WO2015101118A1 - 一种基于三维结构纳米阵列生物芯片的制备方法及其应用 - Google Patents

一种基于三维结构纳米阵列生物芯片的制备方法及其应用 Download PDF

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WO2015101118A1
WO2015101118A1 PCT/CN2014/091814 CN2014091814W WO2015101118A1 WO 2015101118 A1 WO2015101118 A1 WO 2015101118A1 CN 2014091814 W CN2014091814 W CN 2014091814W WO 2015101118 A1 WO2015101118 A1 WO 2015101118A1
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solution
biochip
gold film
film
gold
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PCT/CN2014/091814
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French (fr)
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孙树清
凡勇
丁宇
何永红
马辉
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清华大学深圳研究生院
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic

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  • the present invention relates to a three-dimensional structure nano-array biochip and a preparation method thereof, and in particular, the present invention relates to a three-dimensional structure silica nanotube array biochip and a preparation method thereof, and a biochip thereof Application of biomolecule-specific detection.
  • biosensing based on fluorescent signal detection can be very sensitive, and even individual biomolecules can be detected.
  • fluoro-based biosensing has some drawbacks.
  • labeling biomolecules with fluorescent groups is very expensive and time consuming, and this method is not even possible in some applications.
  • labeling the fluorescent group may affect certain functions of the original biomolecule. Therefore, it is more important to use a non-labeled method to directly detect biomolecule sensors.
  • non-labeled biosensors based on different types of nanostructures have been extensively studied, and these sensors have benefited from the characteristics of nanotechnology.
  • biosensors based on nanowires, nanopores, nanotubes or nanocolumns there are biosensors based on nanowires, nanopores, nanotubes or nanocolumns.
  • Different types of non-labeled biosensors can simply and quickly convert the response of the interaction between the probe molecule and the target molecule into an optical signal, an electrical signal, a thermal signal, or an acoustic signal.
  • signal conversion methods fall into two main categories: optical interference and surface plasmons.
  • An optical waveguide sensor based on a nano-film structure is a typical optical interference biosensor. It has two main advantages.
  • the electromagnetic field of the incident light is confined in the nano-film layer in an enhanced optical waveguide mode, and can effectively cover the biomolecules adsorbed in the thin layer.
  • the nano-film has a large structural characteristic of adsorbing the surface area of the biomolecule, thereby enriching the biomolecule, the response of the biosensor can be further improved.
  • the sensitivity of unlabeled biosensors is not very high relative to biosensors based on fluorescent signal detection. Accordingly, how to improve the response of unlabeled biosensors requires further research.
  • the invention provides a biochip based on a three-dimensional silica nanotube array structure and a preparation method thereof, and an application thereof in the aspect of biomolecule-specific detection.
  • the invention provides a preparation method of a three-dimensional structure nano-array biochip, the method comprising the following steps:
  • the present invention also provides a biochip of a three-dimensional silica nanotube array structure prepared by the above method.
  • the invention also provides the application of the biochip of the three-dimensional silica nanotube array structure prepared by the above method in biomolecule-specific detection.
  • the gold film is treated with a silane solution containing a mercapto group, and then treated with an acid solution to obtain a hydrophilic surface, and then the through-anode aluminum oxide film is attached to the gold film, so that the gold film is pro
  • the water can enhance the connection between the film and the gold film, and then obtain a silica tube (column) by alternately immersing in a mixture of silicon tetrachloride (SiCl 4 ), n-hexane, n-hexane and methanol, ethanol and water for a certain number of times.
  • the obtained gold substrate is immersed in an acid solution to obtain a biochip of a three-dimensional silica nanotube array structure.
  • the method provided by the invention is easy to prepare a three-dimensional array of nanotubes (columns) on a substrate, and the size of the tube (column) is controllable, and can be used for biosensor to perform high-sensitivity, non-labeled biological detection.
  • Example 1 is a scanning electron micrograph (SEM) of a biochip interface of a three-dimensional silica nanotube array structure prepared in Example 1 of the present invention
  • Example 2 is a surface scanning electron micrograph (SEM) of an anodized aluminum film of a through hole used in Example 1 of the present invention
  • FIG. 3 is a perspective view of a reflectance of a biochip-specific detection streptavidin of a three-dimensional silica nanotube array structure prepared according to the method of Application Example 1 of the present invention
  • the invention provides a preparation method of a three-dimensional structure nano-array biochip, the method comprising the following steps:
  • the gold film obtained by the step (3) is sequentially immersed in a mixture of silicon tetrachloride (SiCl 4 ), n-hexane, n-hexane and methanol, ethanol and water using a sol-gel method (this process) Can be cycled multiple times);
  • the gold substrate obtained in the step (4) is treated with an acid solution to remove the aluminum oxide film.
  • the gold film is a glass base material having a gold layer on its surface.
  • the base material may be plated with a thickness of 35-50 nm, preferably 40-45 nm, using magnetron sputtering evaporation or thermal evaporation.
  • the mercaptosilane solution is a (3-mercaptopropyl)trimethoxysilane solution, and the solution has a concentration of 1 to 30 mM, preferably 18 to 22 mM.
  • the soaking time in the solution is 3-12 hours, preferably 3 hours.
  • the mercaptosilane can be dissolved in an organic solvent to form an organic solution of mercaptosilane, wherein the organic solvent for dissolving the mercaptosilane is not particularly required, and may be an organic solvent well known in the art, for example, methanol.
  • One or more of ethanol or acetone preferably, may be methanol.
  • the acid solution may be an aqueous hydrochloric acid solution having a concentration of 0.05 to 0.2 mol, preferably 0.1 mol.
  • the treatment time in the solution is from 1 to 20 hours, preferably from 10 to 12 hours, and the temperature is room temperature.
  • the anodized aluminum oxide film of the through hole and the gold film substrate obtained by the step (2) may be immersed in a mixed solution of acetone or acetone and water.
  • the solution is a mixture of acetone and water
  • the volume ratio of acetone to water may be from 1 to 3:1, preferably 1:1.
  • the alumina film is coated on the gold film substrate and then placed in a dry box at room temperature for 24-48 hours, preferably 24-30 hours.
  • the gold film substrate treated in the step (3) is sequentially immersed in a mixture of silicon tetrachloride (SiCl 4 ), n-hexane, n-hexane and methanol, ethanol and water.
  • the immersion time in the silicon tetrachloride is 1-2 minutes, and in other solutions, it is 3-5 minutes, preferably 5 minutes.
  • the volume ratio of n-hexane to methanol was 1:1.
  • Step (4) is a cycle of the surface sol-gel method.
  • the acid solution used is a phosphoric acid solution at a concentration of 5-15. % by mass, preferably 10 to 12% by mass.
  • the temperature is 20-40 °C.
  • the present invention also provides a biochip of a three-dimensional silica nanotube array structure prepared by the above method.
  • the invention also provides the application of the biochip of the three-dimensional silica nanotube array structure prepared by the above method in biomolecule-specific detection.
  • 3-aminopropyltriethoxysilane was purchased from Alfa Aesar; biotin (Sulfo-NHS-LC-LC-Biotin) was purchased from Shanghai Haoran Biotechnology Co., Ltd.; streptavidin ( Streptavidin) was purchased from Beijing Boaosen Biotechnology Co., Ltd.
  • This example is intended to illustrate a biochip for preparing a three-dimensional silica nanotube array structure using the method of the present invention.
  • K9 glass piece was placed in a mixed solution of H 2 SO 4 (98% by weight) and H 2 O 2 (30% by weight) in a volume ratio of 7:3, and washed for 1 hour, and then used Rinse with deionized water.
  • the cleaned K9 glass piece is vapor-deposited by a magnetron sputtering method with a layer of 40 nm thick gold, that is, the gold substrate used in this embodiment;
  • the gold substrate after the step (2) is treated with a 0.1 mol hydrochloric acid solution, and the treatment time is 10 hours, at room temperature;
  • a through-hole aluminum oxide film is attached to the gold substrate: the anodized aluminum film of the through hole and the gold substrate obtained by the step (3) can be immersed in a mixed solution of acetone and water, wherein the volume ratio of acetone to water is 1 : 1.
  • the alumina film was applied to a gold substrate and then placed in a dry box at room temperature for 24 hours.
  • the surface scanning electron micrograph (SEM) of the anodized aluminum film is shown in Fig. 2.
  • S4800 indicates the type of scanning electron microscope
  • 5.0kV indicates the voltage applied when the sample is observed
  • 9.1mm ⁇ 50.0k indicates the electron gun and The distance of the sample and the magnification multiple
  • 2013-3-13 indicates the date of photographing
  • 1.00 ⁇ m indicates the scale.
  • the gold substrate obtained in the step (4) is immersed in silicon tetrachloride for 2 minutes, and then immersed in a mixture of n-hexane and 1:1 volume ratio of n-hexane and methanol. , ethanol and water for 5 minutes each.
  • a group is a surface sol gel cycle. For this example, 6 surface sol gel cycles were used.
  • the biochip of the three-dimensional silica nanotube array structure prepared in Example 1 was measured as shown in FIG.
  • 10 is a gold film
  • 20 is a silica nanotube array
  • S4800 is a model of a scanning electron microscope
  • 5.0 kV is a voltage applied when observing a sample
  • 9.9 mm ⁇ 50.0 k is a distance between an electron gun and a sample, respectively.
  • the multiple of 2013-7-3 indicates the date of photographing, and 1.00 ⁇ m indicates the scale.
  • This application example uses a biochip of a three-dimensional silica nanotube array structure prepared according to the method of Example 1.
  • the detection device is based on the Kretchmann structure.
  • the incident light is coupled to the waveguide mode in the nanotube array layer, the light reflected by the biochip will appear as a resonant groove in the reflectance angle spectrum.
  • the angular position of this resonant groove is related to the effective refractive index of the nanotube array layer.
  • any change in the effective refractive index of the nanotube array layer by adsorbing biomolecules into the nanotube array layer or due to minor changes in the refractive index of the solution to be tested will be amplified by the waveguide mode, most Finally, the movement of the resonant groove in the reflectance angle spectrum is reflected. In addition, if the incident light angle is fixed near the angle of the resonant groove to measure the reflectance, the change in the effective refractive index of the nanotube array layer can also be quantified by the change in refraction.
  • the biochip connection method for detecting biomolecules was as follows: The biochip was placed in a 5% by weight solution of 3-aminopropyltriethoxysilane for 24 hours.
  • the 3-aminopropyltriethoxysilane can be attached to the hydroxyl group on the surface of the silica at one end and can react with the functional group containing -NHS at the other end.
  • the same sample was used to flow into the NHS-modified biotin (Sulfo-NHS-LC-LC-Biotin) solution for 1 hour, and then changed to 10 mmol of phosphate buffer for a certain period of time, and then the intensity of the reflected light was measured with incidence.
  • the change in angle is shown in Figure 3. In Fig.
  • the vertical axis is the reflectance
  • the horizontal axis is the incident angle of the incident
  • the unit is the degree.
  • the curve of the data point is a diamond shape obtained by using the chip to detect a buffer solution. Reflectance angle spectrum, the curve of the data point is a circular curve of the reflectance measured after 500 ⁇ mol biotin solution (500 ⁇ mol biotin) is reached, and the data point is square. The curve is 100 nmol chain enzyme. The azimuth (100 nmol streptavidin) reached the reflectance angle spectrum measured after adsorption saturation.
  • the time point of 100 nmol streptavidin (100 nmol streptavidin), 4 indicates the time point of passage into the buffer.
  • the intensity of the reflected light is measured as a function of the incident angle. As shown in Fig. 3, the angle of the resonant groove moves from 62.08° after modification of biotin to 62.72° of binding to streptavidin.
  • the method for detecting biomolecules by biochip connection is as follows: for detecting streptavidin molecules, first 10 mmol of phosphate buffer is used to flow into the surface of the sample through the PDMS flow channel to establish a background baseline, and then 100 nmol of streptavidin solution is introduced into the sample. Surface, in this process, the change in reflectance was measured at the angle of the resonance groove measured after the phosphate buffer solution was introduced. As seen from Fig. 4, the change in reflectance was almost negligible with respect to Application Example 1.
  • the present invention relates to the use of biomolecule-specific detection in biomolecule-specific detection and is not limited to the above method, and other end-customized alkyl groups may be attached to the silica tube and the corresponding biomolecule joining method may be used. .
  • the linked biomolecules are also not limited to this embodiment.

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Abstract

一种基于三维结构纳米阵列生物芯片的制备方法及其应用，该方法包括以下步骤：（1）将金膜（10）用含有巯基的硅烷溶液处理；（2）将经步骤（1）处理后的金膜（10）用酸溶液处理；（3）将通孔的阳极氧化铝薄膜贴于经步骤（2）处理后的金膜上；（4）将经步骤（3）得到的金膜（10）依次浸渍在四氯化硅（SiC14）、正己烷、正己烷与甲醇的混合液、乙醇和水中（此过程可循环多次）；（5）将经步骤（4）得到的金基底用酸溶液处理。本方法易于在基底上制备三维结构的纳米管（柱）阵列，并且管（柱）的尺寸可控，可用作生物芯片，并可进行高灵敏度、非标记的生物检测。

Description

一种基于三维结构纳米阵列生物芯片的制备方法及其应用
优先权
本申请要求申请号为201310754275X、名称为《一种基于三维结构纳米阵列生物芯片的制备方法及其应用》的中国专利申请的优先权。
技术领域
本发明涉及一种基于三维结构纳米阵列生物芯片及其制备方法，具体地，本发明涉及一种在金膜上合成三维结构二氧化硅纳米管阵列生物芯片及其制备方法，以及该生物芯片在生物分子特异性检测方面的应用。
背景技术
高灵敏度的生物传感在许多领域中已经得到广泛的应用，包括在生物医药研究、环境监控、蛋白质组学、基因组学、药剂学、医疗诊断等领域，同时其也获得了越来越广泛的认可。例如，基于荧光信号检测的生物传感的灵敏度就可以很高，甚至可以检测到单个的生物分子。然而基于荧光法的生物传感也有些弊端，如用荧光集团标记生物分子价格非常的昂贵并且很耗时，而且这种方法在某些应用中甚至不可能实现。另外，标记荧光集团后可能会影响原有生物分子的某些功能。所以采用非标记的方法去直接检测生物分子的传感器相比就显得比较重要。
在过去的一二十年中，很多基于不同类型纳米结构的非标记生物传感器已经得到广泛的研究，这些传感器都得益于纳米技术的特点。例如有基于纳米线、纳米孔、纳米管或纳米柱类型的生物传感器等。不同类型的非标记的生物传感器可以简单和快速的将探针分子和靶分子之间的相互作用的响应转化成光信号、电信号、热信号或是声信号等。其中对于非标记的光学生物 传感器来说，信号转变方法主要分为两类：光学干涉和表面等离子体。基于纳米薄膜结构的光学波导传感器就是一种典型的光学干涉的生物传感器。它具有两个主要的优点。首先、入射光的电磁场以增强的光波导模式被限制在纳米薄膜层中，并且能够有效地覆盖到薄层中所吸附的生物分子。其次、由于纳米薄膜具有很大的吸附生物分子表面积的结构特点，进而导致生物分子的富集，所以可以进一步提高生物传感器的响应。虽然如此，相对于基于荧光信号检测的生物传感器，非标记的生物传感器的灵敏度还不是很高。相应的，如何提高非标记的生物传感器的响应需要进一步的研究。
发明内容
本发明是提供了一种基于三维二氧化硅纳米管阵列结构的生物芯片及其制作方法，以及其在生物分子特异性检测方面中的应用。
本发明提供了一种基于三维结构纳米阵列生物芯片的制备方法，该方法包括以下步骤：
(1)将金膜用含有巯基的硅烷溶液处理；
(2)将经步骤(1)处理后的金膜用酸溶液处理；
(3)将通孔的阳极氧化铝薄层膜贴于经步骤(2)处理后的金膜上；
(4)将经步骤(3)得到的金膜顺序的浸渍在四氯化硅(SiCl4)、正己烷、正己烷与甲醇的混合液、乙醇和水中(此过程可循环多次)；
(5)将经步骤(4)得到的金基底用酸溶液处理。
本发明还提供了由上述所述方法制备的三维二氧化硅纳米管阵列结构的生物芯片。
本发明还提供了由上述所述方法制备的三维二氧化硅纳米管阵列结构的生物芯片在生物分子特异性检测方面的应用。
在本发明中，将金膜用含有巯基的硅烷溶液处理，再用酸溶液处理，可以得到亲水性的表面，然后将通孔的阳极氧化铝薄膜贴于金膜上，这样金膜的亲水性可以增强薄膜与金膜的连接，再通过交替浸入在四氯化硅(SiCl4)、正己烷、正己烷和甲醇的混合液、 乙醇和水中一定次数得到二氧化硅管(柱)，最后将得到的金基底浸泡于酸溶液中得到三维二氧化硅纳米管阵列结构的生物芯片。本发明提供的方法易于在基底上制备三维结构的纳米管(柱)阵列，并且管(柱)的尺寸可控，可用作于生物芯片进行高灵敏度、非标记的生物检测。
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1是本发明实施例1制备的三维二氧化硅纳米管阵列结构的生物芯片界面的扫描电子显微镜图(SEM)；
图2是本发明实施例1中使用的通孔的阳极氧化铝薄膜的表面扫描电子显微镜图(SEM)；
图3是本发明按照应用例1的方法制备的三维二氧化硅纳米管阵列结构的生物芯片特异性检测链酶亲和素(streptavidin)的反射率角度谱图；
图4是按照应用例1和对比例1的方法制备的利用三维二氧化硅纳米管阵列结构的生物芯片检测链酶亲和素(streptavidin)的动力学曲线图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
本发明提供了一种基于三维结构纳米阵列生物芯片的制备方法，该方法包括以下步骤：
(1)将金膜用含有巯基的硅烷溶液处理；
(2)将经步骤(1)处理后的金膜用酸溶液处理；
(3)将通孔的阳极氧化铝薄膜贴于经步骤(2)处理后的金膜上；
(4)将经步骤(3)得到的金膜使用溶胶凝胶法，依次顺序的浸渍在四氯化硅(SiCl4)、正己烷、正己烷与甲醇的混合液、乙醇和水中(此过程可循环多次)；
(5)将经步骤(4)得到的金基底用酸溶液处理，除去氧化铝薄膜。
根据本发明，在步骤(1)中，所述金膜为表面具有金层的玻璃基底材料。在本发明，可以使用磁控溅射蒸镀法或热蒸镀法在该基底材料上镀上一层具有35-50nm厚度的金，优选为40-45nm。
根据本发明，在步骤(1)中，所述的巯基硅烷溶液为(3-巯基丙基)三甲氧基硅烷溶液，所述溶液浓度为1-30mM，优选为18-22mM。溶液中浸泡时间为3-12小时，优选为3小时。本发明中，可将巯基硅烷溶解于有机溶剂中配成巯基硅烷有机溶液，其中对溶解所述巯基硅烷的有机溶剂没有特殊要求，可以为本领域所熟知的有机溶剂，例如，可以为甲醇、乙醇或丙酮中的一种或多种，优选地，可以为甲醇。
根据本发明，在步骤(2)中，所述酸溶液可以为盐酸水溶液，浓度为0.05-0.2mol，优选为0.1mol。溶液中处理时间为1-20小时，优选为10-12小时，温度为室温。
根据本发明，在步骤(3)中，通孔的阳极氧化铝薄膜和经步骤(2)所得金膜基底可浸渍于丙酮或丙酮与水的混合溶液中。当所述溶液为丙酮与水的混合液时，丙酮与水用量体积比可为1-3∶1，优选为1∶1。氧化铝薄膜覆盖到金膜基底上，然后干燥箱中常温放置24-48小时，优选为24-30小时。
根据本发明，在步骤(4)中，经步骤(3)处理的金膜基底依次顺序的浸渍于四氯化硅(SiCl4)、正己烷、正己烷与甲醇的混合液、乙醇和水中。其中在四氯化硅中浸渍时间为1-2分钟，其他溶液中为3-5分钟，优选为5分钟。正己烷与甲醇用量的体积比为1∶1。步骤(4)为表面溶胶凝胶法的一次循环。
根据本发明，在步骤(5)中，所用的酸溶液为磷酸溶液，浓度为5-15 质量％，优选为10-12质量％。温度为20-40℃。
本发明还提供了由上述所述方法制备的三维二氧化硅纳米管阵列结构的生物芯片。
本发明还提供了由上述所述方法制备的三维二氧化硅纳米管阵列结构的生物芯片在生物分子特异性检测方面的应用。
以下将通过实施例对本发明进行详细描述，但本发明的保护范围并不仅限于这些实施例。
在以下实施例和对比例中：
3-氨丙基三乙氧基硅烷(3-aminopropyltriethoxysilane)购自于Alfa Aesar公司；生物素(Sulfo-NHS-LC-LC-Biotin)购自上海浩然生物技术有限公司；链酶亲和素(streptavidin)购自北京博奥森生物技术有限公司。
实施例1
本实施例用于说明采用本发明的方法制备三维二氧化硅纳米管阵列结构的生物芯片。
(1)金膜的制备：将K9玻璃片置于体积比为7∶3的H2SO4(98重量％)和H2O2(30重量％)混合溶液中，清洗1小时，然后用去离子水冲洗干净。将清洁后的K9玻璃片用磁控溅射法蒸镀一层40nm厚的金，即本实施例使用的金基底；
(2)金基底的巯基硅烷溶液处理：用甲醇配置20mmol的(3-巯基丙基)三甲氧基硅烷溶液。将经步骤(1)得到的金基底浸入上述溶液中溶液3小时。
(3)金基底的酸溶液处理：使用0.1mol的盐酸溶液处理经步骤(2)后的金基底，处理时间为10小时，常温；
(4)通孔氧化铝薄膜贴于金基底上：通孔的阳极氧化铝薄膜和经步骤(3)所得金基底可浸入于丙酮与水的混合溶液，其中丙酮与水的用量体积比为1∶1。让氧化铝薄膜贴于金基底上，然后干燥箱中常温放置24小时。其中阳极氧化铝薄膜的表面扫描电子显微镜图(SEM)如图2所示，图中S4800表示扫描电子显微镜的型号，5.0kV表示观测样品时所加的电压，9.1mm×50.0k分别表示电子枪与样品的距离以及放大的倍数，2013-3-13表示拍照的日期，1.00μm表示标尺。
(5)二氧化硅纳米管的制备：将经步骤(4)得到的金基底浸入四氯化硅中2分钟，然后再依次浸入正己烷、1∶1体积比的正己烷与甲醇的混合液、乙醇和水中各5分钟。这样一组是一次表面溶胶凝胶循环。对于本实施例，使用了6次表面溶胶凝胶循环。
测得实施例1制备的三维二氧化硅纳米管阵列结构的生物芯片如图1所示。图1中10为金膜，20为二氧化硅纳米管阵列，S4800表示扫描电子显微镜的型号，5.0kV表示观测样品时所加的电压，9.9mm×50.0k分别表示电子枪与样品的距离以及放大的倍数，2013-7-3表示拍照的日期，1.00μm表示标尺。
应用例1
特异性检测链酶亲和素
本应用例使用按照实施例1方法制备的三维二氧化硅纳米管阵列结构的生物芯片。检测装置是基于Kretchmann结构。当满足入射光与在纳米管阵列层中的波导模式耦合时，经过生物芯片反射光将在反射率角度谱上以一个共振凹槽的形式出现。这个共振凹槽的角度位置与纳米管阵列层的有效折射率有关。任何通过吸附生物分子到纳米管阵列层中或者由于待测溶液折射率的微小改变而导致纳米管阵列层的有效折射率改变都将被波导模式放大，最 终体现在反射率角度谱中共振凹槽的移动。另外，如果把入射光角度固定在共振凹槽的角度附近去测量反射率，这种纳米管阵列层的有效折射率的改变也可以通过折射的变化来量化。
生物芯片连接探测生物分子的方法如下：将生物芯片置于5重量％的3-氨丙基三乙氧基硅烷(3-aminopropyltriethoxysilane)溶液中24小时。3-氨丙基三乙氧基硅烷一端可以连接二氧化硅表面的羟基，另一端可以与含-NHS的官能团进行反应。此后，同一样品上利用PDMS流道流入NHS修饰的生物素(Sulfo-NHS-LC-LC-Biotin)溶液1小时后换成10mmol磷酸缓冲液(buffer)一定时间，然后测量反射光的强度随入射角度的变化，如图3所示。图3中，纵轴为反射率(Reflectivity)，横轴为光入射角度(Incident Angle)，单位为度(degree)，数据点为菱形的曲线为使用该芯片检测缓冲溶液(buffer)所得到的反射率角度谱图，数据点为圆形的曲线为通入500μmol的生物素溶液(500μmol biotin)达到吸附饱和以后所测量的反射率角度谱图，数据点为方形的曲线是通入100nmol链酶亲和素(100nmol streptavidin)达到吸附饱和后所测的反射率角度谱图。对于特异性检测链酶亲和素分子，100nmol的链酶亲和素通过PDMS流道流入样品表面90分钟，然后用10mmol磷酸缓冲溶液(buffer)冲洗一段时间，此过程中将入射角度固定在共振角度处(62.08°)，然后记录反射率随着时间的变化，如图4所示。图4中，纵轴为反射率(Reflectivity)，横轴为时间(time)，单位为秒(s)，1表示应用例1的结果曲线，2表示对比例1的结果曲线，3表示通入100nmol链酶亲和素(100nmol streptavidin)的时间点，4表示通入缓冲溶液(buffer)的时间点。最后测量反射光的强度随入射角度的变化，如图3所示，共振凹槽的角度从修饰生物素后的62.08°移动到结合链酶亲和素的62.72°。
对比例1
检测链酶亲和素
生物芯片连接探测生物分子的方法如下：对于检测链酶亲和素分子，先用10mmol的磷酸缓冲液通过PDMS流道流入样品表面建立背景基线，然后通入100nmol的链酶亲和素溶液到样品表面，此过程中在通入磷酸缓冲溶液后测得的共振凹槽角度处测量反射率的变化，从图4中看出，反射率的变化相对于应用例1中几乎可忽略。
本发明应用例1和对比例1可充分说明链酶亲和素分子与生物素分子特异性的结合在一起。另外，也可说明此三维二氧化硅纳米管阵列结构的生物芯片可以应用于生物分子的特异性检测。
本发明涉及在生物分子修饰方面在生物分子特异性检测中的应用并不受限于上述方法，可以使用其他末端订制的烷基连接于二氧化硅管上，并使用相应的生物分子连接方法。连接的生物分子也不受限于此实施例。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种基于三维结构纳米阵列生物芯片的制备方法，其特征是：
    A、将金膜用含有巯基的硅烷溶液处理；
    B、将经步骤A处理后的金膜用酸溶液处理；
    C、将通孔的阳极氧化铝薄膜贴于经步骤B处理后的金膜上；
    D、将经步骤C得到的金膜顺序地浸渍在四氯化硅、正己烷、正己烷与甲醇的混合液、乙醇和水这五种溶液中，浸渍一次或循环多次；
    E、将经步骤D得到的金基底用酸溶液处理，除去氧化铝薄膜，得到三维二氧化硅纳米管阵列结构的生物芯片。
  2. 根据权利要求1所述的方法，其特征是：在步骤A中，所述的含有巯基的硅烷溶液为(3-巯基丙基)三甲氧基硅烷溶液，且所述溶液浓度为1-30mM；溶液中浸泡时间为3-12小时。
  3. 根据权利要求1所述的方法，其特征是：在步骤A中，所述含有巯基的硅烷溶液是将巯基硅烷溶解于有机溶剂中配成的巯基硅烷有机溶液。
  4. 根据权利要求1所述的方法，其特征是：在步骤B中，所述酸溶液为盐酸溶液，浓度为0.05-0.2mol；溶液中处理时间为1-20小时，常温。
  5. 根据权利要求1所述的方法，其特征是：在步骤C中，通孔的阳极氧化铝薄膜和经步骤B所得金膜基底浸渍于丙酮或丙酮与水的混合溶液中；当所述溶液为丙酮与水的混合液时，丙酮与水用量体积比为1-3∶1；氧化铝薄膜覆盖到金膜基底上，然后干燥箱中常温放置24-48小时。
  6. 根据权利要求1所述的方法，其特征是：在步骤D中，在四氯化硅中浸渍时间为1-2分钟，其他溶液中为3-5分钟；正己烷与甲醇用量的体积比为1∶1。
  7. 根据权利要求1所述的方法，其特征是：在步骤E中，所用的酸溶液为磷酸溶液，浓度为5-15质量％；温度为20-40℃。
  8. 根据权利要求1所述的方法，其特征是：在步骤A中，所述金膜附着于玻璃基底材料的表面，金膜的厚度为40-45nm。
  9. 一种基于三维结构纳米阵列生物芯片，其特征是：按照权利要求1-8中任一权利要求中所述的方法制备。
  10. 一种非标记的直接检测生物分子的生物芯片，其特征是：使按照权利要求1-8中任一权利要求中所述的方法制备的生物芯片，用于生物分子的特异性检测。
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