Dr Andrea Krumm discusses the merits of microplate-based ratiometric measurements
Ratiometric measurements detect in a single sample two parameters that are analysed by calculating their ratio. This principle is employed to report on biological events. The most popular ratiometric assays are based on fluorescent (FRET) and luminescent (BRET) resonance energy transfer. However, ratiometric fluorescent probes are on the rise as they offer several advantages.
HOW RADIOMETRIC METHODS WORK
No ratio without two values: one measured value is regarded as reference, while the other is the signal indicating the presence of an analyte/event. A closer look at FRET and BRET assays clarifies the role of the values.
FRET and BRET assays are based on the transfer of energy from a donor to an acceptor fluorophore. The energy originated by a fluorophore (FRET) or luminophore (BRET) is only transferred to its acceptor when both are in close proximity, making BRET and FRET ideal applications to study binding events. For analysis, the acceptor signal is divided by the donor, the resulting ratio indicates their proximity.
The obvious advantage of ratiometric measurements is the normalising role of the ‘reference’ (e.g. the donor), which is detected in the same well as the signal of interest and thus corrects for effects such as bleaching. Secondly, the measurement becomes independent of interfering fluorescence of buffer components, and of fluorophore concentrations. This is of particular importance in cell-based assays, as they are often non-homogenously distributed or differently concentrated. These characteristics make ratiometric measurements more robust than single measurements.
HOW RADIOMETRIC ASSAYS ARE DETECTED
Ratiometric signals are detected in the same way in luminescence and fluorescence. For luciferase-based BRET measurements, donor and acceptor signals are recorded over a short time (e.g. one second). Filters transmitting light only at the wavelength of luciferase or fluorophore emission are used to separate the two different emissions. Typically, filters with a wide bandwidth are chosen. These are transmissive for <50nm around their centre wavelength and allow the highest possible light yield to hit the detector. Most monochromators are not recommended for BRET measurements, as they are restricted in their bandwidth and have poor light transmission. There is one exception: the Linear Variable Filter (LVF) monochromator of the Clariostar microplate reader. It is based on filters and allows bandwidths up to 100nm and highest light transmission. In ratiometric assays entirely based on fluorescence (e.g. FRET), either filters or monochromators can be used to separate donor and acceptor signals. In contrast to BRET, excitation at a specific wavelength is required to excite the donor. Furthermore, the signal is captured almost simultaneously with the excitation.
Irrespectively of their luminescent or fluorescent nature, the two signals can be detected sequentially or simultaneously, with simultaneous detection being approximately twice as fast. Simultaneous emission is only possible on plate readers equipped with two detectors. Accordingly, instruments such as the Pherastar FSX performing simultaneous and fastest detection are mainly chosen when time is critical, for instance in high-throughput screens.
Processes measured by ratiometric assays range from calcium signalling to binding events and pH determinations. Here are two examples for cell-based ratiometric assays.
DETERMINE PROTEIN-PROTEIN INTERACTIONS WITH BRET
BRET was used to study the binding of VEGF to its receptor in live cells. VEGF is a growth factor implicated in cancer and its receptor-ligand interaction is a target for anti-cancer therapy. The VEGF-receptor was fused to nano-luciferase whereas the ligand was labelled with the acceptor fluorophore TAMRA. When the ligand binds to its receptor, energy transfer takes place leading to acceptor emission and an increase in BRET ratio. This is calculated by dividing acceptor by donor emission. The increase in BRET ratio after addition of different ligand concentrations is shown in Fig 1.
DETECT PHARMACEUTICALS IN WASTEWATER WITH THE RATIOMETRIC PROBE RO-GFP
Residual drugs increasingly contaminate wastewater and are a potential environmental threat. A ratiometric probe based on redox-sensitive green fluorescent protein (roGFP) reports on non-steroidal anti-inflammatory drugs (NSAIDs). In presence of NSAIDs, the fluorescent probe is reduced and has an excitation peak at around 480nm. In absence of NSAIDs, it is oxidised and is excitable at 400nm. To detect NSAIDs in wastewater, cells expressing the roGFP sensor are exposed to either wastewater or diclofenac, a typical NSAID, and fluorescence is detected with excitation at 400 and 480nm. The ratio of both indicates the presence of drugs and allows calculation of diclofenac equivalents (Fig. 2).
Microplate-based ratiometric measurements are popular because of their robustness: they compensate for background fluorescence, bleaching or differences in probe concentration. They are often used to monitor cellular reactions and the list of probes and assays is steadily growing. In combination with flexible microplate readers, ratiometric approaches complete the assay repertoire of each life sciences laboratory.
Dr Andrea Krumm is with BMG Labtech
