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

Abstract

Investigating the binding interaction of small molecules to large ligands is a compelling task for the field of drug development, as well as agro-biotechnology, since a common trait of drugs and toxins is often a low molecular weight (MW). Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly multiplexed biosensor, to perform small-molecule screening. In this work, characterization of small molecules binding to immobilized probes in a microarray format is demonstrated, with a limit of detection of 1 pg/mm 2 in mass density. First, as a proof of concept to show the impact of spatial and temporal averaging on the system noise, detection of biotin (MW = 244.3 Da) binding to a streptavidin-functionalized chip is performed and the parameters are tuned to achieve maximum signal-to-noise ratio (SNR ≈ 34). The optimized system is then applied to the screening of a 20-multiplexed antibody chip against fumonisin B1 (MW = 721.8 Da), a mycotoxin found in cereal grains. The simultaneously recorded binding curves yield an SNR ≈ 8. Five out of twenty antibodies are also screened against the toxin in a lateral flow assay, obtaining consistent results. With the demonstrated noise characteristics, further sensitivity improvements are expected with the advancement of camera sensor technology.

Bulk-Effect-Free Method for Binding Kinetic Measurements Enabling Small-Molecule Affinity Characterization

“…The black horizontal line corresponds to the binding signal for biotin obtained with the described bulk-effect-minimization method, consistent with theoretically expected values in Chiodi, et al. 11 The refractive index difference between the analyte and buffer solution used in this study created a bulk-effect signal that is about 2 times the size of the biotin-binding signal measured here with the IRIS technique. The same refractive index difference in SPR (as simulated using a Kretcshmann configuration) would result in a bulk-effect signal that is 30 times as large as the same biotin-binding signal measured in SPR.…”

“…The power of the LEDs was adjusted using adjustable LED drivers (Thorlabs LEDD1B) based on the bulk-effect signal captured in Figure 6 , and the bulk-effect minimization was confirmed with another run of 1× PBS, 1% DMSO in 1× PBS, and 1× PBS. The resulting signal is shown in Figure 8 b showing a clear binding step as expected of biotin and confirmed in similar studies, 11 and results were consistent across many trials of this experiment. Due to the high affinity of biotin to streptavidin, the binding interaction is mass-transport-limited (further discussed in Supporting Information ) and, therefore, the data serve only as a proof of concept and do not show actual kinetic information.…”

“…Biotin was flowed across the streptavidin spots at 1 μM concentration for 10 min, at a flow rate of 200 μL/min. Temporal and spatial averaging were implemented to minimize system noise; 11 averaging 100 frames and 60 spots, each spot ∼270 μm in diameter. The resulting signal is shown in Figure 7 .…”

“…IRIS (Interferometric Reflectance Imaging Sensor), which offers a sensitivity comparable to SPR and better than SPR imaging, 11 is not based on evanescent fields and does not suffer from this limitation. While other technologies collect the signal and background together in each measurement, IRIS produces spectral reflectivity information that allows for extracting surface binding and the solution refractive index as separate quantities.…”

Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics

“…Accumulation of biological mass on the sensor surface increases the effective thickness from the top surface to the reference Si surface and alters the reflectance. In IRIS measurements, the molecular weight of the analyte can vary from small molecules [14] to macroparticles such as viruses [15]. The reflectance coming from the surface assumes the form:…”