Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

Abstract

Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion.

Label‐Free Dynamic Mass Redistribution and Bio‐Impedance Methods for Drug Discovery

“…DMR, for example, is excellent for studying G protein signaling whether the signaling network is activated from surface receptors or intracellularly by interaction with effector proteins (Blättermann et al., ; Grundmann & Kostenis, ; Schrage et al., ; Schröder et al., ). However, it is also suited to detect cell responses mediated by tyrosine kinase signaling, ion channel modulation, or cell adhesion (Du et al., ; Sun et al., ; Zaytseva et al., ). Because DMR and CDS access the same phenotypic behavior, i.e., cell shape change, it is not surprising that CDS detects G protein signaling with comparable high accuracy and sensitivity to DMR (Grundmann et al., ; Hennen et al., ).…”

“…Because signal transduction translates into morphological alterations in the cellular structure, DMR and CDS are able to recognize signaling events by sensing whole‐cell responses at the level of cellular shape change. As neither DMR nor CDS‐based readouts are biased toward a specific signaling event, they are capable of detecting any cell activation irrespective of the investigated target as long as the signaling event translates into a cytoskeletal rearrangement (Du et al., ; Schröder et al., ; Sun et al., ; Tran & Fang, ; Zaytseva et al., ). Among the many possible drug targets, G protein–coupled receptors (GPCRs) have been investigated most extensively using label‐free methods.…”

Total Internal Reflection Fluorescence Quantification of Receptor Pharmacology

“…RWG-based TIRFM (rTIRFM) uses a subwavelength diffractive nanograting to couple light into, and travel along, a thin film dielectric transparent waveguide, so an evanescent wave is generated to excite near-field fluorescence ( Figure 1 c) [ 24 , 39 , 40 ]. The resonance effect occurs when the grating couples incident light to the specific modes of the waveguide.…”

“…Combining label-free with TIRFM results can offer confirmative insights into receptor signaling. For instance, combining RWG-enabled DMR profiling with TIRF imaging can be used to ascertain the impact of receptor signaling on cell adhesion [ 24 ]. Results showed that β 2 -AR agonists, but not its antagonists or partial agonists, were capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of HEK293-β 2 AR-GFP cells onto fibronectin-coated surfaces.…”

“…These cell phenotypic assays have gained increasing acceptance in both basic research [ 20 , 21 ] and early drug discovery processes [ 22 , 23 ]. Inspired by the success of these biosensors, we had hypothesized and demonstrated recently that TIRFM, in particular a microplate-compatible TIRF instrument, can be used to characterize receptor pharmacology [ 24 , 25 ].…”