Studies On Integrated Microdevice For Nucleic Acids Extraction, Amplification And Online Detection

Microfluidic chip-based microanalytical systems is one of the most important areas in the field of micro total analysis systems (μTAS) or Lab-on-a-chip, the ultimate objective of which is to integrate all main analytical functions, including sample introduction, sample pretreatment, reactions, separation and detection operations, on a single chip. At present, one of the important field in microfluidic-based microanalytical systems is genetic analysis on chip, which aims at achieving a fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. The present work is focused on developing an integrated glass microdevice for nucleic acid purification, amplification, and online detection.In Chapter 1, the microfluidic chip-based nucleic acids extraction and its integration with subsequent genetic analysis units are reviewed. Various methods for nucleic acid extraction on chip, PCR and isothermal amplification of DNA on chip and intergration of different functional units, are comprehensively described.In Chapter 2, a method for nucleic acid extraction using glass SPE bed (gSPE bed) is described. We make use of the property that glass consists mainly of silica to capture NAs which adsorb to silica in the presence of high concentration of the chaotropic agent, the extracted nucleic acids are eluted with an aqueous low-salt buffer. The DNA were extracted from commercially availableλDNA and some crude biological samples like whole blood and cell suspension to evaluate the capability of gSPE bed for nucleic acid purification. Furthermore, a comparison of the efficiency of DNA extraction using gSPE bed with that using a commercial kit was performed. It is showed that the gSPE bed could successfully extract DNA from 1μL of whole blood and 3200 cells. The advantages of the gSPE bed include simple fabrication, high extraction efficiency and easily integration with other genetic analysis units.In Chapter 3, the feasibility of integrating the nucleic acid extraction with isothermal amplification was evaluated. The structure of the reaction chamber was optimized, and the on-chip mixing of eluted DNA and amplification reagent was tested.The Chapter 4, a glass microdevice for genetic analysis system, which integrated DNA extraction with the loop-mediated isothermal amplification (LAMP) and online detection, was developed based on the researches described in Chapter 2 and 3. The nucleic acids was extracted using gSPE bed because the glass consists mainly of silica and therefore the nucleic acids could be captured on the channel surface. The LAMP which performs under isothermal condition was adopted or (?)leic acid amplification, and the concentration of the DNA fluorescent dye was optimized for oniine detection. An integrated glass microdevice for genetic analysis with the sample-in-answer-out mode was successfully developed. The analytical performance was evaluated usingλDNA as model sample. The whole analytical time from the sample lysis to the final result readout could be accomplished within 2 h.