Optical biosensors can provide real-time analysis of biological binding events without the interference caused by labeling. However, in all label-free techniques, optical waves that propagate along the surface of interest have penetrating evanescent fields that also might be sensitive to undesirable signals, such as changes in the volume refractive index caused by temperature fluctuations.Valery N. Konopsky and Elena V. Alieva of the Russian Academy of Sciences in Troitsk have developed a method that overcomes this problem, separating surface interactions from changes in the volume refractive index by simultaneously detecting two optical waves.To detect both surface and volume signal contributions, the two waves must be registered on the same spot on the sensing surface but have different penetration depths. This is achieved when two parallel beams from a 532-nm Nd:YAG laser are passed through a glass prism and focused with different incident angles to the same point on the sensing surface. To create the sensor, the researchers fabricated a seven-layer photonic crystal on a glass substrate comprising three layers each of alternating 154-nm-thick SiO2 and 89.4-nm-thick Ta2O5, followed by a layer of 638.5-nm SiO2 and water.Exciting one surface wave very close to the angle of total internal reflection of the water caused it to have a longer penetration depth and an increased sensitivity to changes in the overall refractive index. A Hamamatsu photodiode array detected the intensity distributions of the reflected beams to measure the absolute angles of the surface wave excitations. The researchers then manipulated the detected data to derive changes in the refractive index and adlayer thickness.The sensitivity of the model was compared with existing label-free methods with free biotin binding to a streptavidin monolayer. Initial streptavidin binding to the biotinylated surface increased the adlayer thickness by 6.2 nm. A biotin injection at 1500 s caused the streptavidin layer thickness to increase sharply, then decrease slowly between 1501 and 1600 s. The change in thickness might be a necessary postbinding conformation for a successful streptavidin-biotin interaction. This phenomenon did not occur during a second biotin injection because most of the streptavidin binding pockets had already been occupied, and no further interaction was possible.Measurement times of one second per point record the immobilization of streptavidin on a biotinylated surface (a) and free biotin binding to the streptavidin monolayer (b) (no posterior data averaging and smoothing). The corresponding processes are illustrated in the color insets (da=adlayer thickness).The external medium refractive index decreased sharply at 1500 s during the first biotin injection. It remained constant during the streptavidin conformation and decreased again at the second biotin injection.Streptavidin conformation during biotin binding was detected with a signal-to-noise ratio of ~15 at a 1-s signal accumulation. The achieved sensitivity allowed the detection of a minimum of 200,000 streptavidin molecules and 1.3 × 108 biotin molecules. This translates to detection limits as little as 20 fg and to 50 fg of the analyte on the probed surface. The researchers hope that improving the dielectric multilayer coating or decreasing the laser noise will further decrease the noise of the technique. Many existing sensing methods cannot overcome variations in volume refractive index caused by temperature fluctuations. Independently detecting changes in adlayer thickness and in volume refractive index could be useful for temperature-controlled flow cells that might be used to evaluate the temperature dependency of biochemical reactions on a surface.Analytical Chemistry, June 15, 2007, pp. 4729-4735.