WO2023123142A1 - 一种表面等离子体共振传感芯片及其制备方法和应用 - Google Patents

一种表面等离子体共振传感芯片及其制备方法和应用 Download PDF

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WO2023123142A1
WO2023123142A1 PCT/CN2021/142741 CN2021142741W WO2023123142A1 WO 2023123142 A1 WO2023123142 A1 WO 2023123142A1 CN 2021142741 W CN2021142741 W CN 2021142741W WO 2023123142 A1 WO2023123142 A1 WO 2023123142A1
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solution
graphene oxide
sensor chip
plasmon resonance
surface plasmon
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PCT/CN2021/142741
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English (en)
French (fr)
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郭丽莉
王蓓丽
韩亚萌
李书鹏
桂毅
樊强
宋倩
薛晋美
李亚秀
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北京建工环境修复股份有限公司
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Priority to PCT/CN2021/142741 priority Critical patent/WO2023123142A1/zh
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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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands

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  • the invention belongs to the field of environmental pollutant sensing and detection, and in particular relates to a surface plasmon resonance sensing chip and a preparation method and application thereof.
  • Perfluorinated compounds have thermal stability, chemical stability and good surface activity, and have been widely used in various production and daily necessities such as food packaging bags, non-stick pans, textiles, waterproofing agents, electroplating agents, and foam fire extinguishing agents.
  • Perfluorinated compounds have serious biological toxicity effects and can cause serious harm to the ecological environment and human health.
  • Perfluorinated compounds have become a new type of persistent organic pollutants, and the research on their pollution status in the environment is imminent. Therefore, it is very necessary to establish an effective analytical method for the timely detection of perfluorinated compounds in the environment.
  • the detection methods of perfluorinated compounds are mainly high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry. Although these methods have high sensitivity and good accuracy, they need cumbersome sample pretreatment and consume a lot of For organic solvents, the detection cycle is longer and the detection cost is higher. In recent years, some ultraviolet and fluorescent sensing methods have also been established for the rapid detection of perfluorinated compounds.
  • Chinese patent document CN110749562A discloses a dual-wavelength ratio ultraviolet spectrometry for the determination of perfluorooctane sulfonic acid.
  • perfluorooctane sulfonic acid and toluidine blue have an electrostatic interaction to form an ion association, which has two characteristic peaks at 632nm and 502nm and absorbs ratiometrically, according to the concentration of perfluorooctane sulfonic acid solution
  • a dual-wavelength ratio ultraviolet spectrometry method for the determination of perfluorooctane sulfonic acid was established.
  • Chinese patent document CN103558198B discloses a rapid fluorescence detection method for PFOS in environmental water samples, using PFOS to make the metal complex 4-(methylphenyl)-2,2':6',2" through electrostatic quenching -
  • the fluorescence of terpyridine Zn(II) is quenched, and the rapid fluorescence analysis of PFOS in environmental water samples is realized by measuring the change of fluorescence spectrum intensity.
  • These methods are all based on the generation of perfluorinated compounds by the probe materials in the liquid phase identification, resulting in changes in spectral signals, although it is simpler and faster than mass spectrometry, but the sensitivity is low, the stability of the probe is poor, and the reaction in the liquid phase is also easily interfered by related substances. Therefore, the establishment of a more stable and more Sensitive sensing methods for the rapid detection of PFCs are very necessary.
  • the present invention provides a surface plasmon resonance sensor chip and its preparation method and application.
  • the present invention provides the following technical solutions.
  • the present invention provides a surface plasmon resonance sensor chip, which comprises a substrate, a gold film, a graphene oxide layer and a perfluoroalkane chain functionalized layer from bottom to top.
  • the substrate is a porous glass slide substrate.
  • the thickness of the gold film is 40-60 nm, and the thickness of the graphene oxide layer is 50-100 nm.
  • the present invention provides a method for preparing a surface plasmon resonance sensor chip, comprising:
  • Step 1 plating a layer of gold film on the substrate
  • Step 2 setting the graphene oxide layer on the gold film:
  • Step 3 modifying the perfluoroalkane chain functionalized layer on the graphene oxide layer:
  • the substrate is a porous glass slide substrate
  • step 1 is: plating a gold film in each reaction well pool of the porous glass slide base; preferably, vacuum evaporation is used to plate a gold film in the reaction well pool, and the thickness of the gold film is 40 ⁇ 60nm.
  • step 2 satisfies at least one of the following (1)-(5):
  • the concentration of the mercaptoethylamine ethanol solution is 0.1-0.5 mg/mL, and the dosage is 50-150 ⁇ L;
  • the concentration of the graphene oxide solution is 1-5 mg/mL, and the dosage is 100-200 ⁇ L;
  • the solvent of the graphene oxide solution is at least one of water, methanol, and ethanol, preferably water.
  • step 3 satisfies at least one of the following (1)-(5):
  • the silylating agent is aminopropyltriethoxysilane or aminopropyltrimethoxysilane
  • the solvent of the silylating reagent solution is water
  • the volume fraction of the silylating reagent in the silylating reagent solution is 5%-10%, and the consumption of the silylating reagent solution is 50-150 ⁇ L;
  • the activator is EDC/NHS solution or EDC solution, the concentration of EDC and/or NHS in the activator is 0.1-0.5 mg/mL, and the dosage is 20-80 ⁇ L.
  • step 3 the perfluorosilane solution and catalyst are added to the silanized graphene oxide layer for reaction; said step 3 meets at least one of the following (1)-(5):
  • the volume fraction of perfluorosilane in the perfluorosilane solution is 1%-10%, and the dosage is 100-200 ⁇ L;
  • the catalyst is ammonia water, the mass concentration of ammonia water is 25%-28%, and the dosage is 5-20 ⁇ L;
  • Perfluorosilane is perfluorooctyltriethoxysilane and/or perfluorodecyltriethoxysilane;
  • the solvent of the perfluorosilane solution is an alcohol-water solution, the volume ratio of alcohol to water is (2-4): 1, preferably, the alcohol is ethanol and/or methanol;
  • the present invention provides the application of the above-mentioned surface plasmon resonance sensor chip or the surface plasmon resonance sensor chip prepared according to the above-mentioned preparation method in the detection of perfluorinated compounds.
  • the perfluorinated compound is selected from At least one of perfluorooctanesulfonic acid, perfluorocaproic acid, perfluorononanoic acid, and perfluorooctanecarboxylic acid.
  • SPR Surface plasmon resonance
  • the corresponding incident angle ⁇ is the SPR angle
  • the SPR angle will change with the change of the refractive index of the metal film surface. Variety. If special molecules are immobilized on the metal surface, when the solution flowing through the metal surface contains substances that can interact with it, the interaction between them will cause the refractive index of the metal surface dielectric to change, thereby changing the SPR angle.
  • the invention realizes the quantitative detection of the target substance in the solution by measuring the variation of the SPR angle.
  • the present invention provides a method for detecting perfluorinated compounds, comprising the following steps:
  • the surface plasmon resonance sensor chip is loaded into the surface plasmon resonance sensor, and the initial SPR angle ⁇ 0 is measured after injecting a blank solvent;
  • the solvent is water, ethanol or methanol, more preferably water;
  • ⁇ n has a linear relationship with the concentration of perfluorinated compounds, and a standard curve of concentration-SPR angle change is made;
  • the surface plasmon resonance sensor chip provided by the present invention comprises a substrate, a gold film, a graphene oxide layer and a perfluoroalkane chain functionalized layer from bottom to top.
  • the perfluoroalkane chain functionalized layer can have a specific fluorine-fluorine interaction with the perfluorinated compound in the sample to be tested, and specifically recognize the perfluorinated compound, causing the refractive index of the perfluoroalkane chain functionalized layer on the chip surface to change. Sensitive response, so that the SPR angle changes.
  • SPR sensing generates a recognition response to the target on the surface of the solid chip, and the probe functional layer on the chip surface is more stable, does not require labeling, and has lower background signals or noise signals , not easily disturbed, good stability.
  • the graphene oxide layer can significantly improve the detection sensitivity. Since the change of the refractive index of the chip surface medium is very sensitive, when a low concentration of perfluorinated compound solution passes through the chip surface, it can cause the SPR angle to change, the detection limit can reach pg/L, and the sensitivity is high.
  • the surface plasmon resonance sensor chip provided by the present invention has a porous glass substrate as the substrate, and multiple samples can be detected simultaneously on one sensor chip to realize rapid detection of batch samples; and multiple reaction wells can be detected as needed.
  • the pools are functionally modified for the determination of different targets.
  • the thickness of the gold film is 40-60 nm, and the thickness of the graphene oxide layer is 50-100 nm.
  • the thickness of the functionalized layer on the surface of the SPR sensor chip used can generally produce a more sensitive response within 200nm, and the invention controls the total thickness of the gold film and the graphene oxide layer to be less than 200nm.
  • the preparation method of the surface plasmon resonance sensing chip comprises: step 1, coating a layer of gold film on the substrate; step 2, setting a graphene oxide layer on the gold film: adding mercaptoethylamine ethanol solution Add it on the gold film, react, remove the mercaptoethylamine ethanol solution, and get the gold film modified by mercaptoethylamine; add the graphene oxide solution to the gold film modified by mercaptoethylamine, and remove the graphene oxide solution; step 3, in Modified perfluoroalkane chain functionalized layer on the graphene oxide layer: add the silanization reagent solution and the activator to the graphene oxide layer and react to obtain a silanized graphene oxide layer; in the silanized graphene oxide layer The surface is modified with perfluoroalkane chains to prepare a surface plasmon resonance sensor chip.
  • the detection sensitivity can be significantly improved by adding a graphene oxide layer, and the amount of sample required is very small, avoiding the use of a large amount of organic solvents, and has the advantage of being more environmentally friendly.
  • Graphene oxide contains a large number of carboxyl and hydroxyl groups, making it electronegative and hydrophilic, and has good dispersibility in water.
  • the thiol group in the mercaptoethylamine can form a gold-sulfur bond with the gold film, so that the mercaptoethylamine molecules are fixed on the gold film, and the amino groups in the mercaptoethylamine molecules are exposed on the surface; when the graphene oxide solution When dropped onto the mercaptoethylamine-modified gold film, the negatively charged graphene oxide can interact electrostatically with the amino group, so that the graphene oxide can be fixed on the chip surface, and the graphene oxide can improve the sensitivity of SPR sensing.
  • step 3 of the present invention the amino group of the silylating agent reacts with the carboxyl group on the graphene oxide layer to immobilize the silylating agent on the graphene oxide layer. Since the silanizing agent is easily hydrolyzed in water to form silicon hydroxyl groups, the surface of the silanized graphene oxide prepared by this step contains a large number of silicon hydroxyl groups, and then the perfluoroalkane chain is introduced through the silane polycondensation reaction to produce surface plasmons. Body resonance sensor chip.
  • the present invention uses mercaptoethylamine to form a strong gold-sulfur bond with the surface of the gold film, connects them through chemical action, and evenly assembles mercaptoethylamine molecules onto the gold film, and then fixes graphene oxide on the gold film through electrostatic action.
  • the present invention can make the distribution of graphene oxide more uniform, thereby ensuring the accuracy of SPR signal response during testing.
  • the chip prepared by the layer-by-layer assembly modification method of the present invention modifies the perfluoroalkane chain on the surface of the chip, and utilizes the fluorine-fluorine interaction between the perfluoroalkane chain and the perfluoro compound to realize the detection of the perfluoro compound.
  • the perfluorinated compound produces a recognition response
  • the SPR signal response of the chip of the present invention is more sensitive and the detection accuracy is higher.
  • the preparation method of the SPR sensor chip provided by the invention is easy to operate, low in cost, and can be prepared in batches.
  • step 1 is: plating gold film in each reaction well of the porous glass substrate.
  • the concentration of the graphene oxide aqueous solution is 1-5 mg/mL, and the dosage is 100-200 ⁇ L. Controlling the concentration of graphene oxide can avoid the problem of incomplete surface modification of the chip, affecting further modification in subsequent steps, or the problem that the thickness of the graphene oxide layer modified on the chip surface is relatively large. Since the SPR response of the chip surface is most sensitive within 200nm, it is very important to control the thickness of the modification layer. When 150 ⁇ L of 2 mg/mL graphene oxide solution was added, the thickness of the graphene oxide layer modified on the chip was about 50 nm, and the SPR response at this time was more sensitive.
  • the volume fraction of the silylating reagent solution is 5%-10%, and the dosage is 50-150 ⁇ L.
  • the use of the volume fraction of the silylating agent solution of the present invention can avoid the situation that the surface of the chip cannot be completely modified, or the modification layer is too thick or the silylating agent self-polymerizes in the solution.
  • the volume fraction of perfluorosilane in the perfluorosilane solution is 1%-10%, and the dosage is 100-200 ⁇ L.
  • the use of the perfluorosilane solution in the volume fraction of the present invention can avoid the situation that the surface of the chip cannot be completely modified, or the modification layer is too thick or the perfluorosilane self-polymerizes in the solution.
  • a perfluorinated compound detection method provided by the present invention uses an SPR sensor chip to quickly detect the perfluorinated compound pollution level in environmental water samples, and has good practical application value.
  • Fig. 1 is the schematic diagram of the preparation process of the surface plasmon resonance sensor chip in embodiment 1;
  • Fig. 2 is the concentration-SPR angle variation standard curve obtained in Example 2.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip, as shown in Figure 1, comprising the following steps:
  • Step 1 Coating a layer of gold film 2 in the reaction well pool of the six-hole slide substrate 1 by vacuum evaporation technology, the thickness of the gold film 2 is 50nm.
  • Step 2 modifying the graphene oxide layer 3 on the gold film:
  • Step 3 modifying the perfluoroalkane chain functionalized layer on the graphene oxide layer:
  • EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups, first prepare 1mg/mL EDC solution and 1mg/mL NHS solution were then mixed 1:1 by volume to obtain 0.5mg/mL EDC/NHS solution.
  • the silanization layer on the surface of the chip can undergo polycondensation reaction with perfluorooctyltriethoxysilane in the solution, so that the perfluorooctyltriethoxyalkane chain is grafted to the chip surface.
  • the solution is removed from the chip Take it out, wash it with ethanol for 3 times, and then wash it with ultrapure water for 3 times to prepare a surface plasmon resonance sensor chip modified with a perfluoroalkane chain functionalized layer.
  • This embodiment provides a method for detecting perfluorinated compounds, and the perfluorinated compound used in this embodiment is perfluorooctane carboxylic acid.
  • the surface plasmon resonance sensor chip prepared by Example 1 comprises the following steps:
  • the detection linear range of perfluorooctane carboxylic acid is 0.05-10pg/L.
  • the concentration is lower than 0.05pg/L, the SPR angle cannot change, that is, the detection limit is 0.05pg/L .
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip, as shown in Figure 1, comprising the following steps:
  • Step 1 Coating a layer of gold film 2 in the reaction well pool of the six-hole slide substrate 1 by vacuum evaporation technology, the thickness of the gold film 2 is 40nm.
  • Step 2 modifying the graphene oxide layer on the gold film:
  • Step 3 modifying the perfluoroalkane chain functionalized layer on the graphene oxide:
  • EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups.
  • NHS N-hydroxysuccinimide
  • the solution was Take it out from the chip, wash it with ethanol for 3 times, and then wash it with ultrapure water for 3 times to prepare a surface plasmon resonance sensor chip modified with a perfluoroalkane chain functionalized layer.
  • This embodiment provides a method for detecting perfluorinated compounds, and the perfluorinated compound used in this embodiment is perfluorooctane carboxylic acid.
  • the detection linear range of perfluorooctane carboxylic acid is 0.1-12pg/L.
  • concentration is lower than 0.1pg/L, the SPR angle cannot change, that is, the detection limit of the method is 0.1pg/L.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip, as shown in Figure 1, comprising the following steps:
  • Step 1 Coating a layer of gold film 2 in the reaction well pool of the six-hole slide substrate 1 by vacuum evaporation technology, the thickness of the gold film 2 is 60nm.
  • Step 2 modifying the graphene oxide layer on the gold film:
  • Step 3 modifying the perfluoroalkane chain functionalized layer on the graphene oxide layer:
  • EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups, firstly prepare 0.6mg/ mL EDC solution and 0.6mg/mL NHS solution, and then mixed 1:1 by volume to obtain 0.3mg/mL EDC/NHS solution.
  • the silanized layer on the surface of the chip can undergo polycondensation reaction with perfluorooctyltriethoxysilane in the solution, so that the perfluorooctyltriethoxyalkane chain is grafted to the surface of the chip.
  • the solution is taken out from the chip , washed with ethanol for 3 times, and then washed with ultrapure water for 3 times to prepare a surface plasmon resonance sensor chip modified with a perfluoroalkane chain functional layer.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip.
  • the operation steps are basically the same as in Example 1, the difference is that during the preparation of the sensor chip in this embodiment, in step 2, graphite oxide
  • the concentration of alkene solution is 0.5mg/mL.
  • the sensor chip prepared in this example is used to detect perfluorooctane carboxylic acid.
  • concentration is lower than 36 pg/L, the SPR angle cannot change, that is, the detection limit is 36 pg/L.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip.
  • the operation steps are basically the same as in Example 1, the difference is that during the preparation of the sensor chip in this embodiment, in step 2, graphite oxide
  • the concentration of alkene solution is 8mg/mL.
  • the sensor chip prepared in this embodiment is used for the detection of perfluorooctane carboxylic acid.
  • concentration is lower than 22pg/L, the SPR angle cannot change, that is, the detection limit is 22pg/L.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip.
  • the operation steps are basically the same as in Example 1. The difference is that in the preparation process of the sensor chip in this embodiment, in step 3, perfluoro
  • the volume fraction of the octyltriethoxysilane solution is 0.5%.
  • the sensor chip prepared in this example is used to detect perfluorooctane carboxylic acid.
  • concentration is lower than 34pg/L, the SPR angle cannot change, that is, the detection limit is 34pg/L.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor chip.
  • the operation steps are basically the same as in Example 1. The difference is that in the preparation process of the sensor chip in this embodiment, in step 3, perfluoro
  • the volume fraction of the octyltriethoxysilane solution is 15%.
  • the sensor chip prepared in this example is used to detect perfluorooctane carboxylic acid.
  • concentration is lower than 28pg/L, the SPR angle cannot change, that is, the detection limit is 28pg/L.
  • This comparative example provides a method for preparing a surface plasmon resonance sensor chip, comprising the following steps:
  • Step 1 Coating a layer of gold film 2 in the reaction well pool of the six-hole slide substrate 1 by vacuum evaporation technology, the thickness of the gold film 2 is 50nm.
  • Step 2 modifying mercaptopropyltriethoxysilane on the gold film:
  • Step 3 modifying the perfluoroalkane chain functionalized layer on the chip:
  • the surface plasmon resonance sensor chip prepared above that does not contain a graphene oxide layer is used to detect different concentrations of perfluorooctane carboxylic acid standard solutions, and the detection linear range of perfluorooctane carboxylic acid is 1-120 ⁇ g/L, When the concentration is lower than 1 ⁇ g/L, the SPR angle cannot change, that is, the detection limit is 1 ⁇ g/L.
  • This comparative example provides a method for preparing a surface plasmon resonance sensor chip, the steps of which are basically the same as in Example 1, except that the perfluoroalkane chain functionalized layer is not modified on the graphene oxide layer.
  • the sensor chip prepared in this comparative example was used for the detection of perfluorinated compounds.
  • the measured SPR angle did not change significantly, that is, the sensor chip prepared in this comparative example Chips cannot be used for perfluorinated compound detection.

Abstract

一种表面等离子体共振传感芯片及其制备方法和应用，属于环境污染物传感检测领域，解决了现有全氟化合物的检测方法不稳定、检出限高的问题。该表面等离子体共振传感芯片，由下至上包括六孔玻片基底（1）、金膜（2）、氧化石墨烯层（3）和全氟烷烃链功能化层。

Description

一种表面等离子体共振传感芯片及其制备方法和应用 技术领域
本发明属于环境污染物传感检测领域，具体涉及一种表面等离子体共振传感芯片及其制备方法和应用。
背景技术
全氟化合物具有热稳定性、化学稳定性以及良好的表面活性，在食品包装袋、不粘锅、纺织、防水剂、电镀剂、泡沫灭火剂等多种生产生活用品中获得了广泛的应用。但是，随着对全氟化合物的深入研究发现，它们具有严重的生物毒性效应，可以对生态环境和人类健康造成严重的危害。全氟化合物已经成为一类新型的持久性有机污染物，对其在环境中的污染状况研究迫在眉睫。因此建立有效的分析方法对环境中的全氟化合物进行及时检测是非常必要的。
目前，全氟化合物的检测方法主要是高效液相色谱-质谱联用和高效液相色谱串联质谱技术，虽然这些方法灵敏度较高、准确性较好，但是需要进行繁琐的样品前处理，消耗大量的有机溶剂，检测周期较长，检测成本较高。近年来也建立了一些紫外和荧光传感方法用于全氟化合物的快速检测，中国专利文献CN110749562A公开了一种双波长比率紫外光谱法测定全氟辛烷磺酸，该方法利用在酸性条件下，全氟辛烷磺酸与甲苯胺蓝发生静电作用形成一种离子缔合物，该离子缔合物在632nm和502nm出现两个特征峰并呈比率吸收，根据全氟辛烷磺酸溶液浓度与紫外双波长吸收比率值之间的关系，建立了一种测定全氟辛烷磺酸的双波长比率紫外光谱法。中国专利文献CN103558198B公开了一种环境水样中PFOS的荧光快速检测方法，利用PFOS可通过静电猝灭作用使金属配合物4-(甲基苯基)-2，2′：6′，2″-三联吡啶Zn(Ⅱ)的荧光产生淬灭，通过测定荧光光谱强度的变化，实现对环境水样中PFOS的快速荧光分析。这些方法都是基于液相中的探针材料对全氟化合物产生识别，导致光谱信号发生变化，虽然比质谱法更简便快速，但是灵敏度较低，探针的稳定性较差，在液相中的反应也容易受到相关物质的干扰。因此，建立更稳定、更灵敏的传感方法用于全氟化合物的快速检测是非常必要的。
发明内容
因此，为了解决现有全氟化合物的检测方法不稳定、检出限高的问题，本发明提供一种表面等离子体共振传感芯片及其制备方法和应用。
为此，本发明提供了以下技术方案。
第一方面，本发明提供了一种表面等离子体共振传感芯片，由下至上包括基底、金膜、氧化石墨烯层和全氟烷烃链功能化层。
进一步的，所述基底为多孔玻片基底。
进一步的，所述金膜厚度为40～60nm，所述氧化石墨烯层的厚度为50～100nm。
第二方面，本发明提供了一种表面等离子体共振传感芯片的制备方法，包括：
步骤1、在基底上镀一层金膜；
步骤2、在金膜上设置氧化石墨烯层：
将巯基乙胺乙醇溶液加至金膜上，反应，去除巯基乙胺乙醇溶液，得巯基乙胺修饰的金膜；将氧化石墨烯溶液加至巯基乙胺修饰的金膜上，去除氧化石墨烯溶液；
步骤3、在氧化石墨烯层上修饰全氟烷烃链功能化层：
将硅烷化试剂溶液和活化剂加至氧化石墨烯层上，反应，制得硅烷化的氧化石墨烯层；在硅烷化的氧化石墨烯层表面修饰全氟烷烃链，制得表面等离子体共振传感芯片。
进一步的，所述基底为多孔玻片基底，步骤1为：在多孔玻片基底的各反应孔池内镀金膜；优选地，采用真空蒸镀在反应孔池内镀金膜，所述金膜厚度为40～60nm。
进一步的，步骤2满足以下(1)-(5)中的至少一条：
(1)所述巯基乙胺乙醇溶液的浓度为0.1～0.5mg/mL，用量为50～150μL；
(2)巯基乙胺乙醇溶液与金膜反应2～4h；
(3)氧化石墨烯溶液的浓度为1～5mg/mL，用量为100～200μL；
(4)氧化石墨烯溶液与巯基乙胺修饰的金膜反应2～4h；
(5)氧化石墨烯溶液的溶剂为水、甲醇、乙醇中的至少一种，优选为水。
进一步的，步骤3满足以下(1)-(5)中的至少一条：
(1)所述硅烷化试剂为氨基丙基三乙氧基硅烷或氨基丙基三甲氧基硅烷；
(2)所述硅烷化试剂溶液的溶剂为水；
(3)硅烷化试剂溶液中硅烷化试剂的体积分数为5％-10％，硅烷化试剂溶液的用量为50～150μL；
(4)硅烷化试剂溶液与氧化石墨烯层反应2～3h；
(5)所述活化剂为EDC/NHS溶液或EDC溶液，所述活化剂中EDC和/或NHS的浓度为0.1-0.5mg/mL，用量为20～80μL。
进一步的，步骤3中，将全氟硅烷溶液和催化剂加到硅烷化的氧化石墨烯层上，反应；所述步骤3满足以下(1)-(5)中的至少一项：
(1)全氟硅烷溶液中全氟硅烷的体积分数为1％-10％，用量为100～200μL；
(2)催化剂为氨水，氨水质量浓度为25％～28％，用量为5～20μL；
(3)全氟硅烷为全氟辛基三乙氧基硅烷和/或全氟癸基三乙氧基硅烷；
(4)全氟硅烷溶液的溶剂为醇-水溶液，醇和水的体积比为(2～4)：1，优选地，醇为乙醇和/或甲醇；
(5)全氟硅烷溶液和氧化石墨烯层反应4-8h。
第三方面，本发明提供了上述表面等离子体共振传感芯片或根据上述制备方法制得的表面等离子体共振传感芯片在全氟化合物检测中的应用，优选地，所述全氟化合物选自全氟辛烷磺酸、全氟己酸、全氟壬酸、全氟辛烷羧酸中的至少一种。
表面等离子共振(SPR)是一种发生在界面处的物理光学现象，当光从光密介质射入光疏介质，入射角增大到某一角度时，可以使折射角达到90°，折射光完全消失，只剩下反射光，这种现象叫做全反射。当光在玻璃与金属薄膜界面处发生全反射时，在金属薄膜内会产生消逝波,同时金属薄膜内的自由电子又可以产生一定的等离子波，在入射角θ为某一适当值时,表面等离子体与消逝波可以发生共振，即SPR共振，使发射光强大幅度减弱，导致反射光完全消失，此时对应的入射角θ即为SPR角，SPR角会随金属薄膜表面折射率的变化而变化。如果在金属表面固定特殊的分子，当流过金属表面的溶液中含有能与之作用的物质时,它们之间发生相互作用将导致金属表面电介质的折射率发生变化，从而使SPR角发生改变。本发明通过测定SPR角的变化量，实现对溶液中目标物质的定量检测。
第四方面，本发明提供了一种全氟化合物检测方法，包括以下步骤：
(1)获取浓度-SPR角变化量标准曲线：
将表面等离子体共振传感芯片装入表面等离子体共振传感仪中，注入空白的溶剂后测定初始SPR角α 0；所述溶剂为水、乙醇或甲醇，更优选为水；
制备N份含不同浓度全氟化合物的标准溶液，N≥3；将各标准溶液分别注入表面等离子体共振传感芯片的反应孔池中，测定各标准溶液的SPR角α 1～α n；得到各标准溶液SPR角相对于初始SPR角的变化量Δα n，Δα n＝α n-α 0；
Δα n与全氟化合物浓度成线性关系，制作浓度-SPR角变化量标准曲线；
(2)将待测样品注入表面等离子体共振传感芯片的反应孔池中，测定待测样品的SPR角，得SPR角的变化量，带入浓度-SPR角变化量标准曲线，即得待测样品中全氟化合物的浓度。
本发明技术方案，具有如下优点：
1.本发明提供的表面等离子体共振传感芯片，由下至上包括基底、金膜、氧化石墨烯层和全氟烷烃链功能化层。全氟烷烃链功能化层可与待测样品中的全氟化合物发生特异性的氟-氟相互作用，对全氟化合物进行特异性识别，引起芯片表面全氟烷烃链功能化层介质折射率产生敏感响应，从而使SPR角发生改变。
与液相中的探针传感方法相比，SPR传感是在固相芯片表面对目标物产生识别响应，芯片表面的探针功能化层更稳定、无需标记、背景信号或噪音信号较低，不易受到干扰，稳定性好。
氧化石墨烯层可以显著提高检测灵敏度。由于芯片表面介质折射率变化非常敏感，当低浓度的全氟化合物溶液通过芯片表面时，即可以引起SPR角发生改变，检出限可以达到pg/L，灵敏度高。
2.本发明提供的表面等离子体共振传感芯片，基底为多孔玻片基底，一个传感芯片上可以同时检测多个样品，实现批量样品的快速检测；并且可以根据需要，对多个反应孔池进行不同的功能化修饰，用于对不同目标物的测定。
3.本发明提供的表面等离子体共振传感芯片，金膜厚度为40～60nm，所述氧化石墨烯层的厚度为50～100nm。在SPR传感方法中，采用的SPR传感芯片表面的功能化层厚度一般在200nm以内可以产生较灵敏的响应，本发明控制金膜和氧化石墨烯层的总厚度小于200nm。
4.本发明提供的表面等离子体共振传感芯片的制备方法，包括：步骤1、在基底上镀一层金膜；步骤2、在金膜上设置氧化石墨烯层：将巯基乙胺乙醇溶液加至金膜上，反应，去除巯基乙胺乙醇溶液，得巯基乙胺修饰的金膜；将氧化石墨烯溶液加至巯基乙胺修饰的金膜上，去除氧化石墨烯溶液；步骤3、在氧化石墨烯层上修饰全氟烷烃链功能化层：将硅烷化试剂溶液和活化剂加至氧化石墨烯层上，反应，制得硅烷化的氧化石墨烯层；在硅烷化的氧化石墨烯层表面修饰全氟烷烃链，制得表面等离子体共振传感芯片。
通过加入氧化石墨烯层可以显著提高检测灵敏度，并且所需的样品量非常少，避免使用大量的有机溶剂，具有更绿色环保的优点。氧化石墨烯上含有大量的羧基和羟基，使其具有电负性和亲水性，在水中具有良好的分散性。本发明步骤2中，巯基乙胺中的巯基可以与金膜形成金-硫键，使巯基乙胺分子固定到金膜上，巯基乙胺分子中的氨基暴露在表面；当将氧化石墨烯溶液滴加到巯基乙胺修饰的金膜时，带负电的氧化石墨烯可以与氨基发生静电相互作用，使氧化石墨烯固定到芯片表面，氧化石墨烯可以提高SPR传感的灵敏度。
本发明步骤3中，硅烷化试剂的氨基与氧化石墨烯层上的羧基发生酰胺反应，使硅烷化试剂固定在氧化石墨烯层上。由于硅烷化试剂在水中容易发生水解形成硅羟基，因此通过该步反应后制备得到的硅烷化的氧化石墨烯表面含有大量的硅羟基，然后通过硅烷缩聚反应引入全氟烷烃链，制得表面等离子体共振传感芯片。
本发明利用巯基乙胺与金膜表面形成强的金-硫键，通过化学作用连接，将巯基乙胺分子均匀的组装到金膜上，再通过静电作用将氧化石墨烯固定到金膜上，相较于物理涂敷方法，本发明可以使氧化石墨烯的分布更均匀，从而保障测试时SPR信号响应的精确性。
本发明通过层层组装修饰的方法制备的芯片，将全氟烷烃链修饰到芯片表面，利用全氟烷烃链与全氟化合物之间的氟-氟相互作用，实现对全氟化合物的检测。相较于将全氟硅烷与其他具有粘结作用的粘结剂共混后再旋涂到芯片表面，不能确保全氟烷烃链会露在芯片表面，可能会使其包埋到内部，不能对全氟化合物产生识别响应的情况，本发明芯片SPR信号响应更灵敏，检测精度更高。
本发明提供的SPR传感芯片的制备方法操作简便，成本低廉，可以批量制备。
5.本发明提供的表面等离子体共振传感芯片的制备方法，采用的基底为多孔玻片基底；步骤1为：在多孔玻片基底的各反应孔池内镀金膜。
在现有技术中，对传感芯片进行修饰制备时，是将整个芯片浸没到试剂溶液中，溶液中的功能分子依靠一定的作用力固定到芯片表面，但是这种修饰方法是不可控的。会使芯片表面修饰的功能分子分布不均匀，从而影响芯片的检测性能。在本发明中采用含有多个反应孔池的玻片作为基底，每一个反应孔作为一个独立的传感区域，并对每一个反应孔池进行功能化修饰。在对每个反应孔芯片表面进行修饰过程中，是将每一步的反应溶液滴加到反应孔池内单独进行反应，与将整个芯片浸没到反应溶液相比，这样可以减小反应面积，确保功能分子可以修饰到芯片表面，从而提高反应效率，并且使用的反应溶液量也大大减少，具有更加节能环保的优点。
6.本发明提供的表面等离子体共振传感芯片的制备方法，氧化石墨烯水溶液的浓度为1～5mg/mL，用量为100～200μL。控制氧化石墨烯的浓度可避免芯片表面修饰不完全，影响后续步骤的进一步修饰，或芯片表面修饰的氧化石墨烯层厚度较大的问题。由于芯片表面的SPR响应在200nm内是最灵敏的，因此控制修饰层的厚度是非常重要的。当加入150μL 2mg/mL氧化石墨烯溶液时，修饰到芯片上的氧化石墨烯层厚度在50nm左右，此时的SPR响应较灵敏。
7.本发明提供的表面等离子体共振传感芯片的制备方法，硅烷化试剂溶液的体积分数为5％-10％，用量为50～150μL。采用本发明体积分数的硅烷化试剂溶液，可避免芯片表面不能被完全修饰，或修饰层过厚或硅烷化试剂在溶液中自聚的情况。
8.本发明提供的表面等离子体共振传感芯片的制备方法，全氟硅烷溶液中全氟硅烷的体积分数为1％-10％，用量为100～200μL。采用本发明体积分数的全氟硅烷溶液可避免芯片表面不能被完全修饰，或修饰层过厚或全氟硅烷在溶液中自聚的情况。
9.本发明提供的一种全氟化合物检测方法，采用SPR传感芯片对环境水样中的全氟化合物污染水平进行快速检测，具有良好的实际应用价值。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是实施例1中表面等离子体共振传感芯片的制备过程示意图；
图2是实施例2获取浓度-SPR角变化量标准曲线。
附图标记：
1-六孔玻片基底；2-金膜；3-氧化石墨烯层。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1
本实施例提供了一种表面等离子体共振传感芯片的制备方法，如图1所示，包括以下步骤：
步骤1、采用真空蒸镀技术在六孔玻片基底1的反应孔池内镀上一层金膜2，金膜2的厚度为50nm。
步骤2、在金膜上修饰氧化石墨烯层3：
(1)在5mL乙醇中加入1mg巯基乙胺，配制得到0.2mg/mL的巯基乙胺乙醇溶液，在六孔玻片基底1的六个反应孔池内分别加入100μL巯基乙胺乙醇溶液，静置2h；然后将溶液从反应孔池中取出，用乙醇冲洗3次，再用超纯水冲洗3次，得到巯基乙胺修饰的传感芯片；
(2)将固体氧化石墨烯片分散到超纯水中，超声(超声波频率为40kHz)2h后，得到均匀分散的氧化石墨烯溶液，氧化石墨烯溶液浓度为2mg/mL。
(3)在步骤(1)制备的巯基乙胺修饰的传感芯片的六个孔池内分别加入150μL氧化石墨烯溶液，静置4h，然后将溶液从反应孔池中取出，用超纯水冲洗3次，得到氧化石墨烯修饰的传感芯片；
步骤3、在氧化石墨烯层上修饰全氟烷烃链功能化层：
(1)在4.6mL纯水中加入0.4mL氨基丙基三乙氧基硅烷，配制得到体积分数8％的氨基丙基三乙氧基硅烷溶液，然后在氧化石墨烯修饰的传感芯片的六个孔池内分别加入100μL氨基丙基三乙氧基硅烷溶液，并分别加入50μL 0.5mg/mL的EDC/NHS溶液，静置3h后，将溶液从反应孔池中取出，用超纯水冲洗3次，得到硅烷化的传感芯片。
EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺)、NHS(N-羟基琥珀酰亚胺)是可以促进氨基和羧基反应的活化剂，先配制1mg/mL EDC溶液和1mg/mL NHS溶液，然后按体积1：1混合后得到0.5mg/mL的EDC/NHS溶液。
(2)在4.75mL体积比为3：1的乙醇-水中加入0.25mL全氟辛基三乙氧基硅烷，配制得到体积分数5％的全氟辛基三乙氧基硅烷溶液，在上述芯片的六个反应孔池内分别加入150μL全氟辛基三乙氧基硅烷溶液，并分别加入10μL氨水(氨水为市售的工业氨水，质量浓度为25％～28％)，在氨水的催化作用下，芯片表面的硅烷化层可以与溶液中的全氟辛基三乙氧基硅烷发生缩聚反应，使全氟辛基三乙氧基烷烃链接枝到芯片表面，反应6h后，将溶液从芯片中取出，用乙醇冲洗3次，再用超纯水冲洗3次，制备得到全氟烷烃链功能化层修饰的表面等离子体共振传感芯片。
实施例2
本实施例提供了一种全氟化合物检测方法，本实施例中的全氟化合物选用的是全氟辛烷羧酸。
采用实施例1制备的表面等离子体共振传感芯片，包括以下步骤：
(1)获取浓度-SPR角变化量标准曲线：
将表面等离子体共振传感芯片装入表面等离子体共振传感仪中，在反应孔池内注入纯水空白溶剂后测定初始SPR角α 0＝65.1°；
采用纯水制备8份浓度分别为0.03pg/L、0.05pg/L、0.2pg/L、0.6pg/L、1pg/L、3pg/L、5pg/L、10pg/L的全氟辛烷羧酸标准溶液。
将各标准溶液分别注入10μL到表面等离子体共振传感芯片的反应孔池中，测定各标准溶液的SPR角分别为α 1＝65.1°、α 2＝65.3°、α 3＝65.4°、α 4＝65.7°、α 5＝65.9°、α 6＝66.6°、α 7＝67.2°、α 8＝68.8°；得到各标准溶液SPR角相对于初始SPR角的变化量Δα 1＝0°、Δα 2＝0.2°、Δα 3＝0.3°、Δα 4＝0.6°、Δα 5＝0.8°、Δα 6＝1.5°、Δα 7＝2.1°、Δα 8＝3.7°，制作浓度-SPR角变化量标准曲线，如图2所示。
本实施例对全氟辛烷羧酸的检测线性范围为0.05-10pg/L，本实施例中当浓度低于0.05pg/L时，SPR角不能发生变化，即检出限为0.05pg/L。
(2)将环境水样注入10μL到表面等离子体共振传感芯片的反应孔池中，测定待测样品的SPR角为66.9°，得SPR角的变化量为1.8°，带入浓度-SPR角变化量标准曲线，即得环境水样中全氟辛烷羧酸的浓度为4.3pg/L。
实施例3
本实施例提供了一种表面等离子体共振传感芯片的制备方法，如图1所示，包括以下步骤：
步骤1、采用真空蒸镀技术在六孔玻片基底1的反应孔池内镀上一层金膜2，金膜2的厚度为40nm。
步骤2、在金膜上修饰氧化石墨烯层：
(1)在5mL乙醇中加入0.5mg巯基乙胺，配制得到0.1mg/mL的巯基乙胺乙醇溶液，在六孔玻片基底1的六个反应孔池内分别加入50μL巯基乙胺乙醇溶液，静置2h；然后将溶液从反应孔池中取出，用乙醇冲洗3次，再用超纯水冲洗3次，得到巯基乙胺修饰的传感芯片；
(2)将固体氧化石墨烯片分散到超纯水中，超声2h后，得到均匀分散的氧化石墨烯溶液，氧化石墨烯溶液浓度为1mg/mL。
(3)在步骤(1)制备的巯基乙胺修饰的传感芯片的六个孔池内分别加入100μL氧化石墨烯溶液，静置4h，然后将溶液从反应孔池中取出，用超纯水冲洗3次，得到氧化石墨烯修饰的传感芯片；
步骤3、在氧化石墨烯上修饰全氟烷烃链功能化层：
(1)在4.75mL纯水中加入0.25mL氨基丙基三乙氧基硅烷，配制得到体积分数5％的氨基丙基三乙氧基硅烷溶液，然后在氧化石墨烯修饰的传感芯片的六个孔池内分别加入50μL氨基丙基三乙氧基硅烷溶液，并分别加入50μL 0.1mg/mL的EDC/NHS溶液，静置3h后，将溶液从反应孔池中取出，用超纯水冲洗3次，得到硅烷化的传感芯片。
EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺)、NHS(N-羟基琥珀酰亚胺)是可以促进氨基和羧基反应的活化剂，先配制0.2mg/mL EDC溶液和0.2mg/mL NHS溶液，然后按体积1：1混合后得到0.1mg/mL的EDC/NHS溶液。
(2)在4.5mL体积比为2:1的乙醇-水中加入0.5mL全氟辛基三乙氧基硅烷，配制得到体积分数10％的全氟辛基三乙氧基硅烷溶液，在上述芯片的六个反应孔池内分别加入100μL全氟辛基三乙氧基硅烷溶液，并分别加入5μL氨水(氨水为市售的工业氨水，质量浓度为25％～28％)，反应6h后，将溶液从芯片中取出，用乙醇冲洗3次，再用超纯水冲洗3次，制备得到全氟烷烃链功能化层修饰的表面等离子体共振传感芯片。
实施例4
本实施例提供了一种全氟化合物检测方法，本实施例中的全氟化合物选用的是全氟辛烷羧酸。
采用实施例3制备的表面等离子共振传感芯片，包括以下步骤：
(1)获取浓度-SPR角变化量标准曲线：
将表面等离子体共振传感芯片装入表面等离子体共振传感仪中，在反应孔池内注入纯水空白溶剂后测定初始SPR角α 0＝64.9°；
采用纯水制备8份浓度分别为0.05pg/L、0.1pg/L、0.2pg/L、0.5pg/L、1pg/L、4pg/L、8pg/L、12pg/L的全氟辛烷羧酸标准溶液。
将各标准溶液分别注入10μL到表面等离子体共振传感芯片的反应孔池中，测定各标准溶液的SPR角分别为α 1＝64.9°、α 2＝65°、α 3＝65.2°、α 4＝65.4°、α 5＝65.5°、α 6＝66.2°、α 7＝67.1°、α 8＝68.3°；得到各标准溶液SPR角相对于初始SPR角的变化量Δα 1＝0°、Δα 2＝0.1°、Δα 3＝0.3°、Δα 4＝0.5°、Δα 5＝0.6°、Δα 6＝1.3°、Δα 7＝2.2°、Δα 8＝3.4°，制作浓度-SPR角变化量标准曲线。本实施例对全氟辛烷羧酸的检测线性范围为0.1-12pg/L，当浓度低于0.1pg/L时，SPR角不能发生变化，即方法的检出限为0.1pg/L。
(2)将环境水样注入10μL到表面等离子体共振传感芯片的反应孔池中，测定待测样品的SPR角为67.3°，得SPR角的变化量为2.4°，带入浓度-SPR角变化量标准曲线，即得环境水样中全氟辛烷羧酸的浓度为8.3pg/L。
实施例5
本实施例提供了一种表面等离子体共振传感芯片的制备方法，如图1所示，包括以下步骤：
步骤1、采用真空蒸镀技术在六孔玻片基底1的反应孔池内镀上一层金膜2，金膜2的厚度为60nm。
步骤2、在金膜上修饰氧化石墨烯层：
(1)在5mL乙醇中加入2.5mg巯基乙胺，配制得到0.5mg/mL的巯基乙胺乙醇溶液，在六孔玻片基底1的六个反应孔池内分别加入150μL巯基乙胺乙醇溶液，静置3h；然后将溶液从反应孔池中取出，用乙醇冲洗3次，再用超纯水冲洗3次，得到巯基乙胺修饰的传感芯片；
(2)将固体氧化石墨烯片分散到超纯水中，超声(超声波频率为40kHz)2h后，得到均匀分散的氧化石墨烯溶液，氧化石墨烯溶液浓度为5mg/mL。
(3)在步骤(1)制备的巯基乙胺修饰的传感芯片的六个孔池内分别加入200μL氧化石墨烯溶液，静置2h，然后将溶液从反应孔池中取出，用超纯水冲洗3次，得到氧化石墨烯修饰的传感芯片；
步骤3、在氧化石墨烯层上修饰全氟烷烃链功能化层：
(1)在4.5mL纯水中加入0.5mL氨基丙基三乙氧基硅烷，配制得到体积分数10％的氨基丙基三乙氧基硅烷溶液，然后在氧化石墨烯修饰的传感芯片的六个孔池内分别加入150μL氨基丙基三乙氧基硅烷溶液，并分别加入80μL 0.3mg/mL的EDC/NHS溶液，静置2h后，将溶液从反应孔池中取出，用超纯水冲洗3次，得到硅烷化的传感芯片。
EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺)、NHS(N-羟基琥珀酰亚胺)是可以促进氨基和羧基反应的活化剂，先配制0.6mg/mL EDC溶液和0.6mg/mL NHS溶液，然后按体积1：1混合后得到0.3mg/mL的EDC/NHS溶液。
(2)在4.9mL体积比4：1的乙醇-水中加入0.1mL全氟辛基三乙氧基硅烷，配制得到体积分数2％的全氟辛基三乙氧基硅烷溶液，在上述芯片的六个反应孔池内分别加入200μL全氟辛基三乙氧基硅烷溶液，并分别加入20μL氨水(氨水为市售的工业氨水，质量浓度为25％～28％)，在氨水的催化作用下，芯片表面的硅烷化层可以与溶液中的全氟辛基三乙氧基硅烷发生缩聚反应，使全氟辛基三乙氧基烷烃链接枝到芯片表面，反应8h后，将溶液从芯片中取出，用乙醇冲洗3次，再用超纯水冲洗3次，制备得到全氟烷烃链功能化层修饰的表面等离子体共振传感芯片。
实施例6
本实施例提供了一种表面等离子体共振传感芯片的制备方法，操作步骤与实施例1基本相同，不同之处在于，本实施例中传感芯片的制备过程中，步骤2中，氧化石墨烯溶液浓度为0.5mg/mL。
将本实施例制备的传感芯片用于对全氟辛烷羧酸的检测，当浓度低于36pg/L时，SPR角不能发生变化，即检出限为36pg/L。
实施例7
本实施例提供了一种表面等离子体共振传感芯片的制备方法，操作步骤与实施例1基本相同，不同之处在于，本实施例中传感芯片的制备过程中，步骤2中，氧化石墨烯溶液浓度为8mg/mL。
将本实施例制备的传感芯片用于对全氟辛烷羧酸的检测，当浓度低于22pg/L时，SPR角不能发生变 化，即检出限为22pg/L。
实施例8
本实施例提供了一种表面等离子体共振传感芯片的制备方法，操作步骤与实施例1基本相同，不同之处在于，本实施例中传感芯片的制备过程中，步骤3中，全氟辛基三乙氧基硅烷溶液的体积分数为0.5％。
将本实施例制备的传感芯片用于对全氟辛烷羧酸的检测，当浓度低于34pg/L时，SPR角不能发生变化，即检出限为34pg/L。
实施例9
本实施例提供了一种表面等离子体共振传感芯片的制备方法，操作步骤与实施例1基本相同，不同之处在于，本实施例中传感芯片的制备过程中，步骤3中，全氟辛基三乙氧基硅烷溶液的体积分数为15％。
将本实施例制备的传感芯片用于对全氟辛烷羧酸的检测，当浓度低于28pg/L时，SPR角不能发生变化，即检出限为28pg/L。
对比例1
本对比例提供了一种表面等离子体共振传感芯片的制备方法，包括以下步骤：
步骤1、采用真空蒸镀技术在六孔玻片基底1的反应孔池内镀上一层金膜2，金膜2的厚度为50nm。
步骤2、在金膜上修饰巯基丙基三乙氧基硅烷：
在4.5mL乙醇中加入0.5mL巯基丙基三乙氧基硅烷，配制得到10％的巯基丙基三乙氧基硅烷乙醇溶液，在六孔玻片基底1的六个反应孔池内分别加入100μL巯基丙基三乙氧基硅烷乙醇溶液，静置2h；然后将溶液从反应孔池中取出，用乙醇冲洗3次，再用超纯水冲洗3次，得到巯基丙基三乙氧基硅烷修饰的传感芯片。
步骤3、在芯片上修饰全氟烷烃链功能化层：
在4.75mL体积比3：1的乙醇-水中加入0.25mL全氟辛基三乙氧基硅烷，配制得到体积分数5％的全氟辛基三乙氧基硅烷溶液，在上述芯片的六个反应孔池内分别加入150μL全氟辛基三乙氧基硅烷溶液，并分别加入10μL氨水，在氨水的催化作用下，芯片表面的硅烷化层可以与溶液中的全氟辛基三乙氧基硅烷发生缩聚反应，使全氟辛基三乙氧基烷烃链接枝到芯片表面，反应6h后，将溶液从芯片中取出，用乙醇冲洗3次，再用超纯水冲洗3次，制备得到全氟烷烃链功能化层修饰的表面等离子体共振传感芯片。
采用上述制备的不含有氧化石墨烯层的表面等离子体共振传感芯片检测不同浓度的全氟辛烷羧酸标准溶液，得到对全氟辛烷羧酸的检测线性范围为1-120μg/L，当浓度低于1μg/L时，SPR角不能发生变化，即检出限为1μg/L。
对比例2
本对比例提供了一种表面等离子体共振传感芯片的制备方法，与实施例1的步骤基本相同，不同之处在于没有在氧化石墨烯层上修饰全氟烷烃链功能化层。采用本对比例中制备的传感芯片用于全氟化合物检测，当在芯片的反应孔池内注入全氟化合物溶液时，测得的SPR角没有发生明显改变，即该对比例中制备的传感芯片不能用于全氟化合物检测。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种表面等离子体共振传感芯片，其特征在于，由下至上包括基底、金膜、氧化石墨烯层和全氟烷烃链功能化层。
  2. 根据权利要求1所述的表面等离子体共振传感芯片，其特征在于，所述基底为多孔玻片基底。
  3. 根据权利要求1所述的表面等离子体共振传感芯片，其特征在于，所述金膜厚度为40～60nm，所述氧化石墨烯层的厚度为50～100nm。
  4. 一种表面等离子体共振传感芯片的制备方法，其特征在于，包括：
    步骤1、在基底上镀一层金膜；
    步骤2、在金膜上设置氧化石墨烯层：
    将巯基乙胺乙醇溶液加至金膜上，反应，去除巯基乙胺乙醇溶液，得巯基乙胺修饰的金膜；将氧化石墨烯溶液加至巯基乙胺修饰的金膜上，去除氧化石墨烯溶液；
    步骤3、在氧化石墨烯层上修饰全氟烷烃链功能化层：
    将硅烷化试剂溶液和活化剂加至氧化石墨烯层上，反应，制得硅烷化的氧化石墨烯层；在硅烷化的氧化石墨烯层表面修饰全氟烷烃链，制得表面等离子体共振传感芯片。
  5. 根据权利要求4所述的表面等离子体共振传感芯片的制备方法，其特征在于，所述基底为多孔玻片基底，步骤1为：在多孔玻片基底的各反应孔池内镀金膜；优选地，采用真空蒸镀在反应孔池内镀金膜，所述金膜厚度为40～60nm。
  6. 根据权利要求4所述的表面等离子体共振传感芯片的制备方法，其特征在于，步骤2满足以下(1)-(5)中的至少一条：
    (1)所述巯基乙胺乙醇溶液的浓度为0.1～0.5mg/mL，用量为50～150μL；
    (2)巯基乙胺乙醇溶液与金膜反应2～4h；
    (3)氧化石墨烯溶液的浓度为1～5mg/mL，用量为100～200μL；
    (4)氧化石墨烯溶液与巯基乙胺修饰的金膜反应2～4h；
    (5)氧化石墨烯溶液的溶剂为水、甲醇、乙醇中的至少一种，优选为水。
  7. 根据权利要求4所述的表面等离子体共振传感芯片的制备方法，其特征在于，步骤3满足以下(1)-(5)中的至少一条：
    (1)所述硅烷化试剂为氨基丙基三乙氧基硅烷或氨基丙基三甲氧基硅烷；
    (2)所述硅烷化试剂溶液的溶剂为水；
    (3)硅烷化试剂溶液中硅烷化试剂的体积分数为5％-10％，硅烷化试剂溶液的用量为50～150μL；
    (4)硅烷化试剂溶液与氧化石墨烯层反应2～3h；
    (5)所述活化剂为EDC/NHS溶液或EDC溶液，所述活化剂中EDC和/或NHS的浓度为0.1-0.5mg/mL，用量为20～80μL。
  8. 根据权利要求7所述的表面等离子体共振传感芯片的制备方法，其特征在于，步骤3中，将全氟硅烷溶液和催化剂加到硅烷化的氧化石墨烯层上，反应；所述步骤3满足以下(1)-(5)中的至少一项：
    (1)全氟硅烷溶液中全氟硅烷的体积分数为1％-10％，用量为100～200μL；
    (2)催化剂为氨水，氨水质量浓度为25％～28％，用量为5～20μL；
    (3)全氟硅烷为全氟辛基三乙氧基硅烷和/或全氟癸基三乙氧基硅烷；
    (4)全氟硅烷溶液的溶剂为醇-水溶液，醇和水的体积比为(2～4)：1，优选地，醇为乙醇和/或甲醇；
    (5)全氟硅烷溶液和氧化石墨烯层反应4-8h。
  9. 权利要求1-3任一项所述的表面等离子体共振传感芯片或根据权利要求4-8任一项所述的制备方法制得的表面等离子体共振传感芯片在全氟化合物检测中的应用，优选地，所述全氟化合物选自全氟辛烷磺酸、全氟己酸、全氟壬酸、全氟辛烷羧酸中的至少一种。
  10. 一种全氟化合物检测方法，其特征在于，包括以下步骤：
    (1)获取浓度与SPR角变化量标准曲线：
    将表面等离子体共振传感芯片装入表面等离子体共振传感仪中，注入空白溶剂后测定初始SPR角α 0；所述溶剂为水、乙醇或甲醇，更优选为水；
    制备N份含不同浓度全氟化合物的标准溶液，N≥3；将各标准溶液分别注入表面等离子体共振传感芯片的反应孔池中，测定各标准溶液的SPR角α 1～α n；得到各标准溶液SPR角相对于初始SPR角的变化量Δα n，Δα n＝α n-α 0；
    Δα n与全氟化合物浓度成线性关系，制作浓度与SPR角变化量标准曲线；
    (2)将待测样品注入表面等离子体共振传感芯片的反应孔池中，测定待测样品的SPR角，得SPR角的变化量，带入浓度与SPR角变化量标准曲线，即得待测样品中全氟化合物的浓度。
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