An Integrated Nucleic Acid Extraction Microchip for Real-time PCR Micro Total Analysis

Abstract

Abstract A real-time polymerase chain reaction (PCR) micro total analysis system (μ-TAS) was constructed by integrating nucleic acid extraction and PCR amplification with real-time fluorescent detection on a same microfluidic chip, allowing fully automated and on-chip analysis. This approach has the advantages such as low sample consumption, fast analysis and simple operation. 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 by which micro fluidic velocity (0–10 mL min −1 ) could be adjusted, a TEC platform with a precision of temperature control of 0.1 °C, and a CCD detection module. The DNA of human blood was extracted using a silica gel membrane method on the microfluidic chip. The DNA extraction and detection were preset in the μ-TAS. Human blood lysate (20 μL) was loaded into the extraction chamber and then washed at a speed of 2 mL min −1 . DNA and PCR reagents were mixed and then driven into the PCR chamber at a speed of 1 mL min −1 . The reference gene GAPDH in extracted genome DNA was amplified by PCR and verified by melting curve analysis. The results of nucleic acid extraction method on the chip were compared with those obtained using a standard manual centrifuge extraction method. The on-chip PCR amplifications gave obvious amplification curves, with C T values of 25.3 and 26.9 respectively. The melting temperature of all the amplification products was 89.9 °C. The results validated that the chip-based method and corresponding device could realize the extraction, amplification and detection of nucleic acid automatically.