Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

Abstract

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

Enhanced protein adsorption and cellular adhesion using transparent titanate nanotube thin films made by a simple and inexpensive room temperature process: Application to optical biochips

“…Waveguide sensors were recently exploited in several novel directions; such as the on-line monitoring of oriented protein adsorption [41], extracellular vesicles [42] and human primary immune [16] and cancer [17] cell adhesion in a completely labelfree manner. Here we report on the preparation of TNT coatings on surface-sensitive optical waveguide-based biosensors for quantitative adsorption and adhesion studies.…”

Sample handling in surface sensitive chemical and biological sensing: A practical review of basic fluidics and analyte transport

“…Surface-sensitive biosensors enable various activities of adherent cells to be monitored with high sensitivity and high temporal resolution [14,30,67,68]. Cell-based studies performed on labelfree biosensors can basically be classified as adhesion, signaling, or mechanotransduction assays, each of which requires different sample handling strategies.…”

“…For example, adsorption and desorption rates at model surfaces, or kinetic rates in affinity binding can be determined [40,41], which contribute to a better understanding of the mechanism underlying the investigated surface process. Kinetic analysis of experimental data is 9 furthermore the key to a detailed characterization of cell adhesion [14], including the separation of the governing cellular processes from less relevant ones, and the quantification of the differences arising from different cell types, substrata, media and other environmental factors [14,42,43,44,45,46]. However, kinetic data is often misinterpreted.…”

“…Biosensing technologies enabling label-free detection at a solid-liquid interface include surface plasmon resonance (SPR) (and SPR imaging, SPRi) [3,4,5], quartz crystal microbalance (QCM) [6], dual polarization interferometry (DPI) [7,8], grating-coupled interferometry (GCI) [9,10,11,12], the high-throughput compatible resonant waveguide grating biosensing [13,14,15], and optical waveguide lightmode spectroscopy (OWLS) [8,16,17,18]. OWLS instruments are currently among the most sensitive commercial label-free biosensors which enable the monitoring of various processes accompanied by refractive index changes 100-200 nm above a sensor surface .…”

Bulk and surface sensitivity of a resonant waveguide grating imager

“…[9][10][11] To overcome throughput limitation posed by SPR and OWLS, RWG employs nano-grating waveguide biosensors embedded in the Society for Biomolecular Screening compatible microplates, e.g., 96-well, 384-well, and 1536-well microplates. 9,10 The RWG readers, in particular the recently developed imager, 5,12 permit high-throughput and highly sensitive drug profiling and screening, and in-depth analysis of receptor signaling pathways in living cells. 10 The RWG imager uses a tunable light source to interrogate simultaneously all biosensors in the microplate with a temporal resolution of 3 s and a spatial resolution of $80 lm.…”