What is Ultra-sensitive FRET Spectroscopy? | Andor

Ultra-sensitive FRET Spectroscopy

Introduction to Ultra-sensitive FRET

Fluorescence spectroscopy is a widespread and powerful technique that can be applied to studies on large biomolecules and their interactions. In particular techniques using the modality of fluorescence resonance energy transfer (FRET) combined with encoded nanocrystal fluorescent microbeads are now being explored as a potential means of developing early diagnostic devices/systems formedical applications.

A schematic of an example application utilizing fluorescence spectroscopy with a micro-bead assay is illustrated in figure 1 below. This experiment sets out to demonstrate how nanocrystals combined with FRET spectroscopy, could act as a detector for the presence of auto-antibodies. Auto-antibodies can act as markers of diseases developing in healthy individuals.

Ultra-sensitive FRET Spectroscopy
Figure 1: Schematic of the FRET process

This example [from ref. 1] looks at the human antibody, topoisomerase or Topo I (shown as green molecule in figure 1).The test is to detect the presence of anti-Topo I antibodies (blue molecules in the figure).

Samples are prepared such that the Topo I antibody molecules (green) are attached to the microbeads in proximity to the nanocrystals.When these are incubated with anti-Topo I antibodies (blue), the latter attach themselves to the Topo I molecules. By further incubation with a solution containing dye labeled (Alexafluor633) secondary antibodies (brown), further binding occurs, as illustrated in the figure, which facilitates a FRET path between the donor excited nanocrystals and the receptor dye molecules.

When illuminated with the broadband lamp (450nm-500nm), the emission from the dye molecules (red) is mediated via FRET due to close proximity from the binding; if the anti-Topo I antibodies are absent no binding will occur and there will be no emission from the dye molecules attached to the secondary antibodies which will move freely in the solution.The presence of the latter can be confirmed by direct excitation with the laser source at 532nm.

In-depth details on this proof-of-principle technique, as demonstrated by Alyona Sukhanova et.al. can be found in reference 1.

Ultra-sensitive FRET Spectroscopy
Figure 2: Configuration of a typical setup for FRETspectroscopy

A typical setup is illustrated in figure 2 below, showing the main components of a spectrograph, high sensitive detector, microscope with adjustable x-y sample stage, a selection of excitation sources and a means of coupling the light from the output of the microscope into the spectrograph ( in this case via a fibre). Other configurations include a scanning confocal microscope setup and coupling of the light signal, after the confocal aperture, into a fibre for delivery to the spectrograph, whilst others configurations use direct coupling of the light via relay optics from the exit port of the microscope to the entrance slits of the spectrograph.

Reference Material

Alyona Sukhanova, Andrei S. Susha, Alpan Bek, Sergiy Mayilo, Andrey L. Rogach, Jochen Feldmann, Vladimir Oleinikov, Brigitte Reveil, Beatrice Donvito, Jacques H.M. Cohen, and Igor Nabiev, ‘Nanocrystal-Encoded Fluorescent Microbeads for Proteomics: Antibody Profiling and Diagnostics of Aitoimmune Diseases’, Nano Lett., 7 (8), 2322 -2327, 2007. 10.1021/nl070966+ S1530-6984(07)00966-6

Medintz IL, Clapp AR, Mattoussi H, Goldman ER, Fisher B, Mauro JM, ‘Self-assembled nanoscale biosensors based on quantum dot FRET donors’, NATURE MATERIALS 2 (9): 630-638 SEP 2003

Marcel Bruchez Jr., Mario Moronne, Peter Gin, Shimon Weiss, A. Paul Alvisatos, ‘Semiconductor Nanocrystals as Fluorescent Biological Labels’, Science 281, 2013 (1998)

Multimedia Library
Application Images (1)
Application Movies (1)
Publications Database
Evaluation of genetic damage in tobacco and arsenic exposed population of Southern Assam, India using buccal cytome assay and comet assay
Chloroquine-induced glioma cells death is associated with mitochondrial membrane potential loss, but not oxidative stress
Optimising the signal-to-noise ratio in measurement of photon pairs with detector arrays
Statistics of twin-beam states by photon-number resolving detectors up to pump depletion
The Einstein-Podolsky-Rosen paradox in twin images
Spatial properties of twin-beam correlations at low-to high-intensity transition
Coherence properties of high-gain twin beams generated in pump-depletion regime
The influence of non-imaging detector design on heralded ghost-imaging and ghost-diffraction examined using a triggered ICCD camera
Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis
Dramatic effect of single-base mutation on the conformational dynamics of human telomeric G-quadruplex
Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells
Human Rad52-mediated homology search and annealing occurs by continuous interactions between overlapping nucleoprotein complexes
The Structural and Functional Implications of Linked SNARE Motifs in SNAP25
Forster Transfer Recovery Reveals That Phospholamban Exchanges Slowly From Pentamers but Rapidly From the SERCA Regulatory Complex
Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic ‘fingerprinting’
Nanocrystal-Encoded Fluorescent Microbeads for Proteomics: Antibody Profiling and Diagnostics of Autoimmune Diseases
Quantum dots as resonance energy transfer acceptors for monitoring biological interaction
GTP Hydrolysis by the Rho Family GTPase TC10 Promotes Exocytic Vesicle Fusion
Folding of the Adenine Riboswitch

Sign up for the Andor Newsletter Now!

Receive articles like this one, product launches, press releases and more with our quarterly newsletter focusing on either Physical Science or Life Science. It's free to subscribe and you can opt out at any time.


Physical ScienceLife ScienceSUBMIT