基于紫外光化学反应的高聚物微流控芯片制备技术研究及应用
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摘要
微流控芯片是微全分析系统的核心。它是在方寸大小的微芯片上集成通道、反应池、检测器等微元件,用以执行采样、稀释、加样、反应、分离、检测等单元操作功能,结合一定的外部设备实现“试样进、数据出”的快速、自动化学分析或者生化分析。因此,微流控芯片的制备是实现微全分析的关键。
芯片的制备通常采用微机电加工技术,需要昂贵的仪器设备和洁净实验室以及娴熟的制作技能,不便在普通化学和生化实验室中推广。
基于紫外光化学反应的表面处理技术具有光源简单易得、操作方便、无污染、易于实现高精度图案化处理等优点,因而受到微流控芯片研究者的青睐。目前,紫外光表面处理技术多用于微流控芯片表面化学修饰以改善其分析性能。而本论文则以芯片的制备技术为目标,系统地研究基于紫外光化学反应的制备高聚物微(纳)流控芯片及集成化器件的新方法和新技术,并对所制备的芯片开展应用研究。论文共分为六章。
第一章主要介绍了与本论文相关的紫外光与有机高聚物的作用机理,并对近年来紫外光在微流控芯片加工中的应用现状进行综述。
第二章研究在聚苯乙烯(PS)上基于紫外光诱导活化的区域化学镀方法,旨在为具有良好生物相容性的PS芯片上集成微电极等金属微器件,提高芯片的综合功能。首先考察了不同的紫外光源对PS表面的改性效果,发现365nm紫外光对PS表面几乎没有改性效果；254nm紫外光可以在2h内使PS表面产生羧基等含氧活性基团而使之变为亲水。以主要辐射254nm紫外光的紫外消毒灯(低压汞灯)为光源,研究建立了在PS表面通过紫外光诱导活化-区域化学镀制备金膜微器件的方法及相关原理。利用所建立的方法在PS芯片上制备了金薄膜电化学传感微电极和电加热器。通过对PS片上所制备的金膜微器件进行物理和化学性质表征,发现这些金膜微器件具有良好的附着性能、耐酸耐碱性能以及电化学性能。将集成有金膜微电极的PS盖片与带有通道网络结构的PS基片通过等离子体辅助热压封合组成毛细管电泳-安培检测的全PS芯片,应用于神经递质多巴胺和儿茶酚的分离分析,在238V/cm的场强下,分离效率达到1.3×104塔板/m(多巴胺),多巴胺和儿茶酚检测限分别为0.5和0.7μmol/L。五次连续进样分离检测,两者迁移时间的RSD分别为0.5%和0.4%,峰高的RSD分别为1.1%和2.7%。与同类的PDMS-玻璃杂交电泳芯片相比,该芯片具有分离效率高、检测限低、精密度高、分析时间短等优点。
第三章针对塑料芯片中最常用的材料聚甲基丙烯酸甲酯(PMMA)难以借用第二章方法在其上制备金属微器件的难题,研究PMMA的紫外光化学反应行为,以建立可应用于PMMA的紫外光诱导活化的区域化学镀技术。考察了UV、O3以及UV结合03(UV/O3)对PMMA的表面改性效果,观察到只有采用UV/O3对PMMA光照处理才能在其表面形成含氧活性基团。在此基础上,以UV/O3为光源,系统研究了UV/O3区域活化结合化学镀在PMMA上制备金属器件的方法和相关的化学原理,并对所制备的金(铜)薄膜微电极和金薄膜电热器等金属微器件进行了物理和化学性能表征。所制备的金属微器件尺寸精确,具有较强的附着强度,良好的电学和电化学性能。
第四章针对目前制备纳米通道多采用的高能粒子束干法刻蚀技术难以推广普及的问题,研究建立了一种基于紫外光降解作用的、在PMMA上制备一维(1-D)纳流控芯片的制备方法,包括紫外光直接干法刻蚀纳米/亚微米通道和UV/O3甫助低温封合纳米通道两个关键技术,方法新颖、简单、易于批量化生产。研究比较了不同光源对PMMA的刻蚀效果,发现UV/O3具有更好的光降解刻蚀效果。以UV/O3为光源,配合石英掩膜的使用,研究了刻蚀深度与辐照剂量的关系,实验数据经数学拟合,证实两者符合二次函数关系。同时发现,干刻的纳米通道水洗后其深度会加深20%左右。利用UV/O3对PMMA表面的光化学表面改性作用,实现了PMMA纳米通道的低温封合。首先采用UV/O3对带有纳米通道的PMMA基片与空白PMMA盖片的拟封合表面进行整体辐照,在它们表面形成亲水活性基团,然后在流动的水中将两片PMMA贴合,在低温低压(45℃,1.2×105Pa)下放置35min,即可实现不可逆封合。上述封合工艺适用于深宽比大于1：1000的1-D纳米通道,封合后通道的塌陷率为(13±9)%,封合强度可达6.71±2.50MPa。利用建立的方法,制备了PMMA微-纳复合通道芯片,成功应用于异硫氰酸酯荧光素(FITC)阴离子的电驱动富集与消散。
第五章针对由PDMS弹性体与硬质热塑性高聚物(塑料)组成的PDMS-塑料复合芯片很难实现不可逆封合的问题,研究建立了一种简单易行的、基于硅烷化-紫外光活化的PDMS-PS复合芯片的不可逆封合方法。研究表明,经过UV/O3处理后PS表面形成羧基、羟基等活性基团,利用这些基团作为锚点,通过硅烷化反应,将3-氨丙基三乙氧基硅烷(APTES)键合到PS表面,再通过UV/O3的光降解作用,使PS表面的硅烷化层降解,形成残留于PS表面的硅羟基。同样通过UV/O3的表面活化处理,使PDMS表面产生硅羟基。将经UV/O3处理后的硅烷化PS表面与UV/O3处理后的PDMS表面贴合,借助硅羟基之间的缩合反应,实现PDMS与PS两者的不可逆封合。利用PDMS具有的良好透气性,结合PS所具有的极佳生物相容性,设计制备了复合材料的PDMS-PS细胞培养芯片,试用于人宫颈癌细胞的培养时,观察到,PDMS-PS复合芯片的培养效果优于全PS和全PDMS芯片。
第六章针对两相液滴微流控分析系统中液滴内容物在线检测的难题,研究在两相液滴微流芯片上集成安培检测器的方法,并对两相液滴-安培检测系统的电化学行为开展基础性研究。利用第二章所建立的方法,在PS芯片上制备了金属薄膜微电极阵列,研究建立了一个适用于油包水(W/O)两相液滴流系统中水相液滴内容物的在线安培检测系统,并以H2O2为模型待测物,研究了该系统的电化学响应行为。研究发现,W/O两相液滴流的安培信号(i-t曲线)由尖锋形的前沿和较尖锋低的平台形后沿所组成,且信号的轮廓与流速、相比等因素有关。通过系统的研究,对空白磷酸缓冲液液滴和H202液滴的i-t信号的特征和归属作了分析和论证。研究还发现,两相液滴流的信号远远小于相同浓度水相连续流的信号,指出包裹在水相液滴外的油膜对液滴内容物的电化学响应有较大的阻碍作用。通过系统研究流速、水油比、电极构型、电极宽度对液滴流响应的影响,初步优化了该系统,液滴中H2O2的检测灵敏度达到22.2pA/(mol/L),线性范围为10-500μmol/L,同一芯片日内检测250μmol/L H2O2液滴的RSD为3.4%(n=9),日间RSD为8.9%(n=3)。此研究为两相液滴流的在线安培检测在化学与生化分析中奠定了一定的理论和技术基础。
本论文的主要创新点：
1.研究建立了两种基于紫外光诱导活化-区域化学镀技术的在PS和PMMA芯片上集成微金属膜器件的新方法。发现主要辐射254nm紫外光的无臭氧低压汞灯,可以对PS进行有效的光化学改性使之表面产生足量的羧基,但不能有效的改性PMMA。要使PMMA表面也产生足量的羧基,需采用有臭氧的低压汞灯作为光源进行改性。以改性后表面产生的羧基为锚点进行一系列的化学反应后,在光改性区域可以沉积纳米金粒子用于催化后续化学镀的进行。
2.创造性的提出并建立了一种PMMA纳流控芯片的制备方法,包括由低压汞灯发射的深紫外光及其诱导产生的臭氧(UV/O3)通过石英掩膜干法刻蚀制备一维纳米通道,和利用UV/O3对PMMA的表面改性作用实现纳米通道的低温、低压、高强度的封合。该方法无需昂贵的实验设备,为化学和生物化学研究者在普通实验室制备PMMA纳流控或者复合微-纳流控芯片提供了一种新颖的、易于推广的技术平台。
3.研究攻克了PDMS-塑料复合芯片不可逆封接的难题,建立了基于紫外光辅助硅烷化-紫外光活化的PDMS-PS复合芯片的不可逆封接技术。制备的PDMS-PS复合芯片兼有PDMS的弹性、透气性和PS的刚性、生物兼容性,为制备性能优良的PDMS-塑料微流芯片提供了技术支撑。
4.构建了一个集成化的、适用于两相液滴流中水相液滴内电活性物质的在线实时检测的两相液滴-安培检测系统。揭示了该系统对H202液滴的电化学行为,并对所观察到的现象提出了自己的见解,为两相液滴-安培检测系统在化学分析和生化分析中的应用提供了技术和理论基础。
Microfluidic chips, the core of the micro total analysis systems (μ-TAS), are micro-chips integrated with micro channels, micro reaction cells, micro detectors and other micro components to perform the operations in traditional laboratories such as sampling, dilution, reaction, seperation, detection and so on. With the help of a few external equipments, the microfluidic chips can lead to fast, automated chemical or biochemical total analyses featuring the so-called "sample in and data out". Therefore, the preparation of microfluidic chips is the key to realize the micro total analyses.
Preparation of microfluidic chips with traditional micro electro-mechanical systems (MEMS) needs expensive equipments, clean rooms, and skilled operational technicians. Therefore, it is hardly possible to prepare microfluidic chips via MEMS technology in ordinary chemistry or biochemistry laboratories.
Surface treatment of various substrates via UV-photochemical reaction has recently attracted the attention of micrfluidic chip researchers due to its advantages such as pollution-free, easiness to perform highly-accurately region-selective surface treatment, and needless of expensive equipments but a simple UV light source. So far, however, the UV-based surface treatment of microfluidic chips reported by literatures is mainly focused on modifying the surface properties of the chip channels, in-turn, to improve their analytical performance. The present work intends to develop new UV-photoreaction-based methods and techniques used to fabricate micro/nano fluidic chips, to prepare on-chip integrated micro-devices, and to study the possible applications of developed chips in various chemical or biochemical analyses.
This thesis is composed of six chapters:
In chapter1, a brief introduction to the mechanisms of photochemical reactions between UV lights and polymers was made, and the recent progress in the application of UV lights for fabrication of micrfluidic chips was reviewed.
In chapter2, a novel method was developed for preparing of micro-electrodes on polystyrene (PS), one of the most biocompatible polymer materials, by electroless gold plating after UV-induced regional activation of the PS surface. First, several types of UV light soucres were investigated into their performance in hydrophilic modification of PS surface. The high-pressure mercury lamp that mainly emits365nm UV lights did almost no effect on hydrophilic modification of PS, while the low-pressure mercury UV-lamp that mainly emits254nm UV lights could effectively improve the wettability of PS surface. Based on this observation, a low-pressure mercury lamp was selected as the UV light source for photo-modification of PS surface. Micro gold film devices of micro-heater and micro electrochemical sensing electrodes were electrolessly plated on the UV-exposed PS surface after the regionally UV-exposed PS surface was subjected to a series of chemical reactions. The gold films plated with developed method possessed strong adhesion, good resistance to acid and alkali, and excellent elctrochemical properties.
The PS cover sheet with micro-electrodes and PS substrate with micro-channels were bonded after plasma treatment, forming a full-PS capillary electrophoresis chip with integrated amperometric detector. With dopamine (DA) and catechol (CA) as model analytes, the separation efficiency obtained at an electric field of238V/cm was1.3×104plates/m for DA. The detection limits were0.5(DA) and0.7(CA)μmol/L respectively. Five consecutive runs of a standard solution containing100μmol/L DA and100μmol/L CA showed RSDs of0.5%(DA) and0.4%(CA) in migration time and RSDs of1.1%(DA) and2.7%(CA) in peak height of signals, respectivly. Compared with the analytical performances previously reported by using a PDMS-glass hybrid electrophoresis chip, the present full PS chip possessed higher separation efficiency, better signal precision and shorter analysis time.
Polymethylmethacrylate (PMMA) is one of the most widely used polymer material for microfluidic chips. However, it is hardly possible to prepare micro metal devices on the surface of PMMA with the electroless plating method similar to the protocol developed in chapter2. Thus, in chapter3, an investigation into the photochemical reaction between UV lights and PMMA surface was carried out. Deep UV-lights, ozone, and deep UV-lights in cooperation with ozone (UV/O3) were compared with respect to the efficiency of hydrophilization of the PMMA surface. It was observed that only UV/O3can generate oxygen-containing moieties on the PMMA surface, consequently, turn the PMMA surface from hydrophobic to hydrophilic effectively. Based on the observation, a novel method for preparation of micro metal devices on the PMMA surface was established by using UV/O3as the UV-light source. The prepared micro metal devices showed accurate size, strong adhesion to PMMA substrate, good electric and electrochemical properties.
In chapter4, a novel and facile method was developed for preparation of PMMA fluidic chips with1-dimension (1-D) nano-channels. First,1-D nano-or sub-micro channels were etched by photoresist-free UV-lithography based on the UV photodegradation of the PMMA. Then the channels were sealed by UV-assisted low temperature bonding. Different UV light sources were compared with respect to the efficiency of etching of PMMA. It was found that UV/O3has a faster etch rate than UV, so UV/O3was selectived as the UV light source. The etched depth increased with the UV-exposed time, with the depth-time profile fitting into a quadric polynomial curve. The depth of the etched channel increased by around20%after vigorously flushed with water. To bond the chip with nano-channels, the PMMA substrate with open nano-channels and a flat PMMA cover sheet were firstly treated with UV/O3, forming hydrophilic moieties such as hydroxyl and carboxylic acid groups. Then the two UV-exposed PMMA were brought into intimate contact under running water. The bonding was completed under a temperature of45℃and a pressure of1.2×10Pa for35min. The bonding protocol worked well for nanochanels whose aspect ratio was greater than1:1000, and the depth of the bonded nanochannels was decreased by (13+9)%in comparison to that afore-bonded. The bonding strength was very strong, the bonded chips can bear a tensile of6.71±2.50MPa. A PMMA chip with hybrid micro-and nano-fluidic channels was prepared with the developed method, and it was demonstrated for the electrokinetically-driven enrichment and depletion of fluorescein isothiocyanate isomer (FITC) dye anions.
In some circumstances, hybrid microfluidic chips composed of elastic, gas-permeable polydimethylsiloxane (PDMS) and rigid plastics are needed. However, It is very difficult to irreversiblely bond PDMS with plastics such as PS. In chapter5, a novel method for bonding PDMS to PS was proposed based on silanization of PS surface in combination of UV-activating of the to-be-bonded surfaces. Firstly, the PS substrate was exposed to UV/O3to produce hydroxyl and carboxylic acid moieties on its surface. The UV/O3treated PS was silanized with (3-aminopropyl)triethoxysilane (APTES) through the reaction between the hydroxyl and carboxylic acid moieties on the PS surface and the molecules of APTES. Then both the silanized-PS and PDMS surfaces were treated with UV/O3to generate silicon hydroxyl moieties on both surfaces. Finally, the UV-activated PDMS was intimately contacted to the UV-activated PS surface, and irreversibly bonding occurred between PS and PDMS after pressed and stayed for one hour due to the condensation reaction between the silicon hydroxyl moieties on the PS surface and those on PDMS surface. A hybrid PDMS-PS microfluidic chip composed of gas-permeable PDMS substrate with channel network and excellently biocompatible PS cover sheet was fabricated for cell culture. The results showed that the cells cultured in the hybrid PDMS-PS chip grew significantly better than those cultured in a full-PS chip or in a full-PDMS chip.
In chapter6, a novel on-chip-integrated micro electrochemical detection system for the determination of electro-active species contained in aqueous droplets of water in oil (W/O, n-octanol as the oil) two-phase flow was developed and studied. Three micro-electrodes for amperometric detection were prepared on the PS chip with W/O droplet generator network by using the protocol developed by chapter2. With H2O2as the model analyte, the electrochemical behavior of the detector towards the W/O droplet-based system was studied. It was found that the recorded current-time profile of the amperometric signal consisted of peak-shaped front and platform-shaped trailing edge. The attributions of both the charge current and Farady current to signal profiles of either the phosphate blank droplets or H2O2analyte droplets were characterized. The current signals generated by H2O2-containing W/O droplet flow were much lower than that produced by H2O2-containing continuous flow. This could be ascribed to the fact that the oil film packaging the water droplet prevented the droplet from direct contac to the sensing electrodes. The effect of the flow rates, phase ratio of the water to oil, configuration of the electrode system and the width of the electrode on the current signals of the droplets was examined. After optimization, a detection sensitivity of22.2pA/(mol/L) for H2O2concentration ranging from10to500μmol/L was achieved. The RSDs of the current signals observed with W/O droplets containing250μmol/L H2O2were3.2%for inter-day (n=9) and8.9%for intra-day (n=3), respectively.
The main novelties of the present work are summarized as:
1. Two novel approaches have been established for preparation of micro metal film devices on polymeric chips via UV-directed region-selective electroless metal plating, one for PS chips and the other for PMMA chips. It has been revealed that the low pressure mercury lamp emitting254nm UV-light without generation of ozone is effective for photochemical formation of carboxyl moieties on PS surface but it does not work for PMMA. To produce the same moieties on PMMA surface, a low pressure mercury lamp emitting254nm UV-light in company of ozone generation is required. With the carboxyl moieties serving as the anchor, gold nano-particle catalysts for the followed region-selectively electroless plating can be deposited onto the UV-exposed area.
2. A novel approach has been established for fabrication of nanofluidic PMMA chips. The approach includes preparation of1-D nanochannels on PMMA substrates by means of UV/O3lithography and bonding of PMMA substrates with nanochannels at low temperature and low pressure after UV/03-photochemical modification of the to-be-bonded PMMA surfaces. Without needs of expensive equipments, the established approach provides chemists or biochemists with a facile and innovative technical platform for fabricating PMMA nano-or hybrid nano/micro-fluidic chips in common chemistry laboratories.
3. A novel and easy-to-do method has been developed for irreversibly bonding of an elastic PDMS slab to a PS substrate based on UV-assisted silanization of the PS surface followed by UV-photochamical activation of both the PDMS slab and silanized-PS substrate immediately before bonding. With the developed method, hybrid PDMS-PS microchips possessing both the elastic, gas-permeable properties of PDMS and the rigid, biocompatible properties of PS can be fabricated in chemistry or biochemistry laboratories.
4. A novel PS microfluidc chip has been fabricated, which possesses the functions of on-chip generation of droplet-based two-phase flow and on-chip, real-time electrochemical detection of the electroactive species contained inside the W/O droplets. The electrochemical behaviour of the on-chip integrated amperometric detector for in-droplet-contained H2O2has been comprehensively studied, and some phenomenons specifically relevant to the two-phase droplet system revealed.
芯片的制备通常采用微机电加工技术,需要昂贵的仪器设备和洁净实验室以及娴熟的制作技能,不便在普通化学和生化实验室中推广。
基于紫外光化学反应的表面处理技术具有光源简单易得、操作方便、无污染、易于实现高精度图案化处理等优点,因而受到微流控芯片研究者的青睐。目前,紫外光表面处理技术多用于微流控芯片表面化学修饰以改善其分析性能。而本论文则以芯片的制备技术为目标,系统地研究基于紫外光化学反应的制备高聚物微(纳)流控芯片及集成化器件的新方法和新技术,并对所制备的芯片开展应用研究。论文共分为六章。
第一章主要介绍了与本论文相关的紫外光与有机高聚物的作用机理,并对近年来紫外光在微流控芯片加工中的应用现状进行综述。
第二章研究在聚苯乙烯(PS)上基于紫外光诱导活化的区域化学镀方法,旨在为具有良好生物相容性的PS芯片上集成微电极等金属微器件,提高芯片的综合功能。首先考察了不同的紫外光源对PS表面的改性效果,发现365nm紫外光对PS表面几乎没有改性效果；254nm紫外光可以在2h内使PS表面产生羧基等含氧活性基团而使之变为亲水。以主要辐射254nm紫外光的紫外消毒灯(低压汞灯)为光源,研究建立了在PS表面通过紫外光诱导活化-区域化学镀制备金膜微器件的方法及相关原理。利用所建立的方法在PS芯片上制备了金薄膜电化学传感微电极和电加热器。通过对PS片上所制备的金膜微器件进行物理和化学性质表征,发现这些金膜微器件具有良好的附着性能、耐酸耐碱性能以及电化学性能。将集成有金膜微电极的PS盖片与带有通道网络结构的PS基片通过等离子体辅助热压封合组成毛细管电泳-安培检测的全PS芯片,应用于神经递质多巴胺和儿茶酚的分离分析,在238V/cm的场强下,分离效率达到1.3×104塔板/m(多巴胺),多巴胺和儿茶酚检测限分别为0.5和0.7μmol/L。五次连续进样分离检测,两者迁移时间的RSD分别为0.5%和0.4%,峰高的RSD分别为1.1%和2.7%。与同类的PDMS-玻璃杂交电泳芯片相比,该芯片具有分离效率高、检测限低、精密度高、分析时间短等优点。
第三章针对塑料芯片中最常用的材料聚甲基丙烯酸甲酯(PMMA)难以借用第二章方法在其上制备金属微器件的难题,研究PMMA的紫外光化学反应行为,以建立可应用于PMMA的紫外光诱导活化的区域化学镀技术。考察了UV、O3以及UV结合03(UV/O3)对PMMA的表面改性效果,观察到只有采用UV/O3对PMMA光照处理才能在其表面形成含氧活性基团。在此基础上,以UV/O3为光源,系统研究了UV/O3区域活化结合化学镀在PMMA上制备金属器件的方法和相关的化学原理,并对所制备的金(铜)薄膜微电极和金薄膜电热器等金属微器件进行了物理和化学性能表征。所制备的金属微器件尺寸精确,具有较强的附着强度,良好的电学和电化学性能。
第四章针对目前制备纳米通道多采用的高能粒子束干法刻蚀技术难以推广普及的问题,研究建立了一种基于紫外光降解作用的、在PMMA上制备一维(1-D)纳流控芯片的制备方法,包括紫外光直接干法刻蚀纳米/亚微米通道和UV/O3甫助低温封合纳米通道两个关键技术,方法新颖、简单、易于批量化生产。研究比较了不同光源对PMMA的刻蚀效果,发现UV/O3具有更好的光降解刻蚀效果。以UV/O3为光源,配合石英掩膜的使用,研究了刻蚀深度与辐照剂量的关系,实验数据经数学拟合,证实两者符合二次函数关系。同时发现,干刻的纳米通道水洗后其深度会加深20%左右。利用UV/O3对PMMA表面的光化学表面改性作用,实现了PMMA纳米通道的低温封合。首先采用UV/O3对带有纳米通道的PMMA基片与空白PMMA盖片的拟封合表面进行整体辐照,在它们表面形成亲水活性基团,然后在流动的水中将两片PMMA贴合,在低温低压(45℃,1.2×105Pa)下放置35min,即可实现不可逆封合。上述封合工艺适用于深宽比大于1：1000的1-D纳米通道,封合后通道的塌陷率为(13±9)%,封合强度可达6.71±2.50MPa。利用建立的方法,制备了PMMA微-纳复合通道芯片,成功应用于异硫氰酸酯荧光素(FITC)阴离子的电驱动富集与消散。
第五章针对由PDMS弹性体与硬质热塑性高聚物(塑料)组成的PDMS-塑料复合芯片很难实现不可逆封合的问题,研究建立了一种简单易行的、基于硅烷化-紫外光活化的PDMS-PS复合芯片的不可逆封合方法。研究表明,经过UV/O3处理后PS表面形成羧基、羟基等活性基团,利用这些基团作为锚点,通过硅烷化反应,将3-氨丙基三乙氧基硅烷(APTES)键合到PS表面,再通过UV/O3的光降解作用,使PS表面的硅烷化层降解,形成残留于PS表面的硅羟基。同样通过UV/O3的表面活化处理,使PDMS表面产生硅羟基。将经UV/O3处理后的硅烷化PS表面与UV/O3处理后的PDMS表面贴合,借助硅羟基之间的缩合反应,实现PDMS与PS两者的不可逆封合。利用PDMS具有的良好透气性,结合PS所具有的极佳生物相容性,设计制备了复合材料的PDMS-PS细胞培养芯片,试用于人宫颈癌细胞的培养时,观察到,PDMS-PS复合芯片的培养效果优于全PS和全PDMS芯片。
第六章针对两相液滴微流控分析系统中液滴内容物在线检测的难题,研究在两相液滴微流芯片上集成安培检测器的方法,并对两相液滴-安培检测系统的电化学行为开展基础性研究。利用第二章所建立的方法,在PS芯片上制备了金属薄膜微电极阵列,研究建立了一个适用于油包水(W/O)两相液滴流系统中水相液滴内容物的在线安培检测系统,并以H2O2为模型待测物,研究了该系统的电化学响应行为。研究发现,W/O两相液滴流的安培信号(i-t曲线)由尖锋形的前沿和较尖锋低的平台形后沿所组成,且信号的轮廓与流速、相比等因素有关。通过系统的研究,对空白磷酸缓冲液液滴和H202液滴的i-t信号的特征和归属作了分析和论证。研究还发现,两相液滴流的信号远远小于相同浓度水相连续流的信号,指出包裹在水相液滴外的油膜对液滴内容物的电化学响应有较大的阻碍作用。通过系统研究流速、水油比、电极构型、电极宽度对液滴流响应的影响,初步优化了该系统,液滴中H2O2的检测灵敏度达到22.2pA/(mol/L),线性范围为10-500μmol/L,同一芯片日内检测250μmol/L H2O2液滴的RSD为3.4%(n=9),日间RSD为8.9%(n=3)。此研究为两相液滴流的在线安培检测在化学与生化分析中奠定了一定的理论和技术基础。
本论文的主要创新点：
1.研究建立了两种基于紫外光诱导活化-区域化学镀技术的在PS和PMMA芯片上集成微金属膜器件的新方法。发现主要辐射254nm紫外光的无臭氧低压汞灯,可以对PS进行有效的光化学改性使之表面产生足量的羧基,但不能有效的改性PMMA。要使PMMA表面也产生足量的羧基,需采用有臭氧的低压汞灯作为光源进行改性。以改性后表面产生的羧基为锚点进行一系列的化学反应后,在光改性区域可以沉积纳米金粒子用于催化后续化学镀的进行。
2.创造性的提出并建立了一种PMMA纳流控芯片的制备方法,包括由低压汞灯发射的深紫外光及其诱导产生的臭氧(UV/O3)通过石英掩膜干法刻蚀制备一维纳米通道,和利用UV/O3对PMMA的表面改性作用实现纳米通道的低温、低压、高强度的封合。该方法无需昂贵的实验设备,为化学和生物化学研究者在普通实验室制备PMMA纳流控或者复合微-纳流控芯片提供了一种新颖的、易于推广的技术平台。
3.研究攻克了PDMS-塑料复合芯片不可逆封接的难题,建立了基于紫外光辅助硅烷化-紫外光活化的PDMS-PS复合芯片的不可逆封接技术。制备的PDMS-PS复合芯片兼有PDMS的弹性、透气性和PS的刚性、生物兼容性,为制备性能优良的PDMS-塑料微流芯片提供了技术支撑。
4.构建了一个集成化的、适用于两相液滴流中水相液滴内电活性物质的在线实时检测的两相液滴-安培检测系统。揭示了该系统对H202液滴的电化学行为,并对所观察到的现象提出了自己的见解,为两相液滴-安培检测系统在化学分析和生化分析中的应用提供了技术和理论基础。
Microfluidic chips, the core of the micro total analysis systems (μ-TAS), are micro-chips integrated with micro channels, micro reaction cells, micro detectors and other micro components to perform the operations in traditional laboratories such as sampling, dilution, reaction, seperation, detection and so on. With the help of a few external equipments, the microfluidic chips can lead to fast, automated chemical or biochemical total analyses featuring the so-called "sample in and data out". Therefore, the preparation of microfluidic chips is the key to realize the micro total analyses.
Preparation of microfluidic chips with traditional micro electro-mechanical systems (MEMS) needs expensive equipments, clean rooms, and skilled operational technicians. Therefore, it is hardly possible to prepare microfluidic chips via MEMS technology in ordinary chemistry or biochemistry laboratories.
Surface treatment of various substrates via UV-photochemical reaction has recently attracted the attention of micrfluidic chip researchers due to its advantages such as pollution-free, easiness to perform highly-accurately region-selective surface treatment, and needless of expensive equipments but a simple UV light source. So far, however, the UV-based surface treatment of microfluidic chips reported by literatures is mainly focused on modifying the surface properties of the chip channels, in-turn, to improve their analytical performance. The present work intends to develop new UV-photoreaction-based methods and techniques used to fabricate micro/nano fluidic chips, to prepare on-chip integrated micro-devices, and to study the possible applications of developed chips in various chemical or biochemical analyses.
This thesis is composed of six chapters:
In chapter1, a brief introduction to the mechanisms of photochemical reactions between UV lights and polymers was made, and the recent progress in the application of UV lights for fabrication of micrfluidic chips was reviewed.
In chapter2, a novel method was developed for preparing of micro-electrodes on polystyrene (PS), one of the most biocompatible polymer materials, by electroless gold plating after UV-induced regional activation of the PS surface. First, several types of UV light soucres were investigated into their performance in hydrophilic modification of PS surface. The high-pressure mercury lamp that mainly emits365nm UV lights did almost no effect on hydrophilic modification of PS, while the low-pressure mercury UV-lamp that mainly emits254nm UV lights could effectively improve the wettability of PS surface. Based on this observation, a low-pressure mercury lamp was selected as the UV light source for photo-modification of PS surface. Micro gold film devices of micro-heater and micro electrochemical sensing electrodes were electrolessly plated on the UV-exposed PS surface after the regionally UV-exposed PS surface was subjected to a series of chemical reactions. The gold films plated with developed method possessed strong adhesion, good resistance to acid and alkali, and excellent elctrochemical properties.
The PS cover sheet with micro-electrodes and PS substrate with micro-channels were bonded after plasma treatment, forming a full-PS capillary electrophoresis chip with integrated amperometric detector. With dopamine (DA) and catechol (CA) as model analytes, the separation efficiency obtained at an electric field of238V/cm was1.3×104plates/m for DA. The detection limits were0.5(DA) and0.7(CA)μmol/L respectively. Five consecutive runs of a standard solution containing100μmol/L DA and100μmol/L CA showed RSDs of0.5%(DA) and0.4%(CA) in migration time and RSDs of1.1%(DA) and2.7%(CA) in peak height of signals, respectivly. Compared with the analytical performances previously reported by using a PDMS-glass hybrid electrophoresis chip, the present full PS chip possessed higher separation efficiency, better signal precision and shorter analysis time.
Polymethylmethacrylate (PMMA) is one of the most widely used polymer material for microfluidic chips. However, it is hardly possible to prepare micro metal devices on the surface of PMMA with the electroless plating method similar to the protocol developed in chapter2. Thus, in chapter3, an investigation into the photochemical reaction between UV lights and PMMA surface was carried out. Deep UV-lights, ozone, and deep UV-lights in cooperation with ozone (UV/O3) were compared with respect to the efficiency of hydrophilization of the PMMA surface. It was observed that only UV/O3can generate oxygen-containing moieties on the PMMA surface, consequently, turn the PMMA surface from hydrophobic to hydrophilic effectively. Based on the observation, a novel method for preparation of micro metal devices on the PMMA surface was established by using UV/O3as the UV-light source. The prepared micro metal devices showed accurate size, strong adhesion to PMMA substrate, good electric and electrochemical properties.
In chapter4, a novel and facile method was developed for preparation of PMMA fluidic chips with1-dimension (1-D) nano-channels. First,1-D nano-or sub-micro channels were etched by photoresist-free UV-lithography based on the UV photodegradation of the PMMA. Then the channels were sealed by UV-assisted low temperature bonding. Different UV light sources were compared with respect to the efficiency of etching of PMMA. It was found that UV/O3has a faster etch rate than UV, so UV/O3was selectived as the UV light source. The etched depth increased with the UV-exposed time, with the depth-time profile fitting into a quadric polynomial curve. The depth of the etched channel increased by around20%after vigorously flushed with water. To bond the chip with nano-channels, the PMMA substrate with open nano-channels and a flat PMMA cover sheet were firstly treated with UV/O3, forming hydrophilic moieties such as hydroxyl and carboxylic acid groups. Then the two UV-exposed PMMA were brought into intimate contact under running water. The bonding was completed under a temperature of45℃and a pressure of1.2×10Pa for35min. The bonding protocol worked well for nanochanels whose aspect ratio was greater than1:1000, and the depth of the bonded nanochannels was decreased by (13+9)%in comparison to that afore-bonded. The bonding strength was very strong, the bonded chips can bear a tensile of6.71±2.50MPa. A PMMA chip with hybrid micro-and nano-fluidic channels was prepared with the developed method, and it was demonstrated for the electrokinetically-driven enrichment and depletion of fluorescein isothiocyanate isomer (FITC) dye anions.
In some circumstances, hybrid microfluidic chips composed of elastic, gas-permeable polydimethylsiloxane (PDMS) and rigid plastics are needed. However, It is very difficult to irreversiblely bond PDMS with plastics such as PS. In chapter5, a novel method for bonding PDMS to PS was proposed based on silanization of PS surface in combination of UV-activating of the to-be-bonded surfaces. Firstly, the PS substrate was exposed to UV/O3to produce hydroxyl and carboxylic acid moieties on its surface. The UV/O3treated PS was silanized with (3-aminopropyl)triethoxysilane (APTES) through the reaction between the hydroxyl and carboxylic acid moieties on the PS surface and the molecules of APTES. Then both the silanized-PS and PDMS surfaces were treated with UV/O3to generate silicon hydroxyl moieties on both surfaces. Finally, the UV-activated PDMS was intimately contacted to the UV-activated PS surface, and irreversibly bonding occurred between PS and PDMS after pressed and stayed for one hour due to the condensation reaction between the silicon hydroxyl moieties on the PS surface and those on PDMS surface. A hybrid PDMS-PS microfluidic chip composed of gas-permeable PDMS substrate with channel network and excellently biocompatible PS cover sheet was fabricated for cell culture. The results showed that the cells cultured in the hybrid PDMS-PS chip grew significantly better than those cultured in a full-PS chip or in a full-PDMS chip.
In chapter6, a novel on-chip-integrated micro electrochemical detection system for the determination of electro-active species contained in aqueous droplets of water in oil (W/O, n-octanol as the oil) two-phase flow was developed and studied. Three micro-electrodes for amperometric detection were prepared on the PS chip with W/O droplet generator network by using the protocol developed by chapter2. With H2O2as the model analyte, the electrochemical behavior of the detector towards the W/O droplet-based system was studied. It was found that the recorded current-time profile of the amperometric signal consisted of peak-shaped front and platform-shaped trailing edge. The attributions of both the charge current and Farady current to signal profiles of either the phosphate blank droplets or H2O2analyte droplets were characterized. The current signals generated by H2O2-containing W/O droplet flow were much lower than that produced by H2O2-containing continuous flow. This could be ascribed to the fact that the oil film packaging the water droplet prevented the droplet from direct contac to the sensing electrodes. The effect of the flow rates, phase ratio of the water to oil, configuration of the electrode system and the width of the electrode on the current signals of the droplets was examined. After optimization, a detection sensitivity of22.2pA/(mol/L) for H2O2concentration ranging from10to500μmol/L was achieved. The RSDs of the current signals observed with W/O droplets containing250μmol/L H2O2were3.2%for inter-day (n=9) and8.9%for intra-day (n=3), respectively.
The main novelties of the present work are summarized as:
1. Two novel approaches have been established for preparation of micro metal film devices on polymeric chips via UV-directed region-selective electroless metal plating, one for PS chips and the other for PMMA chips. It has been revealed that the low pressure mercury lamp emitting254nm UV-light without generation of ozone is effective for photochemical formation of carboxyl moieties on PS surface but it does not work for PMMA. To produce the same moieties on PMMA surface, a low pressure mercury lamp emitting254nm UV-light in company of ozone generation is required. With the carboxyl moieties serving as the anchor, gold nano-particle catalysts for the followed region-selectively electroless plating can be deposited onto the UV-exposed area.
2. A novel approach has been established for fabrication of nanofluidic PMMA chips. The approach includes preparation of1-D nanochannels on PMMA substrates by means of UV/O3lithography and bonding of PMMA substrates with nanochannels at low temperature and low pressure after UV/03-photochemical modification of the to-be-bonded PMMA surfaces. Without needs of expensive equipments, the established approach provides chemists or biochemists with a facile and innovative technical platform for fabricating PMMA nano-or hybrid nano/micro-fluidic chips in common chemistry laboratories.
3. A novel and easy-to-do method has been developed for irreversibly bonding of an elastic PDMS slab to a PS substrate based on UV-assisted silanization of the PS surface followed by UV-photochamical activation of both the PDMS slab and silanized-PS substrate immediately before bonding. With the developed method, hybrid PDMS-PS microchips possessing both the elastic, gas-permeable properties of PDMS and the rigid, biocompatible properties of PS can be fabricated in chemistry or biochemistry laboratories.
4. A novel PS microfluidc chip has been fabricated, which possesses the functions of on-chip generation of droplet-based two-phase flow and on-chip, real-time electrochemical detection of the electroactive species contained inside the W/O droplets. The electrochemical behaviour of the on-chip integrated amperometric detector for in-droplet-contained H2O2has been comprehensively studied, and some phenomenons specifically relevant to the two-phase droplet system revealed.
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