Cell Separation, PCR Amplification And DNA Sequencing Method Development Based On Blood Sample And Microfluidic Chip Technology

Life science is the one of scientific frontiers in this century. The development of it calls for the advanced analytical methods. The analytical instrument and analytical science are reforming to micromation, integration and portable. Microfluidic technology has become the hardcore in microfluidic analytical system and the most dynamic field ofμTAS. Blood is an important sample for bioanalysis and harbors a massive amount of information about the functioning of all tissues and organs in the body. Consequently, blood sampling and analysis are of prime interest for both medical and science applications. Among the new technologies with an increasingly broader impact in biology, microfluidics and miniaturized lab-on-a-chip-type devices are extremely attractive for blood analysis. A microfluidic system was made for blood analysis on cell level and DNA level. All manipulations were developed including blood cells separation on the glass microfluidic chip based on high gradient magnetic separation, whole blood direct amplification on microfluidic chip, and SNP typing of blood DNA on pyrosequencing microfluidic system. The microfluidic chip is a good platform for bioanalysis.Chapter 1 of the thesis reviewed blood cells manipulation on microfluidic chip and PCR amplification chip and pyrosequencing microfluidic chip.Chapter 2 of the thesis developed a glass microfluidic chip based on high gradient magnetic separation for high efficient separation of red blood cells (RBCs) from whole blood based on their native magnetic properties. The glass chip was fabricated by photolithography and thermal bonding. It consisted one inlet and three outlets, and a nickel wire of 69μm diameter was positioned in the center of a separation channel with 149μm top width and 73μm depth by two parallel ridges (about 10μm high). The two ridges were formed simultaneously during the wet etching of the channels. The nickel wire for generating the magnetic gradient inside the separation channel was introduced from the side of the chip through a guide channel. The external magnetic field was applied by a permanent magnet of 0.3 T placed by the chip side and parallel to the main separation channel. The RBCs were separated continuously from the 1:40 (v/v) diluted blood sample at a flow rate in the range of 0.12～0.92μL/min (9-74 mm/min) with the chip, and up to 93.7% of the RBCs were collected in the middle outlet under flow rate of 0.23μL/min. The cell sedimentation was alleviated by adjusting the specific density of the supporting media with bovine serum albumin. Quantum dots labeling was introduced for visual fluorescence tracking of the separation process. The uneven distribution phenomenon of the blood cells around the nickel wire was reported and discussed.In Chapter 3 of the thesis, direct whole blood PCR amplification on a static chip thermostat without any purification was introduced. The method was independent of desktop facilities, and problems such as cross interferences and contaminations can be avoided. The amplification conditions such as the composition of reagents and thermal programs were investigated systematically by a Gene Amp PCR system with a native p53 gene segment (-543 bp) of human genome and an exterior lambda DNA segment (-500 pb) as targets. Direct amplifications of p53 and K-ras (～157 bp) genes segments from 0.5μL blood samples were successfully demonstrated by a static PCR chip of ITO glass substrate. Fuzzy proportion integration differentiation (PID) algorithms were adopted in controlling temperature programs of the chip. The chip thermostat was typically 25 mm×25 mm effective area, and a piece of polyethylene tube was used as the PCR reaction vial on glass surface of the chip. Only on chip thermal procedures were involved during the whole process. This work demonstrated that chip PCR for field test without desktop facilities is feasible either for point of care test or for forensic analysis.In Chapter 4 of the thesis, SNP typing of blood DNA on flow pyrosequencing capillary microfluidic system platform, a flow pyrosequencing method on capillary was built for blood assay on DNA level. The DNA template was amplified from blood DNA with PCR amplification. In capillary, the beads with DNA template were fixed by permanent magnet and a magnetic shielding box was made to reduce the magnetic effect on PMT. It is very easy to build this platform without micromachining. An autosampling device holding samples and reagents with horizontally fixed slotted microvials, which manipulation is simple and convenient, was used and to meet the needs of sampling on solid pyrosequencing. The BAMPER method is a kind of highly sensitive method for SNP typing based on pyro-sequencing. SNP typing, which is rs8130833 on chromosome 21, was tested on this system platform with BAMPER method. It is fast, convenient and saving reagents to SNP typing based on pyrosequencing microfluidic system.