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Highly multiplexed label-free imaging sensor for accurate quantification of small molecule binding kinetics
* 1 , 1 , 1 , 1 , 2 , 2 , 1, 3
1  Department of Electrical Engineering, Boston University
2  Neogen, INC.
3  Department of Biomedical Engineering, Boston University (registering DOI)

Investigating the binding kinetics of small molecule analytes to larger ligands, such as proteins and antibodies, is a compelling task for the field of drug and biomarker development, as well as the food industry and agro-biotechnology. In 2019, the FDA approved 48 new drugs, 73% of which belong to the category of small molecules, with a molecular weight (MW) below 1KDa. On the same level, most of the fungi-produced toxins that are commonly known to affect crops also fit this description. Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly-multiplexed biosensor, to perform real-time affinity measurement of small molecules binding to immobilized antibodies in a microarray format. As the analytes bind to the surface probes, the biomass accumulation on the surface is quantified by measuring the optical reflectance from the layered Si/SiO2 chip through the solution, in a common-path interferometer configuration. As a proof of concept, label-free detection of biotin molecules binding to immobilized streptavidin probes is performed, achieving 1pg/mm2 sensitivity through signal averaging in a shot noise limited operation. Furthermore, we apply the optimized sensor to the screening of a 20-multiplexed antibody chip (MW~150kDa ligands) against Fumonisin B1 (MW = 721.8Da), one of the most prevalent mycotoxins found in many cereal grains such as corn and wheat. The simultaneously recorded binding curves of the toxin to the multiplexed sensor yield a signal-to-noise ratio of ≈8 when noise reduction methods of spatial and temporal averaging are utilized.

Keywords: Label-free; small molecules; binding kinetics; imaging sensor; IRIS; affinity measurements; multiplexed screening; mycotoxins;