Fluorescence Resonance Energy Transfer (FRET)

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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 (lt 100 A)
• Close ?0 -gt ?2
• Separated ?0 -gt ?1
• 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

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FRET video

• http//www.youtube.com/watch?vpMH8zcWa7WA

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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.