Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte
Transcrição
To detect ligand-analyte interactions using biolayer interferometry, BLI, begin with a hydrated fiber-optic biosensor fixed onto a BLI instrument mount, set to the desired program.
The biosensor tip is coated with a biocompatible layer immobilized with nickel-nitrilotriacetic acid, Ni-NTA, groups. Submerge the biosensor tip in buffer.
The instrument emits white light toward the biosensor, which gets reflected from two interfaces: the internal reference or optical layer, and the biocompatible layer. The reflected beams interfere constructively or destructively at different light wavelengths. A photodetector detects the resulting interference pattern.
Following baseline interferometric response measurement, add a histidine-tagged ligand solution. Histidine tags bind to nickel cations on the biosensor, immobilizing ligands on the tip.
Following association, the emitted white light gets reflected from the optical layer, and the biocompatible layer immobilized with ligands with increased optical thickness at the tip. This increases the distance between the reflective layers, causing interference pattern shift and a wavelength shift.
Wash the tip, removing unbound ligands. Incubate with target analyte solution. The analyte binds to the ligand, further increasing the optical thickness at the tip, causing an increased wavelength shift.
Position the biosensor tip in the buffer. Record the wavelength shift during analyte dissociation from the ligand immobilized on the tip.
Monitoring the change in wavelength shift over time facilitates the study of molecular interactions between ligands and analytes.
