CN104406881B - A kind of piezoelectric sound wave biology sensor based on micro-nano structure - Google Patents
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Abstract
本发明公开了一种基于微纳结构的压电声波生物传感器，包括石英晶振片和频率计，所述石英晶振片包括石英晶片、设置在石英晶片上部的工作电极、设置在石英晶片下部的下电极，所述频率计连接工作电极和下电极，所述石英晶振片表面设置微米结构或纳米结构。与现有技术相比，本发明通过微纳加工工艺可以在石英晶振片表面精确制作不同尺寸、不同形貌的微米纳米结构，可以根据不同的用途来调整微米纳米结构，不仅体现了本发明的应用灵活性，而且提供一种新型的传感器件，增强了这种QCM生物传感器的应用范围。
The invention discloses a piezoelectric acoustic wave biosensor based on a micro-nano structure, which includes a quartz crystal vibrating plate and a frequency meter. The quartz crystal vibrating plate includes a quartz wafer, a working electrode arranged on the upper part of the quartz wafer, and a lower The frequency meter is connected to the working electrode and the lower electrode, and the surface of the quartz crystal oscillator is provided with a microstructure or a nanostructure. Compared with the prior art, the present invention can accurately manufacture micro-nano structures of different sizes and shapes on the surface of the quartz crystal oscillator through the micro-nano processing technology, and can adjust the micro-nano structures according to different purposes, which not only embodies the advantages of the present invention Application flexibility, and providing a new type of sensing device, enhances the application range of this QCM biosensor.
Description
技术领域technical field
本发明涉及一种基于微纳结构的压电声波生物传感器。The invention relates to a piezoelectric acoustic wave biosensor based on a micro-nano structure.
背景技术Background technique
石英晶体微天平(QCM)是基于石英晶体的压电效应对其电极表面质量变化进行测量的仪器。压电效应是由Pierre Curie和Jacques Curie兄弟在1880年发现的，即在石英晶片加一电场，晶片会产生机械变形。相反，若在晶片上施加机械压力，则在晶片相应的方向上产生一定的电场。由于其具有纳克级的质量响应灵敏度，使得QCM广泛运用于工业镀膜、分析化学、分子生物学、免疫学、遗传学、环境科学以及其它一些涉及质量、密度及粘度等检测领域。瑞典的Q-Sense公司的QCM仪器性能在同行业中处于领先地位，在分子生物学领域中主要用于研究生物活性分子之间的相互作用，比如蛋白质吸附动力学、抗原/抗体相互作用、DNA杂交、适配体-蛋白相互作用等方面；在生物医学领域中用于研究或检测抗原、抗体、血细胞、病原微生物、核酸及蛋白质等。Quartz crystal microbalance (QCM) is an instrument based on the piezoelectric effect of quartz crystal to measure the quality change of its electrode surface. The piezoelectric effect was discovered by brothers Pierre Curie and Jacques Curie in 1880, that is, when an electric field is applied to a quartz wafer, the wafer will produce mechanical deformation. On the contrary, if a mechanical pressure is applied on the wafer, a certain electric field will be generated in the corresponding direction of the wafer. Due to its nanogram-level mass response sensitivity, QCM is widely used in industrial coatings, analytical chemistry, molecular biology, immunology, genetics, environmental science and other detection fields involving mass, density and viscosity. The performance of the QCM instrument of Q-Sense company in Sweden is in the leading position in the same industry. It is mainly used in the field of molecular biology to study the interaction between biologically active molecules, such as protein adsorption kinetics, antigen/antibody interaction, DNA Hybridization, aptamer-protein interaction, etc.; in the field of biomedicine, it is used to study or detect antigens, antibodies, blood cells, pathogenic microorganisms, nucleic acids, and proteins.
作为一种高灵敏度的生物传感器而言，一个重要的参数就是其检测的灵敏度。对于QCM，其质量灵敏度与它的共振频率有关，共振频率越高，其质量灵敏度就越高。要提高QCM仪器本身的检测灵敏度，一方面可以通过采用较高共振频率的石英晶振片，目前市场上常见的石英晶振片的频率为5MHz。如果采用更高共振频率的石英晶振片，虽然理论上可以得到较高的质量灵敏度，但是晶振片的厚度跟它的共振频率成反比，即共振频率越高，其晶振片的厚度越薄。然而，晶振片在使用时是通过“O”型橡胶圈压紧密封或者粘接的方式将其固定在液体反应流通池中，如果采用较薄的石英晶振片将导致一些问题，一方面是密封或粘贴固定的方式对于薄的石英晶振片将产生额外的机械应力，这将严重影响QCM的信号及其检测灵敏度；另外一方面，薄的石英晶振片在操作时更容易损坏，使用成本变高。对此，商业化的QCM目前目前主要采取两种方式提高检测灵敏度，比如Q-Sense公司采用5MHz晶振片的高次谐振频率(15MHz、25MHz、35MHz)方式，德国3T公司的QCM则采用10MHz的石英晶振片。As a highly sensitive biosensor, an important parameter is its detection sensitivity. For QCM, its mass sensitivity is related to its resonance frequency, the higher the resonance frequency, the higher its mass sensitivity. To improve the detection sensitivity of the QCM instrument itself, on the one hand, a quartz crystal oscillator with a higher resonance frequency can be used. The frequency of the common quartz crystal oscillator on the market is 5MHz. If a quartz crystal oscillator with a higher resonance frequency is used, although higher mass sensitivity can be obtained theoretically, the thickness of the crystal oscillator is inversely proportional to its resonance frequency, that is, the higher the resonance frequency, the thinner the thickness of the crystal oscillator. However, when the crystal oscillator is used, it is fixed in the liquid reaction flow cell by pressing and sealing or bonding the "O" rubber ring. If a thinner quartz crystal oscillator is used, it will cause some problems. On the one hand, the sealing Or the method of pasting and fixing will generate additional mechanical stress for thin quartz crystal oscillators, which will seriously affect the signal of QCM and its detection sensitivity; on the other hand, thin quartz crystal oscillators are more likely to be damaged during operation, and the cost of use becomes higher . In this regard, commercial QCM currently mainly adopts two methods to improve detection sensitivity. For example, Q-Sense uses the high-order resonance frequency (15MHz, 25MHz, 35MHz) of 5MHz crystal oscillator, and the QCM of German 3T company uses 10MHz. Quartz crystal oscillator.
要提高QCM仪器本身的检测灵敏度的另外一种方法是增加单位面积内结合在石英晶振片中的生物分子的数量。然而，目前商品化QCM仪器用于生物类检测的石英晶振片都采用抛光的石英晶振片，抛光的石英晶振片表面平滑，粗糙度较小，可以看做是平面型的反应表面；而未抛光的石英晶振片表面粗糙度较大，虽然可以在一定程度上提高晶振片的表面积，但是其粗糙结构的形貌和大小无法控制，这是由抛光工艺所决定的，很难得到高一致性的粗糙表面。Another way to improve the detection sensitivity of the QCM instrument itself is to increase the number of biomolecules bound in the quartz crystal oscillator per unit area. However, currently commercialized QCM instruments use polished quartz crystal oscillators for biological detection. Polished quartz crystal oscillators have a smooth surface and low roughness, which can be regarded as a planar reaction surface; The surface roughness of the quartz crystal oscillator is relatively large. Although the surface area of the crystal oscillator can be increased to a certain extent, the shape and size of the rough structure cannot be controlled. This is determined by the polishing process, and it is difficult to obtain high consistency. Rough surface.
发明内容Contents of the invention
本发明要解决的技术问题是增加石英晶片的表面积，以此来增加单位面积内结合在石英晶振片中的生物分子的数量，从而提高QCM生物传感器的检测灵敏度。The technical problem to be solved by the present invention is to increase the surface area of the quartz wafer, so as to increase the quantity of biomolecules combined in the quartz crystal oscillator per unit area, thereby improving the detection sensitivity of the QCM biosensor.
本发明是通过以下技术方案来实现：The present invention is achieved through the following technical solutions:
一种基于微纳结构的压电声波生物传感器，包括石英晶振片和频率计，所述石英晶振片包括石英晶片、设置在石英晶片上部的工作电极、设置在石英晶片下部的下电极，所述频率计连接工作电极和下电极，所述石英晶振片表面设置微米结构或纳米结构。A piezoelectric acoustic wave biosensor based on a micro-nano structure, including a quartz crystal oscillator and a frequency meter, the quartz crystal oscillator includes a quartz wafer, a working electrode arranged on the upper part of the quartz wafer, and a lower electrode arranged on the lower part of the quartz wafer. The frequency meter is connected to the working electrode and the lower electrode, and the surface of the quartz crystal oscillator is provided with microstructure or nanostructure.
进一步，在石英晶振片上旋涂一层光刻胶，经曝光、显影后得到微米结构或纳米结构。Further, a layer of photoresist is spin-coated on the quartz crystal oscillator, and a microstructure or nanostructure is obtained after exposure and development.
微米结构的制作方法是：将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层六甲基二硅氮烷，然后旋涂一层光刻胶，光刻胶厚度在0.2～5μm之间，90℃下烘5～30min，接着，将涂有光刻胶的石英晶振片进行紫外或深紫外曝光，，曝光时间5s～3min，然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶微米结构。The fabrication method of the microstructure is as follows: the quartz crystal vibrating plate is ultrasonically dried with acetone and ethanol for 10 minutes respectively, then dried with oxygen plasma for 5 minutes; then a layer of hexamethyldisilazane is spin-coated, and then a layer of photolithography The thickness of the photoresist is between 0.2-5μm, and it is baked at 90°C for 5-30 minutes. Then, the quartz crystal oscillator coated with the photoresist is exposed to ultraviolet or deep ultraviolet, and the exposure time is 5s-3min. Then, the exposed The high-quality quartz crystal oscillator is immersed in the developing solution, and the positive or negative microstructure is obtained after development.
微米结构或纳米结构的制作方法是：将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层粘附剂聚乙烯吡咯烷酮或三甲氧基硅烷，然后将其放入乙醇和去离子水中各清洗10min，之后在120℃下烘5min；然后旋涂一层电子束光刻胶，电子束光刻胶厚度在0.2～2μm之间，120℃下烘5～15min，接着，将涂有电子束光刻胶的石英晶振片进行电子束曝光，然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶纳米结构或微米结构。The production method of the microstructure or nanostructure is as follows: the quartz crystal vibrating plate is ultrasonically dried with acetone and ethanol for 10 minutes, and then treated with oxygen plasma for 5 minutes; then spin-coated with a layer of adhesive polyvinylpyrrolidone or trimethoxysilane, Then wash it in ethanol and deionized water for 10 minutes, and then bake it at 120°C for 5 minutes; then spin-coat a layer of electron beam photoresist, the thickness of the electron beam photoresist is between 0.2 and 2 μm, and bake it at 120°C 5-15 minutes, then, expose the quartz crystal oscillator coated with electron beam photoresist to the electron beam, then immerse the exposed quartz crystal oscillator in the developing solution, and obtain positive or negative resist nanostructure or microstructure after development .
与现有技术相比，本发明通过微纳加工工艺可以在石英晶振片表面精确制作不同尺寸、不同形貌的微米纳米结构，可以根据不同的用途来调整微米纳米结构，不仅体现了本发明的应用灵活性，而且提供一种新型的传感器件，增强了这种QCM生物传感器的应用范围。Compared with the prior art, the present invention can accurately manufacture micro-nano structures of different sizes and shapes on the surface of the quartz crystal oscillator through the micro-nano processing technology, and can adjust the micro-nano structures according to different purposes, which not only embodies the advantages of the present invention Application flexibility, and providing a new type of sensing device, enhances the application range of this QCM biosensor.
附图说明Description of drawings
图1为本发明的结构示意图。Fig. 1 is a structural schematic diagram of the present invention.
具体实施方式detailed description
下面结合附图和实施例对本发明作进一步说明：Below in conjunction with accompanying drawing and embodiment the present invention will be further described:
如图1所示，一种基于微纳结构的压电声波生物传感器，包括石英晶振片和频率计1，所述石英晶振片包括石英晶片2、设置在石英晶片2上部的工作电极3、设置在石英晶片2下部的下电极4，所述频率计1连接工作电极3和下电极4，所述石英晶振片表面设置微米结构5或纳米结构5。As shown in Figure 1, a kind of piezoelectric acoustic wave biosensor based on micro-nano structure comprises quartz crystal vibrating plate and frequency meter 1, and described quartz crystal vibrating plate comprises quartz wafer 2, is arranged on the working electrode 3 of quartz wafer 2 top, is set On the lower electrode 4 at the lower part of the quartz wafer 2 , the frequency meter 1 is connected to the working electrode 3 and the lower electrode 4 , and the surface of the quartz crystal oscillator is provided with microstructures 5 or nanostructures 5 .
使用时，石英晶振片清洗后，进行化学修饰，然后将其浸入2.5％的戊二醛溶液中反应1小时；然后就可以进行生物分子识别元件6(抗体、DNA、酶等生物分子等)的固定，之后进行待测生物分子的检测。During use, after cleaning the quartz crystal oscillator, carry out chemical modification, then immerse it in 2.5% glutaraldehyde solution and react for 1 hour; After fixation, the detection of the biomolecules to be tested is carried out.
实施例1Example 1
将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层六甲基二硅氮烷，然后旋涂一层光刻胶，光刻胶厚度为5μm，90℃下烘30min，接着，将涂有光刻胶的石英晶振片进行紫外或深紫外曝光，曝光时间5s，然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶微米结构。The quartz crystal vibrating plate was ultrasonically dried with acetone and ethanol for 10 minutes respectively, and then treated with oxygen plasma for 5 minutes; then a layer of hexamethyldisilazane was spin-coated, and then a layer of photoresist was spin-coated. The thickness of the photoresist was 5μm, baked at 90°C for 30min, then, expose the quartz crystal oscillator coated with photoresist to ultraviolet or deep ultraviolet for 5s, then immerse the exposed quartz crystal oscillator in the developing solution, and obtain positive or negative photoresist after development. Glue microstructure.
实施例2Example 2
将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层六甲基二硅氮烷，然后旋涂一层光刻胶，光刻胶厚度为0.5μm，90℃下烘5min，接着，将涂有光刻胶的石英晶振片进行紫外或深紫外曝光，曝光时间3min，然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶微米结构。The quartz crystal vibrating plate was ultrasonically dried with acetone and ethanol for 10 minutes respectively, and then treated with oxygen plasma for 5 minutes; then a layer of hexamethyldisilazane was spin-coated, and then a layer of photoresist was spin-coated. The thickness of the photoresist was 0.5 μm, bake at 90°C for 5 minutes, then expose the quartz crystal oscillator coated with photoresist to ultraviolet or deep ultraviolet for 3 minutes, then immerse the exposed quartz crystal oscillator in the developing solution, and obtain a positive resist or Negative photoresist microstructure.
实施例3Example 3
将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层粘附剂聚乙烯吡咯烷酮或三甲氧基硅烷，然后将其放入乙醇和去离子水中各清洗10min，之后在120℃下烘5min；然后旋涂一层电子束光刻胶，电子束光刻胶厚度为2μm，120℃下烘15min，接着，将涂有电子束光刻胶的石英晶振片进行电子束曝光。然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶纳米级结构。Use acetone and ethanol to ultrasonically dry the quartz crystal oscillator for 10 minutes in turn, and then treat it with oxygen plasma for 5 minutes; then spin-coat a layer of adhesive polyvinylpyrrolidone or trimethoxysilane, and then put it into ethanol and deionized water Wash for 10 minutes each, and then bake at 120°C for 5 minutes; then spin-coat a layer of electron beam photoresist with a thickness of 2 μm, and bake at 120°C for 15 minutes. Then, the quartz coated with electron beam photoresist The crystal oscillator is exposed to electron beams. Then, immerse the exposed quartz crystal oscillator in a developing solution to obtain a positive or negative nanoscale structure after development.
实施例4Example 4
将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层粘附剂聚乙烯吡咯烷酮或三甲氧基硅烷，然后将其放入乙醇和去离子水中各清洗10min，之后在120℃下烘5min；然后旋涂一层电子束光刻胶，电子束光刻胶厚度为0.2μm，120℃下烘5min，接着，将涂有电子束光刻胶的石英晶振片进行电子束曝光。然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶纳米级结构。Use acetone and ethanol to ultrasonically dry the quartz crystal oscillator for 10 minutes in turn, and then treat it with oxygen plasma for 5 minutes; then spin-coat a layer of adhesive polyvinylpyrrolidone or trimethoxysilane, and then put it into ethanol and deionized water Wash for 10 minutes each, and then bake at 120°C for 5 minutes; then spin-coat a layer of electron beam photoresist with a thickness of 0.2 μm, and bake at 120°C for 5 minutes, and then apply the electron beam photoresist Electron beam exposure of the quartz crystal oscillator. Then, immerse the exposed quartz crystal oscillator in a developing solution to obtain a positive or negative nanoscale structure after development.
实施例5Example 5
将石英晶振片依次用丙酮、乙醇各超声10min后吹干，用氧气等离子体处理5min；然后旋涂一层粘附剂聚乙烯吡咯烷酮或三甲氧基硅烷，然后将其放入乙醇和去离子水中各清洗10min，之后在120℃下烘5min；然后旋涂一层电子束光刻胶，电子束光刻胶厚度为1μm，120℃下烘10min，接着，将涂有电子束光刻胶的石英晶振片进行电子束曝光。然后，将曝光后的石英晶振片浸入显影液中，显影后得到正胶或负胶微纳米级结构。Use acetone and ethanol to ultrasonically dry the quartz crystal oscillator for 10 minutes in turn, and then treat it with oxygen plasma for 5 minutes; then spin-coat a layer of adhesive polyvinylpyrrolidone or trimethoxysilane, and then put it into ethanol and deionized water Wash for 10 minutes each, and then bake at 120°C for 5 minutes; then spin-coat a layer of electron beam photoresist with a thickness of 1 μm, and bake at 120°C for 10 minutes. Then, the quartz coated with electron beam photoresist The crystal oscillator is exposed to electron beams. Then, the exposed quartz crystal oscillator is immersed in a developing solution, and a positive or negative micro-nano-scale structure is obtained after development.
实施例6Example 6
采用SiO2类型的光刻胶的10HMz石英晶振片，用0.1％APTES的甲苯溶液或乙醇溶液进行氨基化修饰，然后将其浸入2.5％的戊二醛水溶液中反应1小时；然后就可以进行生物分子识别元件比如抗体、DNA、酶等生物分子的固定，之后进行待测生物分子的检测。Use 10HMz quartz crystal oscillator of SiO2 type photoresist, carry out amination modification with 0.1% APTES toluene solution or ethanol solution, and then immerse it in 2.5% glutaraldehyde aqueous solution to react for 1 hour; then you can carry out biological Molecular recognition elements such as antibodies, DNA, enzymes and other biomolecules are immobilized, and then the biomolecules to be tested are detected.
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