ABSTRACT
A real-time polymerase chain reaction ( PCR ) micro total analysis system (μ-TAS ) integrated nucleic acid extraction, PCR amplification and real-time-fluorescent PCR detection on a same microfluidic chip was prepared for the fully automated and on-chip analysis of nucleic acid. The proposed method had the advantage such as low sample consumption, fast analysis and simple operation and so on. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A polydimethylsiloxane (PDMS) substrate with 3D channels was manufactured by a combination of molds and an injection molding method. The glass substrate and the chip were bonded together using a plasma treatment. The μ-TAS included a microfluidic control device whose micro fluidic velocity ( 0-10 mL/min ) could be adjusted, a TEC platform which the precision of temperature control was 0. 1℃ and a CCD detection module. The DNA of human blood was extracted by using a silica gel membrane method on the microfluidic chip. The processes of DNA extraction and detection were preset in the μ-TAS. Human blood lysate ( 20 μL ) were driven to the extraction chamber and was then washed. The fluidic drive speed was 2 mL/min. DNA and PCR reagents were mixed and then were driven into the PCR chamber. The fluidic drive speed was 1 mL/min. The GAPDH gene in extracted genome DNA was amplified by PCR and detected. The amplified product was verified by melting analysis. The results of nucleic acid extraction method on the chip were compared to those obtained using a standard manual centrifuge extraction method. The amplification curves were obvious. Ct values of the chip method were 25 . 3 and 26 . 9 . The denaturation temperature of all the melting was 89 . 9 ℃. The results validated that the chip-based method and device realized the extraction of nucleic acid, amplification and detection automatically.