Fluorescence Resonance Energy Transfer FRET Xingwei Wang 1
- Slides: 20
Fluorescence Resonance Energy Transfer (FRET) Xingwei Wang 1
FRET based immunosensor From ref [1] 2
Principle n n Two fluorophores: Donor & acceptor In close proximity q q q n n the donor absorbs energy from the source transfers the energy to the acceptor emits fluorescent energy Distance dependent property Detect conformational changes when antibodies combine with their respective antigens 3
Principle (2) n n 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 4
Application I: Monitor early markers of myocardial infarction n n 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 5
Principle n n Försters distance: the distance where energy transfer from the donor to acceptor fluorophore is 50% (< 100 A) Close: λ 0 -> λ 2 Separated: λ 0 -> λ 1 Conformational Change 6
Performance n n n Detection limit: 27 n. M 600 µm diameter silica core optical fibers Taper end: q q n n 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 7
Emission Spectrum From ref [1] 8
Spectrum Methods n n The donor fluorophore excitation light: 540 nm Peak 1 (P 1), is the donor emission spectrum with maximum peak intensity at 570 nm. Peak 2 (P 2), is the acceptor emission spectrum with maximum peak intensity at 610 nm. Rather than analyzing intensity of the emission curves q n susceptible to instrumental baseline shifts Using q q the maximum area under each emission spectrum The ratio of the maximum donor to acceptor area (P 1/P 2) 9
Results n A decrease in the P 1/P 2 ratio after antigen addition is indicative of energy transfer. 10
Problem n n - High STD Different tapering angles - different amounts of photons being captured back Different exposed surface areas - different antibody-Protein immobilized – different signal strength 11
Applications II: Food safety: Detection of Listeria n U. S. each year q q q n n 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. 12
Advantage n n n Detect only viable analytes Reduce false positives Listeria antigen detection limits: 2. 0µg/ml 13
Schematic of the FRET Immunosensor 14
Spectrum n n 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 15
Measuremet With no antigen present (baseline) With specific or nonspecific antigen present Ratio used to determine change 16
Detection limit: 2. 0 µg/ml 17
Advantages n n n Portable On-site analysis of samples Reduce the large economical burden by food products recalls and medical treatments 18
FRET video n http: //www. youtube. com/watch? v=p. MH 8 zc. W a 7 WA 19
References n n Development of a FRET based fiber-optic biosensor for early detection of myocardial infarction Pierce, M. E. ; Grant, S. A. ; Engineering in Medicine and Biology Society, 2004. EMBC 2004. Conference Proceedings. 26 th Annual International Conference of the Volume 1, 2004 Page(s): 2098 - 2101 Vol. 3 Development of a novel FRET immunosensor for detection of listeria Ko, S. ; Grant, S. A. ; Sensors, 2003. Proceedings of IEEE Volume 1, 22 -24 Oct. 2003 Page(s): 288 - 292 Vol. 20
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