带有化学或生物传感器的高聚物微流动注射安培检测芯片的研制及应用
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摘要
近年来,以分析仪器集成化、微型化、自动化为目标的微流控分析芯片得到了迅猛发展。高聚物微流控芯片以其性能优良、易于批量制备、成本低、适合“一次性”使用等优势,在微流控分析领域得到了广泛的重视,已经成为未来微流控芯片的发展趋势。检测器是微流控分析系统中的核心组成部分,将高灵敏度、高选择性、小型化的检测器集成于芯片之上是微流控分析领域的一个研究热点。安培检测器灵敏度高、选择性好、设备简单、传感电极易集成在芯片上,在高聚物芯片上构建集成化安培型化学和生物传感器,是制备“一次性”芯片的最佳选择之一。快速、高精密度、低消耗的流动注射分析法已经为分析工作者广泛接受。近年来,以微流控芯片为基础的微流动注射分析系统也得到了广大研究者的重视。但是,在微流控芯片上集成适合微型流动注射分析的低成本、高灵敏度、高选择性的安培型化学和生物传感器还面临着许多技术挑战。例如,如何对集成化微薄膜电极进行化学或生化修饰前的无损表面处理；如何在集成于微通道内的微膜电极表面固定生物活性物质；如何消除微流控芯片上多组份生物传感器阵列电极间的交叉干扰等等,都是研制微流动注射安培传感芯片过程中的一些亟待解决的问题。
本文旨在研制集成有化学或生物传感器的高聚物微流动注射安培检测芯片,并将它们应用于生物相关组分的测定。首先针对高聚物芯片上采用化学镀技术制备的集成化金薄膜微电极,建立了一种简单、无损的表面预处理方法；然后,采用适当的通道设计和修饰工艺,在封合后的高聚物芯片上将金基薄膜工作电极修饰成具有巯基丙酸自组装单分子层的化学修饰电极,结合稳定、高通量的试样引入和重力驱动技术,实现了微流控芯片上的高灵敏流动注射安培法测定多巴胺；在此基础上,进一步在封合后的高聚物芯片上将三个金基薄膜电极分别修饰成葡萄糖酶电极、Ag/AgCl参比电极、Pt对电极,组成集成化安培型生物传感三电极体系,应用于流动注射安培法测定葡萄糖；最后,研制了一种具有双通道、集成化双酶工作电极阵列的复合高聚物芯片,利用通道的空间间隔消除交叉干扰,实现了葡萄糖和乳酸的同时测定。
全文共分四章：
第一章,评述了近年来涉及微流控芯片的微流动注射分析系统、安培检测器、电化学酶传感器的研究进展。
第二章,主要目标是建立一种自动化、快速、高灵敏度、高选择性检测多巴胺(DA)的微流动注射安培检测系统。首先针对以化学镀技术制备在聚碳酸酯(PC)微流控芯片之上的集成化金薄膜微电极,考察了自组装修饰前对金薄膜微电极进行表面预处理的方法和条件,研究了空气等离子体处理和电化学处理对金膜微电极的清洁效果。研究发现,通过空气等离子体预处理后的金膜微电极能够得到一个非常洁净的表面,在其上进行巯基化合物单分子层自组装,可以修饰上致密的自组装单分子层,覆盖率较不做任何处理或仅经电化学处理后的电极表面显著提高。在此基础上,在封合后的PC芯片上,利用巯基丙酸(MPA)对金膜微电极进行自组装修饰,制成了具有集成化化学修饰工作电极的PC微流控芯片,结合缺口管自动进样系统和重力驱动技术,构建集成有化学修饰电极的芯片流动注射安培检测系统,并应用该系统对多巴胺类化合物进行选择性检测。对该系统的检测电位、流速、进样量进行了优化。在优化的条件下,分析速度达每小时180样,检测限达74 nmol L-1,试样消耗在nL级。100μmol L-1的多巴胺溶液连续19次进样,峰高的相对标准偏差(RSD)为0.9%。自组装电极抗干扰能力较强,能抵御10倍于多巴胺浓度的抗坏血酸的干扰。所建立的方法已应用于新鲜尿样中加标多巴胺及药物制剂中多巴胺的测定。
第三章,主要目标是制备集成有酶传感电极、Ag/AgCl参比电极和Pt对电极三电极系统的一体化PC微流动注射生物传感芯片。由于一体化PC芯片的封合通常需要在较高的温度和压强下进行,而酶电极无法经受如此严酷的封合条件。因此本文重点考查了在芯片封合后,如何分别把集成在芯片上的三个金基电极修饰成葡萄糖氧化酶工作电极、Ag/AgCl参比电极和Pt对电极,并确保修饰过程中每个电极的专用修饰溶液不污染其他两个电极。实验发现,首先对金工作电极进行MPA自组装修饰,接着对另外两个金电极基底分别电镀Ag/AgCl薄膜和Pt薄膜作为参比电极和对电极,并在电镀过程中向通道内注入流动的缓冲液以保护MPA修饰的工作电极不被电镀液污染,最后再把酶固定到MPA修饰的金工作电极上,以这样的顺序分别修饰三个金基微膜电极,能够保证三个电极在修饰过程中不被交叉污染。将所制备的集成有三电极体系葡萄糖酶传感器的PC微流控芯片与重力驱动和自动进样装置相结合,构成微流动注射安培检测分析系统,用来检测葡萄糖的含量。分析性能较常规流动注射安培检测大为改善,系统检测限为4.1μmol L-1,试样消耗量为130 nL,分析通量达每小时116样品,100μmol L-1的葡萄糖溶液的日内和日间重现性分别达到了0.7%和1.8%,芯片与芯片之间的重现性也小于10%。该流动注射安培检测系统已经应用于葡萄糖-NaCl注射液中葡萄糖含量的测定。
第四章,目标为研制可同时测定葡萄糖和乳酸的双通道微流动注射高聚物芯片,为发展微流控多通道电化学生物传感芯片提供技术平台。以带有主/支通道网络结构的PDMS基片和带有微电极阵列的PC盖片组成复合微流控芯片,将葡萄糖酶电极和乳酸酶电极分别设置在两条支通道内,利用支通道的空间隔离作用消除相同酶促产物之间的交叉干扰。实验中发现,在疏水性高聚物芯片中,流体由主通道向两支通道分流的流量不均匀,甚至出现某一支通道断流的现象,严重影响了安培检测器的正常工作。本章对通道构型、芯片表面性质等影响支通道流量均一性的因素进行了考察。研究表明,芯片的通道构型、通道表面的亲疏水性、通道网络的对称性等因素对两分支通道的分流比有一定影响。采用主/支通道截面比为2：1的Y型通道构型、对PDMS通道基片用等离子体预处理,能在一定程度上提高分流的均匀性。但由于手工加工的两支通道出口,其几何尺度和打孔质量均有一定的不确定性,上述措施尚不能使分流完全均匀。在分支通道出口连接背压调节管,通过调节该管的液位使两支通道内流体所受到的背压基本平衡,能有效地解决两支通道分流不均匀的问题,实现了分支通道内流体的可操控。在上述研究基础上,制备了集成有葡萄糖氧化酶和乳酸氧化酶传感电极阵列的PDMS/PC复合芯片,用于葡萄糖和乳酸的同时检测。通过实验对检测电位、流速等条件进行了优化。在优化的条件下,葡萄糖和乳酸的检测限分别为78μmolL-1和127μmol L-1, 11次葡萄糖和乳酸连续进样重现性分别达0.9%和0.8%。本系统抗干扰能力较强,除葡萄糖和乳酸不会发生交叉干扰以外,0.1 mmol L-1的抗坏血酸和0.5 mmol L-1的尿酸均不产生干扰。该双通道流动注射安培检测芯片应用于人血清实际样品中葡萄糖和乳酸的测定,测定结果和参考值并无明显误差。
本论文的主要创新点：
1.建立了一种以空气等离子体无损清洗集成于高聚物芯片上的化学镀金薄膜微电极的方法,实现了在封合后的芯片中,通过巯基化合物的动态自组装制备化学修饰电极。所研制的带有化学修饰电极的聚碳酸酯芯片,与缺口管自动进样装置和重力驱动相结合,实现了高通量的微流动注射安培法选择性检测多巴胺。
2.建立了一种PC微流控芯片封合之后,在通道内分别把三个金膜微电极修饰成葡萄糖氧化酶修饰工作电极、Ag/AgCl参比电极、Pt对电极的方法。通过设计合理的修饰顺序,结合采用保护性液流,确保了各电极在整个修饰过程中不被其他修饰液所污染。利用本方法制备的带有集成化三电极系统的一体化PC微流动注射安培检测芯片具有很好的分析性能。
3.研制了可同时测定葡萄糖和乳酸含量的双通道微流动注射安培检测芯片,利用分支通道的几何构型来消除由于同种酶促产物相互扩散引起的交叉干扰,并且通过在分支通道末端连接背压管来平衡两支通道内背压,以保障支通道分流的均一性。该芯片可以应用于实际样品测定,测定结果与参考值并无误差。
Microfluidic analytical systems aiming at integration, miniaturization and automation of analytical instruments have been developing rapidly in recently years. Microfluidic chips made of polymeric materials have been widely employed because they have such advantages as easy to be mass produced and less expensive, in turn suitable for disposable usages. Thus, polymeric chips will be the developing trend for microfluidic chips in the future. Detector is the key component of microfluidic analytical system. The development of miniaturized, on-chip integrated, high sensitive and selective detectors are the research interest of the microfluidic field. Amperometric detector (AD) offers chip-based analytical systems the advantages including inherent miniaturization and integration, high sensitivity, low-power requirements, and low cost. It will be the best selection for developing of disposable chips that amperometric sensor is to be integrated on polymeric microfluidic chips. Flow injection analysis (FIA) has been accepted by analysts as a powerful, automatic sampling and sample-pretreatment technique, and been wide used in conventional analysis procedures. Efforts have been devoted to the development of microfluidic chip-based FIA (μFIA) devices. However, there are many technical challenges to integrate low cost, high sensitive and selective amperometric chem-and bio-sensors on microfluidic chips for p.FIA, for example, to establish a non-damaging surface polishing method for the gold film microelectrodes integrated in the polymeric chips, to immobilize bioactive species on the gold film microelectrodes that have been sealed inside the microchannel and to eliminate the cross-talk between sensors when multiple analytes have to be detected simultaneously using a sensor array.
The present work is aimed to develop high performance polymeric microfluidic chips with integrated chem- or bio-sensors for micro flow injection amperometric determination of biologically relevant analytes. First, a non-damaging surface polishing method for on-chip integrated gold film microelectrodes was developed. Then, the gold film microelectrode was modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) via appropriate channel design and modified technology. Cooperated with a high throughput sample introduction system and a gravity pump, theμFIA chip integrated MPA modified electrode was used to rapid and high sensitive determination of dopamine (DA). Then, three gold electrode bases sealed in the bonded polymeric chip were modified to a glucose oxidase working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. The fabricated micro flow-injection biosensor chip was used for determination of glucose. Finally, a two-channel PDMS/PC microchip integrated two enzyme-modified electrodes was developed and applied to determine glucose and lactate simultaneously.
The thesis is composed four chapters:
In chapter 1, recent research progress in microfluidic chip based flow injection analysis systems, amperometric detectors and electrochemical enzyme sensors are reviewed.
In chapter 2, a novel chip-based FIA system has been developed for automatic, rapid and selective determination of DA in the presence of ascorbic acid (AA). Methods such as air plasma treatment and electrochemical treatment were compared to clean the surface of electroless gold microelectrodes. It was observed that the gold film electrodes subjected to air plasma treatment could be perfectly modified with high quality SAMs of thio-compounds, the average coverage of the SAMs of thio-compounds on plasma-treated gold surface was significantly higher than that observed on the electrochemically treated electrode. Based on these observations, PC microfluidic chip integrated with MPA modified working electrode was fabricated and was used for selective determination of dopamine in cooperation with slotted-vial sample introduction techniques and gravity pump. The effects of detection potential, flow rate, and sampling volume on the performance of the chip-based FIA-amperometric system were studied. Under optimum conditions, a detection limit of 74 nmol L-1 for DA was achieved at the sample throughput rate of 180 h-1. A relative standard deviation (RSD) of 0.9% for peak heights was observed for 19 runs of a 100μmol L-1 DA solution. Interference-free determination of DA could be conducted if the concentration ratio of AA to DA was no more than 10. The microchip basedμFIA-AD system was applied for spike-recovery test carried out with diluted urine and determination of DA content in pharmaceutical injections of dopamine hydrochloride.
In chapter 3, a microfluidic electrochemical biosensor chip made of full PC sheets was fabricated for micro flow-injection amperometric determination of glucose. The microfluidic electrochemical biosensor chip integrated a glucose oxidase working electrode, an Ag/AgCl reference electrode and a platinum counter electrode. Bonding of the PC chip required high temperature and pressure, which the enzyme modified electrode could not survive. Therefore, this work was intended to develop a method for the post-bonding, in-channel modification of individual gold electrode base of a three-gold-electrode array into a GOD enzyme working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. It was observed that the sequence of first modification of the gold working electrode with MPA SAM, second electrochemical deposition of Ag/AgCl and platinum onto the gold bases for reference and counter electrodes and final immobilization of the enzyme onto the MPA-modified gold working was the one that caused little contamination of the modified electrodes. To protect the MPA-SAM modified working electrode from being contaminated by the plating electrolytes that might diffuse from the detection cell into the microchannel, the microchannel was flushed with a steam of phosphate buffer solution, in the direction from the sampling probe to the detection cell, during the electroplating process. Cooperated with a gravity pump and an automatic sample dispenser, the fabricatedμFIA biosensor chip was used for determination of glucose. A detection limit of 4.1μmol L-1 was achieved at the sampling volume of about 130 nL, and sample throughput rate reached 116 h-1. Inter- and intra-day precisions (RSD) for the determination of a 100μmol L-1 glucose solution were 0.7% and 1.8%, respectively. Chip-to-chip reproducibility was less than 10% (RSD). The developedμFIA biosensor system was successfully applied to the determination of glucose content in pharmaceutical injections.
In chapter 4, a two-channel polymericμFIA-AD chip used for simultaneous determination of glucose and lactate was developed. A hybridized chip made of a PDMS substrate with mcirochannel network and a PC sheet with integrated electrode array was prepared. A micro channel network with mail/branched channels was designed, and the glucose oxidase modified electrode and lactate oxidase modified electrode were respectively placed in each of the two branched channels. Such an arrangement was intended to eliminate the possible cross-talk between the two enzyme electrodes due to the fact that both enzyme electrodes generate a common electro-active species (H2O2). However, it was found that the fluid from the main channel can't split uniformly into two branch channels. Sometimes, the all fluid effused from the main channel flowed into one branch channel but not to another branch. This phenomenon seriously affected the normal operation of amperometric detector. Studies showed that the channel design, the hydrophilicity of the channel and the symmetry of the microchannel network could affect the split ratio of the two branch channel. It was observed that the split uniformity could be improved by using the Y-shaped channel network with the cross-section ratio of main to branch channel was 2:1, and by using an air plasma treated PDMS substrate. Even so, the split ratio could not be the exactly same for the two branches. It was observed that the physical dimension and quality of the waste-drawing holes (outlet holes) of the branch channels can't be exactly the same, consequently, the back pressure generated inside the two branches could not be balanced, which led to the uniformity of the split ratio. So we connected two backpressure adjusting tubes to the outlet holes of the branched channels. The backpressures that the fluids in the two branch channels subjected to could be balanced by adjusting the liquid level difference of the two backpressure adjusting tubes. Based on the studies mentioned above, a PDMS/PC chip with integrated glucose oxidase and lactate oxidase modified electrode array was developed to determine glucose and lactate simultaneously. The effects of detection potential and flow rate on the performance of the chip were studied. Under optimized conditions, the detection limits for glucose and lactate were 78 p.mol L-1 and 127μmol L-1, respectively. RSDs of 0.9% and 0.8% for peak heights were observed for 11 runs of glucose and lactate, respectively. No cross-talk between glucose and lactate was observed in the developed system. In the presence of 0.1 mmol L-1 AA or 0.5 mmol L"1 UA, no inference was found for the determination of 2 mmol L"1 glucose and 2 mmol L-1 lactate. The two-channelμFIA-AD chip was applied to determine the glucose and lactate contents in human serum samples and no significant difference (at the 95% confidence level) was found between the results obtained with the developed method and those observed with pharmacopeia-regulated method.
The main novelty of the present work is summarized as:
1. A non-damaging method was established for cleaning of finely patterned electroless gold film microelectrodes prepared on polycarbonate sheets. A dynamic approach was used to modify the gold working microelectrode that was sealed inside the microchip after chip bonding. Cooperated with slotted-vial sample introduction technique and gravity pump, the developedμFIA-AD system showed excellent analytical performance for the selective determination of dopamine.
2. A method was established for individual modification of three gold microelectrode bases sealed in the channel into a GOD enzyme working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. A reasonable modification sequence cooperated with protective liquid flow made the modification process contamination-free for the modified electrodes. The developed full PCμFIA-AD chip integrated with three-electrode system demonstrated good analytical performance.
3. A two-channelμFIA-AD chip was prepared for simultaneous determination of glucose and lactate. The cross-talk between glucose and lactate caused by the mass-transfer of the same enzymatic product H2O2 was be eliminated by the geometric configuration of the branched channels. Furthermore, the backpressure of the two branched channels could be balanced by adjusting the liquid level difference of the two backpressure adjusting tubes. The developed chip was used for determination of glucose and lactate in real samples and results got by the proposed method were in good agreement with those observed with the pharmacopeia-regulated method.
本文旨在研制集成有化学或生物传感器的高聚物微流动注射安培检测芯片,并将它们应用于生物相关组分的测定。首先针对高聚物芯片上采用化学镀技术制备的集成化金薄膜微电极,建立了一种简单、无损的表面预处理方法；然后,采用适当的通道设计和修饰工艺,在封合后的高聚物芯片上将金基薄膜工作电极修饰成具有巯基丙酸自组装单分子层的化学修饰电极,结合稳定、高通量的试样引入和重力驱动技术,实现了微流控芯片上的高灵敏流动注射安培法测定多巴胺；在此基础上,进一步在封合后的高聚物芯片上将三个金基薄膜电极分别修饰成葡萄糖酶电极、Ag/AgCl参比电极、Pt对电极,组成集成化安培型生物传感三电极体系,应用于流动注射安培法测定葡萄糖；最后,研制了一种具有双通道、集成化双酶工作电极阵列的复合高聚物芯片,利用通道的空间间隔消除交叉干扰,实现了葡萄糖和乳酸的同时测定。
全文共分四章：
第一章,评述了近年来涉及微流控芯片的微流动注射分析系统、安培检测器、电化学酶传感器的研究进展。
第二章,主要目标是建立一种自动化、快速、高灵敏度、高选择性检测多巴胺(DA)的微流动注射安培检测系统。首先针对以化学镀技术制备在聚碳酸酯(PC)微流控芯片之上的集成化金薄膜微电极,考察了自组装修饰前对金薄膜微电极进行表面预处理的方法和条件,研究了空气等离子体处理和电化学处理对金膜微电极的清洁效果。研究发现,通过空气等离子体预处理后的金膜微电极能够得到一个非常洁净的表面,在其上进行巯基化合物单分子层自组装,可以修饰上致密的自组装单分子层,覆盖率较不做任何处理或仅经电化学处理后的电极表面显著提高。在此基础上,在封合后的PC芯片上,利用巯基丙酸(MPA)对金膜微电极进行自组装修饰,制成了具有集成化化学修饰工作电极的PC微流控芯片,结合缺口管自动进样系统和重力驱动技术,构建集成有化学修饰电极的芯片流动注射安培检测系统,并应用该系统对多巴胺类化合物进行选择性检测。对该系统的检测电位、流速、进样量进行了优化。在优化的条件下,分析速度达每小时180样,检测限达74 nmol L-1,试样消耗在nL级。100μmol L-1的多巴胺溶液连续19次进样,峰高的相对标准偏差(RSD)为0.9%。自组装电极抗干扰能力较强,能抵御10倍于多巴胺浓度的抗坏血酸的干扰。所建立的方法已应用于新鲜尿样中加标多巴胺及药物制剂中多巴胺的测定。
第三章,主要目标是制备集成有酶传感电极、Ag/AgCl参比电极和Pt对电极三电极系统的一体化PC微流动注射生物传感芯片。由于一体化PC芯片的封合通常需要在较高的温度和压强下进行,而酶电极无法经受如此严酷的封合条件。因此本文重点考查了在芯片封合后,如何分别把集成在芯片上的三个金基电极修饰成葡萄糖氧化酶工作电极、Ag/AgCl参比电极和Pt对电极,并确保修饰过程中每个电极的专用修饰溶液不污染其他两个电极。实验发现,首先对金工作电极进行MPA自组装修饰,接着对另外两个金电极基底分别电镀Ag/AgCl薄膜和Pt薄膜作为参比电极和对电极,并在电镀过程中向通道内注入流动的缓冲液以保护MPA修饰的工作电极不被电镀液污染,最后再把酶固定到MPA修饰的金工作电极上,以这样的顺序分别修饰三个金基微膜电极,能够保证三个电极在修饰过程中不被交叉污染。将所制备的集成有三电极体系葡萄糖酶传感器的PC微流控芯片与重力驱动和自动进样装置相结合,构成微流动注射安培检测分析系统,用来检测葡萄糖的含量。分析性能较常规流动注射安培检测大为改善,系统检测限为4.1μmol L-1,试样消耗量为130 nL,分析通量达每小时116样品,100μmol L-1的葡萄糖溶液的日内和日间重现性分别达到了0.7%和1.8%,芯片与芯片之间的重现性也小于10%。该流动注射安培检测系统已经应用于葡萄糖-NaCl注射液中葡萄糖含量的测定。
第四章,目标为研制可同时测定葡萄糖和乳酸的双通道微流动注射高聚物芯片,为发展微流控多通道电化学生物传感芯片提供技术平台。以带有主/支通道网络结构的PDMS基片和带有微电极阵列的PC盖片组成复合微流控芯片,将葡萄糖酶电极和乳酸酶电极分别设置在两条支通道内,利用支通道的空间隔离作用消除相同酶促产物之间的交叉干扰。实验中发现,在疏水性高聚物芯片中,流体由主通道向两支通道分流的流量不均匀,甚至出现某一支通道断流的现象,严重影响了安培检测器的正常工作。本章对通道构型、芯片表面性质等影响支通道流量均一性的因素进行了考察。研究表明,芯片的通道构型、通道表面的亲疏水性、通道网络的对称性等因素对两分支通道的分流比有一定影响。采用主/支通道截面比为2：1的Y型通道构型、对PDMS通道基片用等离子体预处理,能在一定程度上提高分流的均匀性。但由于手工加工的两支通道出口,其几何尺度和打孔质量均有一定的不确定性,上述措施尚不能使分流完全均匀。在分支通道出口连接背压调节管,通过调节该管的液位使两支通道内流体所受到的背压基本平衡,能有效地解决两支通道分流不均匀的问题,实现了分支通道内流体的可操控。在上述研究基础上,制备了集成有葡萄糖氧化酶和乳酸氧化酶传感电极阵列的PDMS/PC复合芯片,用于葡萄糖和乳酸的同时检测。通过实验对检测电位、流速等条件进行了优化。在优化的条件下,葡萄糖和乳酸的检测限分别为78μmolL-1和127μmol L-1, 11次葡萄糖和乳酸连续进样重现性分别达0.9%和0.8%。本系统抗干扰能力较强,除葡萄糖和乳酸不会发生交叉干扰以外,0.1 mmol L-1的抗坏血酸和0.5 mmol L-1的尿酸均不产生干扰。该双通道流动注射安培检测芯片应用于人血清实际样品中葡萄糖和乳酸的测定,测定结果和参考值并无明显误差。
本论文的主要创新点：
1.建立了一种以空气等离子体无损清洗集成于高聚物芯片上的化学镀金薄膜微电极的方法,实现了在封合后的芯片中,通过巯基化合物的动态自组装制备化学修饰电极。所研制的带有化学修饰电极的聚碳酸酯芯片,与缺口管自动进样装置和重力驱动相结合,实现了高通量的微流动注射安培法选择性检测多巴胺。
2.建立了一种PC微流控芯片封合之后,在通道内分别把三个金膜微电极修饰成葡萄糖氧化酶修饰工作电极、Ag/AgCl参比电极、Pt对电极的方法。通过设计合理的修饰顺序,结合采用保护性液流,确保了各电极在整个修饰过程中不被其他修饰液所污染。利用本方法制备的带有集成化三电极系统的一体化PC微流动注射安培检测芯片具有很好的分析性能。
3.研制了可同时测定葡萄糖和乳酸含量的双通道微流动注射安培检测芯片,利用分支通道的几何构型来消除由于同种酶促产物相互扩散引起的交叉干扰,并且通过在分支通道末端连接背压管来平衡两支通道内背压,以保障支通道分流的均一性。该芯片可以应用于实际样品测定,测定结果与参考值并无误差。
Microfluidic analytical systems aiming at integration, miniaturization and automation of analytical instruments have been developing rapidly in recently years. Microfluidic chips made of polymeric materials have been widely employed because they have such advantages as easy to be mass produced and less expensive, in turn suitable for disposable usages. Thus, polymeric chips will be the developing trend for microfluidic chips in the future. Detector is the key component of microfluidic analytical system. The development of miniaturized, on-chip integrated, high sensitive and selective detectors are the research interest of the microfluidic field. Amperometric detector (AD) offers chip-based analytical systems the advantages including inherent miniaturization and integration, high sensitivity, low-power requirements, and low cost. It will be the best selection for developing of disposable chips that amperometric sensor is to be integrated on polymeric microfluidic chips. Flow injection analysis (FIA) has been accepted by analysts as a powerful, automatic sampling and sample-pretreatment technique, and been wide used in conventional analysis procedures. Efforts have been devoted to the development of microfluidic chip-based FIA (μFIA) devices. However, there are many technical challenges to integrate low cost, high sensitive and selective amperometric chem-and bio-sensors on microfluidic chips for p.FIA, for example, to establish a non-damaging surface polishing method for the gold film microelectrodes integrated in the polymeric chips, to immobilize bioactive species on the gold film microelectrodes that have been sealed inside the microchannel and to eliminate the cross-talk between sensors when multiple analytes have to be detected simultaneously using a sensor array.
The present work is aimed to develop high performance polymeric microfluidic chips with integrated chem- or bio-sensors for micro flow injection amperometric determination of biologically relevant analytes. First, a non-damaging surface polishing method for on-chip integrated gold film microelectrodes was developed. Then, the gold film microelectrode was modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) via appropriate channel design and modified technology. Cooperated with a high throughput sample introduction system and a gravity pump, theμFIA chip integrated MPA modified electrode was used to rapid and high sensitive determination of dopamine (DA). Then, three gold electrode bases sealed in the bonded polymeric chip were modified to a glucose oxidase working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. The fabricated micro flow-injection biosensor chip was used for determination of glucose. Finally, a two-channel PDMS/PC microchip integrated two enzyme-modified electrodes was developed and applied to determine glucose and lactate simultaneously.
The thesis is composed four chapters:
In chapter 1, recent research progress in microfluidic chip based flow injection analysis systems, amperometric detectors and electrochemical enzyme sensors are reviewed.
In chapter 2, a novel chip-based FIA system has been developed for automatic, rapid and selective determination of DA in the presence of ascorbic acid (AA). Methods such as air plasma treatment and electrochemical treatment were compared to clean the surface of electroless gold microelectrodes. It was observed that the gold film electrodes subjected to air plasma treatment could be perfectly modified with high quality SAMs of thio-compounds, the average coverage of the SAMs of thio-compounds on plasma-treated gold surface was significantly higher than that observed on the electrochemically treated electrode. Based on these observations, PC microfluidic chip integrated with MPA modified working electrode was fabricated and was used for selective determination of dopamine in cooperation with slotted-vial sample introduction techniques and gravity pump. The effects of detection potential, flow rate, and sampling volume on the performance of the chip-based FIA-amperometric system were studied. Under optimum conditions, a detection limit of 74 nmol L-1 for DA was achieved at the sample throughput rate of 180 h-1. A relative standard deviation (RSD) of 0.9% for peak heights was observed for 19 runs of a 100μmol L-1 DA solution. Interference-free determination of DA could be conducted if the concentration ratio of AA to DA was no more than 10. The microchip basedμFIA-AD system was applied for spike-recovery test carried out with diluted urine and determination of DA content in pharmaceutical injections of dopamine hydrochloride.
In chapter 3, a microfluidic electrochemical biosensor chip made of full PC sheets was fabricated for micro flow-injection amperometric determination of glucose. The microfluidic electrochemical biosensor chip integrated a glucose oxidase working electrode, an Ag/AgCl reference electrode and a platinum counter electrode. Bonding of the PC chip required high temperature and pressure, which the enzyme modified electrode could not survive. Therefore, this work was intended to develop a method for the post-bonding, in-channel modification of individual gold electrode base of a three-gold-electrode array into a GOD enzyme working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. It was observed that the sequence of first modification of the gold working electrode with MPA SAM, second electrochemical deposition of Ag/AgCl and platinum onto the gold bases for reference and counter electrodes and final immobilization of the enzyme onto the MPA-modified gold working was the one that caused little contamination of the modified electrodes. To protect the MPA-SAM modified working electrode from being contaminated by the plating electrolytes that might diffuse from the detection cell into the microchannel, the microchannel was flushed with a steam of phosphate buffer solution, in the direction from the sampling probe to the detection cell, during the electroplating process. Cooperated with a gravity pump and an automatic sample dispenser, the fabricatedμFIA biosensor chip was used for determination of glucose. A detection limit of 4.1μmol L-1 was achieved at the sampling volume of about 130 nL, and sample throughput rate reached 116 h-1. Inter- and intra-day precisions (RSD) for the determination of a 100μmol L-1 glucose solution were 0.7% and 1.8%, respectively. Chip-to-chip reproducibility was less than 10% (RSD). The developedμFIA biosensor system was successfully applied to the determination of glucose content in pharmaceutical injections.
In chapter 4, a two-channel polymericμFIA-AD chip used for simultaneous determination of glucose and lactate was developed. A hybridized chip made of a PDMS substrate with mcirochannel network and a PC sheet with integrated electrode array was prepared. A micro channel network with mail/branched channels was designed, and the glucose oxidase modified electrode and lactate oxidase modified electrode were respectively placed in each of the two branched channels. Such an arrangement was intended to eliminate the possible cross-talk between the two enzyme electrodes due to the fact that both enzyme electrodes generate a common electro-active species (H2O2). However, it was found that the fluid from the main channel can't split uniformly into two branch channels. Sometimes, the all fluid effused from the main channel flowed into one branch channel but not to another branch. This phenomenon seriously affected the normal operation of amperometric detector. Studies showed that the channel design, the hydrophilicity of the channel and the symmetry of the microchannel network could affect the split ratio of the two branch channel. It was observed that the split uniformity could be improved by using the Y-shaped channel network with the cross-section ratio of main to branch channel was 2:1, and by using an air plasma treated PDMS substrate. Even so, the split ratio could not be the exactly same for the two branches. It was observed that the physical dimension and quality of the waste-drawing holes (outlet holes) of the branch channels can't be exactly the same, consequently, the back pressure generated inside the two branches could not be balanced, which led to the uniformity of the split ratio. So we connected two backpressure adjusting tubes to the outlet holes of the branched channels. The backpressures that the fluids in the two branch channels subjected to could be balanced by adjusting the liquid level difference of the two backpressure adjusting tubes. Based on the studies mentioned above, a PDMS/PC chip with integrated glucose oxidase and lactate oxidase modified electrode array was developed to determine glucose and lactate simultaneously. The effects of detection potential and flow rate on the performance of the chip were studied. Under optimized conditions, the detection limits for glucose and lactate were 78 p.mol L-1 and 127μmol L-1, respectively. RSDs of 0.9% and 0.8% for peak heights were observed for 11 runs of glucose and lactate, respectively. No cross-talk between glucose and lactate was observed in the developed system. In the presence of 0.1 mmol L-1 AA or 0.5 mmol L"1 UA, no inference was found for the determination of 2 mmol L"1 glucose and 2 mmol L-1 lactate. The two-channelμFIA-AD chip was applied to determine the glucose and lactate contents in human serum samples and no significant difference (at the 95% confidence level) was found between the results obtained with the developed method and those observed with pharmacopeia-regulated method.
The main novelty of the present work is summarized as:
1. A non-damaging method was established for cleaning of finely patterned electroless gold film microelectrodes prepared on polycarbonate sheets. A dynamic approach was used to modify the gold working microelectrode that was sealed inside the microchip after chip bonding. Cooperated with slotted-vial sample introduction technique and gravity pump, the developedμFIA-AD system showed excellent analytical performance for the selective determination of dopamine.
2. A method was established for individual modification of three gold microelectrode bases sealed in the channel into a GOD enzyme working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, respectively. A reasonable modification sequence cooperated with protective liquid flow made the modification process contamination-free for the modified electrodes. The developed full PCμFIA-AD chip integrated with three-electrode system demonstrated good analytical performance.
3. A two-channelμFIA-AD chip was prepared for simultaneous determination of glucose and lactate. The cross-talk between glucose and lactate caused by the mass-transfer of the same enzymatic product H2O2 was be eliminated by the geometric configuration of the branched channels. Furthermore, the backpressure of the two branched channels could be balanced by adjusting the liquid level difference of the two backpressure adjusting tubes. The developed chip was used for determination of glucose and lactate in real samples and results got by the proposed method were in good agreement with those observed with the pharmacopeia-regulated method.
引文
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