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Fluorescence Resonance
Energy Transfer (FRET)
Xingwei Wang
1
FRET based immunosensor
From ref [1]
2
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
3
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
4
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
5
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
6
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
7
Emission Spectrum
From ref [1]
8
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)
9
Results

A decrease in the P1/P2 ratio after antigen
addition is indicative of energy transfer.
10
Problem - 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

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.
12
Advantage



Detect only viable analytes
Reduce false positives
Listeria antigen detection limits: 2.0µg/ml
13
Schematic of the FRET Immunosensor
14
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
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



Portable
On-site analysis of samples
Reduce the large economical burden by food
products recalls and medical treatments
18
FRET video

http://www.youtube.com/watch?v=pMH8zcW
a7WA
19
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, 2004. EMBC 2004.
Conference Proceedings. 26th 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.
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