Fluorescence Workshop UMN PhysicsJune 8-10, 2006

Introduction to Fluorescence EssaysJoachim Mueller

Where is Fluorescence Used?

Microscopy Drug Discovery

Chemical Analysis

Enzymatic Assays Contamination Detection

Environmental Monitoring Molecular Organization

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

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

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).

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

Common Errors in Binding Studies

Concentrations Too High !

[Sites] (nM)

Fb Increasing Ligand Concentrations

Site + SiteKeq

Tetin & Hazlett (2000) Methods 20, 341-361

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

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

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

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.

Ion Probes Specificity

(ion-free reference solution)

(Fluorescence: high = red > orange > yellow > green > blue = low).

Molecular probes, www.probes.com

Measuring H+

Probes with titratable groups can have pH dependent spectra

Basic solution Acidic solution

Fluorescein

pKa ~6.4

Molecular probes, www.probes.com

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