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Fluorescence Resonance Energy Transfer (FRET)

Xingwei Wang

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FRET based immunosensor

From ref [1]

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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

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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

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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

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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

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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

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Emission Spectrum

From ref [1]

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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)

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Results

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

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Problem - High STD

Different tapering angles - different amounts of photons being captured back

Different exposed surface areas - different antibody-Protein immobilized – different signal strength

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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.

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Advantage

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

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Schematic of the FRET Immunosensor

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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

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Measuremet

With no antigen present (baseline)

With specific or nonspecific antigen present

Ratio used to determine change

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Detection limit: 2.0 µg/ml

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Advantages

Portable On-site analysis of samples Reduce the large economical burden by food

products recalls and medical treatments

FRET video

http://www.youtube.com/watch?v=pMH8zcWa7WA

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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.