基于微流控芯片激光诱导荧光检测系统的研究与应用
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摘要
微全分析系统的研究是上世纪90年代所提出的全新概念。微流控芯片的加工以微电路加工技术为基础,以单晶硅片为材质。作为一种分析平台,微流控芯片实验室主要以芯片毛细管电泳的形式开始研究。随着微加工技术的不同发展,微流控芯片实验室的研究已经广泛的涉及到了食品卫生、药物分离、环境分析、生化分析等几乎所有的分析领域。微全分析系统已经将化学分析领域所涉及到的各个单元集成在了非常小的芯片上,具有高通量,高精度,高速度的特点。
激光诱导荧光检测器是目前最灵敏,应用最为广泛的检测方法之一。因此也是与微流控芯片相匹配研究最多的检测器。根据光学体统不同,激光诱导荧光检测器可以分为共聚焦型检测系统,非共聚焦型检测系统和正交型检测系统。
本论文基于微流控芯片电泳正交型激光诱导荧光检测的研制与改进工作入手,完善了微流控芯片激光诱导荧光检测系统的研究。将垂直检测,改为侧壁检测,减小了整个分析系统体积；在光电倍增光前端放置了微型小孔,降低了激光器的噪声,提高了信噪比与检测灵敏度；开发了一种新型芯片定位技术,可以避免由于材料或加工芯片的厚度造成的芯片高度定位误差,不需要人工的反复调整也不需要高成本、高技术的自动调节系统,保证每一块芯片相对于光源高度的轻松、简单、正确定位。将分析对象通过嵌入剂或衍生化反应产生荧光,考察了不同筛分介质对DNA片段的筛分性能,检测了一段p53基因的碱基突变,建立了分析牛奶中氨基酸含量的检测方法等。从而实现了微流控芯片激光诱导荧光检测系统的应用。
本论文的主要工作有：
第一章简要地综述了微流控芯片实验室的发展历史、基本原理、分离模式、联用检测技术、应用领域、研究进展以及本论文研究的目的和意义。
第二章完善了一种集成化的、可用于微流控芯片电泳检测的激光诱导荧光检测系统的研究。制备了盖片、基片长度不同的玻璃微流控芯片,并将芯片的侧壁抛光,使仪器由垂直检测改为侧壁检测,减少了仪器整体的体积；在光电倍增光前端放置了微型小孔,降低了激光器的噪声,提高了信噪比与检测灵敏度；并开发了一种利用芯片健合面上,盖片大于基片的部分作为微流控芯片的定位面和支撑面的方法,结合弹簧压片,实现了芯片的精确定位。以四触点高电压系统控制芯片上的进样和电泳分离操作。激光诱导荧光检测系统采用正交光路模式,对荧光染料Cy5的检出限达到10-11mol/L(S/N=3)。
第三章利用自由基反应,制备了一种可以用于微流控芯片上快速筛分DNA片段的共聚物。将聚吡咯烷酮(PVP)和羟乙基纤维素(HEC)形成的共聚物作为筛分介质成功的分离了DNA片段。通过改变PVP在聚合物中的比列,在进样场强为600 V/cm,共聚物摩尔比列为PVP:HEC为3：1时,可以实现FX174-HaeⅢdigest DNA marke的进样分离分析,将72-1353 bp DNA片段总分离分析时间缩短为3min。样品各片段的分离效率达到2.51×105 m-1-3.89×105 m-1。本研究为DNA片段的分离提供了简便、快速、灵敏的方法。
第四章考察了金纳米粒子与聚合物的复合体系对DNA marker的筛分性能。首先将不含金纳米粒子(GNPs)的聚合物混合溶液涂覆在芯片内壁,然后在这一体系中加入不同粒径大小的GNPs再次涂覆在通道内壁,并作为筛分介质,也可以成功的分离FX174-HaeⅢdigest DNA marker,大大提高检测信噪比。研究发现,在简单超声处理后,直接混合的聚合物溶液中,加入直径大小为30 nm的GNPs,可以在4分钟以内,实现样品DNA片段分离检测。样品DNA各个片段迁移时间的RSD值均小于2.51%,分离效率最高8.54×104 m-1。本研究拓展了纳米材料在微流控芯片电泳中的应用。
第五章利用微流控芯片电泳(ME)结合激光诱导荧光检测(LIF)技术,根据单链构象多态性(SSCP)原理,建立了检测人类p53基因突变的方法。设计不同碱基长度的p53单链序列,针对易突变的外显子7,8,9进行SSCP分析,分离正常与突变的单链DNA序列；研究了筛分介质聚乙烯基氧化物(PEO)的浓度,场强对芯片电泳行为的影响。在PEO浓度为0.5%,分离场强为260 V/cm时,100 s之内就可以实现样品p53外显子7,8,9的正常型与突变型碱基的分离检测。本研究为p53外显子的突变检测提供了快速、简单、灵敏的方法。
第六章建立了一种用于测定牛奶中氨基酸成分的微流控芯片电泳-荧光检测方法。以硼砂缓冲溶液为背景电解质,经红敏荧光染料(Cy5)衍生的7种氨基酸在150 s内可以得到很好的分离和测定。考查了各个分离参数对分离的影响,得到的优化条件为：100 mmol/L硼砂-氢氧化钠溶液(pH 9.7)作为缓冲溶液。在20mmol/L硼砂溶液(pH 9.2)中,衍生试剂Cy5与单个氨基酸的化学计量比为1：1,能够获得稳定荧光强度的氨基酸衍生物。各氨基酸成分在1×10-8mol/L-1-5×10-5 mol/L-1的浓度范围内呈良好的线性关系,相关系数R2在0.9904-0.9984之间。保留时间和峰面积的相对标准偏差分别为2.1%-4.5%和2.3%-5.4%。该方法准确可靠,可用于质量控制为目的的牛奶中氨基酸成分的定量测定。
Micro total analysis system is a field that has been growing ever since the early 1990s. Microfluidic devices originated from the integrated circuits (IC) industry, and early microfluidic devices are mostly silicon based analyzers consisting of channels for species separation in a carrier fluid. However, with recent advances in microfabrication and device design innovative components and platforms are coming to fore with applications in biology, medicine, pharmaceutics, and food and environmental monitoring. "Micro total analysis systems" (microTAS) or "lab-on-a-chip" (LOC) are becoming a reality where entire chemical analyses in miniaturized volumes are performed with high sensitivities and in shorter time spans.
Owing to its inherent selectivity and excellent sensitivity, laser-induced fluorescence (LIF) has been becoming the most popular detection scheme in conjunction with microchip electrophoresis. Various LIF detection systems based on different optical arrangements were developed and have been successfully and broadly applied in the microfluidic chip-based analysis systems. The optical arrangements of hitherto three major types of LIF detection system employed in chip devices, the confocal LIF system, nonconfocal LIF detection system and orthogonal optical arrangement system.
In this dissertation, a LIF detection system for microfluidic chips with a simple orthogonal optical arrangemen was developed. The technologies about microchip electrophoresis were systemic studied, including chip microfabrication, separation DNA marker to investigate the performance of the sieve, detection of the mutations in p53 gene and determination of amino acids in milk. The work was involved in the typical manipulation, such as the preconcentration, reaction, separation and detection. And the application of the LIF detection systems was realized.
The topics of this dissertation are presented below:
Chapter 1 reviews the research and development on microchip-based analytical systems. Then the new projects were carried out.
Chapter 2 states an integrated system involving microfluidic chip, high voltage power supply, laser-induced fluorescence (LIF) detection and control system was developed to perform microchip electrophoresis separation. A simple and convenient approach for accurately locating the chip in the integrated system was developed by using a substrate plate as the chip flat added a base spring to support the position of the chip. An orthogonal optical arrangement was employed in the LIF detection system to simplify the structure of the system with a limit of detection of 10-11 mol/L for Cy5 dye.
Chapter 3 states copolymers of poly(vinylpyrrolidone) (PVP) and hydroxyethylcellulose (HEC) were synthesized, with PVP to HEC molar ratios of 3:1, 2:1 and 1:1. The copolymers were tested as separation media in DNA fragment separation analysis by microchip electrophoresis (MCE). Separation efficiency over 3.8x105 for 118 bp has been reached by using the bare channels without the additional polymer coating step. Under optimized separation conditions for longer read length DNA sequencing, the separation ability of the copolymers decreased with decreasing (PVP-co-HEC) molar ratio from 3:1,2:1 and 1:1. In comparison with (PVP-co-HEC) 1:1, the copolymer with (PVP-co-HEC) 3:1 ratio showed high separation efficiency. By using a 2.5% w/v copolymer with (PVP-co-HEC) 3:1 ratio, FX174-HaeⅢdigest DNA marker was successfully separated within 3 min.
Chapter 4 states a simple and robust static adsorptive coating process in glass microchips for DNA separation was developed by using mixed polymer consisted of 2% hydroxyethylcellulose and 4% poly (vinylpyrrolidone) as surface modifier of microfluidic channels. This surface modifier was also used as the sieving matrix for the DNA separation. In this study, FX174-HaeIII digest sample was used to investigate the performance of the polymer mixture in microchip electrophoresis with laser-induced fluorescence detection. It was also found that adding 30 nm gold nanoparticles to the mixed polymer was very useful to achieve better resolution and reproducibility. Under the optimum conditions, the DNA fragments could be successfully separated within 4 min and the relative standard deviation values of the migration times were less than 2.51%(n=5) during the one week.
Chapter 5 states a form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7,8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method for detection of mutations for exons 7,8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 0.5% w/v poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7,8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7-9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng L-1.
Chapter 6 states a method for determination of amino acids in milk by microchip electrophoresis (ME) coupled with the detection of laser-induced fluorescence (LIF) has been developed. The amino acids in milk which derivatized with fluorescein isothiocyanate were finely separated and determinated by ME-LIF with borate buffer. The separation parameters were investigated to give an optimal experimental condition:borate buffer (100 mmol/L, pH 9.7); applied separation fleld 475 V/cm. All amino acids were separated within 150 s. All amino acid-Cy5 derivatives was linear in the appropriate concentration (R20.9904-0.9984). The relative standard deviations (n=5) of the method were 2.1%-4.5% for migration time and 2.3%-5.4% for peak area. This method allows to determining the amino acids in milk with high sensitivity.
激光诱导荧光检测器是目前最灵敏,应用最为广泛的检测方法之一。因此也是与微流控芯片相匹配研究最多的检测器。根据光学体统不同,激光诱导荧光检测器可以分为共聚焦型检测系统,非共聚焦型检测系统和正交型检测系统。
本论文基于微流控芯片电泳正交型激光诱导荧光检测的研制与改进工作入手,完善了微流控芯片激光诱导荧光检测系统的研究。将垂直检测,改为侧壁检测,减小了整个分析系统体积；在光电倍增光前端放置了微型小孔,降低了激光器的噪声,提高了信噪比与检测灵敏度；开发了一种新型芯片定位技术,可以避免由于材料或加工芯片的厚度造成的芯片高度定位误差,不需要人工的反复调整也不需要高成本、高技术的自动调节系统,保证每一块芯片相对于光源高度的轻松、简单、正确定位。将分析对象通过嵌入剂或衍生化反应产生荧光,考察了不同筛分介质对DNA片段的筛分性能,检测了一段p53基因的碱基突变,建立了分析牛奶中氨基酸含量的检测方法等。从而实现了微流控芯片激光诱导荧光检测系统的应用。
本论文的主要工作有：
第一章简要地综述了微流控芯片实验室的发展历史、基本原理、分离模式、联用检测技术、应用领域、研究进展以及本论文研究的目的和意义。
第二章完善了一种集成化的、可用于微流控芯片电泳检测的激光诱导荧光检测系统的研究。制备了盖片、基片长度不同的玻璃微流控芯片,并将芯片的侧壁抛光,使仪器由垂直检测改为侧壁检测,减少了仪器整体的体积；在光电倍增光前端放置了微型小孔,降低了激光器的噪声,提高了信噪比与检测灵敏度；并开发了一种利用芯片健合面上,盖片大于基片的部分作为微流控芯片的定位面和支撑面的方法,结合弹簧压片,实现了芯片的精确定位。以四触点高电压系统控制芯片上的进样和电泳分离操作。激光诱导荧光检测系统采用正交光路模式,对荧光染料Cy5的检出限达到10-11mol/L(S/N=3)。
第三章利用自由基反应,制备了一种可以用于微流控芯片上快速筛分DNA片段的共聚物。将聚吡咯烷酮(PVP)和羟乙基纤维素(HEC)形成的共聚物作为筛分介质成功的分离了DNA片段。通过改变PVP在聚合物中的比列,在进样场强为600 V/cm,共聚物摩尔比列为PVP:HEC为3：1时,可以实现FX174-HaeⅢdigest DNA marke的进样分离分析,将72-1353 bp DNA片段总分离分析时间缩短为3min。样品各片段的分离效率达到2.51×105 m-1-3.89×105 m-1。本研究为DNA片段的分离提供了简便、快速、灵敏的方法。
第四章考察了金纳米粒子与聚合物的复合体系对DNA marker的筛分性能。首先将不含金纳米粒子(GNPs)的聚合物混合溶液涂覆在芯片内壁,然后在这一体系中加入不同粒径大小的GNPs再次涂覆在通道内壁,并作为筛分介质,也可以成功的分离FX174-HaeⅢdigest DNA marker,大大提高检测信噪比。研究发现,在简单超声处理后,直接混合的聚合物溶液中,加入直径大小为30 nm的GNPs,可以在4分钟以内,实现样品DNA片段分离检测。样品DNA各个片段迁移时间的RSD值均小于2.51%,分离效率最高8.54×104 m-1。本研究拓展了纳米材料在微流控芯片电泳中的应用。
第五章利用微流控芯片电泳(ME)结合激光诱导荧光检测(LIF)技术,根据单链构象多态性(SSCP)原理,建立了检测人类p53基因突变的方法。设计不同碱基长度的p53单链序列,针对易突变的外显子7,8,9进行SSCP分析,分离正常与突变的单链DNA序列；研究了筛分介质聚乙烯基氧化物(PEO)的浓度,场强对芯片电泳行为的影响。在PEO浓度为0.5%,分离场强为260 V/cm时,100 s之内就可以实现样品p53外显子7,8,9的正常型与突变型碱基的分离检测。本研究为p53外显子的突变检测提供了快速、简单、灵敏的方法。
第六章建立了一种用于测定牛奶中氨基酸成分的微流控芯片电泳-荧光检测方法。以硼砂缓冲溶液为背景电解质,经红敏荧光染料(Cy5)衍生的7种氨基酸在150 s内可以得到很好的分离和测定。考查了各个分离参数对分离的影响,得到的优化条件为：100 mmol/L硼砂-氢氧化钠溶液(pH 9.7)作为缓冲溶液。在20mmol/L硼砂溶液(pH 9.2)中,衍生试剂Cy5与单个氨基酸的化学计量比为1：1,能够获得稳定荧光强度的氨基酸衍生物。各氨基酸成分在1×10-8mol/L-1-5×10-5 mol/L-1的浓度范围内呈良好的线性关系,相关系数R2在0.9904-0.9984之间。保留时间和峰面积的相对标准偏差分别为2.1%-4.5%和2.3%-5.4%。该方法准确可靠,可用于质量控制为目的的牛奶中氨基酸成分的定量测定。
Micro total analysis system is a field that has been growing ever since the early 1990s. Microfluidic devices originated from the integrated circuits (IC) industry, and early microfluidic devices are mostly silicon based analyzers consisting of channels for species separation in a carrier fluid. However, with recent advances in microfabrication and device design innovative components and platforms are coming to fore with applications in biology, medicine, pharmaceutics, and food and environmental monitoring. "Micro total analysis systems" (microTAS) or "lab-on-a-chip" (LOC) are becoming a reality where entire chemical analyses in miniaturized volumes are performed with high sensitivities and in shorter time spans.
Owing to its inherent selectivity and excellent sensitivity, laser-induced fluorescence (LIF) has been becoming the most popular detection scheme in conjunction with microchip electrophoresis. Various LIF detection systems based on different optical arrangements were developed and have been successfully and broadly applied in the microfluidic chip-based analysis systems. The optical arrangements of hitherto three major types of LIF detection system employed in chip devices, the confocal LIF system, nonconfocal LIF detection system and orthogonal optical arrangement system.
In this dissertation, a LIF detection system for microfluidic chips with a simple orthogonal optical arrangemen was developed. The technologies about microchip electrophoresis were systemic studied, including chip microfabrication, separation DNA marker to investigate the performance of the sieve, detection of the mutations in p53 gene and determination of amino acids in milk. The work was involved in the typical manipulation, such as the preconcentration, reaction, separation and detection. And the application of the LIF detection systems was realized.
The topics of this dissertation are presented below:
Chapter 1 reviews the research and development on microchip-based analytical systems. Then the new projects were carried out.
Chapter 2 states an integrated system involving microfluidic chip, high voltage power supply, laser-induced fluorescence (LIF) detection and control system was developed to perform microchip electrophoresis separation. A simple and convenient approach for accurately locating the chip in the integrated system was developed by using a substrate plate as the chip flat added a base spring to support the position of the chip. An orthogonal optical arrangement was employed in the LIF detection system to simplify the structure of the system with a limit of detection of 10-11 mol/L for Cy5 dye.
Chapter 3 states copolymers of poly(vinylpyrrolidone) (PVP) and hydroxyethylcellulose (HEC) were synthesized, with PVP to HEC molar ratios of 3:1, 2:1 and 1:1. The copolymers were tested as separation media in DNA fragment separation analysis by microchip electrophoresis (MCE). Separation efficiency over 3.8x105 for 118 bp has been reached by using the bare channels without the additional polymer coating step. Under optimized separation conditions for longer read length DNA sequencing, the separation ability of the copolymers decreased with decreasing (PVP-co-HEC) molar ratio from 3:1,2:1 and 1:1. In comparison with (PVP-co-HEC) 1:1, the copolymer with (PVP-co-HEC) 3:1 ratio showed high separation efficiency. By using a 2.5% w/v copolymer with (PVP-co-HEC) 3:1 ratio, FX174-HaeⅢdigest DNA marker was successfully separated within 3 min.
Chapter 4 states a simple and robust static adsorptive coating process in glass microchips for DNA separation was developed by using mixed polymer consisted of 2% hydroxyethylcellulose and 4% poly (vinylpyrrolidone) as surface modifier of microfluidic channels. This surface modifier was also used as the sieving matrix for the DNA separation. In this study, FX174-HaeIII digest sample was used to investigate the performance of the polymer mixture in microchip electrophoresis with laser-induced fluorescence detection. It was also found that adding 30 nm gold nanoparticles to the mixed polymer was very useful to achieve better resolution and reproducibility. Under the optimum conditions, the DNA fragments could be successfully separated within 4 min and the relative standard deviation values of the migration times were less than 2.51%(n=5) during the one week.
Chapter 5 states a form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7,8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method for detection of mutations for exons 7,8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 0.5% w/v poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7,8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7-9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng L-1.
Chapter 6 states a method for determination of amino acids in milk by microchip electrophoresis (ME) coupled with the detection of laser-induced fluorescence (LIF) has been developed. The amino acids in milk which derivatized with fluorescein isothiocyanate were finely separated and determinated by ME-LIF with borate buffer. The separation parameters were investigated to give an optimal experimental condition:borate buffer (100 mmol/L, pH 9.7); applied separation fleld 475 V/cm. All amino acids were separated within 150 s. All amino acid-Cy5 derivatives was linear in the appropriate concentration (R20.9904-0.9984). The relative standard deviations (n=5) of the method were 2.1%-4.5% for migration time and 2.3%-5.4% for peak area. This method allows to determining the amino acids in milk with high sensitivity.
引文
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