Used In Disease Diagnosis And Environmental Toxicology Testing New Methods Of Microarray And Microfluidic Chips

Miniaturization and integration are important developing directions to modern analytical chemistry. To improve the sensitivity of analysis & detection throughput, fill in the demand of in-field analysis and decrease the detection cost and consumption of sample, how to realize the miniaturization of analytical equipment has become one of the most important frontiers in the field of analytical chemistry. Micro analysis systems that have close relationship to analytical chemistry include microarray chip and microfluidic chip. Both two have the following advantages: low consumption of sample and reagent, fast analysis and easy to realize integration, and have been paid much attention by researchers. Based on above research background, this dissertation focuse on how to make the micro array chip and microfluidic chip to be applied into real sample analysis and achieve the combination between research works in the lab and the detection demand in real life. The title of the dissertation is "Studies of microarray and microfluidic chips facing to detection of clinical disease & environmental toxin." This dissertation has great original and frontier innovation and includes six chapters, which are introduced separately as the following:In chapter one, the origin, class and progress in micro analysis systems have been reviewed. It mainly focuses on the most important developing frontiers in microarray chips & microfluidic chip, and on the frequently used detection methods. At the last, the outline and innovation of this dissertation have been discussed.In chapter two, novel immunoassays for screening of disease markers in human serum are presented by miniaturizing interdigitated array (IDA) of microelectrodes via MEMS on a silicon chip for multi-channel electrochemical measurement. Different selected antibodies (Abs) are incorporated site-specifically into the electrochemically deposited polypyrrole (PPy) formed on the IDA of the silicon chip. By using the strategy presented here, three liver fibrosis markers, hyaluronic acid (HA), lamin (LN) and collagen type IV (IV-C), are detected simultaneously and specifically at the surface of the chip at ng/mL concentration level in a tiny amount of volume, ～ 50 μL. The chip-based immunoassay shows the advantages of high sensitivity, good specificity, high throughput,low sample consumption, and the stability offered via batch production by MEMS as well, which is expected to benefit the multi-target screening of desired clinical analytes.In chapter three, a novel, simple and label-free multianalyte immunoassay system is presented using arrayed electrodes integrated on a silicon chip via MEMS. The chip consisted of six Au disk electrodes, an Au counter electrode and an Ag/AgCl reference electrode. Semi-insulating poly(o-phenylenediamine) (PoPD) was utilized to copolymerize and immobilize antibodies at the arrayed Au electrodes, and enlarged linear detection range and lower detection limit were obtained than those prepared with completely insulating PoPD. In addition, the arrayed electrodes offered exact control of the deposition position via electrochemical operation, allowing selectively immobilization of different antibodies at desired positions on a single chip. And the results obtained via the chips were well consistent with those obtained by commercial radio immunoassays (RIA).In chapter four, a novel strategy for site-specific protein immobilization via combining chip IEF with low-temperature sol-gel technology, called IEF-GEL here, in the channel of a modified poly(methyl methacrylate) (PMMA) microfluidic chip has been proposed. The IEF-GEL process involves firstly IEF for homogenously dissolved protein in phosphate buffer saline (PBS) containing alumina sol and carrier ampholyte with prearranged pH gradient, and then gelation locally for protein encapsulation. The proposed system holds the advantages of IEF and low temperature sol-gel technologies, that is concentrating protein to be focused and retaining the biological activity for the gel embedded protein, thus realizes site-specific immobilization of low-concentration protein at nL volume level. At last, AChE was immobilized in the microfluidic chip, and the glancing response of this enzyme microreactor was obtained. Detection of OPs pesticide in the environment utilizing this enzyme microreactor can be realized in the future work.In chapter five, the screen printed electrodes that integrated 3-electrodes system has been developed first to reinforce above works. During the preparation of the screen printed electrodes, carbon ink was replaced by silver ink, which decreases the cost efficiently. Trace lead was detected with DPASV utilizing this screen printed electrodes, which demonstrates that screen printed electrodes that integrated 3-electrodes systemhave excellent electrode property. Then, a mediator-free amperometric biosensor for screening OPs in FIA system based on anticholinesterase activity of OPs to immobilized Acetylcholinesterase Enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al₂O₃ sol-gel matrix screen-printed on this integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al₂O₃-AChE biosensor effectively detects the substrate at 0.25 V (vs. Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al₂O₃-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.In chapter six, the conclusion of the above works has been obtained firstly, then some research directions that can be further achieved have been proposed, mainly focuse on protein recognition arrays, microfluidic chips, heavy metal ions sensors and OPs detection. The comments shown here may be helpful to further research works.