Optical fluorescent detection system for Microfluidic applications

The principle of fluorescence polarisation was described by Perrin (1926). The mechanism of the assay takes into account that a molecule in solution randomly rotates. Molecular size is the main factor influencing the rate of rotation that is inversely related.

Thus a small molecule rotates faster than a large molecule. If a molecule is labelled with a fluorochrome, the time of rotation through an angle of 68.5° is determined by measuring polarised light intensity in vertical and horizontal planes.

Thus a large molecule is confined to emitting more light to a single plane (more polarised) than a small molecule rotating faster and emitting more depolarised light. The following technique can be implemented for microfluidic applications.

The resultant fluorescence is collected and directed into two channels that measure the intensity of the fluorescence polarized both parallel and perpendicular to that of the excitation beam. With these two measurements, the fluorescence anisotropy, r, can be found from The advantage of such a measuring method is the low requirement for signal processing.

In addition, the output of fluorescence anisotropy is dependant only upon the fluorochrome properties and its absolute quantities. It is not dependant upon the solvent properties (if no interaction between the fluorochrome and the solvent takes place).

Finally, the absolute value of the fluorescence anisotropy, r, can be used to detect the system status and the conditions of the optical alignment. This method has a disadvantage when the fluorochrome has a fast rotational relaxation, resulting from low molecular weight and/or low solvent viscosity.