Modern trends in fluorescence and luminescence microplate assays

1st April 2013

The development of both highly sensitive, flexible and fast instrumentation for effective and robust assay technologies has evolved quite dramatically in response to the needs of the pharmaceutical industry as it applies to drug discovery and development. Here, Silke Angersbach gives an overview of novel assay technologies used in drug discovery and high throughput screening.

Much biochemical assay work involves labelling and detecting molecules as they interact with each other and their surroundings. For decades, radioisotopes have been the method of choice for this task. However, increasing constraints on the use of radioisotopes along with increased demands for speed and sensitivity result in a shift towards alternative technologies compared with existing ones.

There is a rapid surge of interest in new ways of applying fluorescence and luminescence inmolecular biology and, more specifically, in the drug development process. Although fluorescence and luminescence methods are often considered functionally interchangeable there are important differences that determine their suitability for specific applications.

Luminescence technology:

Chemiluminescent assays utilise chemical reactions to produce light. The most established probes for intracellular detection are the bioluminescent substrate for luciferase, luciferin, which is widely used as a reporter gene, and the Ca2+ sensitive photoprotein aequorin. The primary advantage of luminescent indicators is that the background signal is limited to only detector and instrument noise. Consequently, luminescence assays are very sensitive and have a very large dynamic range. On the other hand bioluminescent systems produces relatively low light output that correspond to roughly 0.1­2 photons per second and may limit luminescent applications for highly miniaturised assays.

aGlow' type reagents (Luciferin) reach their peak intensity within two to 20 min and glow for up to one hour. aFlash' type reagents (Aequorin) reach their peak intensity within 0.1-2 seconds rapidly decaying to insignificance within one to 20 seconds. The latter reactions makes it necessary to measure light emission immediately after addition of triggering reagent. The LUMIstar Galaxy allows the detection of both aglow' and aflash' reaction due to the integrated variable volume injectors which dispenses the triggering reagent into the well that is being measured (Fig. 1). The injection timings are fully programmable as well as the incubation temperature and the plate shaking mode. Depending on the application, the microplate can be measured from above or below.

Fluorescence technology: the latest shift towards exploiting fluorescence is due to a combination of recent developments: fluorescent probe molecules which can be added easily to biological material for molecular labelling and better instrumentation with improved methods of detecting and analysing the fluorescent signals. As a result of this, the range of applications for fluorescence assays is now enormous and growing rapidly.

Applications which can be performed with the POLARstar Galaxy range from fluorescent ELISA assays, molecular binding assays and DNA probe assays (eg PCR product quantification) to studies of cell membranes, ion transport, cell death and enzyme kinetics. Fluorescence chemistry and instrumentation now play a growing part in high throughput analysis such as gene expression and drug screening.

In addition to the high sensitivity of fluorescent measurements, an equally important facet of these techniques is the ability to use different aspects of fluorescence output (for example, life time, brightness, anisotropy and energy transfer) to construct assays that do not require separation steps and that have an intrinsically higher information content.

Fluorescence intensity assays: During Fluorescence Intensity assays the change in steady-state total light output is monitored, for example in enzyme kinetic assays or DNA and protein quantification. Dyes with relatively long wavelengths are used in these assays, shifted well away from interfering chromophores and fluorophores in test compound systems.

In some advanced applications (FRET-Assays) the excitation energy of a fluorophore (donor) is transferred to a suitable energy acceptor molecule and fluorescence from the acceptor and the donor are detected spectroscopically (Fig. 2). This nonradiative energy transfer has long been recognised as a useful means to detect molecular proximities.

Time-resolved fluorescence assays: Conventional fluorescent probes suffer from a serious sensitivity limitation which is caused by interference from natural fluorescence contributed by various compounds in biological material. This problem is solved by developing probes with high fluorescence intensity and long decay times, the lanthanide chelate labels (eg europium).

With the POLARstar Galaxy you can measure the delayed fluorescence decay of these fluorophores by defining the lag time after the excitation flash to start the measurement system. The background from the rapidly decaying fluorophores of biological origin do not disturb the measurement of the much slower fluorescent decay of the lanthanide label, since the decay time of the former are of the order of 1­20 nanoseconds as compared with the much longer decay times of 10­1000 microseconds of the latter. The sensitivity of the time-resolved fluorescence method with lanthanide chelates used as labels is for this reason several orders of magnitude better than that of standard fluorescence. More recently, homogeneous assay systems based on time-resolved energy transfer have been implemented.

Fluorescence polarisation assays: Another powerful method is fluorescence polarisation (FP) which can be used to analyse most molecular interactions, including DNA-protein,peptide-protein, receptor-ligand and antibody-antigen binding. In addition the action of degradative enzymes, such as proteases, DNases and RNases can be followed by FP.

This method gives a direct, nearly instantaneous measure of a tracer's bound/free ratio, even in the presence of free tracers. Most other methods require the physical separation of free and bound tracers. FP experiments are done in solution, without solid support, allowing true equilibrium analysis down to the low nanomolar range with the POLARstar.

In addition, because FP measurements are taken in areal-time', experiments are not limited to equilibrium binding studies. Kinetic analysis of association and dissociation reactions are routine with fluorescence polarisation.

The theory of FP is based on the observation that fluorescently labelled molecules in solution, excited with plane-polarised light, will emit light back into a fixed plane if the molecules remain stationary during the fluorophore's period of excitation. The polarisation of a molecule is proportional to molecule's rotational relaxation time. This time is related to viscosity, absolute temperature, molecular volume and the gas constant. Therefore, if viscosity and temperature are held constant, polarisation is directly related to the molecular volume (ie molecular size). Changes in molecular volume result from the binding or dissociation of two molecules, from degradation, or from conformational changes.

Cell-based assays: New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Intracellular detection assays have the advantage that they can be miniaturised to increase screening throughput and reduce costs while at the same time delivering ainformation-rich' data.

One main application is the measurement of intracellular Calcium. Intracellular Calcium is a universal secondary messenger that can be used to give a direct indication of receptor activity and to analyse cellular signal transduction pathways. Increasing numbers of compounds (from combinatorial synthesis) and targets (expanding genomic data) have increased the need for rapid and broadly applicable methods to screen receptors as potential drug targets and to study regulation of intracellular free Ca2+ levels by living organisms.

The typical approach to using these ion indicator dyes includes either a fluorescent microscope equipped with a fluorescence excitation source and a highly sensitive camera, or a cuvette system equipped with photodiodes for detection of emitted fluorescent light.

The NOVOstar offers the equipment necessary for cell-based assays and is the first compact microplate reader with integrated programmable pipetting system (Fig. 3). The NOVOstar has a dual microplate carrier that allows the user to designate a reagent and measurement microplate.

A transfer pipet is incorporated into the plate-to-plate transfers along with access to three reagent stations providing assay flexibility. In addition, the unit has two injectors that offer variable delivery volume and injection speed. Incubation, shaking, multi-colour detection, user-defined kinetic sampling rates, and bottom reading, are all standard.


Dr Silke Angersbach is with BMG Labtechnologies, Offenburg,





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