WO2021114551A1 - 一种应用于发酵过程原位检测的新型无酶葡萄糖传感器 - Google Patents

一种应用于发酵过程原位检测的新型无酶葡萄糖传感器 Download PDF

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WO2021114551A1
WO2021114551A1 PCT/CN2020/087355 CN2020087355W WO2021114551A1 WO 2021114551 A1 WO2021114551 A1 WO 2021114551A1 CN 2020087355 W CN2020087355 W CN 2020087355W WO 2021114551 A1 WO2021114551 A1 WO 2021114551A1
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glucose
sensor
enzyme
fermentation process
gold
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高学金
杜欣钊
高慧慧
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北京工业大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/05—Metallic powder characterised by the size or surface area of the particles
    • B22F1/054—Nanosized particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/07—Metallic powder characterised by particles having a nanoscale microstructure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Definitions

  • the invention belongs to the field of in-situ glucose detection in a fermentation process, and relates to a preparation method of an electrochemical sensor for glucose detection, in particular to a preparation method of an enzyme-free glucose biosensor with a gold-nickel core-shell structure.
  • glucose is the main carbon source for bacterial growth and product synthesis, and its concentration in the fermentation broth must be controlled.
  • Nickel is a common metal material prepared for enzyme-free sensors. Nickel-based sensors have much higher electrocatalytic activity, anti-toxicity and stability than other metal sensors.
  • the voltage of nickel-based electrodes for electrocatalytic oxidation of glucose is generally ⁇ 0.5v (vs SCE), many impurities in the fermentation broth can be catalyzed and oxidized under the high voltage to produce a current response, which leads to a decrease in the detection accuracy of the sensor.
  • the problem to be solved by the present invention is to provide a method for preparing an enzyme-free glucose sensor that is simple to prepare and can be applied to in-situ glucose detection in the fermentation process.
  • the present invention puts two metal precursors of gold and nickel into oleylamine step by step. In, the reduction reaction occurs after heating, and the gold-nickel bimetallic enzyme-free glucose sensor with a thin nickel shell is prepared by controlling the metal ratio.
  • the sensor has the advantages of good selectivity, anti-pollution, anti-poisoning, high temperature resistance and high stability.
  • Step 1 Put gold chloride in oleylamine, heat to 110°C and continue stirring for 4 hours to form gold nanoparticles;
  • Step 2 Add nickel acetylacetonate to the flask, raise the temperature to 220°C-240°C and continue stirring for 1 hour to form a core-shell structure of gold-nickel sodium.
  • Step 3 Cool down the mixed solution for aging, and then cool to room temperature;
  • Step 4 Take the mixed solution, add n-hexane and ethanol for centrifugal washing to remove residual oleylamine on the surface of the composite, and then disperse it in n-hexane;
  • Step 5 Add activated carbon and n-hexane solution, ultrasonic and stir, so that the gold-nickel nanocomposite is evenly dispersed on the carbon carrier;
  • Step 6 Add ethanol and collect by centrifugation, dissolve in acetic acid solution and heat to completely remove the oleylamine on the surface of the complex.
  • Step 7 Add ethanol to collect by centrifugation, and place in an oven to dry overnight.
  • Step 8 Prepare the membrane solution of the nanocomposite, drop it on the surface of the glassy carbon electrode, and dry at room temperature to prepare the Au@Ni/C enzyme-free glucose sensor.
  • the gold and nickel elements are added with different masses of metal precursors according to the molar ratio of 1:1, and the loading ratio of the gold-nickel bimetallic nanocomposite on the carbon matrix is 20wt%. It should be pointed out that the three substances of the present invention are in this ratio
  • the sensor prepared under synthesis has the best performance.
  • the mass of activated carbon added in step 5 is 120 mg.
  • the steps 1-3 are prepared in a nitrogen environment to avoid oxidation of the nanocomposite after reduction of the metal precursor.
  • the membrane solution in step 8 is mixed with 950 ⁇ l of ethanol, 50 ⁇ l of ionized water, and 100 ⁇ l of Nafion membrane solution, and the mass of the added complex is 14 mg.
  • the glassy carbon electrode in step 8 needs to be pretreated before use.
  • the performance detection method of the above Au@Ni/C enzyme-free glucose sensor is as follows:
  • the glassy carbon electrode loaded with nanocomposites is used as the working electrode, the saturated calomel electrode (SCE) is the reference electrode, the platinum plate is the counter electrode, and the three electrodes are inserted into a sodium hydroxide solution with a concentration of 0.1 mol/L;
  • SCE saturated calomel electrode
  • Experiment 1 Selectivity test under different working voltages: Measure the working voltage of the sensor to oxidize glucose by cyclic voltammetry (CV) method, and add different concentrations of glucose and impurities in the fermentation broth to observe the sensor selection under this voltage Sex.
  • CV cyclic voltammetry
  • the present invention uses a high-temperature oleylamine reduction method to prepare Au@Ni/ with a thin nickel shell structure by controlling the molar ratio of gold and nickel metal precursors.
  • the C nanocomposite has simple preparation method and high batch yield;
  • the Au@Ni/C enzyme-free glucose electrochemical sensor of the present invention forms a layer of gold-nickel nanocomposite on the surface of gold nanoparticles, which is compared with traditional gold -Based glucose sensor improves its surface kinetic reaction, reduces the detection voltage of the sensor, makes the sensor electrocatalytically oxidize glucose at a lower working voltage, and is not affected by impurities in the fermentation broth;
  • the glucose sensor of the present invention Due to the presence of the nickel shell on the surface, it is not affected by the oxidation intermediate product and the chloride ion adsorption poisoning in the fermentation broth, and has good anti-toxicity;
  • the enzyme-free sensor has the following advantages: (1) The present invention uses a high-temperature oleylamine reduction method
  • Figure 1 is a scanning electron microscope image and a line scan image of the Au@Ni/C nanocomposite prepared in Example 1 of the present invention.
  • Fig. 2 is the cyclic voltammetry curve of the sensor of Example 2 of the present invention in an electrolyte solution with and without glucose.
  • Fig. 3 shows the influence of impurities such as fructose, sucrose, lactose, and ascorbic acid interferents in the fermentation broth on the detection of the enzyme-free glucose electrochemical sensor of the present invention under different voltages.
  • Fig. 4 is a test graph of current density-time for glucose detection by the sensor of the present invention, and the inset is a linear relationship curve between corresponding response current and glucose concentration.
  • Figure 5 is the cyclic voltammetry curve of the sensor of the present invention in electrolyte solutions containing the same concentration of glucose and different concentrations of Cl -.
  • Fig. 6 is a graph showing changes in current response of the sensor of the present invention to measure the same concentration of glucose within a week.
  • Example 1 (1) Preparation of Au@Ni/C nanocomposite materials: Weigh 60.67mg AuCl 3 into a 25ml three-necked flask, add 10ml oleylamine and a rotor, heat to 110°C and continue stirring for 4h; then weigh 51.38 Add mg Ni(acac) 2 to the flask, heat to 240°C and keep stirring for 1h; cool to 160°C and age for 1h, then cool to room temperature, add n-hexane and ethanol (proportion) to centrifuge and wash and disperse in 10ml n-hexane; add 120mg Activated carbon and n-hexane solution (the proportion of nanocomposite is 20wt%), stir at room temperature for 2h after ultrasound, remove the supernatant after layering, add ethanol and collect by centrifugation, dissolve in acetic acid solution and heat to completely remove the compound Surface oleylamine.
  • Figure 1 is the TEM, HRTEM and line scan images of Au@Ni nanoparticles and Au@Ni/C/GCE nanocomposites prepared in Example 1 of the present invention. It can be seen from Figure 1 A and B that the prepared nanoparticles The particle size distribution is concentrated around 10nm and evenly distributed on the carbon support, confirming that the nano composite particles have no agglomeration and have good electrocatalytic activity due to the large number of surface active sites. It can be seen from C and D of Figure 1 that the nanoparticles are composed of a thin layer of gold-nickel nanocomposite wrapped with a gold core, which confirms that the final nanocomposite is in accordance with the theory.
  • the Au@Ni-C/GCE electrode in Example 1 is used as the working electrode, and the saturated calomel electrode (SCE) and platinum electrode are used.
  • the senor is at 0.05-0.20V, 0.55V vs. .SCE electrocatalytic oxidation of glucose. Then select 0.05V, 0.10V, 0.15V, 0.20V and 0.55V as the working voltages, and add 1mM glucose, 0.2mM sucrose, 0.2mM lactose, 0.2mM fructose and 0.02mM ascorbic acid to the electrolyte solution at intervals of 50s. , Test the current density-time curve of the sensor under different detection voltages.
  • the working voltage corresponding to 0.55V in Figure D is similar to that of the nickel-based sensor.
  • the electrocatalytic activity for glucose is much higher than the voltage range of 0.05-0.20V.
  • the interference substances in the fermentation broth are at this detection voltage.
  • the bottom is also electrocatalytically oxidized, with poor selectivity.
  • the sensor has good electrocatalytic activity and selectivity, and the current response generated by the interfering substances in the fermentation broth is negligible. Especially at the detection voltage of 0.10V, the sensor has the best performance.
  • the Au@Ni/C/GCE electrode obtained in Example 1 was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum plate was used as the counter electrode.
  • the three electrodes were inserted into the 0.1M sodium hydroxide solution, and the time-current test was used.
  • Technology applying 0.10V detection voltage, adding different volumes of glucose solution dropwise to 600r/min sodium hydroxide solution to detect response electrical signals.
  • the test result shown in Figure 4(A) shows that with the increase of glucose concentration, the response current has an upward trend, and it has a linear relationship in the range of 0.5-10mM glucose concentration.
  • the sensitivity is 23.17 ⁇ A mM -1 cm -2 , linear
  • the correlation coefficient is 0.993; it can be seen from Figure 4(B) that the lowest detection limit of the sensor is 10 ⁇ M.
  • the above-mentioned performance means that the sensor prepared by the present invention has a large detection current density for glucose detection, which is easy for subsequent signal processing, the current response changes caused by the change of glucose concentration are obvious, and the discrimination is high.
  • the Au@Ni/C/GCE electrode obtained in Example 1 was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum plate was used as the counter electrode.
  • the three electrodes were inserted into the 0.1M sodium hydroxide solution and analyzed by cyclic voltammetry The method, in the voltage range of -0.8V-0.8V and the scan rate of 50mv/s -1 , tested it in the blank electrolyte solution, the electrolyte solution of 5mmol/L glucose and the CL containing 5mmol/L glucose and different concentrations - under the current response, compared to the Au / C / SCE electrode cyclic voltammetry measurement (FIG.
  • the Au@Ni/C/GCE electrode obtained in Example 1 was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum sheet was used as the counter electrode.
  • the three electrodes were inserted into the 0.1M sodium hydroxide solution for half a day within a week It is the time interval to measure the current response of the sensor to 5mM glucose. From the measurement results in Figure 6, it can be seen that the peak current density measured by the sensor in the continuous test maintains more than 90% of the initial current response, which proves the Au@Ni-C of the present invention
  • the /GCE enzyme-free sensor has excellent long-term stability.

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Abstract

本发明公开了一种应用于发酵过程原位检测的新型无酶葡萄糖传感器，可以对发酵液中的葡萄糖浓度进行检测。包括以下内容：采用高温油胺还原的方法制备具有核壳结构的Au@Ni/C纳米复合物，并以此制备工作电极，构建三电极检测系统，三电极捕获工作电极电催化氧化葡萄糖产生的电信号，获得对应葡萄糖浓度的数据，经过计算得到葡萄糖浓度。与现有的酶葡萄糖生物传感器相比，本发明传感器无需复杂的固定化技术；耐高温，可用于原位检测；精度高，催化活性不受发酵环境如温度、湿度的影响；稳定性强，可适用于长期的发酵过程在线检测环境。与现有的无酶葡萄糖生物传感器相比，本发明传感器在较低的工作电压下工作，选择性高；不受发酵液中大量存在的氯离子的吸附影响，抗中毒性强。因此成为应用于发酵过程原位葡萄糖检测的无酶电化学传感器。

Description

一种应用于发酵过程原位检测的新型无酶葡萄糖传感器 技术领域
本发明属于发酵过程原位葡萄糖检测领域，涉及一种用于葡萄糖检测的电化学传感器的制备方法，具体涉及到一种具有金镍核壳结构的无酶葡萄糖生物传感器的制备方法。
背景技术
葡萄糖的定量检测在化学工业、临床医学、发酵过程、食品安全中都起着至关重要的作用。葡萄糖传感器占有生物传感器市场85％的比重，在发酵过程中葡萄糖作为菌体生长和产物合成的主要碳源，必须控制其在发酵液中的浓度。
现今，发酵工厂大多采用，通过酶基传感器离线测定，或是通过测定PH值、溶氧量等因素间接评估的方式测定葡萄糖浓度，原因在于，在发酵过程中为了防止染菌，在线葡萄糖生物传感器及其与发酵罐间管路必须能承受高温(～120℃)蒸汽灭菌，而葡萄糖氧化酶的作用温度为30-60℃，限制了酶基传感器的原位在线检测；通过其他因素间接测定葡萄糖的方式势必导致检测精度较低的问题，而基于葡萄糖氧化酶(GOD)的电化学传感器，其固定化技术复杂、成本高昂且活性易受温度、湿度的影响且稳定性较差不适用于长期的发酵过程在线检测环境下。
金属材料具有良好的耐高温性及稳定性，自纳米材料出现以来，利用葡萄糖在金属纳米电极表面上的高效电催化氧化特性，研制可以解决GOD传感器应用中所受限制，实现原位检测的无酶葡萄糖传感器是一种极具吸引力的替代技术。其中，对于葡萄糖电催化氧化反应，金、镍是极具吸引力的金属之一。与其他金属相比，金在碱性和中性介质中具有高效电催化活性和良好选择性，其无毒性也是作为构建应用于发酵液中葡萄糖检测的关键因素。但是，在发酵过程中进行葡萄糖的检测，使用Au电极的葡萄糖传感器易受待测液中氧化的中间物质和大 量存在的氯离子的吸附中毒影响。而镍作为无酶传感器制备的常用金属材料，镍基传感器具有远高于其他金属传感器的电催化活性、抗中毒性及稳定性，但由于镍基电极电催化氧化葡萄糖的电压一般在～0.5v(vs SCE)，发酵液中很多杂质可在该高电压下催化氧化产生电流响应，导致传感器的检测精度下降。
基于以上考虑，需要一种技术将上述各材料的优点集合起来，开发出一款可用于发酵过程原位葡萄糖检测并具有良好选择性及稳定性的无酶葡萄糖传感器。
发明内容
本发明所要解决的问题是提供一种制备简单可应用于发酵过程葡萄糖原位在线检测的无酶葡萄糖传感器的制备方法，本发明通过将金、镍两种金属前驱体分步放入到油胺中，加热后发生还原反应，通过控制金属比例制备具有薄镍壳的金镍双金属无酶葡萄糖传感器，该传感器具有选择性好、抗污染、抗中毒、耐高温及稳定性高等优势。
本发明采用的技术方案如下：
步骤1：将氯化金放入油胺中，加热至110℃并持续搅拌4h，形成金纳米颗粒；
步骤2：将乙酰丙酮镍加入烧瓶，升温至220℃-240℃并持续搅拌1h以形成核壳结构的金镍纳步骤3：将混合溶液降温老化，后冷却至室温；
步骤4：取混合溶液添加正己烷及乙醇离心洗涤，以去除复合物表面残留油胺，后分散至正己烷中；
步骤5：加入活性炭及正己烷溶液，超声并搅拌，使得金镍纳米复合物均匀分散到碳载体上；
步骤6：加入乙醇离心收集，溶于乙酸溶液中并加热，彻底去除复合物表面油胺。
步骤7：加入乙醇离心收集，并放置在烘箱中干燥一夜。
步骤8：制备纳米复合物的膜溶液，滴涂于玻碳电极表面，室温晾干，制备Au@Ni/C无酶 葡萄糖传感器。
作为优选，金镍元素根据摩尔比率为1:1添加不同质量的金属前驱体，金镍双金属纳米复合物在碳基质的负载比为20wt％，需要指出，本发明该三种物质在该比例合成下制备的传感器具有最优的性能。
作为优选,步骤5中加入活性炭的质量为120mg。
作为优选，所述步骤1-3在氮气环境下制备，避免金属前体还原后的纳米复合物氧化。
作为优选，步骤8中的膜溶液由950μl乙醇、50μl离子水、100μl Nafion膜溶液混合而成，添加的复合物质量为14mg。
作为优选，步骤8玻碳电极在使用前需要预处理，过程为：将玻碳电极(GCE＝5mm)依次用0.3、0.05μm氧化铝粉末抛光，随后用乙醇和去离子水超声清洗。
上述Au@Ni/C无酶葡萄糖传感器的性能检测方法如下所示：
实验环境：将负载纳米复合物的玻碳电极作为工作电极，饱和甘汞电极(SCE)为参比电极，铂片为对电极，将三电极插入浓度为0.1mol/L的氢氧化钠溶液；
实验一：不同工作电压下的选择性测试：通过循环伏安分析(CV)方法测量传感器氧化葡萄糖的工作电压，并在该电压下，加入不同浓度的葡萄糖及发酵液中存在的杂质观察传感器选择性。
实验二：灵敏度及检测范围测试：在0.10V vs.SCE的检测电压下，以固定的时间间隔向电解质溶液中滴加葡萄糖溶液，观察检测结果的时间-电流曲线。
实验三：抗中毒性测试：在0.10V vs.SCE的检测电压下，采用时间-电流测试技术，测量加入相同浓度的葡萄糖、不同浓度的Cl -下电流响应间的差别。
实验四：稳定性实验：在一周时间内，以半天为时间间隔测量传感器对加入的相同浓度葡萄糖的电流响应变化情况。
上述实验检测结果显示，制备的无酶葡萄糖传感器随着葡萄糖浓度的增大，电流响应成上升趋势，并在一定浓度范围内成线性关系；基于Au@Ni/C纳米复合物的传感器对于发酵过程的葡萄糖检测展现出良好的性能。
本发明与现有技术相比具有的优点是：(1)本发明使用高温油胺还原的方法，通过控制金、镍金属前驱体的摩尔比，制备了具有薄镍壳结构的Au@Ni/C纳米复合物，制备方法简单、批次产量高；(2)本发明的Au@Ni/C无酶葡萄糖电化学传感器在金纳米颗粒表面形成了层金镍纳米复合物，相较于传统金基葡萄糖传感器，改善了其表面动力学反应，降低了传感器的检测电压，使传感器在更低的工作电压下电催化氧化葡萄糖，不受发酵液中杂质的影响；(3)本发明的葡萄糖传感器由于表面镍壳的存在，不受氧化中间产物及发酵液中的氯离子吸附中毒的影响，具有良好的抗中毒性；(4)相较于传统的酶基传感器，该无酶传感器具备良好的稳定性，无需频繁更换，成本低廉，可应用于发酵过程中葡萄糖的长期在线检测。
附图说明
图1为本发明实施例1制备的Au@Ni/C纳米复合物的扫描电镜图及线扫图。
图2为本发明实施例2传感器在有/无葡萄糖的电解质溶液中的循环伏安曲线。
图3为发酵液中存在的杂质如果糖、蔗糖、乳糖、抗坏血酸干扰物在不同电压下对本发明无酶葡萄糖电化学传感器检测的影响。
图4为本发明传感器对于葡萄糖检测的电流密度-时间的测试图，插图为相应的响应电流与葡萄糖浓度的线性关系曲线。
图5为本发明传感器在含有相同浓度葡萄糖及不同浓度Cl -的电解质溶液中的循环伏安曲线。
图6为本发明传感器一周内测量相同浓度葡萄糖的电流响应变化图。
具体实施方式
下面结合具体实施例对本发明做进一步详细的描述，但不认定本发明的实施方式仅限于此。
实施例1：(1)Au@Ni/C纳米复合材料的制备：称取60.67mg AuCl 3放入25ml三口烧瓶，加入10ml油胺及转子，加热至110℃并持续搅拌4h；随后称取51.38mg Ni(acac) 2加入烧瓶，加热至240℃并持续搅拌1h；冷却至160℃老化1h，后冷却至室温，添加正己烷及乙醇(比例)离心洗涤后分散至10ml正己烷中；加入120mg活性炭及正己烷溶液(纳米复合物比重20wt％)，超声后在室温下搅拌2h，静置分层后移除上清液，加入乙醇离心收集，溶于乙酸溶液中并加热，彻底去除复合物表面油胺。随后再次加入乙醇离心收集，并放置在烘箱中120℃干燥一夜，获得Au@Ni/C纳米复合物。(2)修饰电极的制备：取直径为5mm的玻碳电极，依次用0.3、0.05μm Al 2O 3粉末抛光，随后用乙醇和去离子水超声清洗；称取14mg制备的复合物溶于配置的膜溶液(950μl乙醇、50μl离子水、100μl Nafion膜溶液)中，超声30min，取10μl溶液滴涂于GCE电极表面，室温晾干，制得Au@Ni/C/GCE电极。
图1为本发明实例1制备的Au@Ni纳米颗粒及Au@Ni/C/GCE纳米复合物的TEM、HRTEM及线扫图，从图1的A、B中可以看出，制备的纳米颗粒粒径分布集中在10nm左右，并均匀的分布在碳载体上，证实了纳米复颗粒未产生团聚并具备良好的电催化活性由于大量的表面活性位点数。从图1的C、D中可以看出，纳米颗粒由一层薄薄的金镍纳米复合物包裹金核组成，证实了最终制备的纳米复合物与理论符合。
对上述Au@Ni/C无酶葡萄糖传感器的性能进行检测，具体步骤如下：以实施例1中的Au@Ni-C/GCE电极作为工作电极，以饱和甘汞电极(SCE)和铂片电极作为参比电极及对电极，构建三电池检测体系，电解质溶液选择0.1mol/L的氢氧化钠溶液；为了研究制备的传感器对 葡萄糖的电催化氧化行为，采用循环伏安分析的方法，在-1.0V-1.0V电压范围及50mv/s -1的扫描速率下，比较了其在空白的电解质溶液(实线)及含有10mmol/L葡萄糖的电解质溶液(虚线)中的电流响应。同时测试了Au 2@Ni/C/GCE(金镍摩尔比为2:1)电极的循环伏安曲线(图B),从图2中可以看出，传感器在0.05-0.20V、0.55V vs.SCE范围内电催化氧化葡萄糖。随后分别选择0.05V、0.10V、0.15V、0.20V及0.55V作为工作电压，以50s为间隔向电解质溶液中分别添加1mM的葡萄糖、0.2mM蔗糖、0.2mM乳糖、0.2mM果糖及0.02mM抗坏血酸，测试传感器不同检测电压下的电流密度-时间曲线。
如图3所示，D图中对应0.55V的工作电压与镍基传感器的工作电压相近，对于葡萄糖的电催化活性远高于0.05-0.20V电压范围，发酵液中的干扰物质在该检测电压下同样被电催化氧化，选择性较差；从A、B、C三图中可以看出，传感器具备良好的电催化活性及选择性，发酵液中的干扰物质产生的电流响应可忽略不计，尤其是在0.10V检测电压，传感器具有最优的性能，这主要是由于一方面金纳米颗粒本身对于葡萄糖的电催化氧化具有高选择性，另一方面，薄镍壳负载到金纳米颗粒表面，使得金的零电位点电荷负向移动，进而本文传感器与金基传感器相比可在更低的工作电压下电催化氧化葡萄糖，避免了杂质的影响。
将实施例1获得的Au@Ni/C/GCE电极作为工作电极、饱和甘汞电极为参比电极，铂片为对电极，将三电极插入0.1M氢氧化钠溶液中，采用时间-电流测试技术，施加0.10V检测电压，向600r/min的氢氧化钠溶液中逐滴加入不同体积的葡萄糖溶液，进行响应电信号检测。如图4(A)的检测结果显示，随着葡萄糖浓度的增大，响应电流成上升趋势，并在0.5-10mM葡萄糖浓度范围内成线性关系，灵敏度为23.17μA mM -1cm -2，线性相关系数为0.993；从图4(B)中可以看出传感器的最低检测限为10μM。上述性能意味着本发明制备的传感器对于葡萄糖的检测，检测电流密度大易于后续信号处理，葡萄糖浓度的变化导致的电流响应变化明显，区别度高。
将实施例1获得的Au@Ni/C/GCE电极作为工作电极、饱和甘汞电极为参比电极，铂片为对电极，将三电极插入0.1M氢氧化钠溶液中，采用循环伏安分析的方法，在-0.8V-0.8V电压范围及50mv/s -1的扫描速率下，测试了其在空白的电解质溶液、5mmol/L葡萄糖的电解质溶液及含有5mmol/L葡萄糖和不同浓度的CL -下的电流响应，作为比较，制备了Au/C/SCE电极，并以此构建三电极检测体系，测量循环伏安曲线(图5B)；由图5可以看出，本文发明的传感器与金基传感器相比较，氯离子的有无，对于在0.10V左右的电催化行为并无影响，且在多次循环后，溶液中存在葡萄糖催化氧化的中间产物也没有导致电流密度的下降，这证明了本发明的传感器不受电催化产生的中间产物及发酵过程中大量存在的氯离子吸附的影响，传感器具有良好的抗中毒特性。
将实施例1获得的Au@Ni/C/GCE电极作为工作电极、饱和甘汞电极为参比电极，铂片为对电极，将三电极插入0.1M氢氧化钠溶液中，在一周内以半天为时间间隔，测量传感器对于5mM葡萄糖产生的电流响应，由图6的测量结果可知，传感器在连续试验中测量的峰值电流密度保持初始电流响应的90％以上，证明本发明的Au@Ni-C/GCE无酶传感器具有优越的长期稳定性。

Claims (5)

  1. 一种应用于发酵过程原位葡萄糖检测的无酶葡萄糖传感器的制备方法，其特征在于，包括如下步骤：
    步骤1：将氯化金放入油胺中，加热至110℃并持续搅拌4h，形成金纳米颗粒；
    步骤2：将乙酰丙酮镍加入烧瓶，升温至220℃-240℃并持续搅拌1h以形成核壳结构的金镍纳米复合物；
    步骤3：将混合溶液降温老化，后冷却至室温；
    步骤4：取混合溶液添加正己烷及乙醇离心洗涤，去除复合物表面残留油胺，后分散至正己烷中；
    步骤5：加入活性炭及正己烷溶液，超声并搅拌，使金镍纳米复合物均匀分散到碳载体上；
    步骤6：加入乙醇离心收集，溶于乙酸溶液中并加热，彻底去除复合物表面油胺；
    步骤7：加入乙醇离心收集，并放置在烘箱中干燥一夜；
    步骤8：制备纳米复合物的膜溶液，滴涂于玻碳电极表面，室温晾干，制备Au@Ni/C无酶葡萄糖传感器。
  2. 如权利要求1所述的一种应用于发酵过程原位葡萄糖检测的无酶葡萄糖传感器的制备方法，其特征在于：所述步骤1和步骤2中，氯化金和乙酰丙酮镍的摩尔比为1:1，金镍双金属纳米复合物在碳基质的负载比为20wt％。
  3. 如权利要求1所述的一种应用于发酵过程原位葡萄糖检测的无酶葡萄糖传感器的制备方法，其特征在于：所述步骤1-3中还原反应在氮气氛下进行。
  4. 如权利要求1所述的一种应用于发酵过程原位葡萄糖检测的无酶葡萄糖传感器的制备方法，其特征在于：所述步骤5中加入活性炭120mg。
  5. 如权利要求1所述的一种应用于发酵过程原位葡萄糖检测的无酶葡萄糖传感器的制备方法，其特征在于：步骤8中的膜溶液由950μl乙醇、50μl离子水、100μl Nafion膜溶液混合而成，添加的复合物质量为14mg；所述玻碳电极在使用前需要预处理，过程为：将 玻碳电极(GCE＝5mm)依次用0.3、0.05μm氧化铝粉末抛光，随后用乙醇和去离子水超声清洗。
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