超大表面积自驱动微流控芯片的设计与制备
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摘要
目的:利用新型纳米森林材料,构建一种操作简单、检测快速、灵敏度高的用于现场检测的自驱动微流控芯片。方法:利用MEMS加工技术制备出具有优良光学性能和大表面积的石英纳米森林结构微流道,对该纳米森林结构的高度、宽度/横向尺寸、密度、表面积、光学性能、毛细驱动效果、荧光增敏效果做出评价,利用双抗体夹心的方法进行蓖麻毒素的检测。结果:纳米纤维锥底直径200～300nm,高度约1. 0μm,纳米森林的密度约为10个/μm~2,估测表面积比底面积达5∶1以上。其在波长为680nm处的透光率达89. 5%,驱动流速约5mm/s,与平面结构相比,其饱和荧光显色成倍提高。蓖麻毒素的检测限低于10pg/ml,在10～6 250pg/ml范围内具有较好线性关系。结论:基于纳米森林结构,成功构建了一种具有超大表面积和高灵敏度的毛细自驱动微流控芯片。
Objective: A new material with ultra-large surface area named nano-forest is prepared by microelectro-mechanical system( MEMS) processing technology. Based on this material,a new microfluidic chip for point-of-care test with simple operation,rapid detection and high sensitivity is created. Methods: The fabrication of nano-forests in micro-channel on quartz substrate mainly includes,cleaning and drying of quartz substrate;spinning polyimide( PI) coating; re-spinning phenolic resin photoresist on PI coating; photolithography to expose the channel; treating the PI layer with oxygen plasma and argon plasma to synthesize nano-fiber forests structure;nano-fiber-quartz nanoforests are realized by using nano-fiber forests as nanomasks in anisotropic etching of quartz by using reactive ion etching( RIE); the micro-channel with nano-forests structure inside is achieved after removing upper nanofiber forests structure and phenolic resin photoresist coating. The height,width,density and specific surface area of nano-forest are studied and analyzed by scanning electron microscope( SEM). Optical properties are tested by ultraviolet-visible spectrophotometer. The driving force is characterized by the flow rate of PBS solution. The sensitization effect is evaluated by saturated fluorescence test through antibody and AbFluor 680 dye-labeled secondary antibody. The sample pad, bond pad, micro-channel with nano-forests structure,nitrocellulose membrane and absorbent material are assembled on PMMA substrate in sequence,which is the microfluidic chip. The chip based on the sandwich format with a polyclonal antibody and a AbFluor 680 dyelabeled secondary antibody is used to detect ricin toxin( RT). Results: The scanning electron microscope shows that the nanofiber forests structure is formed on quartz substrate after oxygen plasma and argon plasma bombardment. The single nanofiber is upright on the substrate with a diameter of about 50-100 nm,a height of1. 8μm and a density of about 20/μm2. The quartz nano-forests structure can be obtained after RIE with nanofibre forests structure as mask and resist removal. The single structure is shaped like a cone. The diameter of the cone bottom is about 100-200 nm,the height is about 1. 0μm,the density is about 10/μm2,and the surface area to bottom area is more than 5∶ 1. Self-driven test provides information of the flow rate of PBS is to be about 5 mm/s in the micro-channel on the basis of nano-forests structure. The transmittance of the channel is 89. 5% at 680 nm wavelength. It shows that the channel has good transmittance,which makes the loss of excitation light or emission light much less,and is conducive to the sensor capturing more signals. With same surface modification,the planar quartz structure has shortcomings of short lasting effect time and low saturation fluorescence intensity. To the contrary,nano-forests structure with ultra-large surface area has a good sensitization effect in the test. RT can be detected sensitively based on the significantly fluorescent intensity. The linear range of detection is from10 pg/ml to 6 250 pg/ml and the limit of detection( LOD) is lower than 10 pg/ml. Conclusion: The nano-forests structure with good optical properties reduces the requirements of sensor and also makes the choice of fluorescent dyes wider. The three-dimensional structure of the nano-forest has an ultra-large surface area,which increases the amount of antibody compared to the planar structure,and thus improves the sensitivity of detection greatly.Compared with the immunochromatographic test strip,the microfluidic chip has an advantage of high sensitivity,thus the quantitative analysis can be realized within a certain range. Most microfluidic chips require complex equipments to provide driving force,which will make them costly and bulky. Driven by the capillary force,the chip with nano-forests structure inside makes the detection simple and fast. Combined with the miniaturized detection terminal,the platform can be miniaturized,portable,and automated,achieving the goal of simple,fast and efficient analysis. These characteristics make the chip an ideal candidate for the development of rapid detection methods.
Objective: A new material with ultra-large surface area named nano-forest is prepared by microelectro-mechanical system( MEMS) processing technology. Based on this material,a new microfluidic chip for point-of-care test with simple operation,rapid detection and high sensitivity is created. Methods: The fabrication of nano-forests in micro-channel on quartz substrate mainly includes,cleaning and drying of quartz substrate;spinning polyimide( PI) coating; re-spinning phenolic resin photoresist on PI coating; photolithography to expose the channel; treating the PI layer with oxygen plasma and argon plasma to synthesize nano-fiber forests structure;nano-fiber-quartz nanoforests are realized by using nano-fiber forests as nanomasks in anisotropic etching of quartz by using reactive ion etching( RIE); the micro-channel with nano-forests structure inside is achieved after removing upper nanofiber forests structure and phenolic resin photoresist coating. The height,width,density and specific surface area of nano-forest are studied and analyzed by scanning electron microscope( SEM). Optical properties are tested by ultraviolet-visible spectrophotometer. The driving force is characterized by the flow rate of PBS solution. The sensitization effect is evaluated by saturated fluorescence test through antibody and AbFluor 680 dye-labeled secondary antibody. The sample pad, bond pad, micro-channel with nano-forests structure,nitrocellulose membrane and absorbent material are assembled on PMMA substrate in sequence,which is the microfluidic chip. The chip based on the sandwich format with a polyclonal antibody and a AbFluor 680 dyelabeled secondary antibody is used to detect ricin toxin( RT). Results: The scanning electron microscope shows that the nanofiber forests structure is formed on quartz substrate after oxygen plasma and argon plasma bombardment. The single nanofiber is upright on the substrate with a diameter of about 50-100 nm,a height of1. 8μm and a density of about 20/μm2. The quartz nano-forests structure can be obtained after RIE with nanofibre forests structure as mask and resist removal. The single structure is shaped like a cone. The diameter of the cone bottom is about 100-200 nm,the height is about 1. 0μm,the density is about 10/μm2,and the surface area to bottom area is more than 5∶ 1. Self-driven test provides information of the flow rate of PBS is to be about 5 mm/s in the micro-channel on the basis of nano-forests structure. The transmittance of the channel is 89. 5% at 680 nm wavelength. It shows that the channel has good transmittance,which makes the loss of excitation light or emission light much less,and is conducive to the sensor capturing more signals. With same surface modification,the planar quartz structure has shortcomings of short lasting effect time and low saturation fluorescence intensity. To the contrary,nano-forests structure with ultra-large surface area has a good sensitization effect in the test. RT can be detected sensitively based on the significantly fluorescent intensity. The linear range of detection is from10 pg/ml to 6 250 pg/ml and the limit of detection( LOD) is lower than 10 pg/ml. Conclusion: The nano-forests structure with good optical properties reduces the requirements of sensor and also makes the choice of fluorescent dyes wider. The three-dimensional structure of the nano-forest has an ultra-large surface area,which increases the amount of antibody compared to the planar structure,and thus improves the sensitivity of detection greatly.Compared with the immunochromatographic test strip,the microfluidic chip has an advantage of high sensitivity,thus the quantitative analysis can be realized within a certain range. Most microfluidic chips require complex equipments to provide driving force,which will make them costly and bulky. Driven by the capillary force,the chip with nano-forests structure inside makes the detection simple and fast. Combined with the miniaturized detection terminal,the platform can be miniaturized,portable,and automated,achieving the goal of simple,fast and efficient analysis. These characteristics make the chip an ideal candidate for the development of rapid detection methods.
引文
[1]夏启玉,李美英,杨小亮,等.免疫层析试纸条技术及其在转基因检测中的应用.中国生物工程杂志,2017,37(2):101-110.Xia Q Y,Li M Y,Yang X L et al. Immunochromatography test strip and its applications in detection of genetically modified organisms. China Biotechnology,2017,37(2):101-110.
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[8]杨宇东,毛海央,李锐锐,等.双层复合纳米森林结构的制备及其宽光谱高吸收光学特性研究.红外与毫米波学报,2018,37(2):246-250.Yang Y D,Mao H Y,Li R R et al. Fabrication and broadband high absorption features of double-layer hybrid nanoforests. J Infrared Millim Waves,2018,37(2):246-250.
[9] Mao H,Wu D,Wu W,et al. The fabrication of diversiform nanostructure forests based on residue nanomasks synthesized by oxygen plasma removal of photoresist. Nanotechnology,2009,20(44):445304.
[10]Kharisov B I,Kharissova O V,García B O,et al. State of the art of nanoforest structures and their applications. RSC Adv,2015,5(128):105507-105523.
[11] Yang Y,Mao H,Xiong J,et al. Optical features of Nanowire forests prepared by a plasma repolymerization technique. IEEE Trans Nanotechnol,2018,PP(99):452-455.
[12]Zhang H,Yang Y,Li X,et al. Frequency-enhanced transferrin receptor antibody-labelled microfluidic chip(FETAL-Chip)enables efficient enrichment of circulating nucleated red blood cells for non-invasive prenatal diagnosis. Lab Chip,2018,18(18):2749-2756.
[13]Wu W,Mao H,Han X,et al. Fabrication and characterization of Si O2/Si heterogeneous nanopillar arrays. Nanotechnology,2016,27(30):305301.
[14]Sun J,Wang C,Shao B,et al. Fast on-site visual detection of active ricin using a combination of highly efficient dual-recognition affinity magnetic enrichment and a specific gold nanoparticle probe. Anal Chem,2017,89(22):12209-12216.
[15]Wang J,Gao S,Kang L,et al. Development of colloidal goldbased immunochromatographic assay for the rapid detection of ricin toxin in food samples. Food and Agricultural Immunology,2017,22(2):185-193.
[2]Kulinsky L,Noroozi Z,Madou M. Present technology and future trends in point-of-care microfluidic diagnostics. Methods Mol Biol,2013,949:3-23.
[3] Whitesides G M. The origins and the future of microfluidics.Nature,2006,442(7101):368-373.
[4]Pandey C M,Augustine S,Kumar S,et al. Microfluidics based point-of-care diagnostics. Biotechnol J,2018,13(1):1700047
[5] Yu Q,Xue L,Hiblot J,et al. Semisynthetic sensor proteins enable metabolic assays at the point of care. Science,2018,361(6407):1122-1126.
[6] Zarei M. Advances in point-of-care technologies for molecular diagnostics. Biosens Bioelectron,2017,98:494-506.
[7] Mao H,Wu W,She D,et al. Microfluidic surface-enhanced Raman scattering sensors based on nanopillar forests realized by an oxygen-plasma-stripping-of-photoresist technique. Small,2014,10(1):127-134.
[8]杨宇东,毛海央,李锐锐,等.双层复合纳米森林结构的制备及其宽光谱高吸收光学特性研究.红外与毫米波学报,2018,37(2):246-250.Yang Y D,Mao H Y,Li R R et al. Fabrication and broadband high absorption features of double-layer hybrid nanoforests. J Infrared Millim Waves,2018,37(2):246-250.
[9] Mao H,Wu D,Wu W,et al. The fabrication of diversiform nanostructure forests based on residue nanomasks synthesized by oxygen plasma removal of photoresist. Nanotechnology,2009,20(44):445304.
[10]Kharisov B I,Kharissova O V,García B O,et al. State of the art of nanoforest structures and their applications. RSC Adv,2015,5(128):105507-105523.
[11] Yang Y,Mao H,Xiong J,et al. Optical features of Nanowire forests prepared by a plasma repolymerization technique. IEEE Trans Nanotechnol,2018,PP(99):452-455.
[12]Zhang H,Yang Y,Li X,et al. Frequency-enhanced transferrin receptor antibody-labelled microfluidic chip(FETAL-Chip)enables efficient enrichment of circulating nucleated red blood cells for non-invasive prenatal diagnosis. Lab Chip,2018,18(18):2749-2756.
[13]Wu W,Mao H,Han X,et al. Fabrication and characterization of Si O2/Si heterogeneous nanopillar arrays. Nanotechnology,2016,27(30):305301.
[14]Sun J,Wang C,Shao B,et al. Fast on-site visual detection of active ricin using a combination of highly efficient dual-recognition affinity magnetic enrichment and a specific gold nanoparticle probe. Anal Chem,2017,89(22):12209-12216.
[15]Wang J,Gao S,Kang L,et al. Development of colloidal goldbased immunochromatographic assay for the rapid detection of ricin toxin in food samples. Food and Agricultural Immunology,2017,22(2):185-193.