Single Molecule Detection Assays Using Fluorescence ... · December 5, 2008 Intensity as a Function...

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


QuantumXpert TM by Sensor Technologies-FCS Simplified


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


What is FCS?Molecules Move Randomly in Solution


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

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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.


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)


How FCS Works: Hardware

FCS uses confocal optics to measure the motion of fluorescently labeled molecules in a small volume


What is Correlation


Intensity as a Function of Time

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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?


Let’s ask a Stupid Question

Is our intensity data correlated with itself?


The Intensity is Perfectly Correlated with Itself (no surprise)

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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.


Correlation Between Intensity and Itself a Millisecond Later

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Correlation Between Intensity and Itself Six Milliseconds Later

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Correlation Between the Intensity and Itself 48 Milliseconds Later

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Correlation Between the intensity and Itself 96 Milliseconds Later

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Correlation Decays with a Characteristic Time Constant

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Shift in msec


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.


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.


Monomeric Diffusion

•Average particle number = 32

•Diffusion rapid

•Intensity proportional to particle number


Crosslinking Causes Dimerization

•Average particle number = 16

•Diffusion slower

•Intensity unchanged--proportional to 2X particle number


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(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


Particle Number (G(0) = 1 + 1/Np)*

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G(τ

)

τ (s)

*The smaller the particle number the larger the intercept

N=5

N=25


Molecular Size (from the rate of decay)*

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(τ)

τ (s)

*The faster the diffusion the faster the rate of decay

τ=300 ms

τ=0.03 ms


Uncorrelated Diffusion of Independent Monomers


Correlated Diffusion of Dimers


Detection of Molecular Complexingby FCS

No cross-correlationFast autocorrelationFluorescent labels



No cross-correlationSlow autocorrelation

Fluorescent labels boundto different targets



Slow cross-correlationSlow autocorrelation

Fluorescent labels boundto same target


FCS: From Molecules to Bacteria

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r = (MW/((6.02x1023)(4/3)πρ*)1/3 (cm)

1/D

(s/

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Experimental Theoretical


Measuring Diffusion in Viscous Solutions with FCS


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


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)


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)


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


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


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 %


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


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


Stoichiometry: Rh-Fab binding to IgG

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Increase in Slow Diffusing Fraction

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Stoichiometry of Rh-Fab Bound to IgG

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Detecting Molecular Interactions:FCS vs. Conventional Fluorimetry

FluoReporter Biotin Quantitation Assay Kit (Invitroge n #F30751)


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Changes in Fluorescence Intensity Upon Biotin Binding

KD~1.7 nM


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Effect of Biotinylated IgG on Autocorrelation Curves


Effect of [Biotinylated IgG] on Average Number of Particles (Np)

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Biotin-IgG Biotin


Correlation Between Diffusion Time and Biotin-IgG Concentration

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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


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


Detection of Pathogen Simulants


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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


Bacillus subtilis: Antibody Probes

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Bacilli Spores

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Vegetative Bacilli


Bacillus subtilis: DNA Probes

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1000000 •5’ GCTGCCTCCCGTAGGAGT 3’

5’ ACAGATTTGTGGGATTGGCT 3’


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

Single Molecule Detection Assays Using Fluorescence ... · December 5, 2008 Intensity as a Function...

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