Sung-Yi Yang, Kang-Yi Lien, Kao-Jean Huang, Huan-Yao Lei, Gwo-Bin Lee

1. Immunoassayutilizingmagneticbeads for rapidvirus detection in the microfluidic flow cytometer system Professor : Liu, Cheng-Hsien    Student : Olof Sterner Sung-Yi Yang, Kang-Yi Lien, Kao-Jean Huang, Huan-Yao Lei, Gwo-Bin Lee National Cheng Kung University, Tainan Taiwan uTAS2007

2. Agenda • Introduction • Design and Fabrication • Results • Conclusion • Discussion

3. Introduction • In this article: • Integrated fluid chip for rapid virus identification. • Fluid movement is performed by micro circulatory pump driven by airpressure. • Magnetic beads covered with specific antibodies. • Detection of virus is made through fluorescence by a PMT module.

4. Introduction • Current viruses threatening mankind • Influenza • Hepatitis • HIV • Dengue fever • Ebola • Yellow Fever • Etc • Treatment for viral infections are far behind those of bacterial infections • Quick diagnosis crucial

5. Design and Fabrication • Magnetic beads • Covered in specific antibodies toward a certain virus • Virus particle functions as antigen and binds specifically to the primary antibody on the magnetic bead. • Secondary antibody towards the same virus but targeting a different epitope binds to the virus and carries a fluorescent compound.

6. Design and Fabrication • Main detection principle Magnetic beads with primary target antibody Target antigen is added (Virus) Secondary fluorescent antibodies is added Fluidic focusing Detection by means of fluorescense

7. Design and Fabrication • The integrated flow cytometer system A B C Dimensions : 46 mm * 66 mm * 5 mm

8. Design and Fabrication • A – Virus attachement to magnetic beads. Injection of bio sample and magneticbeads Introduction of secfluorescentantibodies Mixing with rotary microfluidic pump

9. Design and Fabrication • Airpressure driven rotary microfluidic pump Biomed Microdevices (2007) 9:545–554 DOI 10.1007/s10544-007-9062-6

10. Design and Fabrication • B – Washing and Pretreatment Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system, Lab Chip, DOI: 10.1039/b700516d, (2007) Cu microcoils induced magnetic field attracts magnetic beads Washing with buffer Release of magnetic beads (Flow generated by rotary microfluidic pump)

11. Design and Fabrication • C – Fluid focusing, detection and sorting Sheath flow injection Flow from rotary microfluidic pump for fluid focusing Detection by PMT module Sorting of magnetic beads using feedback signals from PMT Collection of magnetic beads (Flow generated by rotary microfluidic pump

12. Results • Evaluation of: • Pumping rate vs driving frequency of circulatory pump • Magnetic field and Temperatur vs Applied current • Fluorescence signal amplitude vs time • Virus detection limit

13. Results Magnetic field and Temperature in focusing vs Applied current Pumping rate vs driving frequence of circulatory pump

14. Results Signal amplitude from fluorescence measurement vs time Singal to Noise ratio of 30 Minimum concentration of 104 pfu/ml of Dengue virus could be detected. RT-PCR has detection limit of ~1 pfu/ml

15. Conclusion • Design for rapid virus diagnosis has been proposed

16. Discussion • Easy principle, sandwich immunoassay is well documented • Gate design is not included in article • Sensitivity needs to be enchanced

17. References • Lab Chip, 2007, 7, 868–875, DOI: 10.1039/b700516d • Biomed Microdevices (2007) 9:545–554 DOI 10.1007/s10544-007-9062-6

18. TACK!