WO2016150125A1 - 一种内含内标分子的核壳结构纳米粒子及表面增强拉曼定量检测方法 - Google Patents

一种内含内标分子的核壳结构纳米粒子及表面增强拉曼定量检测方法 Download PDF

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WO2016150125A1
WO2016150125A1 PCT/CN2015/090012 CN2015090012W WO2016150125A1 WO 2016150125 A1 WO2016150125 A1 WO 2016150125A1 CN 2015090012 W CN2015090012 W CN 2015090012W WO 2016150125 A1 WO2016150125 A1 WO 2016150125A1
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internal standard
core
shell
molecule
shell structured
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任斌
沈炜
林旋
刘国坤
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Xiamen University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

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  • the invention relates to a core-shell structure nanoparticle containing an internal standard molecule, in particular to a surface enhanced Raman quantification system.
  • SERS Surface-enhanced Raman
  • the coupling effect of metal nanostructures on the nanometer scale produces enhanced local electromagnetic fields, which is the main source of enhancement of this technology.
  • the enhanced local electromagnetic field is greatly affected by the microscopic size (nanostructure size and coupling pitch, etc.), and the existing nanotechnology is difficult to obtain a uniform SERS-enhanced coupled nanosol or nano-substrate, so that the molecular signal amplification factor It is difficult to achieve agreement, so the molecular concentration cannot be obtained by using the detected molecular signal, that is, quantitative detection cannot be achieved.
  • the SERS detection of the sol system has better signal stability relative to the substrate, and the reproducibility of the agglomerates is improved by some agglomeration control methods (salt control agglomeration, microfluidic control agglomeration, etc.), however, these improvements are still It is not possible to push SERS quantitative detection to practical applications. Factors such as instrument parameters, agglomeration state, and molecular surface coverage are still not reproducible. Therefore, attempts have been made to correct the fluctuation of these parameters by referring to some classical signal feedback methods.
  • the internal standard method has been widely applied to various spectroscopy techniques.
  • the important principle is that the internal standard and the object to be tested are dispersed in the same physical and chemical environment, so the signal of the internal standard can effectively correct the error caused by the detection.
  • the first uneven surface enhancement effect makes the electromagnetic field strength felt by each or each molecule different (physical environment is different), so that the internal standard is difficult to accurately reflect the enhanced state of the object to be tested;
  • There is a difference in the adsorption capacity between the standard and the analyte which may result in the detection of signals with molecules with strong adsorption capacity.
  • the internal standard is difficult to reflect the information of the coverage of the analyte (the chemical environment is different); Due to the difference in chemical properties between the internal standard and the analyte, the adsorption state may change when the pH, ionic strength, and co-adsorbed species of the detection environment change (the chemical environment is different), so the internal standard is difficult to apply.
  • Various experimental conditions In summary, it is difficult to find a universal internal standard molecule that can be applied to different kinds of molecules and different detection environments. It is urgent to develop a new material or a new method to achieve quantitative detection of SERS. SERS can be an accurate, sensitive, reliable and universal analytical method.
  • the object of the present invention is to provide a novel CMS (core@molecular@shell, core@molecules@shell, CMS) nanoparticles to achieve universal, accurate, sensitive, fast, low cost and reliable SERS quantitative detection.
  • metal nanoparticles with surface plasmon resonance effect were prepared as cores and mixed with modified monolayers as internal standards, and then metal shells with surface plasmon resonance effects were grown outside the monolayer to obtain CMS nanoparticles. Finally, the prepared CMS nanoparticles are directly mixed with the analyte to directly detect the SERS signal of the analyte and the internal standard.
  • the invention comprises the following steps:
  • the core is mixed and modified to form a monolayer, the monolayer comprising a skeleton and an internal standard;
  • a metal shell having a surface plasmon resonance effect is grown outside the monolayer as an internal standard to obtain a CMS metal sol;
  • step 5 The signal obtained in step 5 is compared with the standard curve to obtain the content of the analyte.
  • the metal nanoparticles are metal nanoparticles having surface plasmon resonance properties such as gold nanoparticles, silver nanoparticles or copper nanoparticles.
  • the metal nanoparticles are metal nanoparticles of various shapes such as a spherical shape or a rod shape, and the metal nanoparticles have a particle diameter of 10 to 300 nm.
  • the skeleton molecule in the mixed modified monolayer is selected from an adsorption molecule containing a thiol terminal, an amino terminal or a carboxy terminal.
  • These strongly adsorbed molecules include, but are not limited to, mercaptoethylamine, ⁇ -mercaptohexylamine, ethanedithiol, 1,6-hexanedithiol, thioglycolic acid, ⁇ -mercaptohexanoic acid, and the like.
  • the internal standard molecule in the mixed modified monolayer adopts a strongly adsorbed molecule having a Raman scattering cross section containing a thiol terminal, an amino terminal or a carboxy terminal.
  • These molecules include, but are not limited to, mercaptobenzene, 1,4-dimercaptobenzene, 4-mercaptopyridine, 4-mercaptobenzoic acid, and the like.
  • the metal shell layer is a metal shell layer having surface plasmon resonance properties such as silver, gold or copper.
  • the excitation source wavelength detected by the CMS nanoparticle-enhanced Raman spectroscopy method is 400 to 1500 nm.
  • the invention provides a nuclear@molecular@shell (core@molecules@shell, CMS) nanostructure, and its structural specificity has the ability to overcome the existing SERS quantitative bottleneck problem: (1) the molecule between the core-shell structure The layer is not affected by the detection conditions and the environment, and is an ideal stable internal standard; (2) the internal standard molecules are coated inside the nanoparticles, so that the surface sites of the shell can be all supplied to the species to be tested, and the surface is The condition that the target molecule competes with the test species for the surface site can be used for more species to be tested; (3) the CMS particle plays two roles at the same time, that is, it is both the internal standard of SERS and the reinforcing substrate of SERS.
  • the internal standard molecule can effectively feedback the difference in the enhancement effect of the agglomerate during the detection process and a combination of factors, to achieve a more accurate detection of the surface coverage of the molecule to be tested, thereby reflecting the content of the species in the solution, and can construct a good internal standard feedback.
  • the quantitative method based on CMS internal standard particles is a direct detection method without label. The actual test process does not require complicated surface modification and treatment, and the method is simple and effective. Therefore, we prepared CMS nanoparticles and used the nanoparticles as SERS internal standard to achieve accurate, sensitive, reliable and universal quantitative detection of SERS.
  • a monolayer (a strong adsorption molecule containing a thiol terminal, an amino terminal or a carboxy terminal) is mixed and modified, and a strong interaction between the molecule and the metal is obtained.
  • a metal (silver, gold, copper) shell layer can be grown on the molecular layer such that the monolayer is completely embedded within the nanostructure.
  • the internal monolayer does not change due to the adsorption of external molecules, is a very stable SERS internal standard, and can generate a strong SERS signal.
  • the internal internal standard signal can effectively feedback the agglomeration state of the sol, the particle concentration, the surface coverage of the analyte molecule, the instrument state and other parameters, so that the signal after calibration with the internal standard is tested.
  • concentration of the substances is consistent with the Langmuir adsorption isotherm to achieve the purpose of quantitative detection.
  • the present invention has the following outstanding advantages and technical effects:
  • the preparation method and raw material of the CMS nanoparticle of the invention are simple and easy to obtain, wherein the size of the inner core and the outer shell and the thickness of the molecular layer are all precisely controllable.
  • the invention eliminates the competition of the same surface site during the adsorption process of the analyte and the internal standard object, so that the internal standard material can truly and effectively feedback the physical and chemical environment in which the object to be tested is located. , has excellent reproducibility.
  • the invention is applicable to SERS quantitative detection of sol and solid substrates.
  • the invention can be applied to various molecular detections and has excellent universality.
  • the invention broadens the application field of SERS, and lays a foundation for truly making SERS a highly sensitive and quantitative detection technology widely used in scientific research and production.
  • FIG. 1 is a schematic diagram of an experimental procedure for preparing CMS nanoparticles.
  • 2A is a transmission electron micrograph (TEM) of CMS nanoparticles.
  • 2B is a scanning transmission electron micrograph (STEM). The scale in the figure is 20 nm.
  • Figure 3A is an ultraviolet-visible extinction spectrum of monodisperse sol CMS nanoparticles.
  • Figure 3B is a SERS spectrum of monodisperse sol particles.
  • FIG. 4A is a schematic diagram of SERS quantitative detection of CMS nanoparticles in a sol.
  • Figure 4B is a schematic representation of SERS quantitative detection of CMS nanoparticles on a substrate.
  • Figure 5 is a graph showing the quantitative measurement of SERS quantitative detection of 1,4-diisocyanide by the sol quantification method of the present invention.
  • Fig. 5A shows the working curve obtained by using the absolute intensity of the analyte signal and the concentration of the analyte in the small concentration interval
  • Fig. 5B is obtained by using the relative intensity of the analyte and the internal standard in the small concentration interval and the concentration of the analyte. Work curve.
  • Fig. 6 is a quantitative detection of a small concentration range of uric acid molecules by the method of quantitative sol of the present invention.
  • Figure 1 shows a schematic diagram of the experimental procedure for the preparation of CMS nanoparticles.
  • the specific preparation method is:
  • the synthesized 50 nm gold nanoparticles were adjusted (with 15 nm gold nanoparticles as seeds, and grown in two steps to obtain gold nanoparticles of 30 nm and 50 nm, respectively).
  • First adjust the pH of the sol with NaOH It is about 9 and controls the gold nanoparticle concentration to be 30-80 pM, which is 40 pM in this embodiment; then 4-mercaptopyridine molecule is added (the preferred final concentration in the system is 0.05-0.25 ⁇ M, in this embodiment 0.2 ⁇ M) And mercaptoethylamine molecule (the preferred final concentration in the system is 1 - 2 ⁇ M, 1.5 ⁇ M in this example); then adding silver ammonia solution (the preferred final concentration in the system is 0.05-0.5 mM, 0.15 mM in this example), and finally slowly injecting Ascorbic acid solution (preferred final concentration of 0.1-1 mM in the system, 0.2 mM in this example).
  • FIG. 2A is a transmission electron micrograph (TEM) of a CMS nanoparticle
  • FIG. 2B is a scanning transmission electron micrograph (STEM).
  • the TEM image shows that the particle size distribution of the prepared particles is better, and the core-shell structure is obvious. From the elemental imaging diagram of STEM, the micro-core shell structure of the prepared CMS nanoparticles is intact, without defects, and the envisaged The structure is consistent.
  • FIG. 3A is an ultraviolet-visible extinction spectrum of monodisperse sol CMS nanoparticles
  • FIG. 3B is a SERS spectrum of monodisperse sol particles.
  • the UV-visible extinction spectrum shows that the particles have good monodispersity and no obvious agglomeration; the SERS spectrum of the sol which can obtain monodisperse particles reflects that the internal embedded monolayer is in a strong SERS enhancement region, which can provide enough Strong internal standard signal.
  • 4A is a schematic diagram of SERS quantitative detection of CMS nanoparticles in a sol.
  • the prepared Au@CA+Mpy@Ag particles were centrifuged twice at room temperature to remove the residual protective agent and dispersed in half of the original volume.
  • the sol was divided into several parallel samples, 1 ml per sample. Each sample was mixed with an equal volume of different concentrations of 1,4-diisoxonitrile benzene solution, and the final molecular concentration range after mixing was 2-10 nM. After mixing, perform SERS detection. Each concentration was tested 16 times in succession, and the 16 data were used for statistics to obtain the average value and relative deviation. Finally, the working curve is made with the average value and relative deviation and concentration at each concentration.
  • Figure 5 is a graph showing the SERS quantitative detection of 1,4-diisoxonitrile benzene by three batches of CMS nanoparticle sol synthesized by the method of the present invention.
  • Fig. 5A shows the working curve obtained by using the absolute intensity of the analyte signal and the concentration of the analyte
  • Fig. 5B is the working curve obtained by comparing the relative intensity of the analyte with the internal standard with the concentration of the analyte. Comparing the working curves without internal standard calibration and internal standard calibration, it can be seen that the corrected working curve has excellent reproducibility and small error, and the working curve can be used for quantitative detection of unknown concentration samples.
  • the prepared Au@CA+Mpy@Ag particles were centrifuged twice at room temperature to remove residual protective agent and dispersed in the original volume of water.
  • the sol was divided to obtain several parallel samples, 0.5 ml per sample. Each sample was mixed with different concentrations of uric acid solution and NaCl was added to induce agglomeration. The final molecular concentration range after mixing was 69-350 ⁇ M. After mixing, it was stabilized for 10 min and then subjected to SERS detection. Each concentration was tested 16 times in succession, and the 16 data were used for statistics to obtain the average value and relative deviation. Finally, the working curve is made with the average value and relative deviation and concentration at each concentration.
  • Figure 6 is a quantitative measurement of uric acid molecules by the method of sol quantification of the present invention. Comparing the working curves without internal standard calibration and internal standard calibration, it can be seen that the corrected working curve has excellent reproducibility and small error, and the working curve can be used for quantitative detection of unknown concentration samples.
  • the CMS nanoparticle of the invention and the nanoparticle as the SERS internal standard can achieve accurate, sensitive, reliable and universal quantitative detection of SERS.

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Abstract

一种内含内标分子的核壳结构纳米粒子及表面增强拉曼定量检测方法，其特征包括以下步骤：1)制备具有强拉曼信号放大的核@分子@壳层纳米结构，并以夹层中的分子的拉曼信号作为内标；2)将纳米粒子与含待测分子的溶液均匀混合；3)直接检测待测分子与内标分子的拉曼信号；与传统内标法相比，消除了待测物和内标物吸附过程中对同一表面位点的竞争，使得内标物能真实、有效的反馈待测物所处的物理化学环境，有着极好的重现性。

Description

一种内含内标分子的核壳结构纳米粒子及表面增强拉曼定量检测方法 技术领域
本发明涉及一种内含内标分子的核壳结构纳米粒子，尤其是涉及应用于表面增强拉曼定量体系。
背景技术
表面增强拉曼(SERS)技术作为一种非侵入性的检测技术有着极高的表面灵敏度以及分子指纹识别在化学、生物、环境以及食品安全的定性检测中有着广泛的应用。其中金属纳米结构在纳米尺度上的耦合效应产生增强的局域电磁场是该技术增强的主要来源。该增强的局域电磁场受到微观尺寸(纳米结构尺寸和耦合间距等)的影响极大，而现有的纳米技术很难得到均匀SERS增强的耦合的纳米溶胶或者纳米基底，使得分子信号的放大倍数难以达到一致，因此无法用所检测到的分子信号来得到分子浓度，即无法实现定量检测。一般认为溶胶体系的SERS检测有着相对基底更好的信号稳定性，并且通过一些团聚的控制方法(盐控制团聚、微流控系统控制团聚等)来提高团聚体的重现性，然而这些改进仍然无法将SERS定量检测推向实际应用。其中仪器参数、团聚状态以及分子的表面覆盖度等因素仍然无法重现，因此，人们试图通过借鉴一些经典信号反馈方法来试图校正这些参数的波动。
内标法作为一种常见的反馈校正方法，已经广泛的应用于各种谱学技术。其重要原理是内标物和待测物分散于相同的物理化学环境中，因此内标物的信号能够有效的校正检测中带来的误差。然而对于SERS而言，首先不均匀的表面增强效应使得每种或者每个分子所感受到的电磁场强度不同(物理环境不同)，从而使得内标很难准确反映待测物的增强状态；其次由于内标物和待测物的吸附能力存在差别，可能导致检测时只能测到具有较强吸附能力的分子的信号，因此内标很难反映待测物覆盖度的信息(化学环境不同)；最后由于内标和待测物的化学性质的差异，当检测环境的pH、离子强度、共吸附物种等发生变化时可能会导致的吸附状态发生变化(化学环境不同)，因此内标很难应用于各种实验条件。综上所述，目前很难找到一种普适的内标分子能够可适用于不同种类的分子和不同的检测环境，亟待开发一种新材料或者新方法来实现SERS的定量检测，使得 SERS能成为准确、灵敏、可靠和普适的分析方法。
发明内容
本发明目的是提供一种新型的CMS(核@分子@壳层，core@molecules@shell,CMS)纳米粒子来实现普适、准确、灵敏、快速、低成本以及可靠的SERS定量检测。
本发明的技术方案是：
首先制备具有表面等离激元共振效应的金属纳米粒子作为内核并混合修饰上单分子层作为内标，然后在单分子层外生长具有表面等离激元共振效应的金属壳层获得CMS纳米粒子，最后直接将制备的CMS纳米粒子与待测物混合，直接检测待测物和内标物的SERS信号。
本发明包括以下步骤：
1.制备具有表面等离激元共振效应的金属纳米粒子作为内核
2.内核混合修饰上单分子层，所述的单分子层包括骨架和内标；
3.在作为内标的单分子层外生长具有表面等离激元共振效应的金属壳层获得CMS金属溶胶；
4.将获得的金属溶胶与待测物混合；
5.直接检测待测物和内标物的SERS信号；
6.步骤5得到的信号与标准曲线比较，得到待测物的含量。
所述金属纳米粒子为金纳米粒子、银纳米粒子或铜纳米粒子等具有表面等离激元共振性质的金属纳米粒子。
所述金属纳米粒子为球形或棒状等各种形状的金属纳米粒子，所述金属纳米粒子的粒径为10～300nm。
所述的混合修饰单分子层中骨架分子选用含有巯基末端、氨基末端或者羧基末端的吸附分子。这些强吸附分子包括，但不限于巯基乙胺，ω‐巯基己胺，乙二硫醇，1,6‐己二硫醇，巯基乙酸，ω‐巯基己酸等。
所述的混合修饰单分子层中内标分子选用含有巯基末端、氨基末端或者羧基末端的强吸附且有拉曼散射截面的分子。这些分子包括，但不限于巯基苯，1,4‐二巯基苯，4‐巯基吡啶，4‐巯基苯甲酸等。
所述的金属壳层为银、金或者铜等具有表面等离激元共振性质的金属壳层。
所述CMS纳米粒子增强拉曼光谱的方法检测的激发光源波长是400～1500nm。
本发明提供了一种核@分子@壳层(core@molecules@shell,CMS)纳米结构，其结构的特殊性具有克服现有SERS定量瓶颈问题的能力：(1)核壳结构之间的分子层不受检测条件和环境的影响，是一种理想稳定的内标；(2)内标分子被包覆在纳米粒子的内部，使得壳层表面位点可以全部提供给待测物种，表面内标分子与待测物种竞争表面位点的情况，可以用于更多的待测物种；(3)该CMS粒子同时起着两种作用，即本身既是SERS的内标同时也是SERS的增强基底，内标分子能有效的反馈检测过程中团聚体的增强效应的差异和一起因素，实现对待测分子的表面覆盖度更为准确的检测，从而反映溶液中物种的含量，可以构建良好的内标反馈机制；(4)基于CMS内标粒子的定量方法是无标记的直接检测方法，实际测试过程中无需复杂的表面修饰和处理，方法简易有效。因此，我们制备了CMS纳米粒子，并用该纳米粒子作为SERS内标很好的实现了SERS准确、灵敏、可靠和普适的定量检测。
本发明的原理是：
在高SERS活性的金属(金、银、铜)纳米粒子表面混合修饰上单分子层(含有巯基末端、氨基末端或者羧基末端的强吸附分子)，并通过该层分子与金属的强相互作用，使得金属(银、金、铜)壳层能够在该分子层上生长，使得该单分子层完全被包埋在纳米结构内部。内部的单分子层不会因外界分子的吸附而发生变化，是非常稳定的SERS内标物，并且能产生很强的SERS信号。在直接检测待测物时，内部的内标信号能有效的反馈溶胶的团聚状态，粒子浓度，待测物分子的表面覆盖度，仪器状态等参数，使得采用内标校正后的信号与待测物浓度之间符合Langmuir吸附等温式，从而达到定量检测的目的。
与现有的技术相比，本发明具有以下突出的优点和技术效果：
1.本发明的CMS纳米粒子的制备方法和原材料简单易得，其中内核和外壳的大小以及分子层的厚度都是可精确调控的。
2.本发明与传统内标法相比，消除了待测物和内标物吸附过程中对同一表面位点的竞争，使得内标物能真实、有效的反馈待测物所处的物理化学环境，有着极好的重现性。
3.本发明的内标物有较多的选择，能够理性的选择内标种类。
4.本发明适用于溶胶和固体基底的SERS定量检测。
5.本发明能适用于各种分子检测，有着极好的普适性。
6.本发明使得SERS的应用领域扩宽，为真正使SERS这种高灵敏定量的检测技术普遍应用于科研生产中奠定基础。
附图说明
图1是制备CMS纳米粒子的实验流程示意图。
图2A是CMS纳米粒子的透射电镜图(TEM)。图2B是扫描透射电镜图(STEM)。图中标尺为20nm。
图3A是单分散溶胶CMS纳米粒子的紫外‐可见消光光谱。图3B是单分散溶胶粒子的SERS光谱。
图4A是CMS纳米粒子在溶胶中进行SERS定量检测的示意图。图4B是CMS纳米粒子在基底上进行SERS定量检测的示意图。
图5是用本发明溶胶定量方法对1,4‐二异腈苯分子SERS定量检测的工作曲线。图5A在小浓度区间用待测物信号的绝对强度与待测物浓度所得到的工作曲线，图5B在小浓度区间用待测物与内标物的相对强度来与待测物浓度所得到的工作曲线。
图6是用本发明溶胶定量的方法对尿酸分子小浓度区间的定量检测。
具体实施方式
以下实施例将结合附图对本发明作进一步说明。应当理解，此处所描述的具体实施例仅用以解释本发明，不用于限定本发明。
实施例1
CMS纳米粒子的制备：
图1给出CMS纳米粒子制备的实验流程示意图。
以Au@CA+Mpy@Ag为例，其具体制备方法是：
调节合成好的50nm金纳米粒子(以15nm的金纳米粒子为种子，经两步生长，分别得到30nm和50nm的金纳米粒子)。首先用NaOH调控溶胶的pH 约为9，并控制金纳米粒子浓度为30‐80pM，本实施例中为40pM；然后加入4‐巯基吡啶分子(体系内较佳的最终浓度0.05‐0.25μM，本实施例中为0.2μM)和巯基乙胺分子(体系内较佳最终浓度1‐2μM，本实施例1.5μM)；再加入银氨溶液(体系内较佳最终浓度0.05‐0.5mM，本实施例0.15mM)，最后缓慢注入抗坏血酸溶液(体系内较佳最终浓度0.1‐1mM，本实施例0.2mM)。最终合成好的Au@CA+Mpy@Ag溶胶为橙黄色。
图2A是CMS纳米粒子的透射电镜图(TEM)，图2B是扫描透射电镜图(STEM)。TEM图中可以看到所制备的粒子粒径分布较好，核壳结构明显；从STEM的元素成像图来看，所制备的CMS纳米粒子的微观核壳结构完整，没有缺陷，与设想中的结构一致。
图3A是单分散溶胶CMS纳米粒子的紫外‐可见消光光谱，图3B是单分散溶胶粒子的SERS光谱。紫外‐可见消光光谱可以看到粒子的单分散性好，没有明显的团聚；能够获得单分散粒子的溶胶的SERS光谱反映出内部包埋的单分子层处于较强的SERS增强区域，能够提供足够强的内标信号。
实施例2
CMS纳米粒子在溶胶中进行SERS定量检测：
图4A是CMS纳米粒子在溶胶中进行SERS定量检测的示意图。
以1,4‐二异腈苯的定量检测为例，其具体实施方案是：
取所制备的Au@CA+Mpy@Ag粒子室温离心两次去除残留的保护剂后分散到原体积一半的水中。均分溶胶得数个个平行样品，每个样品1ml。每个样品与等体积不同浓度的1,4‐二异腈苯溶液混合，混合后的最终分子浓度区间为2‐10nM。混合后再进行SERS检测。每个浓度连续测试16次，并以这16个数据来做统计，得到平均值和相对偏差。最终以每个浓度下的平均值和相对偏差与浓度来做工作曲线。
图5是用本发明方法合成的3个批次CMS纳米粒子溶胶对1,4‐二异腈苯分子SERS定量检测的工作曲线。图5A用待测物信号的绝对强度与待测物浓度所得到的工作曲线，图5B用待测物与内标物的相对强度来与待测物浓度所得到的工作曲线。对比未用内标校正和内标校正的工作曲线，可以看出校正后的工作曲线重现性极好，误差很小，并且该工作曲线能用于未知浓度样品的定量检测。
实施例3
CMS纳米粒子溶胶用于生物分子的SERS定量检测：
以尿酸的定量检测为例，其具体实施方案是：
取所制备的Au@CA+Mpy@Ag粒子室温离心两次去除残留的保护剂后分散到原体积的水中。均分溶胶得到数个平行样品，每个样品0.5ml。每个样品与不同浓度的尿酸溶液混合并加入NaCl诱导团聚。混合后的最终分子浓度区间为69‐350μM。混合后稳定10min再进行SERS检测。每个浓度连续测试16次，并以这16个数据来做统计，得到平均值和相对偏差。最终以每个浓度下的平均值和相对偏差与浓度来做工作曲线。
图6是用本发明溶胶定量的方法对尿酸分子的定量检测。对比未用内标校正和内标校正的工作曲线，可以看出校正后的工作曲线重现性极好，误差很小，并且该工作曲线能用于未知浓度样品的定量检测。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质说作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
工业实用性
本发明的CMS纳米粒子，并用该纳米粒子作为SERS内标很好的实现了SERS准确、灵敏、可靠和普适的定量检测。

Claims (12)

  1. 一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，包括以下步骤：
    1)制备具有表面等离激元共振效应的金属纳米粒子作为内核；
    2)内核混合修饰上单分子层，所述的单分子层包括骨架和内标；
    3)在作为内标的单分子层外生长具有表面等离激元共振效应的金属壳层，获得CMS金属纳米粒子溶胶；
    4)将获得的金属溶胶与待测物混合；
    5)直接检测待测物和内标物的SERS信号；
    6)步骤5)得到的信号与标准曲线比较，得到待测物的含量。
  2. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤4)待测物与溶胶混合，或者位于固态基底上。
  3. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤1)所述的金属纳米粒子包括金纳米粒子、银纳米粒子或铜纳米粒子。
  4. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤1)所述的内核为球形或棒状。
  5. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤3)所述CMS金属纳米粒子的粒径为10-300nm。
  6. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤2)所述的单分子层中，骨架分子选用含有巯基末端、氨基末端或者羧基末端的吸附分子。
  7. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增强拉曼光谱定量方法，其特征在于步骤2)所述单分子层中，内标分子选用含有巯基末端、氨基末端或者羧基末端的强吸附且有拉曼散射截面的分子。
  8. 如权利要求1所述的一种内含内标分子的核壳结构纳米粒子用于表面增 强拉曼光谱定量方法，其特征在于步骤5)检测步骤中，用于表面增强拉曼光谱的激发光源波长是400-1500nm。
  9. 内含内标分子的核壳结构纳米粒子，其特征在于，包括：
    内核：为具有表面等离激元共振效应的金属纳米粒子；
    在内核上修饰的单分子层：包括骨架和内标；
    壳层：在单分子层上修饰的具有表面等离激元共振效应的金属壳层。
  10. 如权利要求9所述的一种内含内标分子的核壳结构纳米粒子，其特征在于所述的单分子层中的骨架分子选用含有巯基末端、氨基末端或者羧基末端的吸附分子。
  11. 如权利要求9或10所述的一种内含内标分子的核壳结构纳米粒子，其特征在于所述单分子层中内标分子选用含有巯基末端、氨基末端或者羧基末端的强吸附且有拉曼散射截面的分子。
  12. 如权利要求9所述的内含内标分子的核壳结构纳米粒子的用途，其用于基于表面等离激元增强光谱的定量检测，所述定量测量包括表面增强红外和表面增强荧光光谱技术。
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