HTRF^® Chemistry

Successful FRET partners must fulfill certain compatibility criteria. First, their emission spectra must show non-overlapping regions in order to individually measure each partner's fluorescence. Second, the FRET quantum yield - i.e. the amount of energy they can transfer - must be as high as possible. Third, fluorescence emission must occur within a region of the spectrum remote from that produced by proteins; in other words, a red-shifted emission is better for avoiding medium-intrinsic fluorescence. The development of HTRF® by Cisbio Bioassays included extensive research into compatible donor-acceptor pairs and resulted in the selection of different TR-FRET combinations involving four specific fluorophores: Europium or Terbium cryptates as donor and XL665 or d2 as acceptor. Their structures and emission spectra are shown in Figure 1. Due to their high R0 values, both Eu3+ and Lumi4™-Tb cryptates/acceptors pairs lead to an unusually high FRET efficiency (50% to 95% for distances in the 5-10 nm range). Moreover, both cryptate spectra spread over 100 nm. The FRET process tends to concentrate the lanthanide emission energy toward the acceptor. Lumi4™-Tb and d2 match particularly well, due to a higher R0 value with the red shifted acceptor which allows higher FRET efficiency. The Lumi4™-Tb donor is also compatible with a larger choice of acceptors emitting different specific wavelengths. The unique combination of all these properties creates a detection technology with superior performance.

The maximum of the excitation wavelength of Eu3+ or Tb2+ cryptate fits with most energy sources (e.g. nitrogen laser, flashlamp) and is compatible with all HTRF certified readers. The long-lived emission obtained upon excitation (300 µsec to 2.2 msec) is characteristic of Eu3+ and Tb2+ cryptate fluorescence emission between 480 to 720 nm. (Fig. 1).

TR-FRET acceptors are prompt fluorophores which have very short half-lives if no energy transfer occurs from a donor. If the donor transfers energy, then the lifetime of the acceptor's emission is extended due to the long half-lives of the donors.

Example with Europium cryptate and XL665:

Figure 2 shows HTRF® emissions detected at 665nm. Europium Cryptate does not efficiently emit light at 665nm, thus only extremely low fluorescence at 665nm is detected when not associated with XL665. XL665, however, emits light at 665nm quite efficiently, but it is short-lived if not in proximity to Europium Cryptate. When these two fluorophores are brought together by a biomolecular interaction, the long-lived Eu3+ donor induces a long-lived emission from XL665 which can be clearly distinguished from the short-lived XL665 signal using time resolved detection. Additionally, time resolved detection eliminates measurement of background fluorescence which is prompt in nature.

Prof. J.M. Lehn's work, was awarded a Nobel Prize for Chemistry in 1987, for his work on rare earth complexes. These complexes consist of a macrocycle called trisbypyridine, within which a Eu3+ ion is tightly embedded. This cage allows both energy collection and transfer to the Eu3+ ion, which ultimately releases this energy by emitting light with specific spectral properties (Figure 3). Moreover, this type of structure confers long-lived fluorescence, one of Eu3+ cryptate's fundamental properties.

Recently, Lumi4™-Tb, a Terbium complex developed by Lumiphore Inc., was incorporated as a new donor in HTRF assays. Unlike existing terbium chelates, this new complex shares a lot of the properties of rare earth cryptates, including long-lived emissions and high stability.

Cryptates are not subject to the photobleaching that affects a number of more conventional fluorophores. This allows assays to be measured as many times as required without concerns of decreased fluorescence. In addition, the presence of challenging divalent cations or chelators, solvents, or high protein concentrations (e.g. serum) does not affect the fluorescent properties of cryptates. In the case of Eu3+ cryptates, the addition of potassium fluoride (KF) further strengthens this stability and protects Eu3+ ions against non-radiative deactivation by water molecules or quenchers. KF can be added at any time during the assay or just before readout. However, the presence of KF is not mandatory for Lumi4™-Tb, although fluoride ions show some protective action under certain assay configurations (e.g. cell-based).

Cryptates are formed by the inclusion of a cation into a tridimensional cage. The cage acts as a light collecting device and relays the energy to the core lanthanide ion. These properties of the macrocycle favor such a tight association with the lanthanide ion that this interaction becomes virtually unbreakable and leads to an exceptionally inert complex. In Eu3+ trisbipyridine cryptate (TBP Eu3+) for instance, the rigidity given by the bipyridine moieties increases the activation parameters and a ΔG* of approximately 25-30 kcal/mole is reached. Cryptates can therefore be used in harsh chemical conditions, such as solid phase peptidic synthesis using Fmoc chemistry and reverse phase chromatography in the presence of trifluoroacetic acid without the concern of dissociation of the europium ion from its cage. In comparison, the transition state of chelates is easily achieved with a ΔG* of 0-10 kcal/mole, for example by partial protonation of the carboxylate groups. This explains why chelates, unlike cryptates, are not stable in acidic media and prone to exchange their rare earth ions with ions present in the media, like Mn2+.

XL665, a phycobilliprotein pigment purified from red algae, is the first acceptor developed for HTRF®. XL665 is a large heterohexameric structure of 105 kDa that is cross-linked after isolation for better stability and preservation of its fluorescence properties. Its excitation spectrum overlaps that of Eu3+ cryptate emission allowing the donor to excite XL665 with a high quantum yield. XL665's maximum light output is at 665nm, a wavelength region Eu3+ cryptate only weakly emits light. Long-lived fluorescence at 665nm is therefore characteristic of the emission of the acceptor actively engaged in energy transfer.

The second generation d2 acceptor, an organic motif of approximately 1,000 Da, is highly compatible with Eu3+ cryptate. As a much smaller entity, d2 is less likely to cause steric hindrance problems sometimes observed in XL665-based systems. A comparison of d2 and XL665 was conducted by screening 14,700 compounds using an assay for quantifying a phosphorylated peptide (Figure 4). The correlation between the two systems was extremely close, and validated the integration of d2 in a number of different assays, notably cAMP and IP-One. Evaluation has also shown that the new acceptor contributed to significantly greater stability of immuno-competitive assays, and in some cases to better assay sensitivity.