Lanthanide probes
Lanthanide probes are a non-invasive
History
It has been known since the early 1930s that the salts of certain lanthanides are fluorescent.
Techniques
There are two main assaying techniques: heterogeneous and homogeneous. If two lanthanide chelates are used in the analysis one after the other—it is called heterogeneous assaying.
The relaxation of excited molecules states often occurs by the emission of light which is called fluorescence. There are two ways of measuring this emitted radiation: as a function of frequency (inverse to wavelength) or time.[4] Conventionally the fluorescence spectrum shows the intensity of fluorescence at different wavelengths, but since lanthanides have relatively long fluorescence decay times (ranging from one microsecond to one millisecond), it is possible to record the fluorescence emission at different decay times from the given excitation energy at time zero. This is called time resolved fluorescence spectroscopy.[5]
Mechanism
Lanthanides can be used because their small size (ionic radius) gives them the ability to replace metal ions inside protein complex such as calcium or nickel. The optical properties of lanthanide ions such as Ln(III) originate in the special features of their electronic [Xe]4fn configurations.[4] These configurations generate many electronic levels, the number of which is given by [14!/n!(14- n)!], translating into 3003 energy levels for Eu(III) and Tb(III).[1]
The energies of these levels are well defined due to the shielding of the 4f orbitals by the filled 5s and 5p sub-shells,[4] and are not very sensitive to the chemical environments in which the lanthanide ions are inserted. Inner-shell 4f-4f transitions span both the visible and near-infrared ranges.[1] They are sharp and easily recognizable. Since these transitions are parity forbidden, the lifetimes of the excited states are long, which allows the use of time resolved spectroscopy,[4] a definitive asset for bioassays and microscopy. The only drawback of f-f transitions are their faint oscillator strengths which may in fact be turned into an advantage.[1]
The energy absorbed by the organic receptor (ligand) is transferred onto Ln(III) excited states, and sharp emission bands originating from the metal ion are detected after rapid internal conversion to the emitting level.[1] The phenomenon is termed sensitization of the metal centered complex (also referred to as antenna effect) and is quite complex.[4] The energy migration path though goes through the long-lived triplet state of the ligand. Ln(III) ions are good quenchers of triplet states so that photobleaching is substantially reduced. The three types of transitions seen for lanthanide probes are: LMCT, 4f-5d, and intraconfigurational 4f-4f. The former two usually occur at energies too high to be relevant for bio-applications.[1][4]
Applications
Cancer research
Screening tools for the development of new
pH probes
Protonation of basic sites in systems comprising a chromophore and a luminescent metal center leads the way for pH sensors.[4] Some initially proposed systems were based on pyridine derivatives but these were not stable in water.[1] More robust sensors have been proposed in which the core is a substituted macrocycle usually bearing phosphinate, carboxylate or four amide coordinating groups. It has been observed that lanthanide luminescent probe emission increases about six-fold when decreasing the pH of the solution from six to two.[1]
Hydrogen peroxide sensor
Hydrogen peroxide can be detected with high sensitivity by the luminescence of lanthanide probes—however only at relatively high pH values. A lanthanide-based analytical procedure was proposed in 2002 based on the finding that the europium complex with various tetracyclines binds hydrogen peroxide forming a luminescent complex.[1]
Estimating molecule size and atom distances
FRET in lanthanide probes is a widely used technique to measure the distance between two points separated by approximately 15–100 Angstrom.[6] Measurements can be done under physiological conditions in vitro with genetically encoded dyes, and often in vivo as well. The technique relies on a distant- dependent transfer of energy from a donor fluorophore to an acceptor dye. Lanthanide probes has been used to study DNA-protein interactions (using a terbium chelate complex) to measure distances in DNA complexes bent by the CAP protein.[6]
Protein conformation
Lanthanide probes have been used to detect conformational changes in proteins. Recently the Shaker potassium ion channel,[6] a voltage-gated channel involved in nerve impulses was measured using this technique.[7] Some scientist also have used lanthanide based luminescence resonance energy transfer (LRET) which is very similar to FRET to study conformational changes in RNA polymerase upon binding to DNA and transcription initiation in prokaryotes. LRET was also used to study the interaction of the proteins dystrophin and actin in muscle cells. Dystrophin is present in the inner muscle cell membrane and is believed to stabilize muscle fibers by binding to actin filaments. Specifically labelled dystrophin with Tb labelled monoclonal antibodies labeled were used.[6]
Virology
Traditional
Medical imaging
Several systems have been proposed which combine
Biology - Receptor-ligand interactions
Lanthanide probes displays unique fluorescence properties, including long lifetime of fluorescence, large Stokes shift and narrow emission peak. These properties is highly advantageous to develop analytical probes for receptor-ligand interactions. Many lanthanide-based fluorescence studies have been developed for
Instrumentation
The emitted
Light sources which emit short duration pulses can be divided into the following categories:[3]
- Flash tubes
- Mechanically or electronically chopped discharge lamps
- Spark gaps
- Pulsed lasers
The most important factors in the selection of the pulsed light source for are the duration and intensity of the light.[3] Pulsed lasers for the 300 to 500 nm range have now replaced spark caps in fluorescence spectroscopy. There are four general types of pulsing lasers used: lasers with pulsed excitation, lasers with G-switching, mode locked lasers and cavity dumped lasers. Pulsed nitrogen lasers (337 nm) have often been used as an excitation source in time resolved fluorometry.[3]
In time resolved fluorometry the fast photomultiplier tube is the only practical single photon detector. Good single photon resolution is also an advantage in counting photons from long decay fluorescent probes, such as lanthanide chelates.[4]
These commercial instruments are available in the market today:
Ligands
Lanthanide probes'
The chelates which have been studied and utilized to date can be classified into the following groups:[3]
- Tris chelates (three ligands)
- Tetrakis chelates (four ligands)
- Mixed ligand complexes
- Complexes with neutral donors
- Others such as: salicylatecomplexes.
The efficiency of the
See also
- Spectroscopy
- Fluorometry
- Enzymes
- Biosensor
- MRI contrast agent