Utilizing rare-earth lanthanides with long emission half-lives as donor fluorophores, HTRF technology combines standard FRET with the time resolved measurement (TR) of fluorescence [1
]. This powerful combination provides significant benefits to drug discovery researchers in high throughput screening (HTS), including assay flexibility, reliability, increased assay sensitivity, higher throughput, and fewer false positive/false negative results. These features, allied with automated liquid handling and robust detection instrumentation, allow for a broad range of applications in HTS.
The principle behind FRET is based on the transfer of energy between two fluorophores, a donor (long-lived fluorescence) and an acceptor (short-lived fluorescence), when in close proximity [3
]. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer (Fig.
). In the past, organic fluorescent compounds such as fluorescein and rhodamine have been widely used in the regular fluorescence assay. However, these bioassays have great disadvantages in that fluorescent detection because it is dramatically inhibited by noise in the back ground derived from scattered excitation light and significantly interfered by fluorescence from coexisting material in the sample (fluorescent compounds and dust/line), making it difficult to obtain a highly sensitive measurement. Through time-resolved measurement of fluorescence, HTRF allows the elimination of short-lived background fluorescence. Introducing a time delay (50-150 microseconds) between the initial light excitation and fluorescence measurement minimizes the contribution of all non-specific short-lived fluorescence emissions. In contrast, HTRF acceptor fluorophores emit long-lived fluorescence when engaged in the TR-FRET process with a long-lived donor fluorophore, signifying energy transfer through proximity of the labeled biomolecules (Fig.
Fig. (1) The general principle of fluorescence resonance energy transfer technology (FRET). When the donor and acceptor apart, there is no FRET signal. Once they are brought in proximity, the FRET signals are generated (for example, the emission spectrum at 665 (more ...)
Fig. (2) The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, (more ...)
Four specific fluorophores are used in HTRF forming different TR-FRET systems. The central element, the energy donor, is either Europium cryptate (Eu3+
] or Lumi4-Tb (Tb2+
cryptate) (unpublished structure) (Fig.
). These rare earth complexes, based on the work from Nobel Prize laureate, Professor Jean-Marie Lehn, and from Professor Raymond respectively, have a macrocycle within which a Eu3+
ion or Tb2+
is tightly embedded. These ions are not fluorescent on their own; they need a light-collection device (i.e. the cage) to be excited. Similar to functions of the chelate in other luminescent lanthanide technologies, this cage acts as an antenna and allows both energy collection and transfer to the ions, which ultimately releases this energy with a specific fluorescent pattern. In particular, these cryptates are not subject to the photo-bleaching that affects a number of more conventional fluorophores, and the ions are almost inseparable from their macrocycles [5
]. However, due to the unique caged structure, the kinetics stability of the cryptate is extremely higher than the one of those lanthanide chelates [3
]. Rare earth chelates would be dissociated and unstable in acidic media or in the presence of divalent ions like Mn2+
, while rare earth cryptates are extraordinarily stable under wide range of chemical conditions, like reverse phase chromatography in the presence of trifluoroacetic acid, and are not affected by the presence of divalent ions in the media [4
]. It therefore allows for enhanced assay performance in terms of sensitivity, assay window, and robustness, without compromising the features and benefits that HTRF brings to assays: a ‘mix and measure’ non-radioactive format, miniaturizable to uHTS formats, showing excellent robustness and a low compound interference rate (Fig.
). Lumi4-Tb’s structure, a lanthanide tightly embedded in a surrounding macrocycle, remains very much in line with that of previous HTRF cryptates and shows superior stability compared to other Terbium complexes. Terbium is exceptionally bright – 10 to 20 times brighter than Europium that significantly increases the detection sensitivity in assays such as the exploration of cell surface receptors.
Fig. (3) Features of donor cryptate. a. Structure of cryptate trisbipyridine (TBP). Lambda max absorption: 305nm, molar extinction coefficient at 305nm: 30000 M-1 cm-1, molar extinction coefficient 337nm: 4500 M-1 cm-1. b. Detection wavelengths for Europium and (more ...)
The first acceptor developed for HTRF was XL665, a phycobiliprotein pigment purified from red algae [6
]. XL665 is a large heterohexameric edifice of 105 kDa, cross-linked after isolation for better stability and preservation of its photophysical properties in HTRF assays [6
]. Unlike fluorescein, it is fully compatible with Eu cryptates. It is red-shifted and its emission is more likely to be away from possible medium and compound interference. The second-generation acceptor, d2, possesses a series of photophysical properties very similar to those of XL665 but is characterized by organic structures 100 times smaller than XL665. As a much smaller entity, d2 limits the steric hindrance problems sometimes suspected in XL665 based TR-FRET systems. These near-infrared acceptors are also particularly suited for homogeneous assays since their emission is less likely to be disturbed by intrinsic medium or compound autofluorescence arise in the typical compound screen process. The properties of these red acceptors also make them suitable for coupling with Terbium cryptate. Moreover, due to additional peaks in its emission spectrum, Terbium cryptate can be coupled with green acceptors such as fluorescein, emitting in the 520 nm range that may for instance allow designing multiplex assays with two readouts.
HTRF emissions are measured at two different wavelengths, 620nm (donor) and 665nm (acceptor). This feature is extremely advantageous, particularly for reducing well-to-well variations that may arise in homogeneous assay formats. Because wells contain a range of compounds and/or medium additives, each well will have different photophysical properties and cause varying degrees of signal interference and therefore altered signal intensities. This well-to-well signal variation is not due to true differences in light transition, but it can lead to misleading results if only a single emission wavelength is measured. A unique ratio-metric measurement of two emission wavelengths (patent US 5,527,684 and foreign equivalents) corrects for well-to-well variability and signal quenching from assay components and medium variability. Emissions at 620nm (donor fluor) are used as an internal reference while emissions at 665nm (acceptor fluor) are used as an indicator of the biological reaction being assessed. Because both the 620nm and 665nm emissions are decreased by sample interferences, the ratio remains unchanged (Fig. ). In addition, the ratiometric readout can minimize the system errors caused by liquid handling instruments and detectors.