[[File:Bio-layer interferometry wavelength shift due to analyte binding.gif|thumb|Figure 3 - Reflectance signal as a function of wavelength]]
Bio-layer interferometry measures kinetics and biomolecular interactions on a basis of [[wave interference]]. To prepare for BLI analysis between two unique biomolecules, the ligand is first immobilized onto a bio compatible [[biosensor]] while the [[analyte]] is in solution.<ref name=":22">{{cite journal | vauthors = Müller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L | title = Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes | journal = Frontiers in Molecular Biosciences | volume = 7 | pages = 98 | date = 2020-05-27 | pmid = 32528975 | doi = 10.3389/fmolb.2020.00098 | pmc = 7266957 | doi-access = free }}</ref> Shortly after this, the biosensor tip is dipped into the solution and the target molecule will begin to associate with the analyte, producing a layer on top of the biosensor tip. This creates two separate surfaces: i) the substratemolecule immobilized on the biosensor tip itself, and ii) the substrate interactingthat withis thebound moleculeto immobilizedit onfrom the biosensor tipsolution.<ref name="Apiyo_2017" /> This essentially creates a [[thin-film interference]], in which the created layer acts as a thin film bound by these two surfaces. White light from a tungsten lamp is shone onto the biosensor tip and reflected off both surfaces, creating two unique reflection patterns with different [[Luminous intensity|intensities]].<ref name=":22" /> Figure 2 expresses this phenomenon in a more general form. The wavelength shift (Δλ) between these two reflection patterns creates an interference pattern (Figure 3) from which all desired results can be obtained.<ref name="Apiyo_2017" /> Since the wavelength shift is direct measure of the change in thickness of the biological layer and the biological layer thickness will change in response to molecules associating to and dissociating from the biosensor, the interference pattern will allow for real-time monitoring of molecular interactions on the biosensor surface.<ref name=":13">{{cite journal | vauthors = Wallner J, Lhota G, Jeschek D, Mader A, Vorauer-Uhl K | title = Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions | journal = Journal of Pharmaceutical and Biomedical Analysis | volume = 72 | pages = 150–154 | date = January 2013 | pmid = 23146240 | doi = 10.1016/j.jpba.2012.10.008 }}</ref> In short, a positive wavelength shift implies an increase in biolayer thickness and thus more association, while a negative wavelength shift implies a decrease in biolayer thickness and thus more dissociation.<ref name=":13" />
