摘要
芯片毛细管电泳是近年来快速发展和具有广泛应用前景的新技术。该技术是在常规毛细管电泳原理的基础上发展起来的,利用微电子机械系统(MicroElectro Mechanical System,MEMS)技术在玻璃、硅、聚合物等基片上制作一系列微管道等结构单元,利用微芯片体积小、热传导效率高等优点实现对生化样本更加快速、高效的分离分析。PDMS由于具有良好的光学透明性、容易封合、无毒、低电导率、价格低廉、多功能性、固化温度低和生物相容性等优点,在制作微芯片装置中得到了广泛的应用与发展。然而,将PDMS用于微芯片电泳仍需克服其不足,如电渗流(EOF)不稳定、表面疏水性较强、易于吸附分析物等。这些缺点大大限制了可在PDMS上分离的分析物种类,并导致低的分离效率。对PDMS微通道表面进行适当的改性和修饰,可以有效控制EOF,减小分析物和管壁之间的作用,解决分析物在PDMS通道上的吸附问题,我们开展了以下几个方面的工作。
1.绪论部分对芯片毛细管电泳的工作原理、特点、评价标准和研究进展进行了总结。介绍了芯片毛细管电泳中芯片材料种类及制作和各种芯片毛细管联用检测技术。简介了高分子聚合物PDMS芯片毛细管电泳的优缺点,讨论了PDMS表面改性和修饰技术。PDMS表面改性及修饰技术主要有高能氧化、动态修饰、本体修饰和层层组装(LBL)技术等技术。非共价键作用力常被用来构建各种薄膜,最有效的非共价键驱动力是静电相互作用力,被广泛应用于聚离子间的层层组装。本论文中我们采用层层组装技术通过静电作用在通道表面组装了不同物质,并研究了修饰后的芯片的表面性质。
2.利用LBL技术,将壳聚糖(chitosan)和DNA交替组装于PDMS微芯片通道表面,构建了chitosan-DNA生物分子功能化PDMS微芯片通道。采用衰减全反射傅里叶变换红外吸收光谱(ATR-FT-IR)和接触角实验对chitosan-DNA功能化PDMS芯片进行了表征。结果表明,经chitosan-DNA修饰后的PDMS芯片,EOF得到了稳定控制,而且表面亲水性得到了明显改善。以尿酸和抗坏血酸为分离模型体系,对chitosan-DNA功能化PDMS芯片的性能进行了考察。与未经修饰的PDMS芯片相比,尿酸和抗坏血酸在chitosan-DNA修饰PDMS芯片上的分离分析时间大大缩短了,从未修饰时的200 s减少到修饰后的85 s,并且分离效率和分析灵敏度都得到了有效提高,在分离电压为1300V时,尿酸和抗坏血酸的理论塔板数分别为43450和46790N/m。将chitosan-DNA修饰PDMS芯片应用于尿样中尿酸和抗坏血酸的分离和检测,获得满意效果。
3.利用LBL技术将TiO_2 NPs组装到预修饰了一层聚阳离子PDDA的PDMS通道表面,实现了PDMS表面纳米功能化。经PDDA-TiO_2 NPs修饰后的芯片改变了EOF和溶质的迁移速度,提高了分离效率。将该修饰芯片用于神经递质多巴胺和肾上腺素的分离。与未修饰芯片相比,多巴胺和肾上腺素在PDDA-TiO_2NPs修饰芯片上不仅达到了良好的基线分离,而且峰电流显著增大,峰宽变窄,在通道表面的吸附作用得到了有效抑制,分离效率和分析灵敏度也明显提高,在pH 7.0的PBS(40 mM)缓冲液中,分离度由空白芯片上的0.61增加到修饰芯片上的1.55。在1000V的分离电压下,修饰芯片上多巴胺和肾上腺素的理论塔板数分别为1.2534×10~5N/m和9.5757×10~4N/m。多巴胺和。肾上腺素的线性范围均为30-600μM,检测限分别为2.1μM和3.2μM。此外,该修饰方法呈现长期的稳定性和很好的重现性,修饰的PDMS芯片可以连续使用两周。
4.氨基酸是构建许多生物相关分子的基本单元,其在神经信息的传递、维持和调节新陈代谢行为、生物合成蛋白质和多肽、为机体和大脑提供能源等方面起着重要的作用。建立快速、简单的氨基酸分析方法具有十分重要的意义。然而,在电泳分离过程中,氨基酸在PDMS芯片微通道内吸附严重,致使样品峰拖尾,分离效率低下。本文以Cu微盘电极为工作电极,采用柱端安培检测模式,在PDDA-TiO_2 NPs/PDMS修饰芯片上对五种氨基酸进行了分离检测。与未修饰芯片相比,经PDDA-TiO_2 NPs修饰的PDMS芯片在5.0mM硼砂缓冲液中获得了稳定、降低的EOF,这非常有利于在短的分离通道内,分离具有相似迁移时间的氨基酸。实验结果表明,精氨酸、脯氨酸、组氨酸、缬氨酸和苏氨酸在PDDA-TiO_2NPs/PDMS修饰芯片表面的吸附得到了有效抑制,90 s内即得到良好的基线分离,而且分离效率大大提高。
Microchip capillary electrophoresis (MCE) system is a newly rapidly developed research technology which was based on the routine capillary electrophoresis (CE) principle and has extensive application perspective. MicroChannel networks on the microchip are manufactured by the technology of Micro Electro Mechanical System (MEMS) on the substrate of glass, fused silica, and polymers, etc. For its small dimension and high thermal conductivity, MCE system can realize more rapid analysis. PDMS has become a popular material for building microfluidic devices mainly due to its excellent optical transparency, easy sealing with other materials, nontoxicity, low electrical conductivity, low cost, increasing versatility, relatively low curing temperature, and biocompatibility. However, PDMS microfluidic devices employed for electrophoresis show some defects that need to be overcome. These disadvantages include the unstable electroosmotic flow (EOF), extreme hydrophobicity and easy adsorption of samples onto the channel surface, etc. Through modification of appropriate substance on the PDMS surface, the adsorption on the PDMS fabricated microchip can be suppressed. The thesis was composed of four parts:
1. In chapter 1, the working principles, characteristics, evaluation standards and recent developments of microchip capillary electrophosis were reviewed. We introduced the materials and fabrication techniques of microchip and some detection techniques. The advantages and disadavantages of PDMS microchip were mentioned, and the modification techniques on PDMS surface were discussed, which included modification by exposure to energy, dynamic coating, bulk-modification and layer-by-layer (LBL) technique, and so on. Non-covalent interactions were often used to construct various films, and the most effective non-covalent driving force was electrostatic interaction which was widely used in LBL technique between polyions. In this thesis, we assembled different substances via electrostatic interactions through LBL technique, and studied the surface property of modified microchips.
2. A new fabrication of hydrophilic and biologically active PDMS microchip channel based on surface modification with chitosan and DNA using the LBL technique was proposed. The properties of the modifiers were investigated by Fourier transformed infrared adsorption by total attenuated reflection (ATR-FT-IR) spectra of the surface and the contact angle measurement. The results showed that after modification, EOF was more stable and the surface hydrophilicity was improved. Uric acid and ascorbic acid as a group of separation models were used to evaluate the effect of the functional PDMS microfluidic devices. On the chitosan-DNA modified PDMS microchip, the separation time was obviously decreased, and the sensitivity and separation efficiency were greatly enhanced. The separation time for uric acid and ascorbic acid was dramatically decreased from 200 to 85 s on native and chitosan-DNA modified microchips, respectively. The theoretical plate numbers were 43450 and 46790 N/m at the separation voltage of 1300 V for UA and AA, respectively. In addition, this method has been successfully applied to real human urine samples with satisfactory results.
3. TiO_ 2 NPs were employed to construct a nano-structure functional film on the PDMS microchip channel surface through LBL assembly technique on a pre-layer of polycation PDDA. Results showed that on the PDDA-TiO_2 NPs coated microchip, the apparent mobilities of target analytes as well as EOF can be altered, which led to enhanced separation efficiencies. Dopamine and epinephrine served as a model system to evaluate the impact of TiO_2 NPs on EOF and separation. The analytes were well separated on the modified microchip, and it was clearly evident that TiO_2 NPs modification improved the separation efficiencies of dopamine and epinephrine, and the resolution for them was largely enhanced from 0.61 on native PDMS microchip to 1.55 on coated PDMS microchip in 40 mM PBS. The theoretical plate numbers were 1.2534×10~5 N/m and 9.5757×10~4 N/m at the separation voltage of 1300 V for dopamine and epinephrine, respectively. Linear responses of them were obtained both from 25 to 600 uM with detection limits of 2.1μM for dopamine and 3.2μM for epinephrine, respectively. Moreover, the modified PDMS channels have a long-term stability and an excellent reproducibility within two weeks.
4. Amino acids as the main components in organism play an essential role in physiological procedures such as transfer nerve information, regulation metabolic activity, and biosynthesis protein and peptide. Therefore, to establish a rapid and simple method for the analysis of amino acids is very important. However, the strong interactions between PDMS surface and amino acids resulted in unavoidable adsorption on channel surface and poor separation efficiency. In this paper, five amino acids have been detected with an end-channel amperometric detection mode at a copper microdisk electrode on the PDDA-TiO_2 NPs modified microchip. Here, the copper microdisk electrode was used as a working electrode. Compared with the native PDMS microchip, EOF on the PDDA-TiO_2 NPs modified microchip was decreased and more stable, which was favorable for the separation of amino acids since they had similar migration times in the short channels. As a result, the phenomenon of adsorption was well suppressed, and arginine, proline, histidine, valine and serine were successfully separated within 90 s.
引文
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