Real Time Diffractive Optics

Shining another light on multiplex immunoassays!

Diffractive optics technology, or “dot®”, brings together two well-understood technologies: grating-based light diffraction and immobilized capture surfaces. This combination produces a sensitive and very simple technique for the detection of molecular binding and dissociation events without the use of fluorescent labels.

Capture molecules, such as antibodies, are immobilized on an ordered pattern of lines that form a diffraction grating on the surface of the sensor (Figure 1). The sensor surface forms the base of a low volume flow cell. When placed within a diffractive optics instrument, a series of discrete diffraction beams is generated when the patterned molecules are illuminated with a laser. Binding of biomolecules, such as an antigen, to the patterned capture molecules increases the height of the surface pattern, producing an increased phase shift in the reflected beams, which in turn increases diffraction signal intensity which is detected in real time by a photodiode detector below the sensor. Dissociation of the interacting species with a competitive reagent conversely leads to a measurable decrease in signal.

Since the illumination occurs through an integrated optical sensor, the laser beam does not pass though the bulk solution in the flow channel.  This significantly reduces the effects of sample refractive index and color on signal intensity which allows for the direct analysis of crude biological samples.  This property also allows for the use of vigorous mixing or changes in reagent flow during an assay without signal disruption.

Figure 1. Surface binding produces phase shifts that increases the diffraction signal intensity