CN103837528A

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Info

Publication number: CN103837528A
Application number: CN201210483212.0A
Authority: CN
Inventors: 张雪华; 张鑫; 西瓦库玛·帕拉尼萨梅; 贺涛
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2012-11-23
Filing date: 2012-11-23
Publication date: 2014-06-04

Abstract

本发明提供一种检测多巴胺的化学敏感器，所述化学敏感器包含含金纳米颗粒，所述含金纳米颗粒的表面经A类有机功能小分子和B类有机功能小分子修饰；所述A类有机功能小分子包含可以与多巴胺分子中的邻二酚羟基作用的硼酸基团；所述B类有机功能小分子包含可以与多巴胺分子中的质子化端氨基作用的冠醚基团、醛基、丁二酰亚胺活化的羧基、磷酸根离子基团和磺酸根离子基团中的一种或多种。所述A类和B类有机功能小分子还均包括吡啶、巯基、伯胺基和二硫基中的一种或多种，用于修饰所述含金纳米颗粒。还提供了制备该化学敏感器的方法，以及一种检测多巴胺的方法，和该化学敏感器在制备用于检测多巴胺的装置中的应用。

The invention provides a chemical sensor for detecting dopamine, the chemical sensor includes gold-containing nanoparticles, the surface of the gold-containing nanoparticles is modified by a type A organic functional small molecule and a B type organic functional small molecule; the A The class organic functional small molecule contains a boronic acid group that can interact with the ortho-diphenolic hydroxyl group in the dopamine molecule; the B class organic functional small molecule contains a crown ether group that can interact with the protonated terminal amino group in the dopamine molecule, an aldehyde group , one or more of succinimide-activated carboxyl groups, phosphate ion groups and sulfonate ion groups. Both the A-type and B-type organic functional small molecules also include one or more of pyridine, sulfhydryl, primary amino and disulfide groups for modifying the gold-containing nanoparticles. Also provided are a method for preparing the chemical sensor, a method for detecting dopamine, and an application of the chemical sensor in preparing a device for detecting dopamine.

Description

Translated fromChinese

一种检测多巴胺的化学敏感器及其制备方法、检测方法和应用A chemical sensor for detecting dopamine and its preparation method, detection method and application

技术领域technical field

本发明涉及一种化学敏感器，具体涉及一种可用于检测多巴胺的化学敏感器及其制备方法，一种检测多巴胺的方法和该化学敏感器在制备用于检测多巴胺的装置中的应用。The invention relates to a chemical sensor, in particular to a chemical sensor capable of detecting dopamine and a preparation method thereof, a method for detecting dopamine and the application of the chemical sensor in preparing a device for detecting dopamine.

背景技术Background technique

多巴胺（Dopamine，C₆H₃(OH)₂-CH₂-CH₂-NH₂），简称DA，是去甲肾上腺素的前体物质，是一种脑内分泌用来帮助细胞传送脉冲的重要神经递质，对很多生物药理学和生理学过程起着重要的作用，而且一些疾病的产生也与多巴胺的浓度变化有密切关系。多巴胺不足或失调会令人失去控制肌肉的能力，严重时会令病人的手脚不自主地颤动或导致帕金森病、多动症、亨丁顿舞蹈症等疾病。此外，多巴胺为拟肾上腺素药，具有增加肾血流量和使心脏兴奋的功能，已被广泛用于治疗神经紊乱、支气管哮喘、高血压、先天性心脏病和抑郁症等。因此，准确测定神经递质多巴胺在脑、血液、尿和组织中的含量，无论是在生理功能研究还是在临床应用方面都具有重要的意义。Dopamine (Dopamine, C₆ H₃ (OH)₂ -CH₂ -CH₂ -NH₂ ), referred to as DA, is the precursor of norepinephrine, an important neuron used by brain endocrine to help cells transmit pulses Transmitters play an important role in many biopharmacological and physiological processes, and the occurrence of some diseases is also closely related to changes in the concentration of dopamine. Insufficient or imbalanced dopamine will cause people to lose the ability to control muscles. In severe cases, it will cause the patient's hands and feet to vibrate involuntarily or lead to Parkinson's disease, ADHD, Huntington's disease and other diseases. In addition, dopamine is an adrenaline-mimetic drug, which has the function of increasing renal blood flow and stimulating the heart. It has been widely used in the treatment of nervous disorders, bronchial asthma, hypertension, congenital heart disease and depression. Therefore, accurate determination of the content of neurotransmitter dopamine in brain, blood, urine and tissue is of great significance both in physiological function research and clinical application.

多巴胺检测过程中需要解决的主要问题是如何排除共存物质如抗坏血酸和尿酸等的干扰。由于多巴胺具有电化学活性，因此目前检测多巴胺所采用的主要方法是电化学法。但是，抗坏血酸和尿酸的氧化电位与多巴胺的氧化电位非常接近，从而使氧化峰重叠，这对多巴胺的检测产生了一定程度的干扰，影响了检测结果的准确性。而利用化学或物理方法对电极进行修饰，或者其它的检测方法如微透析法、高效液相色谱法、离子色谱法、化学发光法、分光光度法等，可提高检测的选择性。然而，这些方法存在其自身的局限性，例如前处理复杂、成本高、需要衍生、时间长和灵敏度低等缺陷。The main problem to be solved in the process of dopamine detection is how to exclude the interference of coexisting substances such as ascorbic acid and uric acid. Due to the electrochemical activity of dopamine, the main method currently used to detect dopamine is electrochemical method. However, the oxidation potentials of ascorbic acid and uric acid are very close to those of dopamine, so that the oxidation peaks overlap, which interferes with the detection of dopamine to a certain extent and affects the accuracy of the detection results. The use of chemical or physical methods to modify the electrode, or other detection methods such as microdialysis, high performance liquid chromatography, ion chromatography, chemiluminescence, spectrophotometry, etc., can improve the selectivity of detection. However, these methods have their own limitations, such as complex pre-treatment, high cost, need for derivatization, long time and low sensitivity.

比色化学敏感器主要是借助于主客体作用前后颜色的变化作为信号，在无需复杂仪器的情况下，可通过肉眼观察进行检测，应用简单方便，已被广泛用于化学传感器的设计，在痕量底物的分析中有着广泛的应用。The colorimetric chemical sensor mainly uses the color change before and after the host-guest interaction as a signal. It can be detected by naked eyes without complex instruments. The application is simple and convenient, and it has been widely used in the design of chemical sensors. It has a wide range of applications in the analysis of quantitative substrates.

含有贵金属金的纳米颗粒由于具有表面等离子体共振（surface plasmonresonance,SPR）性质，在紫外-可见光波段呈现很强的光谱吸收。表面等离子体共振的响应机理是基于诱导贵金属纳米颗粒的聚集状态改变而引起的吸收光谱变化，与贵金属纳米颗粒的组成、尺寸、形状、粒子间距和周围介质的折射率等因素有密切的关系，可应用于化学和生物传感器领域。Nanoparticles containing noble metal gold exhibit strong spectral absorption in the ultraviolet-visible band due to their surface plasmon resonance (SPR) properties. The response mechanism of surface plasmon resonance is based on the change of absorption spectrum induced by the change of the aggregation state of noble metal nanoparticles, which is closely related to the composition, size, shape, particle distance and refractive index of the surrounding medium of noble metal nanoparticles. It can be applied to the fields of chemical and biological sensors.

目前还需要寻求一种选择性好、操作简便的多巴胺检测方法。At present, it is still necessary to find a method for detecting dopamine with good selectivity and easy operation.

发明内容Contents of the invention

因此，本发明的目的是克服现有的多巴胺检测方法中存在的选择性较差、操作复杂、灵敏度低、设备要求高等缺陷，提供了一种选择性好、灵敏度高、不需依赖大型复杂仪器且操作方便的检测多巴胺的化学敏感器及其制备方法，以及用以检测多巴胺的方法，和该化学敏感器在制备用于检测多巴胺的装置中的应用。Therefore, the purpose of the present invention is to overcome the defects of poor selectivity, complicated operation, low sensitivity and high equipment requirements in the existing dopamine detection method, and provide a kind of method with good selectivity, high sensitivity and no need to rely on large complex instruments. A chemical sensor for detecting dopamine that is easy to operate, a preparation method thereof, a method for detecting dopamine, and an application of the chemical sensor in preparing a device for detecting dopamine.

本发明提供了一种检测多巴胺的化学敏感器，所述化学敏感器包含含金纳米颗粒，所述含金纳米颗粒的表面经A类有机功能小分子和B类有机功能小分子修饰；所述A类有机功能小分子包含硼酸基团；所述B类有机功能小分子包含冠醚基团、醛基、丁二酰亚胺活化的羧基、磷酸根离子基团和磺酸根离子基团中的一种或多种。所述A类有机功能小分子可以与多巴胺分子中的邻二酚羟基作用，所述B类有机功能小分子可以与多巴胺分子中的质子化端氨基作用。The invention provides a chemical sensor for detecting dopamine, the chemical sensor includes gold-containing nanoparticles, the surface of the gold-containing nanoparticles is modified by a type A organic functional small molecule and a B type organic functional small molecule; Type A organic functional small molecules include boric acid groups; the B type organic functional small molecules include crown ether groups, aldehyde groups, carboxyl groups activated by succinimide, phosphate ion groups, and sulfonate ion groups. one or more. The class A organic functional small molecules can interact with the o-diphenolic hydroxyl group in the dopamine molecule, and the class B organic functional small molecules can interact with the protonated terminal amino groups in the dopamine molecule.

根据本发明的化学敏感器，其中，所述A类有机功能小分子还包括吡啶、巯基、伯胺基和二硫基中的一种或多种，用于修饰所述含金纳米颗粒；所述B类有机功能小分子还包括吡啶、巯基、伯胺基和二硫基中的一种或多种，用于修饰所述含金纳米颗粒。According to the chemical sensor of the present invention, wherein, the class A organic functional small molecule further includes one or more of pyridine, mercapto, primary amino and disulfide, for modifying the gold-containing nanoparticles; The class B organic functional small molecules also include one or more of pyridine, sulfhydryl, primary amino and disulfide groups for modifying the gold-containing nanoparticles.

根据本发明的化学敏感器，其中，所述含金纳米颗粒可包括金与银、铜、铂、钯和镓中的一种或多种组成的复合纳米颗粒。作为优选，所述复合纳米颗粒可以为核壳结构的纳米颗粒。According to the chemical sensor of the present invention, the gold-containing nanoparticles may include composite nanoparticles composed of gold and one or more of silver, copper, platinum, palladium and gallium. Preferably, the composite nanoparticles may be nanoparticles with a core-shell structure.

根据本发明的化学敏感器，其中，所述含金纳米颗粒可以为金纳米颗粒。According to the chemical sensor of the present invention, the gold-containing nanoparticles may be gold nanoparticles.

根据本发明的化学敏感器，其中，所述金纳米颗粒可以采用四氯金酸的水溶液通过还原法制得。作为优选，所述还原法包括化学还原法、电化学还原法或辐射还原法。作为更优选，在化学还原法中采用的化学还原试剂为氮，氮-二甲基甲酰胺、柠檬酸、柠檬酸钠和硼氢化钠中的一种或多种。According to the chemical sensor of the present invention, wherein the gold nanoparticles can be prepared by a reduction method using an aqueous solution of tetrachloroauric acid. Preferably, the reduction method includes chemical reduction method, electrochemical reduction method or radiation reduction method. More preferably, the chemical reducing agent used in the chemical reduction method is nitrogen, one or more of nitrogen-dimethylformamide, citric acid, sodium citrate and sodium borohydride.

根据本发明的化学敏感器，其中，所述含金纳米颗粒的形状为纳米球、纳米棒、纳米管、纳米花、纳米片、三角形、四角形、星状或4~24面体，或表面呈刺状。According to the chemical sensor of the present invention, wherein, the shape of the gold-containing nanoparticles is nanospheres, nanorods, nanotubes, nanoflowers, nanosheets, triangles, tetragons, stars, or 4-24-hedrons, or the surface is thorny. shape.

根据本发明的化学敏感器，其中，所述含金纳米颗粒的颗粒尺寸为3.5~25nm，可以优选为10~15nm，可以更优选为13nm。发明人发现，当含金纳米颗粒尺度在以上范围内时，多巴胺会更容易使含金纳米颗粒聚集，从而观察到更加明显的颜色变化。According to the chemical sensor of the present invention, the particle size of the gold-containing nanoparticles is 3.5-25 nm, preferably 10-15 nm, and more preferably 13 nm. The inventors found that when the size of the gold-containing nanoparticles is within the above range, dopamine will more easily aggregate the gold-containing nanoparticles, thereby observing a more obvious color change.

根据本发明的化学敏感器，其中，所述A类有机功能小分子可选自通式如下的化合物中的一种或多种：According to the chemical sensor of the present invention, wherein, the class A organic functional small molecule can be selected from one or more of the compounds of the following general formula:

其中，R=-SH、-NH₂、

x=1~16。作为优选，所述A类有机功能小分子可以为吡啶-4-硼酸和4-巯基苯硼酸中的一种或两种。Among them, R=-SH, -NH₂ ,

x=1~16. Preferably, the class A organic functional small molecule may be one or both of pyridine-4-boronic acid and 4-mercaptophenylboronic acid.

根据本发明的化学敏感器，其中，所述B类有机功能小分子可选自通式如下的化合物中的一种或多种：According to the chemical sensor of the present invention, wherein, the class B organic functional small molecules can be selected from one or more of the compounds of the following general formula:

其中，R₁=-SH、-NH₂、

y=1~16。作为优选，所述B类有机功能小分子可以为3,3'-二硫代二丙酸二(N-羟基丁二酰亚胺酯)和4-巯基苯并18-冠-6中的一种或两种。Among them, R₁ =-SH, -NH₂ ,

y=1~16. Preferably, the B-type organic functional small molecule can be one of 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide ester) and 4-mercaptobenzo 18-crown-6 one or both.

本发明还提供了一种制备本发明的化学敏感器的方法，该方法包括：向沸腾的四氯金酸水溶液中加入柠檬酸钠溶液，并继续加热沸腾30分钟，冷却，离心洗涤，采用孔径为0.2μm的滤膜过滤，制得金纳米颗粒溶胶；向金纳米颗粒溶胶中加入A类有机功能小分子和B类有机功能小分子，搅拌2小时，即得所述化学敏感器。作为优选，所述四氯金酸和柠檬酸钠的摩尔比可以为1:3.5~4。The present invention also provides a method for preparing the chemical sensor of the present invention, the method comprising: adding sodium citrate solution to the boiling tetrachloroauric acid aqueous solution, and continuing to heat and boil for 30 minutes, cooling, centrifugal washing, using the aperture The gold nanoparticle sol is obtained by filtering through a 0.2 μm filter membrane; adding a class A organic functional small molecule and a class B organic functional small molecule into the gold nanoparticle sol, and stirring for 2 hours to obtain the chemical sensor. As preferably, the mol ratio of described tetrachloroauric acid and sodium citrate can be 1:3.5～4.

本发明还提供了一种检测多巴胺的方法，该方法包括：The present invention also provides a method for detecting dopamine, the method comprising:

（1）配制不同浓度的多巴胺溶液；(1) Prepare different concentrations of dopamine solutions;

（2）分别将5μl的不同浓度的多巴胺溶液与3ml的所述化学敏感器进行混合，使所述化学敏感器发生不同的颜色变化；(2) Mixing 5 μl of dopamine solutions of different concentrations with 3 ml of the chemical sensor, so that the chemical sensor undergoes different color changes;

（3）使用紫外-可见光吸收光谱仪测定步骤（2）中不同颜色变化的响应值；(3) Measure the response value of different color changes in step (2) using an ultraviolet-visible light absorption spectrometer;

（4）将5μl待测溶液与步骤（2）中所述3ml的化学敏感器进行混合，测定其颜色变化的响应值，然后与步骤（3）中的响应值相比较，得到待测溶液中多巴胺的浓度。(4) Mix 5 μl of the solution to be tested with the 3ml chemical sensor described in step (2), measure the response value of its color change, and then compare it with the response value in step (3) to obtain the The concentration of dopamine.

根据本发明的方法，其中，步骤（1）中，多巴胺溶液的浓度范围可为2.0~80μM。作为优选，配制浓度分别为2μM，8μM，12μM，16μM，20μM，24μM，28μM，32μM，36μM，40μM，44μM，48μM，52μM，56μM，60μM，64μM，68μM，72μM，76μM，80μM的共20份多巴胺溶液。作为更优选，所述步骤（2）的混合后的溶液中，多巴胺的浓度范围为3.3~133.3nM。According to the method of the present invention, wherein, in step (1), the concentration range of the dopamine solution may be 2.0-80 μM. Preferably, a total of 20 parts of 2 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, 40 μM, 44 μM, 48 μM, 52 μM, 56 μM, 60 μM, 64 μM, 68 μM, 72 μM, 76 μM, 80 μM Dopamine solution. More preferably, in the mixed solution in the step (2), the concentration range of dopamine is 3.3~133.3nM.

根据本发明的方法，其中，步骤（1）中，所述化学敏感器可为溶胶形态。作为优选，在所述化学敏感器中，所述含金纳米颗粒的浓度可为1~100nM。作为更优选，在所述化学敏感器中，所述含金纳米颗粒的浓度可为5~20nM。发明人发现，在该浓度范围内，化学敏感器的灵敏度和准确度大为提高。According to the method of the present invention, wherein, in step (1), the chemical sensor may be in the form of a sol. Preferably, in the chemical sensor, the concentration of the gold-containing nanoparticles may be 1-100 nM. More preferably, in the chemical sensor, the concentration of the gold-containing nanoparticles may be 5-20 nM. The inventors found that within this concentration range, the sensitivity and accuracy of the chemical sensor are greatly improved.

根据本发明的方法，其中，步骤（3）和（4）中的响应值为700nm处与521nm处的吸光度的比值。作为优选，分别以步骤（3）中的比值和多巴胺溶液的浓度为坐标制作标准曲线，然后将步骤（4）中的比值与标准曲线对比，计算得到待测溶液中多巴胺的浓度。According to the method of the present invention, the response value in steps (3) and (4) is the ratio of the absorbance at 700 nm to that at 521 nm. Preferably, a standard curve is prepared using the ratio in step (3) and the concentration of the dopamine solution as coordinates, and then the ratio in step (4) is compared with the standard curve to calculate the concentration of dopamine in the solution to be tested.

上述检测方法的作用在于，在测试过程中，保持敏感器的浓度一致，以及敏感器与多巴胺溶液在混合后的体积一致，而多巴胺的浓度呈现梯度变化，由此对多巴胺的浓度进行对比检测。The function of the above detection method is to keep the concentration of the sensor consistent and the volume of the sensor mixed with the dopamine solution to be consistent during the test, and the concentration of dopamine presents a gradient change, thereby performing a comparative detection of the concentration of dopamine.

本发明还提供了本发明的化学敏感器在制备用于检测多巴胺的装置中的应用。The present invention also provides the application of the chemical sensor of the present invention in the preparation of a device for detecting dopamine.

本发明的化学敏感器和检测方法具有但不限于以下有益效果：The chemical sensor and detection method of the present invention have, but are not limited to, the following beneficial effects:

1.检测灵敏度高，其所能检测到的多巴胺浓度最低可达0.08nM，并且可以在极短的时间内检测出体系中是否含有多巴胺，例如对多巴胺的响应时间可仅为130ms，使检测时间大大缩短。1. High detection sensitivity, the lowest concentration of dopamine that can be detected can reach 0.08nM, and it can detect whether there is dopamine in the system in a very short time, for example, the response time to dopamine can be only 130ms, making the detection time greatly shortened.

2.检测选择性好，相对于尿酸（UA）、抗坏血酸（AA）和一些金属离子，如Sr²⁺、Ca²⁺、Mg²⁺、Ba²⁺、K⁺、Na⁺，本发明的化学敏感器对多巴胺的选择性非常明显，其选择性可高出12倍以上，因而与电化学检测方法相比可获得更准确的检测结果。2. The detection selectivity is good. Compared with uric acid (UA), ascorbic acid (AA) and some metal ions, such as Sr²⁺ , Ca²⁺ , Mg²⁺ , Ba²⁺ , K⁺ , Na⁺ , the chemical The selectivity of the sensor to dopamine is very obvious, and its selectivity can be more than 12 times higher, so compared with the electrochemical detection method, more accurate detection results can be obtained.

3.操作简便，在不需要大型仪器的条件下，可通过颜色变化以肉眼判断体系中是否含有多巴胺。并且在检测多巴胺的具体浓度时，也仅需使用常见的紫外-可见光吸收光谱仪，对检测设备要求较低，因而降低了检测成本。3. It is easy to operate. Without the need of large-scale equipment, it can be judged by the naked eye whether dopamine is contained in the system through the color change. And when detecting the specific concentration of dopamine, it is only necessary to use a common ultraviolet-visible light absorption spectrometer, which requires less detection equipment, thereby reducing the detection cost.

附图说明Description of drawings

以下，结合附图来详细说明本发明的实施方案，其中：Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

图1示出了实施例1中金纳米颗粒溶胶的紫外-可见光吸收光谱。FIG. 1 shows the ultraviolet-visible light absorption spectrum of the gold nanoparticle sol in Example 1.

图2示出了实施例1中金纳米颗粒溶胶的扫描电镜（SEM）照片。FIG. 2 shows a scanning electron microscope (SEM) photo of the gold nanoparticle sol in Example 1.

图3示出了实施例2中的化学敏感器的颜色变化情况。FIG. 3 shows the color change of the chemical sensor in Example 2.

图4和图5分别示出了实施例2中的金纳米颗粒在多巴胺浓度为33.3nM和100nM下的聚集情况SEM照片。Fig. 4 and Fig. 5 respectively show the SEM photographs of the aggregation of gold nanoparticles in Example 2 at dopamine concentrations of 33.3 nM and 100 nM.

图6示出了实施例2中各样品的紫外-可见光吸收光谱。FIG. 6 shows the ultraviolet-visible light absorption spectrum of each sample in Example 2.

图7示出了实施例2中700nm和521nm吸光度随多巴胺浓度而变化的标准曲线。Fig. 7 shows the standard curve of the variation of absorbance at 700nm and 521nm with dopamine concentration in Example 2.

图8示出了实施例2中在向化学敏感器加入33nM多巴胺的前后，其检测灵敏度的对比曲线，其中曲线a为化学敏感器在700nm和521nm处的吸光度比值与加入的多巴胺浓度的关系曲线，而曲线b为加入33nm多巴胺后的关系曲线；Fig. 8 shows before and after adding 33nM dopamine to the chemical sensor inembodiment 2, the comparison curve of its detection sensitivity, wherein curve a is the relationship curve of the absorbance ratio of the chemical sensor at 700nm and 521nm and the concentration of dopamine added , and curve b is the relationship curve after adding 33nm dopamine;

图9示出了实施例3中的化学敏感器的颜色变化情况。FIG. 9 shows the color change of the chemical sensor in Example 3.

图10示出了实施例4中的化学敏感器的颜色变化情况。FIG. 10 shows the color change of the chemical sensor in Example 4.

图11和图12分别示出了实施例5中521nm和700nm处的吸光度变化曲线。Figure 11 and Figure 12 respectively show the absorbance change curves at 521 nm and 700 nm in Example 5.

图13示出了实施例6中化学敏感器对多巴胺和其他物质的检测选择性比较图。FIG. 13 shows a comparative diagram of the detection selectivity of the chemical sensor in Example 6 for dopamine and other substances.

具体实施方式Detailed ways

下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。The present invention will be further illustrated by specific examples below, but it should be understood that these examples are only used for more detailed description, and should not be construed as limiting the present invention in any form.

本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。This section provides a general description of the materials and test methods used in the tests of the present invention. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible. It will be apparent to those skilled in the art that, in the context and context, the materials used and methods of operation used in the present invention are known in the art unless otherwise indicated.

1.纳米颗粒的形状1. Shape of nanoparticles

检测设备：冷场发射扫描电子显微镜（Hitachi S4800），购自日本日立公司。Detection equipment: cold field emission scanning electron microscope (Hitachi S4800), purchased from Hitachi, Japan.

检测方法：测试胶体的扫描电子显微镜SEM图片。Detection method: Scanning electron microscope SEM picture of the test colloid.

2.纳米颗粒的尺寸2. Size of nanoparticles

检测设备：马尔文激光粒度仪（Zetasizer Nano ZS），购自英国马尔文公司。Detection equipment: Malvern laser particle size analyzer (Zetasizer Nano ZS), purchased from Malvern Company, UK.

检测方法：动态光散射技术（dynamic light scatting，DLS）。Detection method: dynamic light scattering (dynamic light scattering, DLS).

3.纳米颗粒的浓度3. Concentration of nanoparticles

检测设备：PerkinElmer(Lambda 750)紫外/可见/近红外分光光度计，购自美国珀金埃尔默公司。Detection equipment: PerkinElmer (Lambda 750) ultraviolet/visible/near-infrared spectrophotometer, purchased from PerkinElmer, USA.

检测方法：通过测试胶体溶液的紫外－可见吸收光谱计算。Detection method: Calculated by testing the ultraviolet-visible absorption spectrum of the colloidal solution.

实施例1Example 1

本实施例用于说明含金纳米颗粒的制备。This example is used to illustrate the preparation of gold-containing nanoparticles.

预先洗涤所有玻璃器皿并在烘箱中干燥后使用，具体可采用王水和去离子水进行洗涤。在250ml的圆底烧瓶中加热100ml 1.0mM的四氯金酸（HAuCl₄·4H₂O）水溶液至沸腾，加入10ml 38.8mM的柠檬酸钠溶液，继续加热沸腾30分钟。在此期间溶液颜色由浅黄色变为酒红色，表明形成了金纳米颗粒。然后将溶液冷却，用去离子水多次离心洗涤，采用孔径为0.2μm的滤膜进行过滤，制得金纳米颗粒溶胶。All glassware is pre-washed and oven-dried before use using aqua regia and deionized water. Heat 100ml of 1.0mM tetrachloroauric acid (HAuCl₄ ·4H₂ O) aqueous solution to boiling in a 250ml round bottom flask, add 10ml of 38.8mM sodium citrate solution, and continue heating and boiling for 30 minutes. The solution color changed from light yellow to wine red during this time, indicating the formation of gold nanoparticles. Then the solution was cooled, centrifuged and washed with deionized water several times, and filtered with a filter membrane with a pore size of 0.2 μm to obtain a gold nanoparticle sol.

测定所制备金纳米颗粒溶胶的吸收光谱，如图1所示，计算其金纳米颗粒的浓度约为11.5nM。该金纳米颗粒溶胶在521nm的波长处有最大吸收，颗粒尺寸约为13nm。该金纳米颗粒溶胶的扫描电镜（SEM）照片如图2所示，观察得知金纳米颗粒基本为球形。The absorption spectrum of the prepared gold nanoparticle sol was measured, as shown in FIG. 1 , and the concentration of the gold nanoparticles was calculated to be about 11.5nM. The gold nanoparticle sol has the maximum absorption at the wavelength of 521nm, and the particle size is about 13nm. The scanning electron microscope (SEM) photo of the gold nanoparticle sol is shown in FIG. 2 , and it is observed that the gold nanoparticles are basically spherical.

实施例2Example 2

本实施例用于说明本发明的化学敏感器及其制备。This example is used to illustrate the chemical sensor of the present invention and its preparation.

在室温下向实施例1中制得的100ml金纳米颗粒溶胶中加入2ml1mM的吡啶-4-硼酸（PDBA）和0.5mM的3,3'-二硫代二丙酸二(N-羟基丁二酰亚胺酯)（DSP）的溶液，搅拌2小时左右，制得化学敏感器，如下式所示：Add 2ml of 1mM pyridine-4-boronic acid (PDBA) and 0.5mM of 3,3'-dithiodipropionic acid bis(N-hydroxybutanedi Imide ester) (DSP) solution, stirred for about 2 hours to prepare a chemical sensor, as shown in the following formula:

配制浓度分别为2μM，8μM，12μM，16μM，20μM，24μM，28μM，32μM，36μM，40μM，44μM，48μM，52μM，56μM，60μM，64μM，68μM，72μM，76μM，80μM的共20份多巴胺溶液，并将5μl的不同浓度的多巴胺溶液与3ml的化学敏感器进行混合，溶液混合后的多巴胺浓度范围为3.3nM～133.3nM，使化学敏感器发生不同的颜色变化，具体如图3所示。A total of 20 dopamine solutions were prepared with concentrations of 2 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, 40 μM, 44 μM, 48 μM, 52 μM, 56 μM, 60 μM, 64 μM, 68 μM, 72 μM, 76 μM, and 80 μM, 5 μl of dopamine solutions of different concentrations were mixed with 3ml of the chemical sensor. The concentration of dopamine after the solution ranged from 3.3nM to 133.3nM, causing the chemical sensor to undergo different color changes, as shown in Figure 3.

通过SEM观察金纳米颗粒在33.3nM和100nM多巴胺浓度下的聚集情况的分别如图4和图5所示。The aggregation of gold nanoparticles observed by SEM at the concentration of 33.3nM and 100nM dopamine is shown in Figure 4 and Figure 5, respectively.

使用紫外-可见光吸收光谱仪测定各样品的紫外-可见光吸收光谱（如图6所示），发明人发现，随着多巴胺浓度的增加，其在521nm处的吸收强度逐渐下降，在700nm处的吸收强度则逐渐增强。计算各样品700nm处与521nm处的吸光度比值，以该比值为纵坐标，以多巴胺溶液的浓度为横坐标制作标准曲线，该曲线如图7所示，计算得其对多巴胺的检测限为0.26nM。Use ultraviolet-visible light absorption spectrometer to measure the ultraviolet-visible light absorption spectrum (as shown in Figure 6) of each sample, the inventor finds, along with the increase of dopamine concentration, its absorption intensity at 521nm place decreases gradually, and the absorption intensity at 700nm place is gradually enhanced. Calculate the absorbance ratio at the 700nm place and the 521nm place of each sample, take this ratio as the ordinate, and take the concentration of the dopamine solution as the abscissa to make a standard curve, as shown in Figure 7, the calculated limit of detection for dopamine is 0.26nM .

发明人还根据图7中的拐点发现，如果在检测前向化学敏感器中预先加入一定量的多巴胺（33nM）后，其检测灵敏度可以得到大幅提高，具体如图8所示。其中，曲线a为化学敏感器在700nm和521nm处的吸光度比值与加入的多巴胺浓度的关系曲线，而曲线b为加入33nm多巴胺后的关系曲线，可观察到曲线斜率明显增大，灵敏度明显提高。The inventors also found based on the inflection point in Figure 7 that if a certain amount of dopamine (33nM) is pre-added to the chemical sensor before detection, its detection sensitivity can be greatly improved, as shown in Figure 8 . Among them, curve a is the relationship curve between the ratio of the absorbance of the chemical sensor at 700nm and 521nm and the concentration of dopamine added, and curve b is the relationship curve after adding 33nm dopamine, and it can be observed that the slope of the curve increases obviously, and the sensitivity improves obviously.

配制浓度为2μM的多巴胺水溶液作为待测溶液。将该5μl待测溶液与3ml上述等量的化学敏感器进行混合，测得其700nm和521nm的吸光度比值，然后与标准曲线对比，计算得其检测浓度为1.98μM，准确度为99%。An aqueous solution of dopamine with a concentration of 2 μM was prepared as the solution to be tested.Mix 5 μl of the solution to be tested with 3ml of the above-mentioned equivalent chemical sensor, measure the ratio of absorbance at 700nm and 521nm, and compare it with the standard curve to calculate the detection concentration to be 1.98μM with an accuracy of 99%.

实施例3Example 3

在室温下向实施例1中制得的100ml金纳米颗粒溶胶中加入2ml 1mM的PDBA和0.01mM的4-巯基苯并18-冠-6（ABCE）的溶液，搅拌2小时左右，制得化学敏感器，如下式所示：Add 2ml of 1mM PDBA and 0.01mM 4-mercaptobenzo18-crown-6 (ABCE) solution to 100ml of gold nanoparticle sol prepared in Example 1 at room temperature, and stir for about 2 hours to obtain chemical sensor, as follows:

按照与实施例2相同的方法，配制不同浓度的多巴胺水溶液，向3ml的化学敏感器中加入5μl不同浓度的多巴胺水溶液，使多巴胺浓度在3.3~133.3nM内呈线性梯度变化，在该过程中可观察到化学敏感器的颜色随多巴胺的加入发生变化（基本上由酒红色变为深蓝色），并且颜色因多巴胺的加入量而不同，具体如图9所示，说明化学敏感器中的金纳米颗粒发生了聚集现象。According to the same method as Example 2, prepare different concentrations of dopamine aqueous solution, add 5 μ l of different concentrations of dopamine aqueous solution to the chemical sensor of 3ml, so that the dopamine concentration is in a linear gradient within 3.3 ~ 133.3nM, in the process can be It was observed that the color of the chemosensor changed with the addition of dopamine (essentially from wine red to dark blue), and the color was different due to the amount of dopamine added, as shown in Figure 9, indicating that the gold nanometer in the chemosensor Agglomeration of particles occurred.

实施例4Example 4

在室温下向实施例1中制得的100ml金纳米颗粒溶胶中加入2ml 0.01mM的4-巯基苯硼酸（MPBA）和0.01mM的ABCE的溶液，搅拌2小时左右，制得化学敏感器，如下式所示：Add 2ml of 0.01mM 4-mercaptophenylboronic acid (MPBA) and 0.01mM ABCE solution to the 100ml gold nanoparticle sol prepared in Example 1 at room temperature, and stir for about 2 hours to obtain a chemical sensor, as follows The formula shows:

按照与实施例2相同的方法，配制不同浓度的多巴胺水溶液，向3ml的化学敏感器中加入5μl的多巴胺水溶液，使多巴胺浓度在3.3~133.3nM内呈线性梯度变化，在该过程中可观察到化学敏感器的颜色随多巴胺的加入发生变化（基本上由酒红色变为深蓝色），并且颜色因多巴胺的加入量而不同，具体如图10所示，说明化学敏感器中的金纳米颗粒发生了聚集现象。According to the same method as in Example 2, prepare different concentrations of dopamine aqueous solution, add 5 μl of dopamine aqueous solution to a 3ml chemical sensor, so that the dopamine concentration changes linearly within 3.3~133.3nM, and can be observed in the process The color of the chemosensor changes with the addition of dopamine (basically from wine red to dark blue), and the color varies with the amount of dopamine added, as shown in Figure 10, which shows that the gold nanoparticles in the chemosensor aggregation phenomenon.

实施例5Example 5

本实施例用于说明本发明的化学敏感器对多巴胺的响应时间。This example is used to illustrate the response time of the chemical sensor of the present invention to dopamine.

使用紫外-可见光吸收光谱仪，采用时间驱动模式测试化学敏感器对多巴胺的响应灵敏性。分别向等量的实施例3中的化学敏感器中滴加5μl的浓度分别为2μM和80μM的多巴胺溶液，立刻检测其在521nm和700nm处的吸光度随时间变化的情况（如图11和图12所示），521nm处的响应时间仅为230ms。The sensitivity of chemosensors to dopamine responses was tested in a time-driven mode using a UV-visible absorption spectrometer. Add 5 μl of dopamine solutions with concentrations of 2 μM and 80 μM respectively to the same amount of the chemical sensor in Example 3, and immediately detect the change of its absorbance at 521 nm and 700 nm with time (as shown in Figure 11 and Figure 12 shown), the response time at 521nm is only 230ms.

实施例6Example 6

分别向3ml的实施例4中的化学敏感器中加入5μl浓度均为13μM的尿酸（UA）、抗坏血酸（AA）、Sr²⁺、Ca²⁺、Mg²⁺、Ba²⁺、K⁺、Na⁺的溶液，以及0.4μM的多巴胺溶液和空白对照溶液（去离子水），使用紫外-可见光吸收光谱仪检测其在700nm处和521nm处的吸光度并计算它们的比值，结果如图13所示。结果显示，本发明的化学敏感器对多巴胺检测的选择性非常好。Add 5 μl of uric acid (UA), ascorbic acid (AA), Sr²⁺ , Ca²⁺ , Mg²⁺ , Ba²⁺ , K⁺ , Na⁺ solution, and 0.4 μM dopamine solution and blank control solution (deionized water), using UV-visible light absorption spectrometer to detect the absorbance at 700nm and 521nm and calculate their ratio, the results are shown in Figure 13. The results show that the selectivity of the chemical sensor of the present invention for the detection of dopamine is very good.

尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。While the invention has been described to a certain extent, it will be obvious that various changes may be made in various conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the described embodiments, but rather falls within the scope of the claims, which include equivalents to each of the elements described.

Claims

Translated fromChinese

1.一种检测多巴胺的化学敏感器，其特征在于，所述化学敏感器包含含金纳米颗粒，所述含金纳米颗粒的表面经A类有机功能小分子和B类有机功能小分子修饰；所述A类有机功能小分子包含硼酸基团；所述B类有机功能小分子包含冠醚基团、醛基、丁二酰亚胺活化的羧基、磷酸根离子基团和磺酸根离子基团中的一种或多种。1. A chemical sensor for detecting dopamine, characterized in that, the chemical sensor comprises gold-containing nanoparticles, and the surface of the gold-containing nanoparticles is modified by class A organic functional small molecules and B class organic functional small molecules; The class A organic functional small molecule contains a boric acid group; the B class organic functional small molecule contains a crown ether group, an aldehyde group, a carboxyl group activated by succinimide, a phosphate ion group and a sulfonate ion group one or more of.

2.根据权利要求1所述的化学敏感器，其特征在于，所述A类有机功能小分子还包括吡啶、巯基、伯胺基和二硫基中的一种或多种，用于修饰所述含金纳米颗粒；所述B类有机功能小分子还包括吡啶、巯基、伯胺基和二硫基中的一种或多种，用于修饰所述含金纳米颗粒。2. The chemical sensor according to claim 1, characterized in that, the class A organic functional small molecule also includes one or more of pyridine, sulfhydryl, primary amino and disulfide, for modifying the The gold-containing nanoparticles; the B-type organic functional small molecules also include one or more of pyridine, sulfhydryl, primary amino and disulfide groups for modifying the gold-containing nanoparticles.

3.根据权利要求1或2所述的化学敏感器，其特征在于，所述含金纳米颗粒包括金与银、铜、铂、钯和镓中的一种或多种组成的复合纳米颗粒；优选地，所述复合纳米颗粒为核壳结构的纳米颗粒。3. The chemical sensor according to claim 1 or 2, wherein the gold-containing nanoparticles comprise composite nanoparticles composed of one or more of gold and silver, copper, platinum, palladium and gallium; Preferably, the composite nanoparticles are nanoparticles with a core-shell structure.

4.根据权利要求1至3中任一项所述的化学敏感器，其特征在于，所述含金纳米颗粒为金纳米颗粒。4. The chemical sensor according to any one of claims 1 to 3, characterized in that the gold-containing nanoparticles are gold nanoparticles.

5.根据权利要求5所述的化学敏感器，其特征在于，所述金纳米颗粒采用四氯金酸的水溶液通过还原法制得；优选地，所述还原法包括化学还原法、电化学还原法或辐射还原法；更优选地，在化学还原法中采用的化学还原试剂为氮，氮-二甲基甲酰胺、柠檬酸、柠檬酸钠和硼氢化钠中的一种或多种。5. chemical sensor according to claim 5, is characterized in that, described gold nanoparticle adopts the aqueous solution of tetrachloroauric acid to make by reduction method; Preferably, described reduction method comprises chemical reduction method, electrochemical reduction method or radiation reduction method; more preferably, the chemical reduction reagent used in the chemical reduction method is nitrogen, one or more of nitrogen-dimethylformamide, citric acid, sodium citrate and sodium borohydride.

6.根据权利要求1至5中任一项所述的化学敏感器，其特征在于，所述含金纳米颗粒的形状为纳米球、纳米棒、纳米管、纳米花、纳米片、三角形、四角形、星状或4~24面体，或表面呈刺状。6. The chemical sensor according to any one of claims 1 to 5, wherein the shape of the gold-containing nanoparticles is nanosphere, nanorod, nanotube, nanoflower, nanosheet, triangle, quadrangular , stellate or 4~24-hedron, or the surface is spiny.

7.根据权利要求1至6中任一项所述的化学敏感器，其特征在于，所述含金纳米颗粒的颗粒尺寸为3.5~25nm，优选为10~15nm，更优选为13nm。7. The chemical sensor according to any one of claims 1 to 6, characterized in that the particle size of the gold-containing nanoparticles is 3.5-25 nm, preferably 10-15 nm, more preferably 13 nm.

8.根据权利要求1至7中任一项所述的化学敏感器，其特征在于，所述A类有机功能小分子选自通式如下的化合物中的一种或多种：8. The chemical sensor according to any one of claims 1 to 7, characterized in that, the class A organic functional small molecule is selected from one or more of the compounds of the following general formula:

其中，R=-SH、-NH₂、

x=1~16；Among them, R=-SH, -NH₂ ,

x=1~16;

优选地，所述A类有机功能小分子为吡啶-4-硼酸和4-巯基苯硼酸中的一种或两种。Preferably, the class A organic functional small molecule is one or both of pyridine-4-boronic acid and 4-mercaptophenylboronic acid.

9.根据权利要求1至8中任一项所述的化学敏感器，其特征在于，所述B类有机功能小分子选自通式如下的化合物中的一种或多种：9. The chemical sensor according to any one of claims 1 to 8, characterized in that, the class B organic functional small molecule is selected from one or more of the following compounds of the general formula:

其中，R₁=-SH、-NH₂、y=1~16；Among them, R₁ =-SH, -NH₂ , y=1~16;

优选地，所述B类有机功能小分子为3,3'-二硫代二丙酸二(N-羟基丁二酰亚胺酯)和4-巯基苯并18-冠-6中的一种或两种。Preferably, the class B organic functional small molecule is one of 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimide ester) and 4-mercaptobenzo 18-crown-6 or two.

10.一种制备权利要求1至9中任一项所述的化学敏感器的方法，其特征在于，该方法包括：向沸腾的四氯金酸水溶液中加入柠檬酸钠溶液，并继续加热沸腾30分钟，冷却，离心洗涤，采用孔径为0.2μm的滤膜过滤，制得金纳米颗粒溶胶；向金纳米颗粒溶胶中加入A类有机功能小分子和B类有机功能小分子，搅拌2小时，即得所述化学敏感器；优选地，所述四氯金酸和柠檬酸钠的摩尔比为1:3.5~4。10. a method for preparing the chemical sensor described in any one of claims 1 to 9, is characterized in that, the method comprises: add sodium citrate solution in the tetrachloroauric acid aqueous solution of boiling, and continue heating boiling 30 minutes, cooling, centrifuging and washing, and filtering with a filter membrane with a pore size of 0.2 μm to obtain a gold nanoparticle sol; add a class A organic functional small molecule and a class B organic functional small molecule to the gold nanoparticle sol, and stir for 2 hours. That is, the chemical sensor is obtained; preferably, the molar ratio of tetrachloroauric acid and sodium citrate is 1:3.5~4.

11.一种检测多巴胺的方法，其特征在于，该方法包括：11. A method for detecting dopamine, characterized in that the method comprises:

（2）分别将5μl的不同浓度的多巴胺溶液与3ml的权利要求1~9中任一项所述的化学敏感器进行混合，使所述化学敏感器发生不同的颜色变化；(2) Mixing 5 μl of dopamine solutions of different concentrations with 3 ml of the chemical sensor according to any one of claims 1 to 9, so that the chemical sensor has different color changes;

12.根据权利要求11所述的方法，其特征在于，步骤（1）中，多巴胺溶液的浓度范围为2.0~80μM；优选地，配制浓度分别为2μM，8μM，12μM，16μM，20μM，24μM，28μM，32μM，36μM，40μM，44μM，48μM，52μM，56μM，60μM，64μM，68μM，72μM，76μM，80μM的共20份多巴胺溶液；更优选地，所述步骤（2）的混合后的溶液中，多巴胺的浓度范围为3.3~133.3nM。12. The method according to claim 11, characterized in that, in step (1), the concentration range of the dopamine solution is 2.0-80 μM; preferably, the prepared concentrations are 2 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, A total of 20 dopamine solutions of 28 μM, 32 μM, 36 μM, 40 μM, 44 μM, 48 μM, 52 μM, 56 μM, 60 μM, 64 μM, 68 μM, 72 μM, 76 μM, and 80 μM; more preferably, in the mixed solution of the step (2) , the concentration range of dopamine is 3.3~133.3nM.

13.根据权利要求11或12所述的方法，其特征在于，步骤（1）中，所述化学敏感器为溶胶形态；优选地，在所述化学敏感器中，所述含金纳米颗粒的浓度为1~100nM，更优选为5~20nM。13. The method according to claim 11 or 12, characterized in that, in step (1), the chemical sensor is in the form of a sol; preferably, in the chemical sensor, the gold-containing nanoparticles The concentration is 1-100 nM, more preferably 5-20 nM.

14.根据权利要求11至13中任一项所述的方法，其特征在于，步骤（3）和（4）中的响应值为700nm处与521nm处的吸光度的比值；优选地，分别以步骤（3）中的比值和多巴胺溶液的浓度为坐标制作标准曲线，然后将步骤（4）中的比值与标准曲线对比，计算得到待测溶液中多巴胺的浓度。14. The method according to any one of claims 11 to 13, wherein the response value in steps (3) and (4) is the ratio of the absorbance at 700nm to that at 521nm; The ratio in (3) and the concentration of the dopamine solution are used as coordinates to make a standard curve, and then the ratio in step (4) is compared with the standard curve to calculate the concentration of dopamine in the solution to be tested.

15.权利要求1至9中任一项所述的化学敏感器在制备用于检测多巴胺的装置中的应用。15. Use of the chemical sensor according to any one of claims 1 to 9 in the preparation of a device for detecting dopamine.