WO2016109911A1 - 一种基于银纳米粒子四面体拉曼多重检测的方法 - Google Patents

一种基于银纳米粒子四面体拉曼多重检测的方法 Download PDF

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WO2016109911A1
WO2016109911A1 PCT/CN2015/000721 CN2015000721W WO2016109911A1 WO 2016109911 A1 WO2016109911 A1 WO 2016109911A1 CN 2015000721 W CN2015000721 W CN 2015000721W WO 2016109911 A1 WO2016109911 A1 WO 2016109911A1
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agnp
raman
silver nanoparticle
tetrahedron
dna1
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徐丽广
郝昌龙
严文静
匡华
胥传来
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江南大学
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  • the invention relates to a method based on silver nanoparticle tetrahedral Raman multiple detection, belonging to the technical field of analytical chemistry.
  • the electromagnetic field enhancement effect on the surface of noble metal nanomaterials causes the Raman enhancement effect on the molecules adsorbed on the surface.
  • the SERS Raman molecular Raman signals may be enhanced 106-fold to achieve single molecule detection of the Raman spectrum.
  • SERS detection can maintain the original state of the sample well, is not affected by the sample mechanism and back ground, the peak width of the spectrum is narrow, has a unique molecular fingerprint, can be used in high temperature, high pressure environment, etc. Used in the fields of pharmaceutical, drug identification, biomedical, food hazard detection.
  • SDM Sulfadimethoxypyrimidine
  • SDM Sulfadimethoxypyrimidine
  • OTA Ochratoxin A
  • Aflatoxin M1 belongs to one of a class of structurally similar compounds of aflatoxin, and has the highest probability of aflatoxin in foods and feeds in hot and humid regions. Aflatoxin M1 is mainly caused by carcinogenicity and mutagenicity, and has a destructive effect on human and animal liver tissues, which can lead to liver cancer and even death.
  • the object of the present invention is to construct a silver nanoparticle tetrahedron and apply it to multi-Raman spectroscopy detection of sulfamethazine (SDM) and aflatoxin M1 (AFM1), ochratoxin (OTA) and the like.
  • SDM sulfamethazine
  • AFM1 aflatoxin M1
  • OTA ochratoxin
  • the technical scheme of the invention a method based on silver nanoparticle tetrahedral Raman multiple detection:
  • Silver nanoparticles with a particle size of 10 nm were synthesized by sodium borohydride reduction silver nitrate method.
  • the synthesized silver nanoparticles and the thiol-modified DNA are coupled to form AgNP-DNA1, AgNP-DNA2, AgNP-DNA3, AgNP-DNA4 complex.
  • Raman beacon molecules 4-aminothiophenol (4-ATP), 4-nitrobenzenethiol (NTP) and 4-methoxybenzyl mercaptan (MATT) were respectively modified to AgNP-DNA1.
  • AgNP-DNA2, AgNP-DNA3 surface obtained AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT.
  • the AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT, and AgNP-DNA4 prepared above were mixed, and a silver nanoparticle tetrahedron was obtained by base-pair pairing hybridization.
  • a series of different concentrations of SDM, AFM1, and OTA standard solutions were added to the silver nanoparticle tetrahedral system prepared in the step (4), and the Raman signals were respectively determined according to the Raman signal intensity of the three different beacons and the object to be tested.
  • the concentration establishes a standard curve.
  • a clean conical flask was placed in an ice bath, followed by 20 mL of ultrapure water, 5 mL of 1% polyvinylpyrrolidone and 0.6 mL of a 0.01 mol/L aqueous solution of sodium borohydride. Then, 5 mL of a 1% polyvinylpyrrolidone and 5 mL of a 1% silver nitrate aqueous solution were simultaneously added to the Erlenmeyer flask at a rate of 30 mL/h, and the solution was changed from colorless to yellow while stirring. The obtained silver nanoparticles had a diameter of 10 nm.
  • Raman beacon molecules 4-aminothiophenol (4-ATP), 4-nitrobenzenethiol (NTP) and 4-methoxybenzyl mercaptan (MATT) were respectively added to AgNP-DNA1.
  • the final concentration of Raman beacon molecules in the solution was 3 ⁇ M.
  • the beacon molecules were added to the system for reaction overnight, and each was centrifuged at 13000 r/min for 10 min to remove the supernatant and then to the system. The original volume was recovered by adding 20 mM Tris-HCl buffer.
  • AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT complexes were prepared.
  • the above prepared AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 were each 100 ⁇ L in a 1.5 mL centrifuge tube and mixed uniformly. 4 ⁇ L of 5 M NaCl solution was added, shaken and mixed, and a 90 ° C water bath was added. After 5 min, it was slowly lowered to room temperature in water vapor to prepare a silver nanoparticle tetrahedron.
  • SDM For the simultaneous detection of SDM, AFM1, and OTA, three substances were added to the system one by one in sequence, with each substance separated by 30 min.
  • the added concentration of SDM was 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5 fM; the concentration of AFM1 was 0, 0.1, 0.5, 1, 5, 10, 50 fM; the concentration of OTA was 0, 0.01. , 0.05, 0.1, 0.5, 1, 5 fM.
  • the Raman spectra of all the three substances were added and the reaction was completed, and the concentration standard curves of SDM, AFM1 and OTA were established according to the intensity of the Raman signals of 4-ATP, NTP and MATT, respectively.
  • the Raman spectroscopy test time was 20 s and the excitation wavelength was 633 nm.
  • a general-purpose method based on silver nanoparticle tetrahedron Raman multiplex detection, Raman multiplex detection of corresponding analytes can be prepared by changing the sequence of nucleic acid aptamer used in the preparation of silver nanoparticle tetrahedron sensor.
  • the Raman enhancement effect of silver nanoparticles is more remarkable than that of other precious metals, and is a good Raman base material.
  • the detection is based on the controllable adjustment of the spatial structure of the nanoparticle tetrahedron. There is no irregular aggregation of nanoparticles during the whole detection process, which reduces the interference of the external environment.
  • the target is caused by the identification of the aptamer.
  • the tetrahedral structure changes and has good specificity.
  • the tetrahedron has six DNA edges, which can be used for simultaneous detection of multiple targets, and can be used for detection of small molecules as well as macromolecules such as proteins. Quantitative testing.
  • Figure 1 is a schematic diagram of the invention based on silver nanoparticle tetrahedral Raman detection.
  • FIG. 1 Silver nanoparticle tetrahedron: (A) TEM image, (B) frozen electron three-dimensional imaging image, (C) spatial configuration diagram; silver nanoparticle tetrahedron after SDM addition: (D) TEM image, ( E) Three-dimensional imaging image of frozen electrons, (F) Schematic diagram of spatial configuration.
  • Figure 3 (A) Silver nanoparticle tetrahedron for Raman fingerprinting of SDM, AFM1 and OTA detection; (B) Simultaneous detection of SDM, AFM1 and OTA based on silver nanoparticle tetrahedron multiplex detection system, in which the concentration of SDM is in turn 0, 0.001, 0.005, 0.01, 0.05, 0.1 fM; the concentration of AFM1 is 0, 0.1, 0.5, 1, 5, 10, 50 fM; the concentration of OTA is 0, 0.01, 0.05, 0.1, 0.5, 1, respectively. 5fM; (C) from left to right: standard curve of SDM concentration and Raman signal intensity, standard curve of AFM1 concentration and Raman signal intensity, standard curve of OTA concentration and Raman signal intensity.
  • Figure 4 shows the Raman spectrum of SDM in milk based on silver nanoparticle tetrahedron.
  • a clean conical flask was placed in an ice bath, followed by 20 mL of ultrapure water, 5 mL of 1% polyvinylpyrrolidone and 0.6 mL of 0.01 molar per liter of aqueous sodium borohydride solution. Then, 5 mL of a 1% polyvinylpyrrolidone and 5 mL of a 1% silver nitrate aqueous solution were simultaneously added to the Erlenmeyer flask at a rate of 30 mL/h, and the solution was changed from colorless to yellow while stirring. The resulting silver nanoparticles were 10 nm in diameter.
  • the above prepared AgNP-DNA1-ATP, AgNP-DNA2-NTP, AgNP-DNA3-MATT, AgNP-DNA4 were each 100 uL and mixed uniformly in a 1.5 mL centrifuge tube, and 4 uL of 5 M NaCl solution was added, shaken and mixed, and a 90 ° C water bath was prepared. After 5 min, it was slowly lowered to room temperature in water vapor to prepare a silver nanoparticle tetrahedron.

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Abstract

一种基于银纳米粒子四面体拉曼多重检测的方法。该方法利用DNA介导纳米粒子自组装技术组装得到银纳米粒子四面体，利用四面体的空间构型，通过在银纳米粒子四面体中引入三段含有待测物磺胺二甲氧嘧啶（SDM）、黄曲霉毒素M1（AFM1）、赭曲霉毒素（OTA）核酸适配体片段的DNA和三种信标分子，构建了基于银纳米粒子四面体的拉曼多重检测体系。当存在待测物时，四面体的空间构型发生改变引起拉曼信号的变化，进而进行检测。三种信标分子拉曼信号的变化对应于三种目标物的浓度，从而实现了对三种目标物SDM，AFM1和OTA的同时检测。该方法的灵敏度高、具有良好的特异性，并且避免了纳米粒子无序聚集，有利于拉曼传感检测技术在实际中的应用。

Description

一种基于银纳米粒子四面体拉曼多重检测的方法 技术领域
本发明涉及一种基于银纳米粒子四面体拉曼多重检测的方法，属于分析化学技术领域。
背景技术
1928年，印度科学家拉曼首次发现了拉曼散射现象，即当一个已知能量或波长、频率的光子和一个分子相互作用时，引起分子振动和能量损失的过程。随后拉曼先生发明了第一台拉曼光谱仪，并因此获得诺贝尔物理学奖。但是由于拉曼信号比较弱，该技术一直没有得到广泛的应用。后来随着激光器、CCD和滤光片的发明使得拉曼光谱仪的性能大大改进，因而才有了后来拉曼光谱仪大范围的普及和应用。表面增强拉曼散射(SERS)是在原有拉曼散射的基础之上，利用贵金属纳米材料(如金、银等)表面的电磁场增强效应，使得吸附在其表面的分子产生拉曼增强效应的现象。一般情况下，SERS可以将拉曼分子的拉曼信号增强106倍，从而实现拉曼光谱的单个分子检测。另外，由于SERS检测能很好的保持样品的原有状态、不受样品机制和背地的影响、图谱峰宽较窄、具有独特的分子指纹图谱、可用以高温、高压环境等优点，目前已广泛用于制药、毒品鉴别、生物医学、食品危害因子检测等领域。
磺胺二甲氧嘧啶(SDM)是一类人工合成广谱抑菌剂药物，添加在饲料中可以增肥家畜，预防和治疗细菌性疾病。这种药物容易残留在动物体内并对人体健康造成严重的影响，如损害脑神经系统，造成溶血性贫血，过敏反应，引发甲状腺癌等。鉴于此，很多国家对动物源性食品中SDM的含量做出规定，中国的限量是100mg/mL。赭曲霉毒素A(OTA)是曲霉菌属和青霉菌属的某些产毒菌株的次级代谢产物，在全球范围内对农作物的污染都比较严重，是一种强烈的肾毒素和肝毒素，还具有免疫抑制性，直接危害人类健康，引起DNA的损伤，有致畸、致癌和致突变的作用。黄曲霉毒素M1属于黄曲霉毒素一类结构相似的化合物中的一种，在湿热地区食品和饲料中出现黄曲霉毒素的机率最高。黄曲霉毒素M1危害主要表现在致癌性和致突变性，对人及动物肝脏组织有破坏作用，可导致肝癌甚至死亡。
发明内容
本发明的目的是构建一种银纳米粒子四面体，并应用于磺胺二甲嘧啶(SDM)及黄曲霉毒素M1(AFM1)、赭曲霉毒素(OTA)等的多重拉曼光谱检测。
本发明的技术方案：一种基于银纳米粒子四面体拉曼多重检测的方法：
(1)10纳米粒径银纳米粒子(AgNP)合成
采用硼氢化钠还原硝酸银法合成粒径为10纳米的银纳米粒子。
(2)银纳米粒子修饰DNA
上述合成的银纳米粒子和巯基修饰的DNA(DNA1，DNA2，DNA3，DNA4)进行偶联形成AgNP-DNA1，AgNP-DNA2，AgNP-DNA3，AgNP-DNA4复合体。
(3)拉曼信标分子的修饰
将三种拉曼信标分子4-氨基苯硫酚(4-ATP)、4-硝基苯硫醇(NTP)、4-甲氧基苄硫醇(MATT)分别对应修饰到AgNP-DNA1、AgNP-DNA2、AgNP-DNA3表面得到AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT。
(4)银纳米粒子四面体的组装
将上述制备的AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT，AgNP-DNA4混合，利用碱基互补配对杂交得到银纳米粒子四面体。
表1 检测目标物核酸适配体的名称和序列
  Sequence(5’-3’)
SDM GAG GGC AAC GAG TGT TTA TAG A
AFM1 ACT GCT AGA GAT TTT CCA CAT
OTA GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA
表2 用于构建银纳米粒子四面体的核酸序列。
Figure PCTCN2015000721-appb-000001
(5)基于银纳米颗粒四面体拉曼传感器的构建与应用
向步骤(4)制备出的银纳米粒子四面体体系中加入一系列不同浓度的SDM、AFM1、OTA标准溶液，分别测定其拉曼信号，根据三种不同信标的拉曼信号强度与待测物浓度建立标准曲线。
具体为：
(1)10nm粒径银纳米粒子(AgNP)合成
取一洁净的锥形瓶置于冰浴中，依次加入20mL超纯水，5mL质量分数为1％的聚乙烯吡咯烷酮和0.6mL0.01mol/L的硼氢化钠水溶液。然后将5mL质量分数为1％的聚乙烯吡咯烷酮和5mL质量分数为1％的硝酸银水溶液同时以30mL/h的速度加入到锥形瓶中，边加入边搅拌，溶液由无色变成黄色。所得的银纳米粒子直径为10nm。
(2)银纳米粒子修饰DNA
取30μL 20nM上述合成的银纳米粒子于PCR管中，加入1μL 10μM的DNA1混匀后，依次向体系中加入5μL 5×tris-硼酸缓冲液和1.25μL 2mol/L NaCl溶液，室温振摇反应12h，13000r/min离心10min，去除上清液，加超纯水至原体积，得AgNP-DNA1。AgNP-DNA2，AgNP-DNA3，AgNP-DNA4复合体的制备方法与AgNP-DNA1类似。
(3)拉曼信标分子的修饰
将三种拉曼信标分子4-氨基苯硫酚(4-ATP)、4-硝基苯硫醇(NTP)、4-甲氧基苄硫醇(MATT)分别对应加入到AgNP-DNA1、AgNP-DNA2、AgNP-DNA3体系中，溶液中拉曼信标分子的终浓度均为3μM，信标分子加入到体系中反应过夜，各自以13000r/min离心10min，去除上清液，再向体系中加入20mM Tris-HCl缓冲液恢复到原体积。制备得到AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT复合体。
(4)银纳米粒子四面体的组装
将上述制备的AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT，AgNP-DNA4各取100μL于1.5mL离心管中混合均匀，加入4μL 5M NaCl溶液，震荡混匀，90℃水浴5min，再在水蒸气中缓慢降到室温，即制备得到银纳米粒子四面体。
(5)基于银纳米颗粒四面体拉曼传感器的构建与应用
对于SDM、AFM1、OTA的同时检测，三种物质按照先后顺序逐个加入到体系中，每种物质中间间隔30min。SDM的添加浓度依次为0，0.001，0.005，0.01，0.05，0.1，0.5fM；AFM1的添加浓度依次为0，0.1，0.5，1，5，10，50fM；OTA的添加浓度依次为0，0.01，0.05，0.1，0.5，1，5fM。三种物质全部加入并反应结束后测体系的拉曼光谱，分别根据4-ATP，NTP及MATT的拉曼信号的强度建立SDM、AFM1、OTA的浓度标准曲线。拉曼光谱测试时间为20s，激发波长为633nm。
一种通用型基于银纳米粒子四面体拉曼多重检测的方法，通过对银纳米粒子四面体制备过程中使用的核酸适配体序列的改变，即可制备得到对应待测物的拉曼多重检测传感器。
本发明的有益效果：首先，银纳米颗粒的拉曼增强效果比其他贵金属明显，是很好的拉曼基底材料。其次，检测是基于对纳米颗粒四面体空间结构的可控调节，在整个检测过程中没有出现纳米颗粒的无规则聚集现象，减少了外界环境的干扰；再次，目标物通过与适配体识别引起四面体结构发生变化，具有很好的特异性；最后，四面体具有六个DNA边，可用于多个目标物的同时检测，既可以用于小分子的检测，也可以用于蛋白质等大分子的定量检测。
附图说明
图1本发明基于银纳米颗粒四面体拉曼检测的原理图。
图2银纳米颗粒四面体的：(A)TEM图，(B)冷冻电子三维成像图，(C)空间构型示意图；加入SDM后的银纳米颗粒四面体的：(D)TEM图，(E)冷冻电子三维成像图，(F)空间构型示意图。
图3(A)银纳米颗粒四面体用于SDM、AFM1和OTA检测的拉曼指纹图谱；(B)基于银纳米颗粒四面体的多重检测体系同时检测SDM、AFM1和OTA，其中SDM的浓度依次为0，0.001，0.005，0.01，0.05，0.1fM；AFM1的浓度依次为0，0.1，0.5，1，5，10，50fM；OTA的浓度依次为0，0.01，0.05，0.1，0.5，1，5fM；(C)从左到右依次为：SDM浓度与拉曼信号强度的标准曲线，AFM1浓度与拉曼信号强度的标准曲线，OTA浓度与拉曼信号强度的标准曲线。
图4基于银纳米颗粒四面体检测牛奶中SDM的拉曼光谱。
具体实施方式
实施例1
(1)10纳米粒径银纳米粒子(AgNP)合成
取一洁净的锥形瓶置于冰浴中，依次加入20mL超纯水，5mL质量分数为1％的聚乙烯吡咯烷酮和0.6mL 0.01摩尔每升的硼氢化钠水溶液。然后将5mL质量分数为1％的聚乙烯吡咯烷酮和5mL质量分数为1％的硝酸银水溶液同时以30mL/h的速度加入到锥形瓶中，边加入边搅拌，溶液由无色变成黄色。所得的银纳米粒子直径为10纳米。
(2)银纳米粒子修饰DNA
取30μL 20nM上述合成的银纳米粒子于PCR管中，加入1μL10μM的DNA1混匀后，依次向体系中加入5μL 5×tris-硼酸缓冲液和1.25μL 2摩尔每升NaCl溶液，室 温振摇反应12h，13000r/min，离心10min，去除上清液，加超纯水至原体积，得AgNP-DNA1。AgNP-DNA2，AgNP-DNA3，AgNP-DNA4复合体的制备方法与AgNP-DNA1类似。
(3)拉曼信标分子的修饰
将三种拉曼信标分子4-氨基苯硫酚(4-ATP)，4-硝基苯硫醇(NTP)和4-甲氧基苄硫醇(MATT)分别加入到AgNP-DNA1，AgNP-DNA2，AgNP-DNA3体系中，溶液中拉曼信标分子的终浓度均为3uM，信标分子加入到体系中反应过夜，各自以13000r/min，离心10min，去除上清液，再向体系中加入20mM Tris-HCl缓冲液恢复到原体积。制备得到AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT复合体。
(4)银纳米粒子四面体的组装
将上述制备的AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT，AgNP-DNA4各取100uL于1.5mL离心管中混合均匀，加入4uL 5M NaCl溶液，震荡混匀，90℃水浴5min，再在水蒸气中缓慢降到室温，即制备得到银纳米粒子四面体。
(5)基于银纳米颗粒四面体拉曼传感器的构建与应用
对于SDM、AFM1、OTA的同时检测，三种物质按照先后顺序逐个加入到体系中，每种物质中间间隔30min。SDM的添加浓度依次为0，0.001，0.005，0.01，0.05，0.1，0.5fM；AFM1的添加浓度依次为0，0.1，0.5，1，5，10，50fM；OTA的添加浓度依次为0，0.01，0.05，0.1，0.5，1，5fM。三种物质全部加入并反应结束后测体系的拉曼光谱，分别根据4-ATP，NTP和MATT的拉曼信号的强度建立SDM、AFM1、OTA的浓度标准曲线。拉曼光谱测试时间为20s，激发波长为633nm。

Claims (6)

  1. 一种基于银纳米粒子四面体拉曼多重检测的方法，其特征在于利用含有待测物SDM，AFM1，OTA核酸适配体片段的DNA组装得到银纳米粒子四面体，当存在待测物时，四面体的空间构型发生改变引起拉曼信号的变化，进而进行检测；工艺步骤为：
    (1)10nm粒径银纳米粒子AgNP合成
    采用硼氢化钠还原硝酸银法合成粒径为10nm的AgNP；
    (2)银纳米粒子修饰DNA
    上述合成的银纳米粒子AgNP分别和巯基修饰的DNA1，DNA2，DNA3，DNA4进行偶联形成AgNP-DNA1，AgNP-DNA2，AgNP-DNA3，AgNP-DNA4复合体；
    构建银纳米粒子四面体的核酸序列：
    DNA1：5’-TTT ATT GAG GGC AAC GAG TGT TTA TAG ACT TTC CCT ATT AGA AGG TCT CAG GTG CGC GTT TCC AGC CAT ACC TTA GGT ACT TCT GCC-3’；
    DNA2：5’-TTT CGC GCA CCT GAG ACC TTC TAA TAG GGT TTG CGA CAG TCG TTC AAC TAG AAT GCC CTT TGG GCT GTT CCG GGT GTG GCT CGT CGG-3’；
    DNA3：5’-TTT ACT GCT AGA GAT TTT CCA CAT GGC TAT TT GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT ACA TTT CC GAC GAG CCA CAC CCG GAA CAG CCC-3’；
    DNA4：5’-TTT GTC TAT AAA CAC TCG TTG CCC TCA ATT TT TGA CGA TCT CTA AAA GGT GTA CCG ATT TTG GGC ATT CTA G TTG AAC GAC TGT CGC-3’；
    (3)拉曼信标分子的修饰
    将三种拉曼信标分子4-氨基苯硫酚4-ATP，4-硝基苯硫醇NTP、4-甲氧基苄硫醇MATT对应修饰到AgNP-DNA1，AgNP-DNA2，AgNP-DNA3表面得到AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT；
    (4)银纳米粒子四面体的组装
    将上述制备的AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT，AgNP-DNA4混合，利用碱基互补配对杂交得到银纳米粒子四面体；
    (5)基于银纳米颗粒四面体拉曼传感器的构建与应用
    向步骤(4)制备出的银纳米粒子四面体体系中加入一系列不同浓度的SDM、AFM1、OTA标准溶液，分别测定其拉曼信号，根据三种不同信标的拉曼信号强度与待测物浓度建立标准曲线。
  2. 根据权利要求1所述的基于银纳米粒子四面体拉曼多重检测的方法，其特征在于10nm粒径银纳米粒子AgNP合成：取一洁净的锥形瓶置于冰浴中，依次加入20mL超纯水，5mL质量分数为1％的聚乙烯吡咯烷酮和0.6mL 0.01mol/L的硼氢化钠水溶液，然后将5mL质量分数为1％的聚乙烯吡咯烷酮和5mL质量分数为1％的硝酸银水溶液同时以30mL/h的速度加入到锥形瓶中，边加入边搅拌，溶液由无色变成黄色，得到10nm粒径银纳米粒子AgNP。
  3. 根据权利要求1所述的基于银纳米粒子四面体拉曼多重检测的方法，其特征在于银纳米粒子修饰DNA：取30μL 20nM所述合成的银纳米粒子AgNP于PCR管中，加入1μL 10μM的DNA1混匀后，依次向体系中加入5μL 5×tris-硼酸缓冲液和1.25μL 2mol/L NaCl溶液，室温振摇反应12h，13000r/min离心10min，去除上清液，加超纯水至原体积，得AgNP-DNA1；AgNP-DNA2，AgNP-DNA3，AgNP-DNA4复合体的制备方法与AgNP-DNA1类似。
  4. 根据权利要求1所述的基于银纳米粒子四面体拉曼多重检测的方法，其特征在于拉曼信标分子的修饰：将三种拉曼信标分子4-氨基苯硫酚4-ATP、4-硝基苯硫醇NTP、4-甲氧基苄硫醇MATT分别对应加入到AgNP-DNA1、AgNP-DNA2、AgNP-DNA3体系中，溶液中拉曼信标分子的终浓度均为3μM，信标分子加入到体系中反应过夜，各自以13000r/min离心10min，去除上清液，再向体系中加入20mM Tris-HCl缓冲液恢复到原体积，制备得到AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT复合体。
  5. 根据权利要求1所述的基于银纳米粒子四面体拉曼多重检测的方法，其特征在于银纳米粒子四面体的组装：将上述制备的AgNP-DNA1-ATP，AgNP-DNA2-NTP，AgNP-DNA3-MATT，AgNP-DNA4各取100μL于1.5mL离心管中混合均匀，加入4μL 5M NaCl溶液，震荡混匀，90℃水浴5min，再在水蒸气中缓慢降到室温，即制备得到银纳米粒子四面体。
  6. 根据权利要求1所述的基于银纳米粒子四面体拉曼多重检测的方法，其特征在于基于银纳米颗粒四面体拉曼传感器的构建与应用：对于SDM、AFM1、OTA的同时检测，三种物质按照先后顺序逐个加入到体系中，每种物质中间间隔30min；SDM的添加浓度依次为0，0.001，0.005，0.01，0.05，0.1，0.5fM；AFM1的添加浓度依次为0，0.1，0.5，1，5，10，50fM；OTA的添加浓度依次为0，0.01，0.05，0.1，0.5，1，5fM；三种物质全部加入并反应结束后测体系的拉曼光谱，分别根据4-ATP，NTP及MATT的拉曼信号的强度建立SDM、AFM1、OTA的浓度标准曲线；拉曼光谱测试时间为20s，激发波长为633nm。
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