Monomer‐excimer systems

• Two or more fluorophores are needed

• F + F* [FF]*

• Excited dimer

• Diffusion controlled

• Homodimer (excimer), heterodimer (exciplex)

• Mainly hydrocarbon fluorophores (pyrene,anthracene etc.)

Energetics of excimer formation

• excimer band is always structureless and red shifted• Monomer / excimer ratio is independent of concentration• two-point-calibration

S1

S0

Basic principles of fluorescence• In sensors: guest induced formation or guestdissociation

Examples

= Cd2+

Energy transfer systems

• Two or more fluorophores areneeded

• A + D* A* + D (vs. Innerfilter effect)

• Spectral overlap, transitionmoments

• Distance (

Energy transfer systems

• Short range (Dexter) mechanism : electron exchange between overlapping molecular orbitals (

Energy transfer systems• Förster type of energy transfer (FRET)

• Strongly distance dependent (molecular ruler)

• Enzyme kinetic measurements

• HOST-GUEST (receptor-ligandum)

• Membrane diffusion / fusion

• Conformational changes

• Colocalisation

• Imaging techniques (resolution increase)

Examples

ExamplesMolecular Beacon

Ion selective synthetic fluorescent chemosensors

General issues

• Cation sensing by chemosensors in vivo• Fluorescent signal is much more sensitive than coloring (histochemical dyes)

• Small size can diffuse through membranes• At present: calcium, magnesium, sodium, potassium, zinc, copper, iron, cadmium, mercury

• Sensor design: as discussed before• Detection: translate binding event into a readable signal

Binding / dissociation constants

• Ka =

• Many factors can affect the ligands coordination sites (e.g. protonation etc.)  apparent stability constant:

• Here M’ and L’ refers to all kinds of M and L species (free, protonated etc.) 

[ ][ ] [ ]yx

yx

LMLM

[ ][ ][ ]'' LM

MLK appa =

Binding / dissociation constants

• The apparent Ka can be altered in cellular environment (eg. Fura‐2 and Ca Kd = 145 nM in vitro and 371 nM in cells)

• The presence of complex equilibria makes it difficult to determine selectivity of one ion over another

• pM values refer to the –lg[M], where [M] is the free aqueous metal ion concentration under given conditions (e.g. pH, [M]tot; [L]tot ) 

• If pKa of the ligand’s donor atom is higher than pH ofsolution, protonation can efficiently compete withbinding

• EDTA has larger Ka for Zn than TPEN

• EDTA (pKa1 = 10.19) TPEN (pKa1 = 7.12)

• The apparent Ka is more affected for EDTA, pM islower (free Zn is higher)

• To judge ion selectivities the use of pM is highlyrecommended

pM relevancy

Signal transduction strategies

• Internal charge transfer (ICT) systems‐ Enable two‐point (ratiometric) detection

• Photoinduced electron transfer (PET) systems– Signal evolution can be detected very sensitively

• Excimers– Mainly for hybridization studies

• Energy transfer systems– Hybridization – Distance measurements

Photoinduced electron transfer systems

• Spacer separated receptor – fluorophore (donor‐acceptor)

• Driving force of PET can be estimated: Rehm‐Weller equation

Redox potentials of A, D, energy of the 0‐0 transition and work related to coulombic stabilization 

• Coordination changes donor potential therefore increases ΔGET 

• FE = φf’ /φf

pET EAAEDDEG ω+Δ−−=Δ−+

00)/()/(

Prediction

Examples

Na+Over Ca, K, Mg, Li)

Cd2+(excimer)

(R)-2-phenylglycinol

Zn2+

Zn2+ and K+(Logic operations)

Na+

Calcium selective indicators ‐modularity

Calcium selective indicators ‐modularity

Green: Autofluorescence of flavoproteines (in mitochondria)

Red: Rhod-2 (free calcium)

Co-localization

Sodium selective indicators 

Find optimal D/A match

Tuning with different EWGs(11b)

Copper selective indicators 

Copper is a soft LA that does not alter the donor’s potential very much – fine tuning is necessary

Internal charge transfer (ICT) sensors

• Receptor and signaling units are integrated

• Guest induces shifted emission maximum

• Ratiometric sensing

Calcium ICT sensors

Abs and em are shifted

EM shift is much weaker Due to dumpening of Ca

Fura‐2 in cells

Zinc ICT sensors

Zn coordinates to A red shifted emission

(Zn in neurobiology)

Zinc ICT sensors

Zinc ICT sensors

510 580580/510

Excimer sensors

Aptamer: A DNA strand that recognizes an analyte

Excimer sensors

Examples

Excimer sensors

FRET sensors

FRET sensors – base pair separation

FRET sensors – base pair separation

Monomer-excimer systemsEnergetics of excimer formationBasic principles of fluorescenceExamplesEnergy transfer systemsEnergy transfer systemsEnergy transfer systemsExamplesExamplesIon selective synthetic fluorescent chemosensorsGeneral issuesBinding / dissociation constantsBinding / dissociation constantsSlide Number 14pM relevancySignal transduction strategiesPhotoinduced electron transfer systemsSlide Number 18ExamplesCalcium selective indicators - modularityCalcium selective indicators - modularitySodium selective indicators Copper selective indicators Internal charge transfer (ICT) sensorsCalcium ICT sensorsFura-2 in cellsZinc ICT sensorsZinc ICT sensorsZinc ICT sensors Excimer sensors Excimer sensorsExamples Excimer sensors FRET sensors FRET sensors – base pair separation FRET sensors – base pair separation