Development of immunoassays using interferometric real-time registration of their kinetics.
A method for effective development of solid-phase immunoassays on a glass surface and for optimization of related protocols by highly sensitive quantitative monitoring of each assay step has been proposed and experimentally implemented. The method is based on the spectral correlation interferometry (SCI) that allows real-time measuring of the thickness of a biomolecular layer bound to the recognition molecular receptors on the sensor chip surface. The method is realized with compact 3-channel SCI-biosensors that employ as the sensor chips standard cover glass slips without deposition of any additional films. Different schemes for antibody immobilization on a glass surface have been experimentally compared and optimized toward a higher sorption capacity of the sensor chips. Comparative characterization of the kinetics of each immunoassay stage has been implemented with the optimized protocols: i) covalent immobilization of antibody on an epoxylated surface and ii) biotinylated antibody sorption on a biotinylated surface via a high-affinity biotin-streptavidin bond. We have shown that magnetic nanoparticles employed as labels with model detection of cardiac troponin I further amplify the SCI signal, resulting in 100-fold improvement of the detection limit. The developed protocols can also be used with the alternative immunoassay platforms, including the label methods based on registration of only the final assay result, which is the quantity of bound labels.
Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov Str., 38, 119991, Moscow, Russia ; Moscow Institute of Physics and Technology, Institutskiy per., 9, 141700, Moscow Region, Dolgoprudny, Russia.
A V
AV
Orlov
Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov Str., 38, 119991, Moscow, Russia ; Moscow Institute of Physics and Technology, Institutskiy per., 9, 141700, Moscow Region, Dolgoprudny, Russia.
A G
AG
Burenin
Moscow Institute of Physics and Technology, Institutskiy per., 9, 141700, Moscow Region, Dolgoprudny, Russia.
V O
VO
Shipunova
Moscow Institute of Physics and Technology, Institutskiy per., 9, 141700, Moscow Region, Dolgoprudny, Russia.
A A
AA
Lizunova
Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov Str., 38, 119991, Moscow, Russia.
B G
BG
Gorshkov
Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov Str., 38, 119991, Moscow, Russia.
P I
PI
Nikitin
Label-free biosensors; cardiac troponin I; efficiency of biomolecular immobilization; immunoassay; interferometry; magnetic nanoparticles; sensor chips; surface biotinylation; surface epoxylation; surface functionalization