Highly Multiplexed Label-Free Imaging Sensor for Accurate Quantification of Small-Molecule Binding Kinetics

Elisa Chiodi,Allison M Marn,Matthew T Geib,John Rejman,David Ankrapp,M Selim Ünlü,Fulya Ekiz Kanik

doi:10.3390/iecb2020-07032

Abstract

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. 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 1 pg/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~150 kDa ligands) against Fumonisin B1 (MW = 721.8 Da), 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.

Highlights

Small molecules are defined as chemical compounds with a molecular weight below 1 kDa [1]
The main reason for that is the kinetic capability of label-free sensors, which allows for the study of the real-time affinity behavior of molecules, without an invasive labeling process
The Interferometric Reflectance Imaging Sensor (IRIS) system is based upon detection of biomass accumulation through interferometric imaging, and the images are acquired with a CMOS camera (FLIR GS3-U3-51S5M-C)

Summary

Introduction

Small molecules are defined as chemical compounds with a molecular weight below 1 kDa [1] The relevance of these molecules in the field of drug development, as well as agronomy and food biotechnology, arises from the fact that most drugs and toxins belong to this category [2]. As a matter of fact, small-sized molecules have easier access to the cellular membrane, as well as being more recognized by membrane-bound antibodies and receptors. For this reason, research on small molecules has been ramping up in the most recent years; just in 2019, 73% of FDA-approved drugs belonged to this category, and 71% in 2018 [3,4]. The main reason for that is the kinetic capability of label-free sensors, which allows for the study of the real-time affinity behavior of molecules, without an invasive labeling process

Methods

Results

Conclusion