A Fully-Integrated Microfluidic Chip for RNA-Virus Detection

1. A Fully-Integrated Microfluidic Chip for RNA-Virus Detection (1) (2) (3) (4) RT-PCR reagents PCR reagents after RT-PCR Computer Microfluidic chip High Voltage Power Supply RT/PCR reaction chamber capillary electrophoresis chip sample preparation PMT RNA Transmission line optical fiber Hg lamp computer photomultiplier tube Optical fiber Microfluidic control module Temperature control module Focus lens Band-pass filter (A) (B) (e) SU-8 mold formation (a) Lithography 1078 1353 872 (b) Platinum deposition 511 (f) PDMS replication 603 310 (c) Gold deposition 281 Fluorescent signal (mV) 271 234 (d) Insulation layer setup (g) O2 plasma bonding 194 118 72 Leads of heaters and sensors Time (min) Capillary electrophoresis PCRreagents RT/PCRreaction chamber Peristaltic micro-pneumatic pump RT-PCRreagents Optical fiber channel Fu-Chun Huang1, Chia-Sheng Liao2, Gwo-Bin Lee1,2 1Department of Engineering Science, 2Institute of Micro-Electro-Mechanical-System Engineering National Cheng Kung University, Tainan, Taiwan 701 • Abstract • Experimental The study reports a new fully-integrated microfluidic device capable of performing Reverse-Transcription Polymerase Chain Reaction (RT-PCR), transportation of amplified molecular samples, capillary electrophoresis (CE) separation and on-line detection of RNA-based virus. Using three major technologies, including molecular biology, micro-fabrication and micro-fluidics, we develop an innovative micro-total-analysis-system (μ-TAS) for rapid clinical diagnosis. We have successfully demonstrated the detection of the Dengue-2 virus using the hand-held system in a shorter period with less consumption of samples and reagents. The system can perform high heating (20℃/sec) and cooling (10℃/sec) rates. The integrated system has successfully carried out the detection of Dengue-2 virus samples. (A) (B) • Typical PCR thermal cycles generated by the PCR chip. • SEM images. (1) A SU-8 mold of two optical fibers and one microchannel, (2) PDMS replication from the SU-8 mold. (3) SU-8 mold of a pneumatic pump, and (4) PDMS replication of the micro-pump. • Design and fabrication Schematic representation of the fully-integrated microfluidic chips for RNA virus detection. • Schematic representation of the experimental setup for CE detection. Note that excitation and emitted lights are transmitted by buried optic fibers. • The PCR and microfluidic control systems. Simplified fabrication process for the fully- integrated microfluidic chip. Electrophoregrams of amplified RNA product of Dengue II virus and Hae III digested x-174 DNA markers. • Conclusions • Fully-integrated microfluidic chips capable of performing DNA amplification from RNA virus, sample transportation, capillary electrophoresis separation, and on-line optical detection have been developed. • The developed system consumes less reagent and sample volumes, and provides higher heating/cooling rates with a more precise temperature control. • The proposed microfluidic chip is a powerful tool for the detection of DNA/RNA samples and has significant potential for implementation in micro total analysis systems. 2006 Photograph of the fully-integrated microfluidic chip after assembly. MML MEMS design and Micro-fabrication Lab