Fluorescence Polarization

Fluorescence Polarization theory was first described in 1926 by Perrin. Weber (1953) greatly expanded the theory and developed the first instrumentation for the measurement of Fluorescence Polarization. Dandliker and co-workers (1961) expanded Fluorescence Polarization into biological systems, such as antigen/antibody reactions and hormone/receptor interactions. Dr. Michael Jolley first developed Fluorescence Polarization as a commercial system for the monitoring of therapeutic drug levels and the detection of drugs of abuse in human body fluids.

All molecules in solution rotate. The rate of rotation of a molecule is inversely proportional to its size. Very few molecules are fluorophores (naturally fluorescent). To make a non-fluorescent molecule fluorescent, a fluorophore must be attached to it. We call the resultant molecule a "tracer". By selecting a fluorophore, whose fluorescence lifetime (the time between absorbing a photon and emitting one) is on the same time scale as the rate of the molecule’s rotation, Fluorescence Polarization can be employed to determine the tracer’s size. Thus Fluorescence Polarization can be used to monitor a change in the size of a tracer and hence to detect its binding to a larger one, such as an antibody or receptor, in real time.

Fluorescence Polarization is a homogeneous technology. As a result, reactions are very rapid, taking seconds to minutes to reach equilibrium. Reagents are stable, allowing large quantities to be prepared, resulting in high reproducibility. Due to these properties, Fluorescence Polarization has proven to be highly automatable, often performed with a single incubation with a single, pre-mixed tracer-receptor reagent. The fact that there are no washing steps increases the precision and speed over heterogeneous technologies and dramatically reduces waste.

Other homogeneous technologies based on fluorescence intensity have been developed. These include energy transfer, quenching, and enhancement assays. Fluorescence Polarization offers several advantages over these alternatives:

• Fluorescence Polarization assays are easier to construct;

• Fluorescence Polarization assays tracers do not have to respond to binding by intensity changes;

• Fluorescence Polarization assays require no tracer;

• Fluorescence Polarization assays can use crude receptor preparation material;

• Fluorescence Polarization assays are independent of intensity; and

• Fluorescence Polarization assays are relatively immune to the inner filter effect allowing use of colored solutions and cloudy suspensions.

Fluorescence Polarization instrumentation requires little or no standardization since Fluorescence Polarization is derived from fundamental properties of a molecule and because reagents are stable. Fluorescence Polarization is relatively insensitive to instrument changes such as drift, gain settings, or lamp changes. Further, fluorescence intensity is obtained in addition to polarization if desired for a specific assay.

Fluorescence Polarization can be used for studying the interactions of antigens and antibodies, hormones and receptors, DNA and DNA, and DNA and DNA binding proteins, amongst others. It can also be used for the study of enzymes, where (usually) a large molecule is broken down to smaller ones. The technique has been used successfully for over twenty years in the research, pharmaceutical, and human clinical diagnostic fields.

For more detailed discussion of Fluorescence Polarization theory and applications refer to http://www.jolley.com/princ.html or to the additional links provided under Related Links.