Fluorescence and Fluorescent Sensors
First used in 1852 by Gabriel Stokes to describe the appearance of a quinine sulfate
solution, the name fluorescence was derived from the mineral fluorite (CaF2), which
commonly contains fluorescent impurities such as europium. Fluorescence is defined as the
emission of light by a substance that has absorbed light of a different wavelength (), with
the most striking examples being compounds that absorb UV light (invisible) and emit in
the visible spectrum.
On a molecular level fluorescence begins with the absorption of a photon. This
absorption results in the promotion of an electron to an excited singlet state which, on the
order of nanoseconds, decays back to the ground state accompanied by the emission of a
photon of lower energy (longer wavelength ()). Nonradiative decay processes, such as heat
loss (typically occurring to the excited-state atom), account for the difference in energy
between the two photons. The absorbance and emission of a fluorescent compound
(fluorophore) correspond to the energy of these electronic transitions. Typical organic
fluorophores contain aromatic groups or other conjugated -systems. This extended
conjugation decreases the HOMO-LUMO gap of a fluorophore resulting in lower energy
electron transitions to produce visible light emissions.
Figure 1. Simplified Jablonski diagram showing fluorescence
Figure 2. Common structural motifs in organic fluorophores
Fluorescence is a useful property with practical applications in analytical chemistry,
biology, earth sciences and electronics (among others). One notable application of this
property is in the development of fluorescent chemosensors: abiotic molecular devices
whose fluorescent properties can respond to external stimuli in a quantifiable manner. The
ability of these materials to function on the molecular level provides numerous advantages
over classical techniques, for example chemosensors allow for the in vivo quantification of
metals involved in cellular signaling. Fluorescence offers desirable attributes for sensing
applications, such as high sensitivity of detection down to the single molecule, “on-off”
switchability, subnanometer spatial resolution, submicron visualization and submillisecond
temporal resolution. Quantitative data can readily be obtained for specific targets by
operationally simple detection schemes exploiting binding-induced perturbations of deexcitation pathways such as photoinduced electron transfer (PET) or intramolecular charge
transfer (ICT). It should be noted that methods such as internal charge transfer (ICT) and
photoinduced electron transfer (PET) typically result in probes that exhibit either
ratiometric or “turn-on” responses, allowing sensitive analyses capable of detecting and
quantifying extremely small quantities of a given analyte. Changes in the intensity and/or
wavelength of light emitted from molecular probes have been used in the detection of heavy
metal ions and anionic metabolites; in the labeling of amino acids, peptides, nucleotides, and
other macromolecules; and for monitoring reactive oxygen/nitrogen species.
Common fluorescence terminology and concepts:
Stokes fluorescence is when the fluorescence emission is of a lower energy (longer ) than
the light absorbed (em > abs and Eabs > Eem). This is the most common type of fluorescence.
Nonradiative decay pathways such as heat loss account for the energy differences.
Anti-Stokes fluorescence is when the fluorescence emission is of a higher energy (shorter
) than the light absorbed (em < abs and Eabs < Eem).
Stokes shift describes the difference between the
emission
wavelength
(em)
and
the
absorbance
wavelength (abs).
Molar absorbance () is the parameter describing the ability of a molecule to absorb light
at a particular wavelength. The absorbance E measured at any wavelength on a
spectrophotometer is proportional to the concentration c (in mol/l) and path length in a
sample l (in cm). This relationship is represented by the Beer-Lambert law equation E = cl.
Fluorescence quantum yield (φF) is defined as photons emitted over photons absorbed.
While the maximum fluorescence quantum yield (φF) is 1 for any given compound, this
value is never observed in solution, and compounds with
quantum yields as low as 0.1 are still considered highly
fluorescent.
Bathochromic shift is the change in a spectral band to a longer wavelength (). This term
is informally called a red shift, however it has no relation to the Doppler shift. Examples of
how the term is used include; spectral changes in substituted series of molecules and
spectral changes due to environmental changes such as polarity (i.e. solvatochromism).
Hypsochromic shift is the change in a spectral band to a shorter wavelength (). This term
is informally called a blue shift, however it has no relation to the Doppler shift. Examples of
how the term is used include; spectral changes in substituted series of molecules and
spectral changes due to environmental changes such as polarity (i.e. solvatochromism).
Photobleaching is the photochemical destruction of a dye or fluorophore.
“Turn-on” fluorescent sensors respond to analytes with an increase in fluorescence
intensity. These sensors commonly utilize a PET mechanism.
Ratiometric fluorescent sensors respond to analytes with changes in the relative
intensities of two emission bands. These sensors, which commonly utilize an ICT
mechanism, allow for obtaining internally self-calibrated response signals.
Photoinduced Electron Transfer (PET)
Photoinduced electron transfer (PET) a commonly employed de-excitation pathway
for development of “turn-on” fluorescence chemosensors. PET systems are composed of
three components, a fluorophore, a linker and a receptor. The receptor component typically
possesses a non-bonding electron pair, typically of a nitrogen atom, whose energy lies
between the HOMO/LUMO gap of the fluorophore. Electron transfer from the non-bonding
lone pair of the receptor to the excited state fluorophore quenches fluorescence. For PET to
occur, the energy of the fluorophores excited state must be sufficient to oxidize the receptor
and reduce the fluorophore. Binding of a cation to the receptor lone pair raises the
oxidation potential, making PET thermodynamically disfavored and restoring fluorescence
to the system. This attribute makes PET systems ideal candidates for the detection of metal
ions as their “turn-on” mechanism is activated by coordination events.
Attributes of PET Sensors:

Receptor and fluorophores are closely linked but separate electronic systems

Relatively insensitive to polarity of local environment

Non-bonding receptor electrons participate in redox chemistry with the
fluorophores quenching fluorescence.

Protonation or coordination lowers the energy of receptor electrons, inhibiting the
PET process and restoring fluorescence.

Most general mechanism for fluorescent sensing
Figure 3. Spectral and visual example of a PET sensor (left) and simplified orbital depiction
of the PET process (right)
*For ease of communication HOMO and LUMO were used. These terms only apply to the
ground state. The figure actually depicts singly occupied molecular orbitals (SOMO).
Intramolecular Charge Transfer (ICT)
Intramolecular charge transfer (ICT) is a common mechanism utilized in ratiometric
fluorescent chemosensors. ICT and PET compounds are structurally distinct from one
another, with ICT compounds including the receptor and fluorophore in the same electronic
system. Correspondingly, ICT systems are characterized as “charge-polarized” states, as
compared to the “charge-separated” states of PET systems. The two sensing mechanisms
are easily differentiated spectroscopically. ICT states (or charge transfer (CT) states) are
fluorescent, as compared to quenched PET states. Typically the CT states show highly
shifted emission bands. Upon binding analyte the CT states become inaccessible and the
normal fluorescence or locally excited (LE) state is restored, resulting in two wavelength
emissions.
Compounds displaying ICT are highly polarized, possessing electron rich and
electron poor regions or functional groups in their conjugated -systems. In the excited
state electron donor groups become more donating and electron withdrawing groups more
withdrawing, which results in a greater excited state molecular dipole. In polar solvents the
dipole is stabilized, resulting in a lower energy CT state and a bathochromic shift in
emissions. ICT sensing can be achieved by binding analytes to either the donor or acceptor
region of the fluorophore. A common example is binding a cation at an electron-donating
dialkylamino group. Binding the nitrogen lone pairs removes their electron donating ability,
making the long wavelength CT state inaccessible and restoring the shorter wavelength LE
state.
Attributes of ICT Sensors:

Receptor and fluorophores are part of the same electronic system

Often sensitive to the polarity of local environments

Polarized molecules containing electron donor and acceptor components

Interactions with analytes influence the polarization of the fluorophore, which can
alter emission intensity and the wavelength of the absorption and emission.

Analyte induced shifts in emission wavelength (em) allow for ratiometric analysis,
providing sensors with their own internal standard.
Figure 4. Spectral and visual example of an ICT sensor (left) and simplified orbital depiction
of the ICT process (right)
Please see Introduction to Fluorescence Sensing by Alexander P. Demchenko (especially
chapters 1 and 6) for more background information.