Fluorescence Workshop UMN Physics - Fitzkee Lab · Fluorescence Workshop UMN Physics June 8-10,...
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Fluorescence Workshop UMN PhysicsJune 8-10, 2006
Introduction to Fluorescence EssaysJoachim Mueller
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Where is Fluorescence Used?
Microscopy Drug Discovery
Chemical Analysis
Enzymatic Assays Contamination Detection
Environmental Monitoring Molecular Organization
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Why is Fluorescence Used?Advantages of Fluorescence Based Analysis Systems
1. Sensitive
• High signal to noise ratio
• Single molecule sensitivity
2. Flexible
• Broad array of possible parameters to measure (Intensity, Spectra Shifts, Anisotropy, Decay parameters, FRET, …)
3. Information can be multidimensional
• Use of multiple probes or probes that report a variety of environmental parameters.
4. Real-time data acquisition
• System dynamics
• Kinetic events
5. Monitoring can be distant
• Chambers for harsh environmental conditions
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Biochemical & Chemical Assays for Detection
The Ideal Assay
1. Appropriate sensitivity for real-world samples
2. Recognition only to the target molecule
3. High signal/noise ratio
4. Low volume requirements
5. Fast reaction completion
6. Fast sampling
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A Robust General System: Immunoassays
Binding site
Fab Fragment
Binding site
Mouse IgG: The two heavy chains are shown in yellow and light blue. The two light chains are shown in green and dark blue..J.Harris, S.B.Larson, K.W.Hasel, A.McPherson, "Refined structure of an intact IgG2a monoclonal
antibody", Biochemistry 36: 1581, (1997).
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Many Recognition Assays Rely on an Equilibrium Condition
Site + SiteKeq
120
100
80
60
40
20
0
Frac
tion
Liga
nd B
ound
10-10
10-9
10-8
10-7
10-6
[Antibody] free (M)Free Binding Sites (M)
Kd
Critical Point
][
]][[
LigandSitesfreeLigandfreeSites
dK•
=
Fb =m ⋅ Sfree
Kd + S free
+ c
What should you titrate?
Adapted from S. Tetin, K. Swift, & , E, Matayoshi
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Common Errors in Binding Studies
Concentrations Too High !
[Sites] (nM)
Fb Increasing Ligand Concentrations
Site + SiteKeq
Tetin & Hazlett (2000) Methods 20, 341-361
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Errors in analysis: data transformation
Scatchard PlotDouble Reciprocal Plot
Untransformed Data
Fb
Fb
1/Fb
[Ligand]
Fb/[L
igan
d]
Theophylline &anti-TheophyllineIgG. Kd = 2 nM & n=1
Kd=1.95 nMn=1.02
Kd=8.5 nMn=2.0
Kd=2.5 nMn=1.1
1/[Ligand]
Tetin & Hazlett (2000) Methods 20, 341-361
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How do we create an assay?
The test samples do not have labeled material, do they?
1. The simple binding competition assay:
Anisotropy = High
Anisotropy = Low
Measurement
Unknown + labeled “tracer” + antibody
Less Labeled Tracer is bound
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Abbott Laboratory Polarization Assay
0.24
0.22
0.20
0.18
0.16
0.14
0.12
100806040200
[Vancomycin] (ng/mL)
Pola
rizat
ion
00.119100160.13950410.16725760.20710910.2235
1000.2340
[Tracer] bound (%)
PolarizationVancomycin(ng/mL)
ConsiderationsSpeedAccuracyBackground contributionsMeasuring errors
Calibration curve for a vancomycinimmunoassay. Vancomycin tracer and varying concentrations unlabeled vancomycin were added to sera and polarization values were collected using the TDx assay instrumentation. Curve was fit to a 4-parameter logistic function (empirical fit).
S.Y. Tetin et. al. Analytical Biochemistry 307 (2002) 84–91
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Solid Support ImmunoassaysNanoparticle Immunoassays employing fluorescence correlation spectroscopy
Competitive Immunoassay
+
ComplexSlower Diffusion & Bright
Fluorescent Antigen& Antigen
Particle with IgG attached
Potential AdvantagesFast separation of soluble, unattached fluorescent antigenLarge, dilute volumes can be assayed (depends on Kd)
Adapted from Meyers-Almes (2001), in Fluorescence Correlation Spectroscopy, Rigler & Elson, Eds.
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Ion Probes Specificity
(ion-free reference solution)
(Fluorescence: high = red > orange > yellow > green > blue = low).
Molecular probes, www.probes.com
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Measuring H+
Probes with titratable groups can have pH dependent spectra
Basic solution Acidic solution
Fluorescein
pKa ~6.4
Molecular probes, www.probes.com
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Fluorescein Data
5
4
3
2
1
01086420
14
12
10
8
6
4
2
0
pHFl
uore
scen
ce
Life
time
(ns)
4 ns