中国生物工程杂志

Objective: A new material with ultra-large surface area named nano-forest is prepared by micro-electro-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 dye-labeled 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 50-100nm, a height of 1.8μm and a density of about 20/μm ². The quartz nano-forests structure can be obtained after RIE with nano-fibre forests structure as mask and resist removal. The single structure is shaped like a cone. The diameter of the cone bottom is about 100-200nm, the height is about 1.0μm, the density is about 10/μm ², 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 5mm/s in the micro-channel on the basis of nano-forests structure. The transmittance of the channel is 89.5% at 680nm 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 from 10pg/ml to 6 250pg/ml and the limit of detection (LOD) is lower than 10pg/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.