Post on 20-Jul-2020
www.fcsxpert.com December 5, 2008
Single Molecule Detection Assays Using Fluorescence Correlation Spectroscopy
David Wolf
The FCSXpert TeamDylan BulsecoCarla Coltharp
Jack HayesChampika Samarasekera
Charlie Blanchard
www.fcsxpert.com December 5, 2008
QuantumXpert TM by Sensor Technologies-FCS Simplified
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Overview
•What is FCS•Assaying for Protein•Assaying Molecular Interactions
•Rh-Fab Binding to IgG: Calculation of stoichiometry•Biotinylated-IgG binding to Dye-strepavidin: FCS vs. Conventional Fluorimetry
•Detecting Specific Pathogen Simulants
FCS Application Examples
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What is FCS?Molecules Move Randomly in Solution
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This Random Motion Causes a Fluctuation in the Number of Molecules in the Confocal Volume
FCS measures the fluctuations in fluorescence intensity as molecules diffuse in and out of the laser beam
0 20 40 60 80 100940
960
980
1000
1020
1040
1060
Inte
nsity
(co
unts
per
100
ns)
Time (usec)
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What is FCS?
• Physical processes are in a state of dynamic equilibrium.
• FCS uses confocal optics to confine the volume of measurement to a small confocal volume
• In a small volume concentration fluctuates about its mean
• FCS measures the fluctuations in fluorescence intensity that result from these concentration fluctuations.
• FCS measures concentration fluctuations, which result from random diffusion or directed flow in and out of the confocal volume as well
as processes which are independent of volume.
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Examples of Processes which Cause Fluctuations
• Diffusion• Directed flow (hydrodynamic and electrophoretic)• Chemical Equilibrium• Intersystem crossing between singlet and triplet states• Nonradiative fluorescence resonance energy transfer
(FRET)
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How FCS Works: Hardware
FCS uses confocal optics to measure the motion of fluorescently labeled molecules in a small volume
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What is Correlation
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Intensity as a Function of Time
0 20 40 60 80 100
9.8
9.9
10.0
10.1
10.2
10.3
Inte
nsity
Time
Let’s consider a fluorescence intensity which is fluctuating about its mean. We note that the fluctuations are not truly random. Rather they fluctuate with some characteristic time constant.
The leads us to ask the question if the intensity is rising or falling now, what is the probability that it will still be rising or falling some time in the future?
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Let’s ask a Stupid Question
Is our intensity data correlated with itself?
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The Intensity is Perfectly Correlated with Itself (no surprise)
9.8 9.9 10.0 10.1 10.2 10.3
9.8
9.9
10.0
10.1
10.2
10.3
Inte
nsity
Intensity
R=1.0
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What is the Correlation Function?
It is no surprise that intensity is correlated with itself. However, let’s ask the slightly more interesting question whether the intensity at any point in time is correlated with itself a millisecond later in time.
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Correlation Between Intensity and Itself a Millisecond Later
9.8 9.9 10.0 10.1 10.2 10.3
9.8
9.9
10.0
10.1
10.2
10.3
Inte
nsity
one
mse
cond
late
r
Intensity
R-0.97
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Correlation Between Intensity and Itself Six Milliseconds Later
9.8 9.9 10.0 10.1 10.2 10.39.8
9.9
10.0
10.1
10.2
10.3
Inte
nsity
Six
Mill
isec
onds
Lat
er
Intensity
R-0.92
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Correlation Between the Intensity and Itself 48 Milliseconds Later
9.8 9.9 10.0 10.1 10.2 10.3
9.8
9.9
10.0
10.1
10.2
10.3
Inte
nsity
48
Mill
isec
onds
Lat
er
Intensity
R-0.50
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Correlation Between the intensity and Itself 96 Milliseconds Later
9.8 9.9 10.0 10.1 10.2 10.3
9.8
9.9
10.0
10.1
10.2
10.3In
tens
ity 9
6 M
inut
es L
ater
Intensity
R-0.00
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Correlation Decays with a Characteristic Time Constant
10 100
0.0
0.2
0.4
0.6
0.8
1.0
Cor
rew
latio
n C
oeffi
cien
tR
(∆t)
Shift in msec
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What is the Autocorrelation Function?
•The correlation coefficient expresses the probability that if the signal is rising or falling now that it will be still rising or falling sometime later.•This probability goes from 1 at time 0, to 0 at time infinity.•Since we are considering the correlation between intensity and itself, we refer to this as the autocorrelation function.•If we perform the same analysis comparing fluctuations in intensity at some wavelength with, say, intensity at a second wavelength we would refer to this as a cross-correlationfunction.
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It is important to recognize that in biology the fluctuations are not really random. Some underlying process with a characteristic time scale is driving them.
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Monomeric Diffusion
•Average particle number = 32
•Diffusion rapid
•Intensity proportional to particle number
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Crosslinking Causes Dimerization
•Average particle number = 16
•Diffusion slower
•Intensity unchanged--proportional to 2X particle number
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10-5 10-4 10-3 10-2 10-1 100 101 102
0.998
1.000
1.002
1.004
1.006
1.008
1.010
1.012
1.014G
(t)
T (s)
τ = 4 msD=w2/4τ
=(1.2x10-4)2 cm2
16x10-3 s= 9x10-7 cm2/s
A Single Autocorrelation Curve
Particle Number,
G(t) = 1 +R(t)/Np
G(0) = 1+ 1/Np
Np =90
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Particle Number (G(0) = 1 + 1/Np)*
10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 101 102 1030.98
1.00
1.02
1.04
1.06
1.08
1.10
1.12
1.14
1.16
1.18
1.20
G(τ
)
τ (s)
*The smaller the particle number the larger the intercept
N=5
N=25
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Molecular Size (from the rate of decay)*
10 -7 10 -6 10 -5 10-4 10 -3 10 -2 10 -1 100 101 102 103
0.98
1.00
1.02
1.04
1.06
1.08
1.10
1.12G
(τ)
τ (s)
*The faster the diffusion the faster the rate of decay
τ=300 ms
τ=0.03 ms
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Uncorrelated Diffusion of Independent Monomers
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Correlated Diffusion of Dimers
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Detection of Molecular Complexingby FCS
No cross-correlationFast autocorrelationFluorescent labels
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Detection of Molecular Complexingby FCS
No cross-correlationSlow autocorrelation
Fluorescent labels boundto different targets
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Detection of Molecular Complexingby FCS
Slow cross-correlationSlow autocorrelation
Fluorescent labels boundto same target
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FCS: From Molecules to Bacteria
1E-8 1E-7 1E-6 1E-5 1E-4
100000
1000000
1E7
1E8
1E9
r = (MW/((6.02x1023)(4/3)πρ*)1/3 (cm)
1/D
(s/
cm2 )
Experimental Theoretical
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Measuring Diffusion in Viscous Solutions with FCS
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Advantages of FCS with the QuantumXpert
•Advantages offered by Autocorrelation•Flexible easy-to-use assay format (e.g. multiwell plates & flow-cells)
•Low sample volumes and concentrations (<10 ul)•Provides a direct measure of particle number and therefore concentration• Does not require any signal amplification•Increased sensitivity (Typically 1000 X over conventional techniques)
•Nonaveraging technique offers simultaneous measurement of different fractions (e.g. bound and unbound)
•Can extract complex signals from high background (S/N advantage)•Does not require washing (no separation of unbound probe required) •Provides a direct measure of stoichiometry•Reduces false negatives
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Advantages of FCS with the QuantumXpert
•Additional Advantages of Cross-correlation•Reduced false-positives•Simple direct assay for complexes •Improved sensitivity•Improved S/N•Multiplexed assays possible (2 excitation wavelengths and 3 detection channels)
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FCS Applications in Solution•Protein-protein interactions
•Protein-nucleic acid interactions
•Protein/peptide Lipid Vesicle
•Ligand-receptor binding
•Enzyme activation and kinetics
•Complex formation
•Protein folding
•Pathogen detection (bacterial, viral)
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Application Examples
•Assaying for Protein•Assaying Molecular Interactions
•Rh-Fab Binding to IgG: Calculation of stoichiometry•Biotinylated-IgG binding to Dye-strepavidin: FCS vs. Conventional Fluorimetry
•Detecting Specific Pathogen Simulants
FCS Application Examples
www.fcsxpert.com December 5, 2008
Quant-iT Protein Assay (Invitrogen)
•Based on merocyanine dye that increases in fluorescence in hydrophobic environments
•Assay buffer contains detergent and proteins become contained inmicelles
•Upon addition of protein:•dye fluorescence increasesdue to binding in hydrophobic regions of the protein containing micelles
•dye diffusion slowsupon binding to protein containing micelles
•particle number reducesdue to multiple dyes binding to protein containing micelles.
Assaying Protein in Solution
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Intensities
Merocyanine Binding to Protein Containing Micelles
Fluorescence Intensity
Blue: Reagent + 200 ng/mL Protein (250,000 cps)
Red: Reagent alone (25,000 cps)
Fluctuation = 48 %
Fluctuation = 7 %
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Merocyanine Binding to Protein Containing Micelles
+ 200 ng/mL ProteinMerocyanine Alone
50 particles 5 particles
10 msec
100 msec
Stoichiometry ~ 50/5 = 10*We have ignored any unbound dye
τD= 2.6 msr= 1.61 nm
τD= 99 msr= 62 nm
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Schematic for Rh-Fab Binding to IgG
N=5Rh-Fab
One Diffusing Component
* Information on stoichiometry can be extracted from the fractions of
slow and fast diffusing components*
Two Diffusing ComponentsN=25
IgG
Rh-Fab
IgG
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Stoichiometry: Rh-Fab binding to IgG
10-3 10-2 10-1 100 101 102 103 104
1.000
1.025
1.050
1.075
1.100
G(τ
)
τ
0.00
Res
idua
ls
Rh-Fab + IgG2-diffusing componentsRh-Fab
1-diffusing component
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Increase in Slow Diffusing Fraction
0 1000 2000 3000 4000
0
1
2
3
4
5
6
7
8
F2N
p
[IgG] nM
Increase in Slow FractionA
A
BB
C
C
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Stoichiometry of Rh-Fab Bound to IgG
0.1 1 10 100 1000 100001
2
3
4
5
6
7
Sto
ichi
omet
ry (
RhF
ab/Ig
G)
log [IgG] nM
Calculated StoichiometryA
BB
CC
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Detecting Molecular Interactions:FCS vs. Conventional Fluorimetry
FluoReporter Biotin Quantitation Assay Kit (Invitroge n #F30751)
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0 50 100 150 200 25030000
40000
50000
60000
70000
80000
90000
100000
Biotin Biotinylated-IgG
Flu
ores
cenc
e In
tens
ity (
cps)
[Biotin] or [Biotinylated IgG] nM
Changes in Fluorescence Intensity Upon Biotin Binding
KD~1.7 nM
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10-5 10-4 10-3 10-2 10-1 100 101 102
0.99
1.00
1.01
1.02
1.03
1.04
G(t
)
τ (s)
1 pM Biotinylated IgG 1 nM Biotinylated IgG
Effect of Biotinylated IgG on Autocorrelation Curves
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Effect of [Biotinylated IgG] on Average Number of Particles (Np)
0 5 10 15 20
0
50
100
150
200
250
300
Np
Biotinylated-IgG (nM)
0 5 10 15 20
0
50
100
150
200
250
300
Np
[Biotin] nM OR [Biotin-IgG] nM
Biotin-IgG Biotin
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Correlation Between Diffusion Time and Biotin-IgG Concentration
0 5 10 15 20
0
50
100
150
200
250
300
350
Tau
D2
(ms)
[Biotin-IgG] nM
1 nM Biotin-IgG
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Summary of FCS Measurement with the FluoReporter Biotin Quantitation Kit
•More information than simply intensity•Macromolecular complexing of multivalent analytes•Differences in diffusion time which is dependent on complex size
•Greater sensitivity•Reagent was used at a 50-fold dilution of the recommended working stock solution•Measurements can be made in as little as 10 µl sample volumes•Intensity is linear into the fmol range•Invitrogen: 40 nM biotinylated protein in 100 µl •QuantumXpert: 10 pM biotinylated protein in 10 µµµµl
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Protein-DNA Interactions
• (Roche 2008) Fluorescence Correlation Spectroscopy of the Binding of Nucleotide Excision Repair Protein XPC-hHr23B with DNA Substrates.J Fluoresc. 18:987–995• %DNA bound determined using fluorescently labeled DNA, which
diffuses slower when bound to proteins
• (Wolke 2003) Analysis of p53 “Latency” and “Activation” by Fluorescence Correlation Spectroscopy. J. Biol. Chem. 278:32587-32595 • %DNA bound determined using fluorescently labeled DNA,• Binding affinities determined using protein titration• Selectivity determined by competing with unlabeled, nonspecific DNA
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Detection of Pathogen Simulants
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10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103
1.000
1.001
1.002
1.003
1.004
1.005
1.006
Ab Alone
Ab + HB101
G(τ
)
τ
0 20 40 60 80 100 1200
50000
100000
150000
200000
250000
300000
Ab Alone
Ab + HB101
Inte
nsity
Time (sec)
Bacterial Detection Using One FCS Channel Signal Extraction From High Background
•Signal/Background ~ 0.4•Antibody in solution•No washing required
E. coli HB101Alexa-546 α E. coli
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Bacillus subtilis: Antibody Probes
10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102
1
2
3
4
5
G(τ
)τ
0 25 50 75 100 1250
1000020000300004000050000
0 25 50 75 100 1250
250005000075000
100000
Bacilli Spores
10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103
1.0
1.5
2.0
2.5
3.0
3.5
G(τ
)
τ
0 25 50 75 100 125 150 175 200 225 250 275 300
0150003000045000
0 25 50 75 100 125 150 175 200 225 250 275 300
0250005000075000
Vegetative Bacilli
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Bacillus subtilis: DNA Probes
10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103
1.0
1.1
1.2
1.3
G(τ
)
τ
0 25 50 75 100 125 150 175 200 225 250 275 300 325
0200000400000600000
0 25 50 75 100 125 150 175 200 225 250 275 300 3250
250000500000750000
1000000 •5’ GCTGCCTCCCGTAGGAGT 3’
5’ ACAGATTTGTGGGATTGGCT 3’
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QuantumXpert TM
Hardware• No user alignment required• Flexible sample handling options
– Manual 8-well – Automated sampling system (standard 96-
well or 384-well format with flow cell and temperature control
Software• Easy to use• Push-button acquisition• Intuitive User Interface
Application-specific kits
An inexpensive integrated system for FCS assays