| 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. |
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