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

FRET video a7WA a7WA 19

20 References 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, EMBC Conference Proceedings. 26th Annual International Conference of the Volume 1, 2004 Page(s): Vol.3 Engineering in Medicine and Biology Society, EMBC Conference Proceedings. 26th Annual International Conference of the Development of a novel FRET immunosensor for detection of listeria Ko, S.; Grant, S.A.; Sensors, Proceedings of IEEE Volume 1, Oct Page(s): Vol. Sensors, Proceedings of IEEE