EVANESCENT WAVE FIBER-OPTIC BIOSENSORS:
Evanescent wave fiber-optical biosensor utilizes the principles of attenuated total reflection (ATR) spectroscopy to detect and measure the interactions of biochemical materials instantaneously. “The basis of ATR is the reflection of light inside the core of a waveguide when the angle of incidence is greater than the critical angle. Waveguides can be slab guides, planar integrated optics or optical fibers. Light waves are propagated along waveguides by the law of total internal reflection (TIR).” At the interface, the intensity of light does not immediately fall to zero; therefore, inducing evanescent electromagnetic field which propagates and decays exponentially into the surrounding dielectric media with lower refractory index .
The distance at which the magnitude of the electric field at the surface decays to its l/e value, is called penetration depth ( and it is measure using the following formula where is the internal incident ray angle with the normal to the (core/cladding) interface .
As it appears from the formula, the wavelength of light, ratio of the refractive indices, and angle of the light at the interface determine the penetration depth. The penetration depth varies typically from 50 to 1000 nm. Therefore, reactions that take place close to the interface perturb the evanescent wave and the changes in signal can be calibrated to the amount of binding between immobilized ligand and fluorescent-labeled target .
Fluorescent measurements are utilized at the surface of the optical biosensors to detect the binding events. Fluorescent probes strongly excite bound-fluorophore adjacent to the fiber surface. This causes a portion of the fluorescent signals to be coupled back into the optical fibers which are consequently picked up by a fluorimeter. Exact antibody/antigen complex can be determined by exploiting the detection of fluorescent-emitting labels .
It has been shown that evanescent wave detecting excites fluorophores primarily bound to the fiber as opposed to those in the bulk solution. Fluorescent radiation transmits back through the fiber in high order modes .
SANDWICH FORMAT FLUOROIMMUNOASSAYS
The evanescent wave fiber-optic biosensor can be utilized to perform sandwich format fluoroimmunoassays. “Capture antibodies immobilized on the waveguide selectively bind specific target antigen. Following incubation with a fluorophore-labeled detection antibody to the
antigen, the fluorophore (typically cyanine-5 or Alexa Fluor647) is excited by a 635-nm laser to generate a detectable signal. Fluorescent molecules within 100–1000 nm of the waveguide surface are excited within the evanescent field, and a portion of their emission energy recouples into the fiber. Only those labeled antigens captured by antibodies within the evanescent wave are excited by the laser light, and background signals from unbound particles contribute little or nothing to the total measured fluorescent signal. ”