理。此外，本发明的拉曼探针壳层的第二层上的缝隙也提供了大量的电磁场增强和化学增强热点。从而使得本发明的拉曼探针，当第一拉曼信号层的拉曼信号分子与第二层上的缝隙中的拉曼信号分子是相同的时，其拉曼信号得到更进一步的增强；当第一拉曼信号层的拉曼信号分子与第二层上的缝隙中的拉曼信号分子是不同的时，具备多指标编码的功能。The first Raman signal layer of the Raman probe of the present invention is located between the core shells, which is equivalent to the core shell with respect to the core-shell structure (nano-nuclear and shell) composed of gold, silver, copper or platinum. The gap layer between the layers, when its size belongs to the sub-nanometer level, is equivalent to the sub-nano gap between the core shells described in the previous paragraph. Therefore, the Raman probe of the present invention can have an enhancement mechanism of the slit-enhanced Raman probe. In addition, the gaps in the second layer of the Raman probe shell of the present invention also provide a large number of electromagnetic field enhancement and chemical enhancement hot spots. Therefore, the Raman probe of the present invention is further enhanced when the Raman signal molecule of the first Raman signal layer is the same as the Raman signal molecule in the gap on the second layer; When the Raman signal molecule of the first Raman signal layer is different from the Raman signal molecule in the gap on the second layer, the function of multi-indicator coding is provided.

本发明的拉曼探针的优点有：近红外区增强性能好，高材料稳定性、高光稳定性、非共振激发下的低光热损伤等。The advantages of the Raman probe of the present invention are: good enhancement performance in the near-infrared region, high material stability, high light stability, low photothermal damage under non-resonant excitation, and the like.

非共振激发下低光热损伤：本发明的拉曼探针的紫外可见光谱吸收峰在590nm左右，在可见光区域。而使用近红外区域的785nm波长的激发光去激发该拉曼探针，避开了其吸收最大处，显著地减少了光吸收引起的热效应，可以极大的降低生物组织的热损伤，尤其适合于生物成像应用。Low photothermal damage under non-resonant excitation: The Raman probe of the present invention has an ultraviolet visible spectrum absorption peak around 590 nm in the visible light region. The excitation light of 785 nm wavelength in the near-infrared region is used to excite the Raman probe, avoiding the maximum absorption, significantly reducing the thermal effect caused by light absorption, and greatly reducing the thermal damage of the biological tissue, especially suitable for For bioimaging applications.

紫外可见光谱的最大吸收峰由金属的形貌决定。传统的选择是激光尽量靠近其最大吸收峰。本发明用了远离最大吸收峰的激光波长来激发。好处是：(1)减少了光吸收引起的热效应，使得材料的光稳定性非常优异。光稳定性指在连续成像的时候拉曼探针的稳定性。(2)降低了对生物组织的光热损伤。The maximum absorption peak of the UV-Vis spectrum is determined by the morphology of the metal. The traditional choice is to have the laser as close as possible to its maximum absorption peak. The present invention uses a laser wavelength away from the maximum absorption peak to excite. The benefits are: (1) The thermal effect caused by light absorption is reduced, making the material excellent in light stability. Light stability refers to the stability of the Raman probe at the time of continuous imaging. (2) Reduce the photothermal damage to biological tissues.

对比本发明的拉曼探针与传统球形拉曼探针(见图4A和图4B)：Comparing the Raman probe of the present invention with a conventional spherical Raman probe (see Figures 4A and 4B):

1、传统球形拉曼探针：其吸收峰在530nm左右，在可见光区域，使用共振波长532nm激光去激发时，拉曼增强性能较好。而使用近红外激光785nm激发，增强效果非常不好。1. Conventional spherical Raman probe: its absorption peak is about 530 nm. In the visible region, when Raman excitation is performed using a resonant wavelength of 532 nm, the Raman enhancement performance is better. The excitation effect is very poor when excited by a near-infrared laser at 785 nm.

2、本发明的拉曼探针：吸收峰在590nm左右，使用吸收较强的532nm激光去激发时，拉曼增强性能不好。而使用吸收非常弱的近红外激光785nm激发，增强效果非常好。2. The Raman probe of the present invention: the absorption peak is about 590 nm, and the Raman enhancement performance is not good when the 532 nm laser with a strong absorption is used for excitation. The 785 nm excitation with a very weak absorption of the near-infrared laser is very effective.

本发明的拉曼探针的新特点：New features of the Raman probe of the present invention:

a)利用内嵌的拉曼信号分子4-硝基苯硫醇既可形成核壳之间的缝隙层，此缝隙层为拉曼探针内部的完整的缝隙层，又可造成壳层第二层上的缝隙(形成具有大量的电磁场“热点”)，这样得到的纳米颗粒内部(核壳之间的缝隙层)和外部(第二层上的缝隙)都具有大量的电磁场“热点”，因此可实现高性能的拉曼增强效果。大量内部和外部电磁场“热点”的结合，实现高增强倍数，目前已实现单颗粒拉曼信号的检测(高灵敏度)。a) using the embedded Raman signal molecule 4-nitrobenzenethiol can form a gap layer between the core shell, which is a complete gap layer inside the Raman probe, and can also cause the shell layer second a gap on the layer (formed with a large number of electromagnetic fields "hot spots"), so that the inside of the nanoparticle (the gap layer between the core shell) and the outside (the gap on the second layer) have a large number of electromagnetic field "hot spots", so High performance Raman enhancement is achieved. A large number of internal and external electromagnetic field "hot spots" combine to achieve high enhancement multiples, and single-particle Raman signal detection (high sensitivity) has been achieved.

b)不同的拉曼信号分子(比如4-硝基苯硫醇、3-硝基苯甲基硫醇和2-巯基-5-硝基苯并咪唑)吸附时间影响核壳之间的缝隙层的结构和第二层上的缝隙的形貌，同时影响最终的拉曼增强性能(电镜照片见图2)。b) Different Raman signal molecules (such as 4-nitrophenylthiol, 3-nitrobenzylthiol and 2-mercapto-5-nitrobenzimidazole) adsorption time affects the gap layer between the core shells The morphology of the structure and the gap on the second layer, while affecting the final Raman enhancement performance (see Figure 2 for an electron micrograph).

c)外部(第二层上的缝隙)电磁场的大量“热点”提供了吸附不同拉曼信号分子的机会，可容易实现多指标检测和成像。c) External (slit on the second layer) A large number of "hot spots" of electromagnetic fields provide the opportunity to adsorb different Raman signal molecules, making it easy to achieve multi-indicator detection and imaging.

本发明的有益效果包括：Advantageous effects of the present invention include:

1、进一步提高了拉曼增强性能。与本申请人的前期研究成果专利CN201610200580.8的核壳结构相比提高1-2个数量级；与普通的纳米金球，纳米金棒等探针相比，增强性能提高4-5个数量级。1. Raman enhancement performance is further improved. Compared with the core-shell structure of the applicant's previous research result patent CN201610200580.8, it is 1-2 orders of magnitude higher; compared with ordinary nano gold balls, nano gold bars and other probes, the enhancement performance is improved by 4-5 orders of magnitude.

2、可以实现单颗粒的拉曼信号检测。2, can achieve single-particle Raman signal detection.

3、50×50像素的细胞层面的拉曼成像时间为6s，其中这6s包括积分时间、激光移动时间和机器对数据的处理输出时间。实现了细胞层面的高分辨率(比如50×50像素)高速(比如6s)的拉曼成像。根据文献结果这是目前全球在高分辨成像下最快的速度。而现有技术中的拉曼探针由于积分时间长(目前已报道的数据最快是30s)，因此即使在激光移动时间和机器对数据的处理输出时间不变的情况下，其50×50像素的细胞层面的拉曼成像时间也远远大于6s。3, 50 × 50 pixel cell level Raman imaging time is 6s, which 6s include integration time, laser movement time and machine processing data output time. High-resolution (such as 50 × 50 pixels) high-speed (such as 6s) Raman imaging at the cell level is achieved. According to the literature, this is the fastest speed in the world under high-resolution imaging. However, the Raman probe in the prior art has a long integration time (the fastest reported data is currently 30 s), so even when the laser moving time and the processing output time of the machine are unchanged, the 50×50 The Raman imaging time at the cell level of the pixel is also much greater than 6 s.

4、3.2cm×2.7cm组织层面的拉曼成像时间是52s。即实现了组织层面大范围(比如3.2cm×2.7cm)的超快速(比如52s)拉曼成像。这52s包括积分时间，样品移动时间，机器对数据的处理输出时间。4. The Raman imaging time of the 3.2 cm × 2.7 cm tissue level is 52 s. That is, ultra-fast (such as 52s) Raman imaging at a large scale (such as 3.2cm × 2.7cm) at the organizational level is achieved. This 52s includes the integration time, the sample movement time, and the machine's processing output time for the data.

5、本发明的拉曼探针稳定、生物相容性好。5. The Raman probe of the present invention is stable and biocompatible.

6、可实现多指标的分子检测和生物细胞和组织成像。6, can achieve multiple indicators of molecular detection and biological cell and tissue imaging.

本发明的拉曼探针具有拉曼信号强、信号重复性好、能够适用于不同拉曼信号分子标记、制备简单等优点，可用于超灵敏拉曼检测技术、多指标分子检测应用、及超快速的生物医学拉曼成像。The Raman probe of the invention has the advantages of strong Raman signal, good signal repeatability, can be applied to different Raman signal molecular markers, simple preparation, and the like, and can be used for ultra-sensitive Raman detection technology, multi-index molecular detection application, and super Rapid biomedical Raman imaging.

本发明所述的“化合物”，包括所有立体异构体、几何异构体、互变异构体和同位素。"Compound" as used in the present invention includes all stereoisomers, geometric isomers, tautomers and isotopes.

本发明所述的“化合物”，可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。本发明中含有不对称碳原子的化合物，可以光学活性纯的形式或外消旋形式被分离出来；光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。"Compounds" as used herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compound containing an asymmetric carbon atom in the present invention can be isolated in optically active pure form or in racemic form; the optically active pure form can be resolved from a racemic mixture, or by using a chiral starting material or a chiral reagent. synthesis.

本发明所述的“化合物”，还包括互变异构体形式；互变异构体形式来源于一个单键与相邻的双键交换并一起伴随一个质子的迁移。The "compound" of the present invention also includes tautomeric forms; the tautomeric form is derived from the exchange of a single bond with an adjacent double bond and accompanied by a proton transfer.

本发明还包括所有同位素的原子，无论是在中间体或最后的化合物；同位素的原子包括具有相同的原子数、但不同质量数的，例如，氢的同位素包括氘和氚。The invention also includes atoms of all isotopes, whether in the intermediate or final compound; atoms of the isotopes include isotopes including the same number of atoms but different mass numbers, for example, hydrogen, including lanthanum and cerium.

本发明中的“治疗”意味着对哺乳动物体内疾病的任何治疗，包括：(1)防止疾病，即造成临床疾病的症状不发展；(2)抑制疾病，即阻止临床症状的发展；(3)减轻疾病，即造成临床症状的消退。"Treatment" in the present invention means any treatment for a disease in a mammal, including: (1) preventing the disease, that is, causing the symptoms of the clinical disease to not develop; and (2) inhibiting the disease, that is, preventing the development of clinical symptoms; Reducing the disease, which causes the clinical symptoms to subside.

附图说明DRAWINGS

图1是本发明的拉曼探针的截面的结构示意图。BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing the structure of a cross section of a Raman probe of the present invention.

图2是本发明所实现的4-硝基苯硫醇在金纳米核上吸附不同时间(0min、5min、10min、20min、30min、60min、960min)生长的核壳结构金颗粒(即实施例1)的形貌，图中标尺为50nm。2 is a core-shell structured gold particle in which 4-nitrobenzenethiol is adsorbed on a gold nano-nucleus at different times (0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 960 min) (ie, Example 1). The shape of the figure is 50 nm in the figure.

图3是本发明所实现的在金纳米核上吸附不同时间((0min、5min、10min、20min、30min、60min、960min)形成的拉曼探针的拉曼光谱图，所用拉曼信号分子为4-硝基苯硫醇。3 is a Raman spectrum of a Raman probe formed on a gold nanocore adsorbed at different times (0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 960 min), and the Raman signal molecule used is 4-nitrobenzenethiol.

图4A是传统球形拉曼探针与本发明的拉曼探针的紫外可见吸收光谱的对比。4A is a comparison of the ultraviolet visible absorption spectrum of a conventional spherical Raman probe with the Raman probe of the present invention.

图4B是传统球形拉曼探针与本发明的拉曼探针的拉曼光谱的对比。4B is a comparison of a Raman spectrum of a conventional spherical Raman probe with a Raman probe of the present invention.

图5A是本发明的拉曼探针与CN201610200580.8专利的拉曼探针(具有双层核壳结构)的形貌的对比，图中的标尺均为50nm，左边图像是CN201610200580.8专利的拉曼探针，右边图像是本发明的拉曼探针。Figure 5A is a comparison of the morphology of the Raman probe of the present invention with the Raman probe of the CN201610200580.8 patent (having a two-layer core-shell structure), the scales in the figure are both 50 nm, and the left image is the CN201610200580.8 patent. Raman probe, the right image is the Raman probe of the present invention.

图5B是本发明的拉曼探针与CN201610200580.8专利的拉曼探针(具有双层核壳结构)的拉曼信号强度对比。Figure 5B is a comparison of the Raman signal intensity of the Raman probe of the present invention with the Raman probe of the CN201610200580.8 patent (having a two-layer core-shell structure).

图6是本发明实施例2所实现的3-硝基苯甲基硫醇在金纳米核上吸附10分钟时生长的核壳结构金颗粒，(A：颗粒的形貌，图中标尺为50nm；B：拉曼光谱)。6 is a core-shell structured gold particle grown by adsorbing 3-nitrobenzylthiol on a gold nano-nucleus for 10 minutes according to the embodiment 2 of the present invention (A: particle morphology, the scale is 50 nm in the figure) ;B: Raman spectroscopy).

图7是本发明实施例3所实现的2-巯基-5-硝基苯并咪唑在金纳米核上吸附10分钟时生长的核壳结构金颗粒，(A：颗粒的形貌，图中标尺为50nm；B：拉曼光谱)。7 is a core-shell structured gold particle grown by adsorbing 2-mercapto-5-nitrobenzimidazole on a gold nanocore for 10 minutes, which is realized in Example 3 of the present invention (A: particle morphology, scale in the figure) 50 nm; B: Raman spectroscopy).

图8是本发明实施例5所实现的可具有多种拉曼信号的拉曼探针的拉曼光谱，所使用的核壳结构内部(即第一拉曼信号层)的拉曼信号分子为4-硝基苯硫醇，金壳层外部(即第二层上的缝隙中)的拉曼信号分子分别为2-苯硫酚，对腈基苯硫醇，3-氟苯硫醇，3、4-二氯苯硫酚。8 is a Raman spectrum of a Raman probe that can have various Raman signals, which is implemented in Embodiment 5 of the present invention, and the Raman signal molecule inside the core-shell structure (ie, the first Raman signal layer) is 4-nitrobenzenethiol, the Raman signal molecule outside the gold shell (ie, in the gap on the second layer) is 2-thiophenol, p-cyanobenzenethiol, 3-fluorobenzenethiol, 3 , 4-dichlorothiophenol.

图9是本发明实施例9-1所实现的拉曼探针的单颗粒拉曼信号检测图(A：颗粒的原子力显微成像图，图中标尺为1μm；B：A中箭头所指的单颗粒的拉曼光谱)。9 is a single-particle Raman signal detection diagram of a Raman probe realized in Embodiment 9-1 of the present invention (A: atomic force microscopic imaging diagram of particles, in which the scale is 1 μm; B: A refers to an arrow Raman spectroscopy of single particles).

图10是本发明实施例10肺癌细胞(H1299)的超快拉曼成像结果图(A：细胞明场图，B：细胞拉曼图)，50×50像素点，每个像素点的积分时间为0.7ms，总成像时间为6s，图中标尺均为10um。Figure 10 is a graph showing ultrafast Raman imaging of lung cancer cells (H1299) of Example 10 of the present invention (A: cell bright field diagram, B: cell Raman diagram), 50 × 50 pixel points, integration time of each pixel For 0.7ms, the total imaging time is 6s, and the scale in the figure is 10um.

图11是本发明实施例11肺癌细胞(H1299)的多指标拉曼成像结果图，使用4种拉曼信号分子的拉曼探针(A：细胞明场图，B：4种拉曼信号分子的拉曼探针分布叠加图，C：拉曼信号分子为2-苯硫酚的拉曼探针在细胞内分布图，D：拉曼信号分子为3、4-二氯苯硫酚的拉曼探针在细胞内分布图，E：拉曼信号分子为3-氟苯硫醇的拉曼探针在细胞内分布图，F：拉曼信号分子为对腈基苯硫醇的拉曼探针在细胞内分布图)，图中标尺为10um。Figure 11 is a graph showing multi-indicator Raman imaging of lung cancer cells (H1299) of Example 11 of the present invention, using Raman probes of four Raman signal molecules (A: cell bright field diagram, B: 4 Raman signal molecules) Raman probe distribution overlay, C: Raman signal molecule is the intracellular distribution of 2-thiophenolic Raman probe, D: Raman signal molecule is 3,4-dichlorothiophenol The distribution of the MANN probe in the cell, E: Raman signal molecule is the intracellular distribution of the Raman probe of 3-fluorobenzenethiol, F: Raman signal molecule is the Raman probe of the nitrile phenyl thiol The needle is distributed within the cell), and the scale is 10um.

图12是本发明实施例12小鼠组织层面成像-腘窝***的超快拉曼成像结果图，A：明场照片，B：明场照片与拉曼叠加，图中标尺均为1cm。Figure 12 is a diagram showing the results of ultrafast Raman imaging of tissue-level axillary lymph nodes in a mouse according to Example 12 of the present invention, A: bright field photograph, B: bright field photograph and Raman stack, the scales are 1 cm in the figure.

具体实施方式detailed description

下面结合实施例对本发明的技术内容做进一步的说明：下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The technical content of the present invention is further described in the following with reference to the embodiments. The following embodiments are illustrative and not limiting, and the scope of the present invention is not limited by the following embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

如图1和3所示，本发明的拉曼探针具有纳米核1、第一拉曼信号层2和壳层3。纳米核1被第一拉曼信号层2包被；第一拉曼信号层2内分布有拉曼信号分子。壳层3具有第一层31和第二层32。第一拉曼信号层2被第一层31包被；第二层32包覆在第一层31外，具有缝隙4。第一层31为封闭结构。As shown in FIGS. 1 and 3, the Raman probe of the present invention has a nanocore 1, a first Raman signal layer 2, and a shell layer 3. The nanocore 1 is coated by the first Raman signal layer 2; Raman signal molecules are distributed in the first Raman signal layer 2. The shell layer 3 has a first layer 31 and a second layer 32. The first Raman signal layer 2 is coated by the first layer 31; the second layer 32 is wrapped outside the first layer 31 and has a slit 4. The first layer 31 is a closed structure.

图1和图3所示的拉曼探针看上去就如同盛开的花朵，花朵的相邻花瓣之间形成了第二层32上的缝隙4；或者如同齿轮，在截面上其壳层3呈齿轮状，齿轮的相邻齿之间形成第二层32上的缝隙4。The Raman probe shown in Figures 1 and 3 looks like a blooming flower, and a gap 4 on the second layer 32 is formed between adjacent petals of the flower; or like a gear, the shell layer 3 is in cross section. In the shape of a gear, a gap 4 in the second layer 32 is formed between adjacent teeth of the gear.

实施例1 制备以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针Example 1 Preparation of a Raman probe using 4-nitrobenzenethiol as a Raman signal molecule in a first Raman signal layer

步骤一：将400uL 1nmol/L的采用种子生长法制备得到的金纳米核颗粒(粒径为25nm)，加入到1mL 0.02mol/L十六烷基氯化铵溶液里，离心分离、重分散在400uL 0.02mol/L十六烷基氯化铵溶液中，得到以十六烷基氯化铵为稳定剂的金纳米核。Step 1: 400uL 1nmol/L gold nano-nuclear particles (particle size 25nm) prepared by seed growth method, added to 1mL 0.02mol/L cetyl ammonium chloride solution, centrifuged and redispersed in Gold nuclei with cetyl ammonium chloride as a stabilizer were obtained in 400 uL of 0.02 mol/L cetyl ammonium chloride solution.

步骤二：在步骤一得到的以十六烷基氯化铵为稳定剂的金纳米核中加入20uL 10mmol/L 4-硝基苯硫醇的乙醇溶液，分别混合震荡(即4-硝基苯硫醇在金纳米核上吸附)0、5、10、20、30、60、960分钟后，离心分离、重分散在200uL 0.1mol/L十六烷基氯化铵溶液中，重复三次，得到在金纳米核的外表面修饰有一层4-硝基苯硫醇拉曼信号分子层(即第一拉曼信号层，其中的拉曼信号分子是4-硝基苯硫醇)的金纳米颗粒。Step 2: Add 20 uL of 10 mmol/L 4-nitrobenzenethiol in ethanol to the gold nanonucleus with cetyl ammonium chloride as the stabilizer obtained in the first step, and mix and vortex (ie 4-nitrobenzene). After the thiol is adsorbed on the gold nano-nucleus for 0, 5, 10, 20, 30, 60, 960 minutes, it is centrifuged and redispersed in 200 uL of 0.1 mol/L cetyl ammonium chloride solution, and repeated three times. A gold nanoparticle having a layer of 4-nitrophenylthiol Raman signal molecule (ie, a first Raman signal layer in which the Raman signal molecule is 4-nitrobenzenethiol) is modified on the outer surface of the gold nanocore. .

步骤三：将步骤二得到的在金纳米核的外表面修饰有一层4-硝基苯硫醇拉曼信号分子层的金纳米颗粒加入到4mL 0.05mol/L十六烷基氯化铵溶液、200uL 4.86mmol/L氯金酸溶液、120uL 40mmol/L抗坏血酸溶液混合的生长液中，振荡搅拌，使得第一拉曼信号层外依次包被上金壳层的第一层和第二层，且第二层上具有缝隙，得到以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针，其截面大体形态如图1所示，真实形貌如图2所示，其拉曼光谱图如图3所示。图3中拉曼探针的拉曼信号强度随着混合震荡时间的延长，呈现出先增加后降低的趋势；最高的拉曼强度出现在5分钟这个时间点，之后随着混合震荡时间的延长，拉曼信号强度逐渐降低。Step 3: adding gold nanoparticles modified with a layer of 4-nitrobenzenethiol Raman signal molecule on the outer surface of the gold nano core obtained in step 2 to 4 mL of 0.05 mol/L cetyl ammonium chloride solution, 200uL 4.86mmol / L chloroauric acid solution, 120uL 40mmol / L ascorbic acid solution mixed growth solution, shaking and stirring, so that the first Raman signal layer is coated with the first layer and the second layer of the gold shell layer in turn, and There is a gap on the second layer, and the Raman probe with 4-nitrobenzenethiol as the Raman signal molecule in the first Raman signal layer is obtained. The general shape of the cross section is as shown in Fig. 1, and the true morphology is shown in Fig. 2. As shown, its Raman spectrum is shown in Figure 3. The Raman signal intensity of the Raman probe in Figure 3 shows a trend of increasing first and then decreasing with the increase of the mixing oscillating time; the highest Raman intensity appears at the time of 5 minutes, and then with the extension of the mixing oscillating time, The Raman signal strength gradually decreases.

4-硝基苯硫醇在金纳米核上吸附0分钟，即加入4-硝基苯硫醇后立即离心分离，不给予混合震荡的时间，这影响了壳层中第二层缝隙的形成，其形貌如图2中0min所示。其拉曼信号强度显著弱于吸附时间5和10min的拉曼探针(如图3所示)。因此，可以推断：第二层的缝隙结构能够增强拉曼信号强度。4-nitrobenzenethiol was adsorbed on the gold nanonucleate for 0 minutes, immediately after the addition of 4-nitrobenzenethiol, without centrifugation, which affected the formation of the second layer of the gap in the shell. Its shape is shown as 0min in Figure 2. The Raman signal intensity is significantly weaker than the Raman probe with adsorption time of 5 and 10 min (as shown in Figure 3). Therefore, it can be inferred that the slit structure of the second layer can enhance the Raman signal strength.

如图3所示，在5分钟时间点，得到的拉曼探针的性能最好。强度排序为：5min＞10min＞20min＞30min≈60min≈960min＞0min。As shown in Figure 3, the Raman probe obtained had the best performance at the 5 minute time point. The order of intensity is: 5 min>10 min>20 min>30 min≈60 min≈960 min>0 min.

对比例1(对应实施例1)制备CN201610200580.8专利中具有双层核壳结构的拉曼探针Comparative Example 1 (corresponding to Example 1) Preparation of a Raman probe having a double-layer core-shell structure in the CN201610200580.8 patent

见专利CN201610200580.8说明书的实施例1：See Example 1 of the specification of the patent CN201610200580.8:

步骤一：将400uL 1nmol/L的采用种子生长法制备得到的金纳米核颗粒(粒径为20nm)，加入到1mL 0.1mol/L十六烷基氯化铵溶液里，离心分离、重分散在400uL 0.1mol/L十六烷基氯化铵溶液中，得到以十六烷基氯化铵为稳定剂的金纳米核；Step 1: 400uL 1nmol/L gold nano-nuclear particles prepared by seed growth method (particle size 20nm), added to 1mL 0.1mol/L cetyl ammonium chloride solution, centrifuged and redispersed in In a 400uL 0.1mol/L cetyl ammonium chloride solution, a gold nanonucleus with cetyl ammonium chloride as a stabilizer is obtained;

步骤二：在金纳米核中加入50uL 2mmol/L对二巯基苯的乙醇溶液，混合震荡30分钟后，离心分离、重分散在200uL 0.1mol/L十六烷基氯化铵溶液中，重复三次，得到在金纳米核的外表面修饰有一层拉曼分子层的第一金纳米颗粒；Step 2: Add 50uL of 2mmol/L p-diphenylbenzene in ethanol solution, mix and shake for 30 minutes, centrifuge and redisperse in 200uL 0.1mol/L cetyl ammonium chloride solution, repeat three times. a first gold nanoparticle having a layer of Raman molecules modified on the outer surface of the gold nanocore;

步骤三：将第一金纳米颗粒加入到4mL 0.1mol/L十六烷基氯化铵溶液、200uL 4.86mmol/L氯金酸溶液、200uL 40mmol/L抗坏血酸溶液混合的生长液中，振荡搅拌，得到在第一金纳米颗粒的外表面贴覆有一层金壳层的第二金纳米颗粒，即双层核壳结构金纳米颗粒。此处所说的双层核壳结构金纳米颗粒从内到外依次包括金纳米核、拉曼分子层和金壳层(金壳层的层数为一层)。Step 3: adding the first gold nanoparticle to a growth solution of 4 mL of 0.1 mol/L cetyl ammonium chloride solution, 200 uL of 4.86 mmol/L chloroauric acid solution, and 200 uL of 40 mmol/L ascorbic acid solution, and stirring with shaking. A second gold nanoparticle having a gold shell layer on the outer surface of the first gold nanoparticle, that is, a double-layer core-shell structure gold nanoparticle is obtained. The double-layered core-shell structure gold nanoparticles referred to herein include a gold nanocore, a Raman molecular layer and a gold shell layer from the inside to the outside (the number of layers of the gold shell layer is one layer).

通过电镜观察对比例1的拉曼探针与实施例1的拉曼探针的形貌上的差别，主要在对比例1的拉曼探针虽然具有实施例1的拉曼探针的纳米核、第一拉曼信号层和壳层中的第一层，但是不具带有缝隙的第二层，如图5A所示。图5A中左边图像是对比例1的拉曼探针的电镜形貌图；右边图像是实施例1的拉曼探针吸附10min时的电镜形貌图(同图2中10min时的图片)。这个明显的形貌差异主要通过第一拉曼信号层上的拉曼信号分子4-硝基苯硫醇产生，并在拉曼强度上表现出巨大差异，如图5B所示。在统一的测试采集时间的条件下，实施例1的拉曼探针的拉曼信号强度非常显著的大于对比例1的拉曼探针的拉曼信号强度。除4-硝基苯硫醇外，其他一些同时含有巯基和硝基的化合物也具备生成如图2所示的拉曼探针形貌，比如：3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑。The difference in morphology between the Raman probe of Comparative Example 1 and the Raman probe of Example 1 was observed by electron microscopy, mainly in the Raman probe of Comparative Example 1, although the nanonucleus of the Raman probe of Example 1 was used. The first Raman signal layer and the first layer in the shell layer, but without the second layer with slits, as shown in FIG. 5A. The left image in Fig. 5A is the electron microscopic topography of the Raman probe of Comparative Example 1. The right image is the electron microscopic topography when the Raman probe of Example 1 is adsorbed for 10 min (the same picture as in Fig. 2 at 10 min). This apparent morphological difference is mainly produced by the Raman signal molecule 4-nitrobenzenethiol on the first Raman signal layer and exhibits a large difference in Raman intensity, as shown in Fig. 5B. The Raman signal intensity of the Raman probe of Example 1 was significantly greater than the Raman signal intensity of the Raman probe of Comparative Example 1 under the conditions of uniform test acquisition time. In addition to 4-nitrobenzenethiol, other compounds containing both sulfhydryl and nitro groups also have the appearance of a Raman probe as shown in Figure 2, such as 3-nitrobenzylthiol, 2- Amino-5-nitrobenzenethiol, o-nitrothiophenol, 2-mercapto-6-nitrobenzothiazole and 2-mercapto-5-nitrobenzimidazole.

实施例2 制备以3-硝基苯甲基硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针Example 2 Preparation of a Raman probe using 3-nitrobenzylthiol as a Raman signal molecule in a first Raman signal layer

步骤二：在步骤一得到的以十六烷基氯化铵为稳定剂的金纳米核中加入20uL 10mmol/L 3-硝基苯甲基硫醇的乙醇溶液，混合震荡10分钟后，离心分离、重分散在200uL 0.1mol/L十六烷基氯化铵溶液中，重复三次，得到在金纳米核的外表面修饰有一层3-硝基苯甲基硫醇拉曼信号分子层(即第一拉曼信号层)的金纳米颗粒。Step 2: Add 20 uL of 10 mmol/L 3-nitrobenzyl mercaptan in ethanol to the gold nanonucleus with cetyl ammonium chloride as the stabilizer obtained in the first step, mix and shake for 10 minutes, and centrifuge. Re-dispersed in 200uL 0.1mol/L cetyl ammonium chloride solution and repeated three times to obtain a layer of 3-nitrobenzyl thiol Raman signal molecule modified on the outer surface of gold nanonucleus (ie, A Raman signal layer) of gold nanoparticles.

步骤三：将步骤二得到的在金纳米核的外表面修饰有一层3-硝基苯甲基硫醇拉曼信号分子层的金纳米颗粒加入到4mL 0.05mol/L十六烷基氯化铵溶液、200uL 4.86mmol/L氯金酸溶液、120uL 40mmol/L抗坏血酸溶液混合的生长液中，振荡搅拌，使得第一拉曼信号层外依次包被上金壳层的第一层和第二层，且第二层上具有缝隙，得到以3-硝基苯甲基硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针，其截面大体形态如图1所示，真实形貌如图6A所示，其拉曼光谱图如图6B所示。Step 3: Adding gold nanoparticles with a layer of 3-nitrobenzylthiol Raman signal molecule modified on the outer surface of the gold nanonucleus obtained in step 2 to 4 mL of 0.05 mol/L cetyl ammonium chloride The solution, 200 uL of 4.86 mmol / L chloroauric acid solution, 120 uL of 40 mmol / L ascorbic acid solution mixed in a growth solution, shaking and stirring, so that the first Raman signal layer is coated with the first layer and the second layer of the gold shell layer And having a gap on the second layer, obtaining a Raman probe using 3-nitrobenzylthiol as the Raman signal molecule in the first Raman signal layer, the general shape of the cross section is as shown in FIG. The appearance is shown in Fig. 6A, and its Raman spectrum is shown in Fig. 6B.

实施例3 制备以2-巯基-5-硝基苯并咪唑作为第一拉曼信号层中拉曼信号分子的拉曼探针Example 3 Preparation of Raman probe with 2-mercapto-5-nitrobenzimidazole as Raman signal molecule in the first Raman signal layer

步骤二：在步骤一得到的以十六烷基氯化铵为稳定剂的金纳米核中加入20uL 10mmol/L 2-巯基-5-硝基苯并咪唑的乙醇溶液，混合震荡10分钟后，离心分离、重分散在200uL 0.1mol/L十六烷基氯化铵溶液中，重复三次，得到在金纳米核的外表面修饰有一层2-巯基-5-硝基苯并咪唑拉曼信号分子层(即第一拉曼信号层)的金纳米颗粒。Step 2: Add 20 uL of 10 mmol/L 2-mercapto-5-nitrobenzimidazole in ethanol to the gold nanonucleus with cetyl ammonium chloride as the stabilizer obtained in the first step, and mix and shake for 10 minutes. Centrifugal separation, redispersion in 200uL 0.1mol / L cetyl ammonium chloride solution, repeated three times, to obtain a layer of 2-mercapto-5-nitrobenzimidazole Raman signal molecule modified on the outer surface of gold nanonucleus Gold nanoparticles of the layer (ie the first Raman signal layer).

步骤三：将步骤二得到的在金纳米核的外表面修饰有一层2-巯基-5-硝基苯并咪唑拉曼信号分子层的金纳米颗粒加入到4mL 0.05mol/L十六烷基氯化铵溶液、200uL 4.86mmol/L氯金酸溶液、120uL 40mmol/L抗坏血酸溶液混合的生长液中，振荡搅拌，使得第一拉曼信号层外依次包被上金壳层的第一层和第二层，且第二层上具有缝隙，得到以2-巯基-5-硝基苯并咪唑作为第一拉曼信号层中拉曼信号分子的拉曼探针，其截面大体形态如图1所示，真实形貌如图7A所示，其拉曼光谱图如图7B所示。Step 3: Adding a gold nanoparticle having a layer of 2-mercapto-5-nitrobenzimidazole Raman signal molecule modified on the outer surface of the gold nanonucleus obtained in the second step to 4 mL of 0.05 mol/L hexadecyl chloride Ammonium solution, 200 uL 4.86 mmol / L chloroauric acid solution, 120 uL 40 mmol / L ascorbic acid solution mixed growth solution, shaking and stirring, so that the first Raman signal layer is coated on the first layer and the first layer of the gold shell layer The second layer has a gap on the second layer, and a Raman probe with 2-mercapto-5-nitrobenzimidazole as the Raman signal molecule in the first Raman signal layer is obtained, and the cross-sectional general shape thereof is as shown in FIG. The true topography is shown in Fig. 7A, and the Raman spectrum is shown in Fig. 7B.

实施例4 制备以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针Example 4 Preparation of a Raman probe using 4-nitrobenzenethiol as a Raman signal molecule in a first Raman signal layer

步骤二：在步骤一得到的以十六烷基氯化铵为稳定剂的金纳米核中加入20uL 10mmol/L 4-硝基苯硫醇的乙醇溶液，混合震荡10分钟后，离心分离、重分散在200uL 0.1mol/L十六烷基氯化铵溶液中，重复三次，得到在金纳米核的外表面修饰有一层4-硝基苯硫醇拉曼信号分子层(即第一拉曼信号层)的金纳米颗粒。Step 2: Add 20 uL of 10 mmol/L 4-nitrobenzenethiol in ethanol to the gold nanonucleus with cetyl ammonium chloride as the stabilizer obtained in the first step, mix and shake for 10 minutes, centrifuge and separate. Disperse in 200uL 0.1mol / L cetyl ammonium chloride solution, repeated three times, to obtain a layer of 4-nitrobenzenethiol Raman signal molecule modified on the outer surface of gold nanonuclei (ie, the first Raman signal Layer) of gold nanoparticles.

步骤三：将步骤二得到的在金纳米核的外表面修饰有一层4-硝基苯硫醇拉曼信号分子层的金纳米颗粒加入到4mL 0.05mol/L十六烷基溴化铵溶液、200uL 4.86mmol/L氯金酸溶液、120uL 40mmol/L抗坏血酸溶液混合的生长液中，振荡搅拌，使得第一拉曼信号层外依次包被上金壳层的第一层和第二层，且第二层上具有缝隙，得到以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子的拉曼探针，其截面大体形态如图1所示，真实形貌类似于图2的60min时间点所示。Step 3: adding the gold nanoparticles modified with a layer of 4-nitrobenzenethiol Raman signal molecule on the outer surface of the gold nano core obtained in step 2 to 4 mL of 0.05 mol/L cetyl ammonium bromide solution, 200uL 4.86mmol / L chloroauric acid solution, 120uL 40mmol / L ascorbic acid solution mixed growth solution, shaking and stirring, so that the first Raman signal layer is coated with the first layer and the second layer of the gold shell layer in turn, and There is a gap on the second layer, and a Raman probe with 4-nitrobenzenethiol as the Raman signal molecule in the first Raman signal layer is obtained. The general shape of the cross section is as shown in Fig. 1, and the real shape is similar to the figure. The 60 min time point is shown.

实施例5 制备多指标的拉曼探针Example 5 Preparation of multi-index Raman probe

步骤一：将2mL 0.2nmol/L的实施例1制备出的拉曼探针(粒径约为70nm)，加入到4mL 0.01mol/L十六烷基氯化铵溶液里，得到以十六烷基氯化铵为稳定剂的拉曼探针溶液，平均分成4份，每份1mL。Step 1: 2 mL of 0.2 nmol/L of the Raman probe prepared in Example 1 (having a particle size of about 70 nm) was added to 4 mL of a 0.01 mol/L cetyl ammonium chloride solution to obtain hexadecane. A Raman probe solution containing ammonium chloride as a stabilizer was divided into 4 parts on average, 1 mL each.

步骤二：分别在上述4份拉曼探针溶液中加入50uL 10mmol/L的2-苯硫酚，对腈基苯硫醇，3-氟苯硫醇，3、4-二氯苯硫酚的乙醇溶液，混合震荡60-360分钟后，离心分离，重分散在1mL 0.05mol/L十六烷基氯化铵溶液中，重复三次，使得拉曼探针第二层缝隙中分别修饰上2-苯硫酚，对腈基苯硫醇，3-氟苯硫醇，3、4-二氯苯硫酚，从而分别得到以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰2-苯硫酚的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰对腈基苯硫醇的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰3-氟苯硫醇的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰3、4-二氯苯硫酚的拉曼探针。Step 2: Add 50 uL of 10 mmol/L 2-thiophenol, p-cyanobenzenethiol, 3-fluorobenzenethiol, 3,4-dichlorothiophenol to the above 4 parts of the Raman probe solution. Ethanol solution, mixed for 60-360 minutes, centrifuged, redispersed in 1mL 0.05mol / L cetyl ammonium chloride solution, repeated three times, so that the second layer of the Raman probe is modified respectively 2 Thiophenol, p-cyanobenzenethiol, 3-fluorobenzenethiol, 3,4-dichlorothiophenol, thereby obtaining 4-nitrobenzenethiol as the Raman signal in the first Raman signal layer Raman probe for modifying 2-thiophenol in the second layer of the molecule, 4-nitrobenzenethiol as the Raman signal molecule in the first Raman signal layer and modification of the p-nitrile benzene in the second layer gap a Raman probe of thiol, 4-nitrobenzenethiol as a Raman signal molecule in the first Raman signal layer and a Raman probe for modifying 3-fluorobenzenethiol in the second layer of the gap, 4- Nitrophenylthiol is used as a Raman signal in the first Raman signal layer and a Raman probe for modifying 3,4-dichlorothiophenol in the second layer of the gap.

从图8可以看出，上述4种第二层缝隙中分别修饰不同拉曼信号分子的拉曼探针分别具有2-苯硫酚的拉曼特征峰(637cm ^-1，1379cm ^-1)，对腈基苯硫醇的拉曼特征峰(1177cm ^-1，2230cm ^-1)，3-氟苯硫醇的拉曼特征峰(876cm ^-1，999cm ^-1)，3、4-二氯苯硫酚的拉曼特征峰(568cm ^-1)，但都同时具有4-硝基苯硫醇的拉曼特征峰(1340cm ^-1)，因此为多指标的拉曼探针。 It can be seen from Fig. 8 that the Raman probes respectively modifying different Raman signal molecules in the above four kinds of second layer gaps respectively have Raman characteristic peaks of 2-phenylthiophenol (637 cm ^-1 , 1379 cm ^-1 ), Raman characteristic peak of nitrile phenyl mercaptan (1177cm ^-1 , 2230cm ^-1 ), Raman characteristic peak of 3-fluorobenzenethiol (876cm ^-1 , 999cm ^-1 ), 3,4-dichlorothiophenol The Raman characteristic peak (568 cm ^-1 ), but both have a Raman characteristic peak of 4-nitrobenzenethiol (1340 cm ^-1 ), and thus is a multi-index Raman probe.

实施例6 制备外层结构为介孔二氧化硅的拉曼探针Example 6 Preparation of a Raman probe having an outer layer structure of mesoporous silica

步骤一：将5mL 0.4nmol/L实施例1制备出的拉曼探针(颗粒粒径为70nm)，加入到5mL 0.1mol/L十六烷基氯化铵溶液里，离心分离、重分散在5mL 0.001mol/L十六烷基氯化铵溶液中，加入0.1mol/L的NaOH溶液30μl将溶液的pH值调整至10-11，得到金纳米颗粒溶液。Step 1: 5 mL of 0.4 nmol/L Raman probe prepared in Example 1 (particle size: 70 nm) was added to 5 mL of 0.1 mol/L cetyl ammonium chloride solution, centrifuged and redispersed in In a 5 mL 0.001 mol/L cetyl ammonium chloride solution, 30 μl of a 0.1 mol/L NaOH solution was added to adjust the pH of the solution to 10-11 to obtain a gold nanoparticle solution.

步骤二：将本实施例步骤一所得的金纳米颗粒溶液中分三次加入含有5％正硅酸四乙酯的甲醇溶液，每次50μl，继续搅拌反应15h，获得第二层外包覆10-15nm介孔二氧化硅层的拉曼探针。Step 2: The gold nanoparticle solution obtained in the first step of the present embodiment is added to a methanol solution containing 5% tetraethyl orthosilicate in three portions, 50 μl each time, and the reaction is further stirred for 15 hours to obtain a second outer coating. Raman probe of 15 nm mesoporous silica layer.

步骤三：将步骤二得到的第二层外包覆10-15nm介孔二氧化硅层的拉曼探针离心，分散于乙醇中，加入6-8粒固体硝酸铵颗粒超声，重复洗涤3-4次后离心分散于乙醇中，以去除十六烷基氯化铵，得到第二层外包覆介孔二氧化硅的拉曼探针，即外层结构为介孔二氧化硅的拉曼探针。Step 3: The second layer of the Raman probe coated with the 10-15 nm mesoporous silica layer obtained in the second step is centrifuged, dispersed in ethanol, and ultrasonically added with 6-8 solid ammonium nitrate particles, and repeatedly washed 3- After 4 times, it was dispersed and dispersed in ethanol to remove cetyl ammonium chloride to obtain a Raman probe of the second layer of mesoporous silica, that is, Raman with outer structure of mesoporous silica. Probe.

本实施例制备的外层结构为介孔二氧化硅的拉曼探针的第二层上具有缝隙，虽然是以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子，具有4-硝基苯硫醇的拉曼特征峰。但是若步骤一中的出发拉曼探针的第一拉曼信号层上是其他拉曼信号分子(比如3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑等)，那么通过本实施例的制备方法获得外层结构为介孔二氧化硅的拉曼探针则具有其他拉曼信号分子的拉曼特征峰。由于制备方法类似，这里不再赘述。The outer layer structure prepared by the present embodiment has a gap on the second layer of the Raman probe of mesoporous silica, although 4-nitrobenzenethiol is used as the Raman signal molecule in the first Raman signal layer. A Raman characteristic peak having 4-nitrobenzenethiol. However, if the first Raman signal layer of the starting Raman probe in step one is other Raman signal molecules (such as 3-nitrobenzylthiol, 2-amino-5-nitrobenzenethiol, ortho Nitrothiophenol, 2-mercapto-6-nitrobenzothiazole and 2-mercapto-5-nitrobenzimidazole, etc., then the outer layer structure is mesoporous silica obtained by the preparation method of the present embodiment The Raman probe has Raman characteristic peaks of other Raman signal molecules. Since the preparation method is similar, it will not be described here.

实施例7 制备外层结构为巯基化合物的拉曼探针Example 7 Preparation of a Raman probe having an outer structure of a mercapto compound

步骤一：将5mL 0.4nmol/L实施例1制备出的拉曼探针(颗粒粒径为70nm)，加入到0.5mL 0.1mol/L十六烷基氯化铵溶液里，离心分离，重分散在0.5mL 0.001mol/L十六烷基氯化铵溶液中，得到金纳米颗粒溶液。Step 1: 5 mL of 0.4 nmol/L of the Raman probe prepared in Example 1 (particle size of 70 nm) was added to 0.5 mL of 0.1 mol/L cetyl ammonium chloride solution, centrifuged, and dispersed. A gold nanoparticle solution was obtained in 0.5 mL of a 0.001 mol/L cetyl ammonium chloride solution.

步骤二：将步骤一所得的金纳米颗粒溶液中加入0.5mL 1-丁基-3-甲基咪唑双三氟甲磺酰亚胺盐，在涡旋震荡仪上涡旋5分钟，使上层液体澄清。Step 2: Add 0.5 mL of 1-butyl-3-methylimidazolium bistrifluoromethanesulfonimide salt to the gold nanoparticle solution obtained in the first step, and vortex on a vortex shaker for 5 minutes to make the upper liquid clarify.

步骤三：向步骤二所得的溶液中加入1mL 0.2mol/L巯基十一烷酸的1-丁基-3-甲基咪唑双三氟甲磺酰亚胺盐溶液，涡旋5分钟，使上层液体变成深红色，加入5mL水将上层的巯基十一烷酸修饰的金纳米颗粒提取出来。Step 3: Add 1 mL of a solution of 0.2 mol/L mercapto undecanoic acid in 1-butyl-3-methylimidazolium bistrifluoromethanesulfonimide salt to the solution obtained in the second step, and vortex for 5 minutes to make the upper layer The liquid turned dark red and the upper decylundecanoic acid modified gold nanoparticles were extracted by adding 5 mL of water.

步骤四：向步骤三所得的上层液体中反复加水提取巯基十一烷酸修饰的金纳米颗粒，直到溶液澄清为止，离心去除残存的1-丁基-3-甲基咪唑双三氟甲磺酰亚胺盐，得到在水中分散性良好的外层结构为巯基化合物的拉曼探针，其中巯基化合物为巯基十一烷酸。Step 4: repeatedly adding water to the upper liquid obtained in the third step to extract the decylundecanoic acid-modified gold nanoparticles until the solution is clear, and removing the remaining 1-butyl-3-methylimidazolium bistrifluoromethanesulfonyl by centrifugation. The imine salt gives a Raman probe in which the outer layer structure having good dispersibility in water is a mercapto compound, wherein the mercapto compound is mercapto undecanoic acid.

除巯基十一烷酸外，当采用其他巯基化合物代替巯基十一烷酸时，则制备得到的外层结构为巯基化合物的拉曼探针中的巯基化合物为其他巯基化合物，比如巯基十一烷醇等。由于方法类似，故这里不再赘述。In addition to mercapto undecanoic acid, when other mercapto compounds are used in place of mercapto undecanoic acid, the mercapto compounds in the Raman probes in which the outer structure is a mercapto compound are prepared as other mercapto compounds, such as nonyldecane. Alcohol, etc. Since the method is similar, it will not be described here.

本实施例制备的外层结构为巯基化合物的拉曼探针的第二层上具有缝隙，虽然是以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子，具有4-硝基苯硫醇的拉曼特征峰。但是若步骤一中的出发拉曼探针的第一拉曼信号层上是其他拉曼信号分子(比如3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑等)，那么通过本实施例的制备方法获得外层结构为巯基化合物的拉曼探针则具有其他拉曼信号分子的拉曼特征峰。由于制备方法类似，这里不再赘述。The outer layer structure prepared in this embodiment has a slit on the second layer of the Raman probe of the mercapto compound, although 4-nitrobenzenethiol is used as the Raman signal molecule in the first Raman signal layer, and has 4- Raman characteristic peak of nitrobenzenethiol. However, if the first Raman signal layer of the starting Raman probe in step one is other Raman signal molecules (such as 3-nitrobenzylthiol, 2-amino-5-nitrobenzenethiol, ortho Nitrothiophenol, 2-mercapto-6-nitrobenzothiazole and 2-mercapto-5-nitrobenzimidazole, etc., then Raman obtained as a mercapto compound with an outer layer structure by the preparation method of this example The probe has Raman characteristic peaks of other Raman signal molecules. Since the preparation method is similar, it will not be described here.

实施例8 制备外层结构为介孔二氧化硅的多指标的拉曼探针Example 8 Preparation of a multi-index Raman probe having an outer structure of mesoporous silica

步骤一：将40mL 0.2nmol/L的实施例1制备出的拉曼探针(粒径约为70nm)，加入到80mL 0.01mol/L十六烷基氯化铵溶液里，得到以十六烷基氯化铵为稳定剂的拉曼探针溶液，平均分成4份，每份20mL。Step 1: 40 mL of 0.2 nmol/L of the Raman probe prepared in Example 1 (having a particle size of about 70 nm) was added to 80 mL of a 0.01 mol/L cetyl ammonium chloride solution to obtain hexadecane. The Raman probe solution containing ammonium chloride as a stabilizer was divided into 4 parts on average, 20 mL each.

步骤二：分别在上述4份拉曼探针溶液中加入800uL 10mmol/L的2-苯硫酚，对腈基苯硫醇，3-氟苯硫醇，3、4-二氯苯硫酚的乙醇溶液，混合震荡60-360分钟后，离心分离，重分散在20mL 0.05mol/L十六烷基氯化铵溶液中，重复三次，使得拉曼探针第二层缝隙中分别修饰上2-苯硫酚，对腈基苯硫醇，3-氟苯硫醇，3、4-二氯苯硫酚，从而分别得到以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰2-苯硫酚的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰对腈基苯硫醇的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰3-氟苯硫醇的拉曼探针，以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子且第二层缝隙中修饰3、4-二氯苯硫酚的拉曼探针。Step 2: Add 800 uL of 10 mmol/L 2-thiophenol, p-cyanobenzenethiol, 3-fluorobenzenethiol, 3,4-dichlorothiophenol to the above 4 parts of Raman probe solution. Ethanol solution, mixed for 60-360 minutes, centrifuged, redispersed in 20mL 0.05mol / L cetyl ammonium chloride solution, repeated three times, so that the second layer of the Raman probe is modified in the second 2- Thiophenol, p-cyanobenzenethiol, 3-fluorobenzenethiol, 3,4-dichlorothiophenol, thereby obtaining 4-nitrobenzenethiol as the Raman signal in the first Raman signal layer Raman probe for modifying 2-thiophenol in the second layer of the molecule, 4-nitrobenzenethiol as the Raman signal molecule in the first Raman signal layer and modification of the p-nitrile benzene in the second layer gap a Raman probe of thiol, 4-nitrobenzenethiol as a Raman signal molecule in the first Raman signal layer and a Raman probe for modifying 3-fluorobenzenethiol in the second layer of the gap, 4- Nitrophenylthiol is used as a Raman signal in the first Raman signal layer and a Raman probe for modifying 3,4-dichlorothiophenol in the second layer of the gap.

步骤三：将上述4份修饰后的拉曼探针溶液，加入到20mL 0.1mol/L十六烷基氯化铵溶液里，离心分离、重分散在5mL 0.001mol/L十六烷基氯化铵溶液中，加入0.1mol/L的NaOH溶液30μl将溶液的pH值调整至10-11，得到金纳米颗粒溶液。Step 3: Add the above 4 modified Raman probe solutions to 20 mL of 0.1 mol/L cetyl ammonium chloride solution, centrifuge and redisperse in 5 mL of 0.001 mol/L hexadecyl chloride. In the ammonium solution, 30 μl of a 0.1 mol/L NaOH solution was added to adjust the pH of the solution to 10-11 to obtain a gold nanoparticle solution.

步骤四：将本实施例步骤三所得的金纳米颗粒溶液中分三次加入含有5％正硅酸四乙酯的甲醇溶液，每次50μl，继续搅拌反应15h，获得第二层缝隙中分别修饰不同拉曼信号分子且第二层外包覆10-15nm介孔二氧化硅层的拉曼探针。Step 4: The gold nanoparticle solution obtained in the third step of the present embodiment is added to a methanol solution containing 5% tetraethyl orthosilicate in three portions, 50 μl each time, and the reaction is further stirred for 15 hours to obtain different modifications in the second layer gap. The Raman signal molecule and the second layer is coated with a Raman probe of a 10-15 nm mesoporous silica layer.

步骤五：将步骤四得到的第二层外包覆10-15nm介孔二氧化硅层的拉曼探针离心，分散于乙醇中，加入6-8粒固体硝酸铵颗粒超声，重复洗涤3-4次后离心分散于乙醇中，以去除十六烷基氯化铵，得到第二层缝隙中分别修饰不同拉曼信号分子且外包覆介孔二氧化硅的拉曼探针，即外层结构为介孔二氧化硅的多指标拉曼探针。Step 5: The second layer of the Raman probe coated with the 10-15 nm mesoporous silica layer obtained in the fourth step is centrifuged, dispersed in ethanol, and ultrasonically added with 6-8 solid ammonium nitrate particles, and repeatedly washed 3- After 4 times, it was dispersed and dispersed in ethanol to remove cetyl ammonium chloride, and a Raman probe in which a different Raman signal molecule was separately modified and mesoporous silica was coated in the second layer gap was obtained. A multi-index Raman probe of the structure mesoporous silica.

本实施例制备的外层结构为介孔二氧化硅的多指标拉曼探针的第二层上具有缝隙，且缝隙内分别修饰了不同的拉曼信号分子。虽然是以4-硝基苯硫醇作为第一拉曼信号层中拉曼信号分子，具有4-硝基苯硫醇的拉曼特征峰。但是若步骤一中的出发拉曼探针的第一拉曼信号层上是其他拉曼信号分子(比如3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑等)，那么通过本实施例的制备方法获得外层结构为介孔二氧化硅的拉曼探针则具有其他拉曼信号分子的拉曼特征峰。由于制备方法类似，这里不再赘述。The second layer of the multi-index Raman probe having the outer layer structure of mesoporous silica prepared in this embodiment has a slit, and different Raman signal molecules are respectively modified in the slit. Although 4-nitrobenzenethiol is used as the Raman signal molecule in the first Raman signal layer, it has a Raman characteristic peak of 4-nitrobenzenethiol. However, if the first Raman signal layer of the starting Raman probe in step one is other Raman signal molecules (such as 3-nitrobenzylthiol, 2-amino-5-nitrobenzenethiol, ortho Nitrothiophenol, 2-mercapto-6-nitrobenzothiazole and 2-mercapto-5-nitrobenzimidazole, etc., then the outer layer structure is mesoporous silica obtained by the preparation method of the present embodiment The Raman probe has Raman characteristic peaks of other Raman signal molecules. Since the preparation methods are similar, they will not be described again here.

实施例9 本发明的拉曼探针在单颗粒检测方面的应用Example 9 Application of Raman Probe of the Invention in Single Particle Detection

实施例9-1Example 9-1

步骤一：将10uL 0.1pmol/L的根据实施例1制备出的拉曼探针滴于硅片上，干燥后将硅片固定在原子力-显微共焦拉曼联用光谱仪上；Step 1: 10 uL 0.1 pmol / L of the Raman probe prepared according to Example 1 was dropped on a silicon wafer, and after drying, the silicon wafer was fixed on an atomic force-microscopic confocal Raman spectrometer;

步骤二：对有拉曼探针的硅片进行原子力显微成像，找到并确认硅片上的多个单颗粒；然后对单颗粒依次进行拉曼光谱采集，积分时间为10s，激光功率为1％，并对结果进行分析。结果如图9所示，单个拉曼探针(即单颗粒)的拉曼强度依然可以明显检测出。图9B为图9A箭头所指的单个拉曼探针的拉曼光谱，表现出明显的4-硝基苯硫醇的拉曼特征峰，且峰形尖锐、信噪比好、识别度高。Step 2: Atomic force microscopy imaging of the silicon wafer with Raman probe, find and confirm a plurality of single particles on the silicon wafer; then carry out Raman spectral acquisition on the single particle in turn, the integration time is 10s, the laser power is 1 % and analyze the results. As a result, as shown in Fig. 9, the Raman intensity of a single Raman probe (i.e., a single particle) can still be clearly detected. Figure 9B is a Raman spectrum of a single Raman probe indicated by the arrow of Figure 9A, showing a distinct Raman characteristic peak of 4-nitrobenzenethiol with sharp peak shape, good signal to noise ratio, and high recognition.

对比例2(对应实施例9-1)Comparative Example 2 (corresponding to Example 9-1)

步骤一：将10uL 0.1pmol/L专利CN201610200580.8中公开的介孔二氧化硅包被的内嵌对二巯基苯的拉曼探针(双层核壳结构)滴于硅片上，干燥后将硅片固定在原子力-显微共焦拉曼联用光谱仪上；Step 1: The mesoporous silica-coated Raman probe (double-layer core-shell structure) coated with mesoporous silica disclosed in 10 uL of 0.1 pmol/L patent CN201610200580.8 was dropped on a silicon wafer, and dried. Fixing the silicon wafer on an atomic force-microscopic confocal Raman spectrometer;

步骤二：对有拉曼探针的硅片进行原子力显微成像，找到并确认硅片上的多个单颗粒；然后对单颗粒依次进行拉曼光谱采集，积分时间为30s，激光功率为100％，并对结果进行分析。拉曼光谱强度弱，信噪比差，识别度低。Step 2: Atomic force microscopy imaging of the silicon wafer with Raman probe, find and confirm a plurality of single particles on the silicon wafer; then carry out Raman spectral acquisition on the single particle in turn, the integration time is 30s, the laser power is 100 % and analyze the results. Raman spectral intensity is weak, signal to noise ratio is poor, and recognition is low.

实施例9-2 应用基于拉曼流式的单颗粒标记液相芯片。Example 9-2 A single particle labeling liquid phase chip based on Raman flow was applied.

步骤一、基于拉曼流式的单颗粒标记液相芯片，单颗粒拉曼探针作为生物标记物，内嵌不同的拉曼信号分子实现编码，得到编码后的单颗粒拉曼探针；然后将每种所述编码后的单颗粒拉曼探针共价交联上针对特定检测物，即靶分子的捕获分子，得到针对不同检测物的编码单颗粒拉曼探针；所述捕获分子包括抗原、抗体和/或核酸探针； Step 1. Based on the Raman flow type single particle labeling liquid phase chip, the single particle Raman probe is used as a biomarker, and different Raman signal molecules are embedded to realize encoding, and the encoded single particle Raman probe is obtained; Each of the encoded single-particle Raman probes is covalently cross-linked to a specific detector, ie, a capture molecule of a target molecule, to obtain a single-particle Raman probe encoding a different detector; the capture molecule includes Antigen, antibody and/or nucleic acid probe;

步骤二、先把多种所述针对不同检测物的编码单颗粒拉曼探针混合，再加入微量待检样本，形成悬液；在所述悬液中，所述待检样本中的靶分子与所述针对不同检测物的编码单颗粒拉曼探针表面交联的捕获分子发生特异性结合；最后使用显微共焦拉曼光谱仪识别所述针对不同检测物的编码单颗粒拉曼探针的编码及与之特异性结合的靶分子，从而识别待检样本。Step 2: first mixing a plurality of the encoded single-particle Raman probes for different detection substances, and then adding a trace amount of the sample to be tested to form a suspension; in the suspension, the target molecule in the sample to be inspected Specific binding to the capture molecules surface-crosslinked by the single-particle Raman probe for different analytes; finally, the single-particle Raman probe for different detectors is identified using a micro-convex Raman spectrometer The target molecule is encoded and specifically bound to identify the sample to be examined.

实施例10 细胞层面超快速的拉曼成像Example 10 Ultra-fast Raman imaging at the cell level

步骤一：将实施例6制备的外层结构为介孔二氧化硅的拉曼探针均匀分散于pH＝7.4的PBS溶液中制成0.05nmol/L的外层结构为介孔二氧化硅的拉曼探针溶液。Step 1: The Raman probe having the outer layer structure of mesoporous silica prepared in Example 6 was uniformly dispersed in a PBS solution of pH=7.4 to prepare an outer layer structure of mesoporous silica of 0.05 nmol/L. Raman probe solution.

步骤二：选用肺癌细胞(H1299)作为研究对象，将无菌的0.05nmol/L的外层结构为介孔二氧化硅的拉曼探针溶液与处于对数生长期的H1299细胞置于细胞培养箱中，以37℃孵育6h，使该拉曼探针进入细胞内部。Step 2: Select lung cancer cells (H1299) as the research object, and place a sterile 0.05 nmol/L Raman probe solution with mesoporous silica as the outer layer and H1299 cells in the logarithmic growth phase. The Raman probe was introduced into the interior of the cell by incubating at 37 ° C for 6 h in the chamber.

步骤三：用拉曼光谱仪对经步骤二处理的肺癌细胞(H1299)进行超快速的拉曼成像，通过拉曼图谱对成像结果进行分析。实验结果如图10所示，共2500个像素点，每个像素点的积分时间为0.7ms，完整细胞成像所用的时间为6s，使用拉曼探针内的拉曼信号分子4-硝基苯硫醇的拉曼特征峰(1340cm ^-1)重构出图像(如图10B所示)，可观察到该拉曼探针聚集在H1299细胞表面和细胞内部，并可实现超快速的成像。该结果与本申请人的前期研究申请的专利CN201610200580.8所涉及的表面增强拉曼探针(不具备第二层上的缝隙结构)相比，每个像素点的积分时间提高了1个数量级，完整细胞成像所用的时间也提高了1个数量级。CN201610200580.8中双层核壳结构(CN201610200580.8说明书的实施例5)的每个像素点的积分时间为10ms，完整细胞成像所用的时间为53s；三层核壳结构(CN201610200580.8说明书的实施例6)的每个像素点的积分时间为1ms，完整细胞成像所用的时间为40s。 Step 3: Ultra-fast Raman imaging of the lung cancer cells (H1299) treated in the second step was performed by Raman spectroscopy, and the imaging results were analyzed by Raman spectroscopy. The experimental results are shown in Fig. 10. A total of 2500 pixels, the integration time of each pixel is 0.7ms, and the time for complete cell imaging is 6s. The Raman signal molecule 4-nitrobenzene in the Raman probe is used. The Raman characteristic peak of the thiol (1340 cm ^-1 ) reconstructed the image (as shown in Fig. 10B), and it was observed that the Raman probe was aggregated on the surface of the H1299 cell and inside the cell, and ultra-fast imaging was possible. This result is one order of magnitude higher for each pixel point than the surface-enhanced Raman probe (without the gap structure on the second layer) of the applicant's patent application CN201610200580.8. The time taken for complete cell imaging has also increased by an order of magnitude. CN201610200580.8 The integration time of each pixel in the double-layer core-shell structure (Example 5 of the specification of CN201610200580.8) is 10ms, and the time for complete cell imaging is 53s; the three-layer core-shell structure (CN201610200580.8 The integration time for each pixel of Example 6) was 1 ms, and the time taken for intact cell imaging was 40 s.

实施例11 多种信号的多指标检测Example 11 Multi-indicator detection of multiple signals

步骤一：将制备的介孔二氧化硅包被的第一拉曼信号层为4-NBT，第二层缝隙内分别为对腈基苯硫醇、3-氟苯硫醇、2-苯硫酚和3、4-二氯苯硫酚这4种拉曼信号分子的拉曼探针(即实施例8制备的外层结构为介孔二氧化硅的多指标的拉曼探针)均匀分散于pH＝7.4的PBS溶液中制成0.05nmol/L的混合拉曼探针溶液，其中每种拉曼探针浓度为0.0125nmol/L。Step 1: The prepared first porous Raman signal layer of the mesoporous silica is 4-NBT, and the second layer of the gap is p-cyanobenzenethiol, 3-fluorobenzenethiol, 2-phenylsulfide The Raman probe of the four Raman signal molecules of phenol and 3,4-dichlorothiophenol (that is, the Raman probe of the multi-index of the mesoporous silica prepared in Example 8) is uniformly dispersed. A 0.05 nmol/L mixed Raman probe solution was prepared in a PBS solution of pH=7.4, wherein each Raman probe concentration was 0.0125 nmol/L.

步骤二：选用肺癌细胞(H1299)作为研究对象，将无菌的0.05nmol/L的混合拉曼探针溶液与处于对数生长期的H1299细胞置于细胞培养箱中，以37℃孵育6h，使该拉曼探针进入细胞内部。Step 2: Lung cancer cells (H1299) were selected as the research object. The sterile 0.05 nmol/L mixed Raman probe solution and H1299 cells in logarithmic growth phase were placed in a cell culture incubator and incubated at 37 ° C for 6 h. The Raman probe is brought into the interior of the cell.

步骤三：用拉曼光谱仪对肺癌细胞(H1299)进行多指标拉曼成像，通过拉曼图谱对成像结果进行分析。实验结果如图11所示，共2500个像素点，每个像素点的积分时间为10ms，完整细胞成像所用的时间为40s。图11A和图11B分别为细胞明场图和4种拉曼信号分子的拉曼探针分布叠加图。分别使用拉曼探针内的拉曼信号分子2-苯硫酚的拉曼特征峰(637cm ^-1)，3、4-二氯苯硫酚的拉曼特征峰(568cm ^-1)，3-氟苯硫醇的拉曼特征峰(999cm ^-1)，对腈基苯硫醇的拉曼特征峰(2230cm ^-1)重构出图像(分别见图11C、D、E、F)，可观察到4种拉曼信号分子的拉曼探针在H1299细胞表面和细胞内部的分布。实现多种信号(上述4种拉曼信号分子)的多指标(第一拉曼信号层中的拉曼信号分子与第二层缝隙中的拉曼信号分子)检测。 Step 3: Multi-indicator Raman imaging of lung cancer cells (H1299) was performed by Raman spectroscopy, and the imaging results were analyzed by Raman spectroscopy. The experimental results are shown in Figure 11, with a total of 2500 pixels, the integration time of each pixel is 10ms, and the time taken for intact cell imaging is 40s. 11A and 11B are a superimposed diagram of a Raman probe distribution of a cell bright field map and four Raman signal molecules, respectively. The Raman characteristic peak of the Raman signal molecule 2-thiophenol in the Raman probe (637 cm ^-1 ), the Raman characteristic peak of 3,4-dichlorothiophenol (568 cm ^-1 ), 3- The Raman characteristic peak of fluorobenzenethiol (999 cm ^-1 ) was reconstructed from the Raman characteristic peak (2230 cm ^-1 ) of nitrile phenyl mercaptan (see Figure 11C, D, E, F, respectively). Distribution of Raman probes to four Raman signaling molecules on the surface and inside the cells of H1299 cells. Multiple indicators (Raman signal molecules in the first Raman signal layer and Raman signal molecules in the second layer gap) of a plurality of signals (the above four Raman signal molecules) are detected.

实施例12 本发明的拉曼探针在生物医学成像、离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用Example 12 Application of the Raman probe of the present invention in biomedical imaging, isolated tissue, isolated organs, dead human body or dead animal body imaging

步骤一：将介孔二氧化硅包被的第一拉曼信号层中内嵌4-硝基苯硫醇的拉曼探针(即实施例5制备的外层结构为介孔二氧化硅的拉曼探针)均匀分散于生理盐水中制成1nmol/L的溶液。Step 1: Raman probe in which 4-nitrobenzenethiol is embedded in the first Raman signal layer coated with mesoporous silica (that is, the outer layer structure prepared in Example 5 is mesoporous silica) The Raman probe was uniformly dispersed in physiological saline to prepare a 1 nmol/L solution.

步骤二：向正常小鼠左下肢爪垫部位皮下注射25ul经超声分散的1nmol/L的步骤一配制的拉曼探针溶液，并按摩注射部位5分钟。Step 2: 25 ul of the ultrasonically dispersed 1 nmol/L Raman probe solution prepared by subcutaneous injection into the left lower extremity paw pad of normal mice, and massage the injection site for 5 minutes.

步骤三：注射24小时后，将小鼠麻醉并暴露出左侧腘窝***，使用拉曼光谱仪对小鼠的左下肢进行超快的拉曼成像并对成像结果进行分析。实验结果如图10所示，使用拉曼探针内的拉曼信号分子4-硝基苯硫醇的拉曼特征峰(1340cm ^-1)重构出图像，可实现对腘窝***位置的快速、精确定位，大范围(3×2.7cm)成像仅需52s。 Step 3: 24 hours after the injection, the mice were anesthetized and exposed to the left axillary lymph nodes, and the ultra-fast Raman imaging of the left lower limb of the mouse was performed using Raman spectroscopy and the imaging results were analyzed. The experimental results are shown in Figure 10. Using the Raman characteristic peak of the Raman signal molecule 4-nitrophenylthiol (1340 cm ^-1 ) in the Raman probe to reconstruct the image, the position of the axillary lymph node can be achieved quickly. Accurate positioning, large-scale (3 × 2.7cm) imaging requires only 52s.

本发明的拉曼探针用于生物医学成像极大地提高了成像的速度，较传统的拉曼成像更有潜力应用于临床。当然也可应用于离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中。对于已经死亡的人体或已经死亡的动物体由于不能进行血液循环和淋巴循环，因此只能对注射部位进行拉曼成像。The Raman probe of the present invention is used for biomedical imaging to greatly improve the speed of imaging, and has more potential for clinical application than conventional Raman imaging. Of course, it can also be applied to the imaging of isolated tissues, isolated organs, dead human bodies or dead animals. For a human body that has died or an animal that has died, Raman imaging can only be performed on the injection site because blood circulation and lymph circulation cannot be performed.

对比例3(对应实施例12)Comparative Example 3 (corresponding to Example 12)

步骤一：将专利CN201610200580.8中公开的介孔二氧化硅包被的内嵌对二巯基苯的拉曼探针(双层核壳结构)均匀分散于生理盐水中制成1nmol/L的溶液。Step 1: uniformly disperse the mesoporous silica-coated Raman probe (double-layer core-shell structure) embedded in the mesoporous silica disclosed in the patent CN201610200580.8 in physiological saline to prepare a solution of 1 nmol/L. .

步骤三：注射24小时后，将小鼠麻醉并暴露出左侧腘窝***，使用拉曼光谱仪对小鼠的左下肢进行超快的拉曼成像并对成像结果进行分析。使用拉曼探针内的拉曼信号分子对二巯基苯的拉曼特征峰(1555cm ^-1)重构出图像，可实现对腘窝***位置的快速、精确定位，大范围(2.6×2.4cm)成像需22分钟。 Step 3: 24 hours after the injection, the mice were anesthetized and exposed to the left axillary lymph nodes, and the ultra-fast Raman imaging of the left lower limb of the mouse was performed using Raman spectroscopy and the imaging results were analyzed. Using the Raman signal molecule in the Raman probe to reconstruct the image of the Raman characteristic peak of dimercaptobenzene (1555cm ^-1 ), the position of the axillary lymph node can be quickly and accurately located, and the range is large (2.6×2.4cm). ) Imaging takes 22 minutes.

本发明的拉曼探针，通过改变核壳金纳米颗粒外部(即第二层上)的拉曼信号分子的种类和/或第一拉曼信号层中的拉曼信号分子的种类，可获得具有不同信号特征的拉曼探针；通过对该拉曼探针进行一定的生物修饰，可用于实现靶向不同肿瘤细胞的多指标成像。The Raman probe of the present invention can be obtained by changing the kind of the Raman signal molecule outside the core-shell gold nanoparticle (ie, on the second layer) and/or the kind of the Raman signal molecule in the first Raman signal layer. Raman probes with different signal characteristics; through the biological modification of the Raman probe, can be used to achieve multi-index imaging targeting different tumor cells.

本发明的拉曼探针在生物医学检测领域方面的应用还包括DNA检测、RNA检测、外泌体检测和抗原抗体检测等。比如在DNA和RNA检测中，将一段特定的DNA/RNA序列标记上该拉曼探针，然后利用碱基互补配对原则去探测待测样品中是否带有与之匹配的DNA/RNA序列。The application of the Raman probe of the present invention in the field of biomedical detection also includes DNA detection, RNA detection, exosome detection and antigen-antibody detection. For example, in DNA and RNA detection, a specific DNA/RNA sequence is labeled with the Raman probe, and then the base complementary pairing principle is used to detect whether the sample to be tested has a matching DNA/RNA sequence.

在外泌体检测中，将外泌体表面特定的标记物标记在该拉曼探针上，利用标记物与外泌体特异性结合的原理去检测待测样品中是否含有相应的外泌体。In exosomal detection, a specific label on the surface of the exosomes is labeled on the Raman probe, and the principle of specific binding of the label to the exosomes is used to detect whether the sample to be tested contains the corresponding exosomes.

在抗原抗体检测中，将特定抗原或抗体标记上该拉曼探针后，利用抗原抗体特异性结合的原理去检测待测样品中是否含有相应的抗体或抗原。In the antigen-antibody detection, after the specific antigen or antibody is labeled with the Raman probe, the principle of specific binding of the antigen-antibody is used to detect whether the sample to be tested contains the corresponding antibody or antigen.

本发明的拉曼探针在肿瘤检测和治疗领域也具有重要的应用价值。在肿瘤检测方面，该拉曼探针可以通过肿瘤部位血管的高滞留通透效应被动富集到肿瘤区域，并对肿瘤区域进行成像检测。因此本发明的拉曼探针能够用于制备肿瘤检测试剂盒、肿瘤治疗试剂盒、肿瘤检测治疗一体化试剂盒或者肿瘤药物。The Raman probe of the invention also has important application value in the field of tumor detection and treatment. In terms of tumor detection, the Raman probe can be passively enriched into the tumor region by the high retention effect of the blood vessel at the tumor site, and the tumor region is imaged and detected. Therefore, the Raman probe of the present invention can be used for preparing a tumor detection kit, a tumor treatment kit, a tumor detection treatment integration kit, or a tumor drug.

进一步地，在肿瘤治疗方面，本发明的拉曼探针可以作为药物的载体，进行肿瘤化疗药物的载带，然后通过肿瘤部位血管的高滞留通透效应被动富集到肿瘤区域，对肿瘤区域进行定点的热化疗。Further, in terms of tumor treatment, the Raman probe of the present invention can be used as a carrier of a drug, carrying a carrier of a tumor chemotherapy drug, and then passively enriching the tumor region through a high retention effect of a blood vessel at a tumor site, and the tumor region is Perform fixed-point thermochemotherapy.

本发明的拉曼探针也可用于防伪领域。比如，使用该拉曼探针制作成不同的商标字体或图案，然后通过拉曼检测来进行真伪辨别。The Raman probe of the present invention can also be used in the field of anti-counterfeiting. For example, the Raman probe is used to make different trademark fonts or patterns, and then the Raman detection is used for authenticity discrimination.

以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。The above has described in detail the preferred embodiments of the invention. It should be understood that many modifications and variations can be made in the present invention without departing from the scope of the invention. Therefore, any technical solution that can be obtained by a person skilled in the art based on the prior art based on the prior art by logic analysis, reasoning or limited experimentation should be within the scope of protection determined by the claims.

Claims

一种拉曼探针，其特征在于，具有纳米核、第一拉曼信号层和壳层；所述纳米核被所述第一拉曼信号层包被；所述第一拉曼信号层内分布有拉曼信号分子；所述壳层具有第一层和第二层；所述第一拉曼信号层被所述第一层包被；所述第二层包覆在所述第一层外，具有缝隙。A Raman probe having a nanocore, a first Raman signal layer and a shell layer; the nanocore being coated by the first Raman signal layer; the first Raman signal layer a Raman signal molecule is distributed; the shell layer has a first layer and a second layer; the first Raman signal layer is coated by the first layer; and the second layer is coated on the first layer In addition, there are gaps.
如权利要求1所述的拉曼探针，其特征在于，所述缝隙为能够增强拉曼光谱信号强度的结构。The Raman probe according to claim 1, wherein the slit is a structure capable of enhancing the intensity of a Raman spectral signal.
如权利要求1所述的拉曼探针，其特征在于，在所述拉曼探针的截面上，所述纳米核、所述第一拉曼信号层和所述壳层组合成盛开的花朵状，花朵的相邻花瓣之间形成所述缝隙。The Raman probe according to claim 1, wherein said nanocore, said first Raman signal layer and said shell layer are combined into a blooming flower on a section of said Raman probe In the shape, the gap is formed between adjacent petals of the flower.
如权利要求1所述的拉曼探针，其特征在于，在所述拉曼探针的截面上，所述壳层呈齿轮状，齿轮的相邻齿之间形成所述缝隙。The Raman probe according to claim 1, wherein in the cross section of the Raman probe, the shell layer has a gear shape, and the slit is formed between adjacent teeth of the gear.
如权利要求2-4任一项所述的拉曼探针，其特征在于，所述缝隙的数目为多个；所述缝隙的大小、形状不完全一致。The Raman probe according to any one of claims 2 to 4, wherein the number of the slits is plural; the size and shape of the slits are not completely identical.
如权利要求1所述的拉曼探针，其特征在于，所述缝隙在所述第一拉曼信号层中的拉曼信号分子的作用下形成。The Raman probe of claim 1 wherein said slit is formed by a Raman signal molecule in said first Raman signal layer.
如权利要求6所述的拉曼探针，其特征在于，所述第一拉曼信号层中的拉曼信号分子包括带有硝基的硫醇化合物。The Raman probe of claim 6 wherein the Raman signal molecule in the first Raman signal layer comprises a thiol compound bearing a nitro group.
如权利要求6所述的拉曼探针，其特征在于，所述第一拉曼信号层中的拉曼信号分子选自同时含有巯基和硝基的化合物。The Raman probe according to claim 6, wherein the Raman signal molecule in the first Raman signal layer is selected from the group consisting of a compound containing both a thiol group and a nitro group.
如权利要求6所述的拉曼探针，其特征在于，所述第一拉曼信号层中的拉曼信号分子选自同时含有巯基、硝基和苯环的化合物。The Raman probe according to claim 6, wherein the Raman signal molecule in the first Raman signal layer is selected from the group consisting of a compound containing a thiol group, a nitro group and a benzene ring.
如权利要求6所述的拉曼探针，其特征在于，所述第一拉曼信号层中的拉曼信号分子选自4-硝基苯硫醇、3-硝基苯甲基硫醇、2-氨基-5-硝基苯硫醇、邻硝基苯硫酚、2-巯基-6-硝基苯并噻唑和2-巯基-5-硝基苯并咪唑中的一种或多种；结构式如下：The Raman probe according to claim 6, wherein the Raman signal molecule in the first Raman signal layer is selected from the group consisting of 4-nitrobenzenethiol, 3-nitrobenzylthiol, One or more of 2-amino-5-nitrobenzenethiol, o-nitrothiophenol, 2-mercapto-6-nitrobenzothiazole, and 2-mercapto-5-nitrobenzimidazole; The structure is as follows:
如权利要求1所述的拉曼探针，其特征在于，所述第一层为封闭结构。The Raman probe of claim 1 wherein said first layer is a closed structure.
如权利要求1所述的拉曼探针，其特征在于，所述缝隙内分布有拉曼信号分子。The Raman probe according to claim 1, wherein a Raman signal molecule is distributed in said slit.
如权利要求12所述的拉曼探针，其特征在于，所述第一拉曼信号层的拉曼信号分子与所述缝隙内的拉曼信号分子是相同的。The Raman probe of claim 12 wherein the Raman signal molecules of said first Raman signal layer are identical to the Raman signal molecules within said gap.
如权利要求12所述的拉曼探针，其特征在于，所述第一拉曼信号层的拉曼信号分子与所述缝隙内的拉曼信号分子是不同的。The Raman probe of claim 12 wherein the Raman signal molecules of said first Raman signal layer are different from the Raman signal molecules within said gap.
如权利要求12所述的拉曼探针，其特征在于，所述缝隙内的拉曼信号分子通过作用力吸附到所述壳层上。The Raman probe according to claim 12, wherein the Raman signal molecules in the slit are adsorbed onto the shell layer by a force.
如权利要求12所述的拉曼探针，其特征在于，所述壳层为金壳层、银壳层、铜壳层或者铂壳层。The Raman probe according to claim 12, wherein the shell layer is a gold shell layer, a silver shell layer, a copper shell layer or a platinum shell layer.
如权利要求16所述的拉曼探针，其特征在于，所述缝隙内的拉曼信号分子包括可与所述壳层产生静电吸附作用或化学共价结合的分子。The Raman probe of claim 16 wherein the Raman signal molecules within the gap comprise molecules that can electrostatically or chemically covalently bond to the shell layer.
如权利要求1所述的拉曼探针，其特征在于，所述纳米核为金纳米核、银纳米核、铜纳米核或铂纳米核。The Raman probe according to claim 1, wherein the nano-nucleus is a gold nano core, a silver nano core, a copper nano core or a platinum nano core.
如权利要求1所述的拉曼探针，其特征在于，还具有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；所述外层结构包被在所述第二层外。The Raman probe according to claim 1, further comprising an outer layer structure; said outer layer structure being mesoporous silica, a mercapto compound or electrostatically adsorbing or chemically cooperating with said shell layer a valence-bound polymeric compound; the outer structure is coated outside the second layer.
一种如权利要求1所述的拉曼探针的制备方法，其特征在于，包括如下步骤：A method for preparing a Raman probe according to claim 1, comprising the steps of:

步骤一、将原料纳米核颗粒加入到表面活性剂的水溶液里，离心，重分散在表面活性剂的水溶液中，得到以表面活性剂为稳定剂的纳米核；Step 1: adding the raw material nano-nuclear particles to the aqueous solution of the surfactant, centrifuging, and redispersing in the aqueous solution of the surfactant to obtain a nano-nucleus with a surfactant as a stabilizer;

步骤二、在所述步骤一得到的以表面活性剂为稳定剂的纳米核中加入拉曼信号分子溶液，离心，重分散在表面活性剂的水溶液中，制备得到在纳米核的外表面包被有所述第一拉曼信号层的纳米颗粒，即在纳米核的外表面修饰有所述拉曼信号分子的纳米颗粒；Step 2: adding a Raman signal molecule solution to the nano-nuclear obtained by using the surfactant as a stabilizer in the first step, centrifuging, redispersing in an aqueous solution of the surfactant, and preparing the outer surface of the nano-nucleus to be coated. The nanoparticles of the first Raman signal layer, that is, the nanoparticles having the Raman signal molecule modified on the outer surface of the nano core;

步骤三、将所述步骤二得到的在纳米核的外表面包被有所述第一拉曼信号层的纳米颗粒加入到含有表面活性剂的水溶液、金属离子化合物溶液、还原剂混合的生长液中，得到具有所述壳层包被在所述第一拉曼信号层外的纳米颗粒，即得到所述拉曼探针；所述金属离子化合物溶液选自氯金酸溶液、硝酸银溶液、氯化铜溶液、硫酸铜溶液和氯铂酸溶液中的一种或多种。Step 3: adding the nanoparticles coated with the first Raman signal layer on the outer surface of the nano core obtained in the step 2 to a growth liquid containing a surfactant, an aqueous solution of a metal ion compound, and a reducing agent. Obtaining a nanoparticle having the shell layer coated outside the first Raman signal layer, thereby obtaining the Raman probe; the metal ion compound solution is selected from the group consisting of a chloroauric acid solution, a silver nitrate solution, and a chlorine One or more of a copper solution, a copper sulfate solution, and a chloroplatinic acid solution.
如权利要求20所述的制备方法，其特征在于，所述步骤二在所述步骤一得到的以表面活性剂为稳定剂的纳米核中加入拉曼信号分子溶液后，混合震荡2-20分钟后，再进行后续操作。The preparation method according to claim 20, wherein the step 2 is performed by adding a Raman signal molecule solution to the nano-nucleus with a surfactant as a stabilizer obtained in the step 1, and mixing and shaking for 2-20 minutes. After that, follow up.
如权利要求21所述的制备方法，其特征在于，混合震荡时间为5-10分钟。The preparation method according to claim 21, wherein the mixing oscillating time is 5 to 10 minutes.
如权利要求20所述的制备方法，其特征在于，在所述步骤三得到的具有所述壳层的纳米颗粒外包覆介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物。The preparation method according to claim 20, wherein the nanoparticle having the shell layer obtained in the step 3 is coated with mesoporous silica, a mercapto compound or electrostatically adsorbed with the shell layer. A polymer compound that acts or chemically covalently binds.
如权利要求20所述的制备方法，其特征在于，还包括步骤四、向所述步骤三得到的具有所述壳层的纳米颗粒中加入拉曼信号分子，离心，重分散在表面活性剂的水溶液中，得到所述第二层的缝隙中修饰有拉曼信号分子的纳米颗粒。The preparation method according to claim 20, further comprising the step of: adding a Raman signal molecule to the nanoparticle having the shell layer obtained in the third step, centrifuging, and redispersing the surfactant. In the aqueous solution, nanoparticles in which the Raman signal molecule is modified in the gap of the second layer are obtained.
如权利要求24所述的制备方法，其特征在于，在所述步骤四得到的所述第二层的缝隙中修饰有拉曼信号分子的纳米颗粒外包覆介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物。The preparation method according to claim 24, wherein the nanoparticle modified with the Raman signal molecule in the gap of the second layer obtained in the fourth step is coated with mesoporous silica, a mercapto compound or A polymer compound which can be electrostatically adsorbed or chemically covalently bonded to the shell layer.
如权利要求20所述的制备方法，其特征在于，所述步骤一中的原料纳米核颗粒的制备方法包括柠檬酸钠热还原法、种子生长法、聚乙烯吡咯烷酮保护还原法或紫外光引发还原法。The preparation method according to claim 20, wherein the preparation method of the raw material nano-nuclear particles in the first step comprises a sodium citrate thermal reduction method, a seed growth method, a polyvinylpyrrolidone protective reduction method or ultraviolet light-induced reduction. law.
如权利要求20所述的制备方法，其特征在于，所述表面活性剂选自十六烷基氯化铵、十六烷基溴化铵、聚乙烯吡咯烷酮中的一种或者多种。The method according to claim 20, wherein the surfactant is one or more selected from the group consisting of cetyl ammonium chloride, cetyl ammonium bromide, and polyvinyl pyrrolidone.
如权利要求20所述的制备方法，其特征在于，所述步骤三中的还原剂选自抗坏血酸、盐酸羟胺、甲醛中的一种或者多种。The method according to claim 20, wherein the reducing agent in the third step is selected from one or more of ascorbic acid, hydroxylamine hydrochloride, and formaldehyde.
如权利要求1所述的拉曼探针在单颗粒检测方面的应用。The use of the Raman probe of claim 1 for single particle detection.
如权利要求29所述的拉曼探针在单颗粒检测方面的应用，其特征在于，所述拉曼探针的第二层外包被有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；包括以下步骤：The use of the Raman probe according to claim 29 for single particle detection, characterized in that the second layer of the Raman probe is overcoated with an outer layer structure; the outer layer structure is mesoporous dioxide a silicon or a mercapto compound or a polymer compound which can be electrostatically adsorbed or chemically covalently bonded to the shell layer; the following steps are included:

A、将所述拉曼探针的溶液滴于硅片上，干燥后将硅片固定在原子力-显微共焦拉曼联用光谱仪上进行单颗粒测试；A, the solution of the Raman probe is dropped on a silicon wafer, and after drying, the silicon wafer is fixed on an atomic force-microscopic confocal Raman spectrometer for single particle test;

B、首先对有拉曼探针的硅片进行原子力显微成像，找到并确认硅片上的多个单颗粒；然后对单颗粒依次进行拉曼光谱采集，并对结果进行分析。B. Firstly, atomic force microscopy imaging of the silicon wafer with Raman probe is performed to find and confirm a plurality of single particles on the silicon wafer; then, the single particles are sequentially subjected to Raman spectroscopy, and the results are analyzed.
如权利要求29所述的拉曼探针在单颗粒检测方面的应用，其特征在于，所述应用基于拉曼流式的单颗粒标记液相芯片；包括以下步骤：The use of the Raman probe according to claim 29 for single particle detection, characterized in that the application is based on a Raman flow type single particle labeling liquid phase chip; comprising the steps of:

a、基于拉曼流式的单颗粒标记液相芯片，单颗粒拉曼探针作为生物标记物，内嵌不同的拉曼信号分子实现编码，得到编码后的单颗粒拉曼探针；然后将每种所述编码后的单颗粒拉曼探针共价交联上针对特定检测物，即靶分子的捕获分子，得到针对不同检测物的编码单颗粒拉曼探针；所述捕获分子包括抗原、抗体和/或核酸探针；a Raman flow-based single-particle labeling liquid phase chip, single-particle Raman probe as a biomarker, embedded with different Raman signal molecules to achieve encoding, to obtain a coded single-particle Raman probe; Each of the encoded single-particle Raman probes is covalently cross-linked to a specific detector, ie, a capture molecule of a target molecule, to obtain a single-particle Raman probe encoding a different analyte; the capture molecule includes an antigen , antibodies and/or nucleic acid probes;

b、先把多种所述针对不同检测物的编码单颗粒拉曼探针混合，再加入微量待检样本，形成悬液；在所述悬液中，所述待检样本中的靶分子与所述针对不同检测物的编码单颗粒拉曼探针表面交联的捕获分子发生特异性结合；最后使用显微共焦拉曼光谱仪识别所述针对不同检测物的编码单颗粒拉曼探针的编码及与之特异性结合的靶分子，从而识别待检样本。b. first mixing a plurality of the encoded single-particle Raman probes for different detection substances, and then adding a trace amount of the sample to be tested to form a suspension; in the suspension, the target molecules in the sample to be tested are The specific molecules of the single-particle Raman probe surface cross-linked capture molecules for different detections are specifically bound; finally, the micro-confocal Raman spectrometer is used to identify the single-particle Raman probes for different detections. The target molecule is encoded and specifically bound to identify the sample to be examined.
如权利要求1所述的拉曼探针在细胞水平成像中的应用。Use of the Raman probe of claim 1 in cell level imaging.
如权利要求32所述的拉曼探针在细胞水平成像中的应用，其特征在于，所述拉曼探针的第二层外包被有外层结构；所述外层结构为介孔二氧化硅、巯基化合物或者可与所述壳层产生静电吸附作用或化学共价结合的高分子化合物；包括以下步骤：The use of the Raman probe according to claim 32 for imaging at a cellular level, characterized in that the second layer of the Raman probe is overcoated with an outer layer structure; the outer layer structure is mesoporous dioxide a silicon or a mercapto compound or a polymer compound which can be electrostatically adsorbed or chemically covalently bonded to the shell layer; the following steps are included:

1)、将细胞与所述拉曼探针的溶液共孵育，使所述拉曼探针进入细胞内部；1) co-incubating the cells with the solution of the Raman probe to allow the Raman probe to enter the interior of the cell;

2)、用拉曼光谱仪对经所述步骤1)处理过的细胞进行拉曼成像，对成像结果进行分析。2) Raman imaging of the cells treated in the step 1) by Raman spectroscopy, and analyzing the imaging results.
如权利要求33所述的拉曼探针在细胞水平成像中的应用，其特征在于，所述步骤1)中将细胞与0.001-100n mol/L的所述拉曼探针的溶液共同置于细胞培养箱中，以37℃孵育0.5-24h，使所述拉曼探针进入细胞内部。The use of the Raman probe according to claim 33 for imaging at a cellular level, characterized in that in the step 1), the cells are co-located with a solution of 0.001-100 nmol/L of the Raman probe. The Raman probe was introduced into the interior of the cell by incubating at 37 ° C for 0.5-24 h in a cell culture incubator.
如权利要求33所述的拉曼探针在细胞水平成像中的应用，其特征在于，所述步骤2)中每个像素点的积分时间为0.7-10ms，激光功率为1％-10％，高分辨率的细胞成像可在3-20s内完成；所述高分辨率的细胞成像指细胞成像的像素点大于等于50×50像素点。The application of the Raman probe according to claim 33 in cell level imaging, wherein the integration time of each pixel in the step 2) is 0.7-10 ms, and the laser power is 1%-10%. High-resolution cell imaging can be accomplished in 3-20 s; the high-resolution cell imaging refers to cell imaging with pixel points greater than or equal to 50 x 50 pixels.
如权利要求1所述的拉曼探针在医学成像中的应用。The use of the Raman probe of claim 1 in medical imaging.
如权利要求36所述的拉曼探针在医学成像中的应用，其特征在于，包括以下步骤：The use of the Raman probe according to claim 36 in medical imaging, comprising the steps of:

I、将所述拉曼探针均匀分散于生理盐水或pH＝7.4的PBS溶液中制成0.01-50n mol/L的拉曼探针溶液；I, the Raman probe is uniformly dispersed in physiological saline or PBS solution of pH=7.4 to prepare a Raman probe solution of 0.01-50 nm mol/L;

II、向试验动物体内局部注射经超声分散的所述步骤I制得的0.01-50n mol/L的拉曼探针溶液；II. Injecting 0.01-50 nmol/L of Raman probe solution prepared by the step I obtained by ultrasonication into the test animal;

III、注射0.5-24h后，使用拉曼光谱仪对经所述步骤II处理的试验动物的感兴趣部位进行拉曼成像，并对成像结果进行分析。III. After 0.5-24 hours of injection, the region of interest of the test animal treated in the step II was subjected to Raman imaging using a Raman spectrometer, and the imaging results were analyzed.
如权利要求37所述的拉曼探针在医学成像中的应用，其特征在于，所述步骤III中每个像素点的积分时间为0.7-100ms。The application of the Raman probe according to claim 37 in medical imaging, characterized in that the integration time of each pixel in the step III is 0.7-100 ms.
如权利要求1所述的拉曼探针在离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用。Use of the Raman probe of claim 1 in the imaging of isolated tissues, isolated organs, dead humans or dead animals.
如权利要求39所述的拉曼探针在离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用，其特征在于，包括以下步骤：The use of the Raman probe according to claim 39 in the imaging of isolated tissues, isolated organs, dead human bodies or dead animals, comprising the steps of:

I、将所述拉曼探针均匀分散于生理盐水或pH＝7.4的PBS溶液中制成0.01-50n mol/L的拉曼探针溶液；I, the Raman probe is uniformly dispersed in physiological saline or PBS solution of pH=7.4 to prepare a Raman probe solution of 0.01-50 nm mol/L;

II、将离体组织、离体器官与经超声分散的所述步骤I制得的0.01-50n mol/L的拉曼探针溶液共孵育10-60分钟，或者向已经死亡的人体或已经死亡的动物体内局部注射经超声分散的所述步骤I制得的0.01-50n mol/L的拉曼探针溶液；II. Incubating the ex vivo tissue and the isolated organ with the 0.01-50 nmol/L Raman probe solution prepared by the step I of the ultrasonic dispersion for 10 to 60 minutes, or to the already dead human body or having died a 0.01-50 nmol/L Raman probe solution prepared by the step I obtained by ultrasonic dispersion;

III、使用拉曼光谱仪对经所述步骤II处理的离体组织、离体器官、已经死亡的人体或已经死亡的动物体的感兴趣部位进行拉曼成像，并对成像结果进行分析。III. Raman imaging was performed on the isolated tissue treated by the step II, the isolated organ, the dead human body or the dead animal body using a Raman spectrometer, and the imaging results were analyzed.
如权利要求40所述的拉曼探针在离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用，其特征在于，所述步骤III中每个像素点的积分时间为0.7-100ms。The use of the Raman probe according to claim 40 for imaging of isolated tissue, isolated organs, dead human body or dead animal body, characterized by integration of each pixel in said step III The time is 0.7-100ms.
如权利要求40所述的拉曼探针在离体组织、离体器官、已经死亡的人体或已经死亡的动物体成像中的应用，其特征在于，所述步骤III为在孵育10-60分钟后，使用拉曼光谱仪对经所述步骤II处理的离体组织、离体器官、已经死亡的人体或已经死亡的动物体的感兴趣部位进行拉曼成像，并对成像结果进行分析。The use of the Raman probe according to claim 40 for imaging of isolated tissues, isolated organs, dead human bodies or dead animals, wherein said step III is for 10 to 60 minutes of incubation. Thereafter, Raman imaging was performed on the isolated tissue treated with the step II, the isolated organ, the dead human body, or the dead animal body using a Raman spectrometer, and the imaging results were analyzed.
如权利要求1所述的拉曼探针在生物医学检测领域中的应用。The use of the Raman probe of claim 1 in the field of biomedical detection.
如权利要求1所述的拉曼探针在DNA检测、RNA检测、外泌体检测和/或抗原抗体检测中的应用。Use of the Raman probe of claim 1 for DNA detection, RNA detection, exosome detection and/or antigen antibody detection.
如权利要求1所述的拉曼探针在制备肿瘤检测试剂盒、肿瘤治疗试剂盒、肿瘤检测治疗一体化试剂盒或者肿瘤药物中的应用。The use of the Raman probe according to claim 1 in the preparation of a tumor detection kit, a tumor treatment kit, a tumor detection treatment integration kit, or a tumor drug.
如权利要求1所述的拉曼探针在防伪领域中的应用。The use of the Raman probe of claim 1 in the field of anti-counterfeiting.