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Novel idea for diagnosis of sickle cell disease
- 1. Novel Idea for diagnosis of Sickle Cell Disease with DNA Hybridization Presentation by: Isaac Hui Gaurang Mahale Akshatha Suresh Under the guidance of Prof.Dr.Tzung K. Hsiai
- 2. What is Sickle Cell Anemia • genetic blood disorder . • characterized by red blood cells that assume an abnormal, rigid, sickle shape
- 3. Outline • Motivation • Objective • Cell Sorting • DNA Hybridization • Fabrication • Anticipated Results • Pitfalls • Future Work
- 4. Motivation • SCD affects 90,000 to100,000 Americans. • SCD occurs among about 1 out of every 500 Black or African-American births. • From 1989 through 1993, an average of 75,000 hospitalizations due to SCD occurred in the United States, costing approximately $475 million. • Current prenatal diagnoses are invasive and pose a 0.5% risk to the child and to the mother.
- 5. Objective • Noninvasive • High sensitivity • High purity • Easy fabrication
- 6. Approach – Cell Sorting Different options for cell sorting • Magnetic Assisted Cell Sorter (MACS) • Fluorescence Activated Cell Sorter (FACS) • Pulse Laser Activated Cell Sorter (PLACS)
- 7. PLACS
- 8. PLACS Setup
- 9. PLACS Advantages • High Purity (97%) • High Throughput (1500 cells s-1) • High Cell Viability after high sorting speeds (90%)
- 10. Chip Design
- 11. Chip Design
- 12. DNA Hybridization • Denaturing: DNA strands separated by high temperature or pH • Renaturing: When temperature again lowered, complementary strands will rebond • Hybridization: Process in which labeled RNA finds and basepairs with complementary strand
- 14. Electrochemical DNA Hybridization • Catalysed reduction and oxidation of DNA bases • Single stranded oligonucleotide (probe) • Complementary DNA sequence (target) • Formation of hybrid • Conversion to analytical signal
- 16. Label-based detection • Based on redox-active label • Solution containing a redox-active and DNA- binding molecule • Electrochemical technique applied to measure surface species • Probe signal and hybrid signal basis for detection of hybridization
- 17. Advantages • Simple • Rapid response times • Low-cost • Suitable for microfabrication • Small sample volumes • Efficiency and high performance
- 18. Fabrication Methods • SOFT LTHOGRAPHY –DNA –hybridization • DRY ETCHING- CELL sorting
- 19. Soft Lithography
- 20. Steps Followed • Air and fluid mother molds were fabricated on silicon wafers by photolithography • molds are baked on a hot plate so that the photoresist could reflow and form a rounded shape • vapor treatment was applied to these molds before each replication process to prevent adhesion to the photoresist • Two pieces are chemically bond with each other and hermetically sealed on glass
- 21. Why Soft Lithography? • PDMS is bio-compatible • Cost effective • Same mold can be used to make numerous stamps • Good optical characteristics
- 22. Dry Etching • Etching is done on glass • T-shaped channels (2 or more outputs) • No cell sticking problems
- 23. Anticipated Results • High sensitivity – 90% detection rate – 85% accuracy • High purity – 90% fetal cells extracted • Fetal Cells extracted from Maternal blood
- 24. Pitfalls • Need for concentrated sample volumes in order to achieve sensitivity • Soft lithography technique produces defects due to the external contamination • Difficult fabrication techniques • Cell Sorting may contain contaminations of mothers blood
- 25. Future Work • Circular channels • Fringe pattern analysis is done and we compare different channels • curved channels have 100 times better sensitivity than others
- 26. Circular Channel by Micro-Machining and Soft Lithography • master mold is obtained from micro-machining • semicircular channels are created and mounted on one top of other to get circular channels
- 27. Advantages • intricate patterns can be obtained • ease of use • good resolution • reusable master mold
- 28. Future Work • Can expand from Sickle Cell Disease to other Diseases • Push for clinically viable application • Cheaper fabrication methods
- 29. Questions?
- 30. References • http://www.biop.dk/biophotonics05/posters/S%C3%B8rens en_poster-4.pdf • http://thebigone.stanford.edu/papers/Theses/HopeThesis. pdf • http://s-space.snu.ac.kr/bitstream/10371/9558/1/85.(2- 1)42-20080331162245.pdf • http://pubs.rsc.org/en/content/articlehtml/2012/lc/c2lc21 084c • http://www.cdc.gov/ncbddd/sicklecell/data.html