1 / 20

Fluorescence Resonance Energy Transfer ( FRET)

Fluorescence Resonance Energy Transfer ( FRET). Xingwei Wang. FRET based immunosensor. From ref [1]. Principle. Two fluorophores: Donor & acceptor In close proximity the donor absorbs energy from the source transfers the energy to the acceptor the acceptor emits fluorescent energy

yoko
Télécharger la présentation

Fluorescence Resonance Energy Transfer ( FRET)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Fluorescence Resonance Energy Transfer (FRET) Xingwei Wang

  2. FRET based immunosensor From ref [1]

  3. Principle • Two fluorophores: Donor & acceptor • In close proximity • the donor absorbs energy from the source • transfers the energy to the acceptor • the acceptor emits fluorescent energy • Distance dependent property • Detect conformational changes when antibodies combine with their respective antigens

  4. Principle (2) • The fluorophores were conjugated to an antibody-Protein A complex • then immobilized to the distal end of an optical fiber. • Conformational changes • Investigate donor and acceptor fluorophore emission spectrum

  5. Application I: Monitor early markers of myocardial infarction • 1.1 million cases of acute myocardial infarction (AMI) occur each year in the United States • Can be modified and inserted subcutaneously to provide early warning of an impending heart attack

  6. Principle • Försters distance: the distance where energy transfer from the donor to acceptor fluorophore is 50% (< 100 A) • Close: λ0 -> λ2 • Separated: λ0 -> λ1 • Conformational Change

  7. Performance • Detection limit: 27nM • 600 µm diameter silica core optical fibers • Taper end: • hydrofluoric acid for 2-4 hours • 12.0 mm of the cladding was removed • Evanescent wave reaches the sensing area of the cladding-stripped fiber tip • Exciting the donor fluorophores located within its penetrating depth

  8. Emission Spectrum From ref [1]

  9. Spectrum Methods • The donor fluorophore excitation light: 540 nm • Peak 1 (P1), is the donor emission spectrum with maximum peak intensity at 570 nm. • Peak 2 (P2), is the acceptor emission spectrum with maximum peak intensity at 610 nm. • Rather than analyzing intensity of the emission curves • susceptible to instrumental baseline shifts • Using • the maximum area under each emission spectrum • The ratio of the maximum donor to acceptor area (P1/P2)

  10. Results • A decrease in the P1/P2 ratio after antigen addition is indicative of energy transfer.

  11. Problem - High STD • Different tapering angles - different amounts of photons being captured back • Different exposed surface areas - different antibody-Protein immobilized – different signal strength

  12. Applications II: Food safety: Detection of Listeria • U.S. each year • 33 million cases of foodborne diseases • more than 5 billion dollars for treatment • about 9,000 deaths • Listeria - one of the main organisms causing the outbreaks of foodborne illnesses • Rapid, accurate methods for detecting pathogens in food processing facilities are needed.

  13. Advantage • Detect only viable analytes • Reduce false positives • Listeria antigen detection limits: 2.0µg/ml

  14. Schematic of the FRET Immunosensor

  15. Spectrum • I(h = 570 nm to 575 nm): the average fluorescence intensity of the donor fluorophore • I(h =608 nm to 613 nm): the average fluorescence intensity of the acceptor

  16. Measuremet With no antigen present (baseline) With specific or nonspecific antigen present Ratio used to determine change

  17. Detection limit: 2.0 µg/ml

  18. Advantages • Portable • On-site analysis of samples • Reduce the large economical burden by food products recalls and medical treatments

  19. FRET video • http://www.youtube.com/watch?v=pMH8zcWa7WA

  20. References • Development of a FRET based fiber-optic biosensor for early detection of myocardial infarctionPierce, M.E.; Grant, S.A.;Engineering in Medicine and Biology Society, 2004. EMBC 2004. Conference Proceedings. 26th Annual International Conference of theVolume 1,  2004 Page(s):2098 - 2101 Vol.3 • Development of a novel FRET immunosensor for detection of listeriaKo, S.; Grant, S.A.;Sensors, 2003. Proceedings of IEEEVolume 1,  22-24 Oct. 2003 Page(s):288 - 292 Vol.

More Related