TEER and Ion Selective Transwell-Integrated Sensors System for Caco-2 Cell Model

Abstract

Monitoring of ions in real-time directly in cell culture systems and in organ-on-a-chip platforms represents a significant investigation tool to understand ion regulation and distribution in the body and ions' involvement in biological mechanisms and specific pathologies. Innovative flexible sensors coupling electrochemical stripping analysis (square wave anodic stripping voltammetry, SWASV) with an ion selective membrane (ISM) were developed and integrated in Transwell™ cell culture systems to investigate the transport of zinc and copper ions across a human intestinal Caco-2 cell monolayer. The fabricated ion-selective sensors demonstrated good sensitivity (1 × 10^-11 M ion concentration) and low detection limits, consistent with pathophysiological cellular concentration ranges. A non-invasive electrochemical impedance spectroscopy (EIS) analysis, in situ, across a selected spectrum of frequencies (10-10⁵ Hz), and an equivalent circuit fitting were employed to obtain useful electrical parameters for cellular barrier integrity monitoring. Transepithelial electrical resistance (TEER) data and immunofluorescent images were used to validate the intestinal epithelial integrity and the permeability enhancer effect of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) treatment. The proposed devices represent a real prospective tool for monitoring cellular and molecular events and for studies on gut metabolism/permeability. They will enable a rapid integration of these sensors into gut-on-chip systems.

Keywords: Caco-2 monolayer; Transwell™ plate; anodic stripping voltammetry; electrochemical impedance spectroscopy (EIS); organ-on-a-chip (OoC); transepithelial electrical resistance (TEER).

Figure 1

Flexible ion selective electrodes. Photograph of the ion selective sensor on flexible Kapton substrate (70 × 4 mm² tapered tip) and close-up schematic image of the sensitive area structure of sensor. Two different working electrodes supported by a common Ag/AgCl reference allow detection of two different ions.

Figure 2

Photographs of sensor-integrated Transwell™ plates. TEER measurement set-up by circular transparent electrodes with FPC-to-DIP adapters (a). Ion-selective flexible sensors inserted in transwell plate, one for each compartment (top and bottom) of the well; each sensor can measure two different ions with a common thin film Ag/AgCl reference electrode (black electrode on flex between the two working electrodes) (b).

Figure 3

Impedance spectroscopy of 21 days cultivation of Caco-2 cells in transwell inserts, before and after EGTA treatment. Schematic representation of the applied equivalent circuit indicating the electrical parameters of the cell layer (a). Nyquist plots of EIS analysis of Caco-2 monolayer between 10 Hz and 100 kHz, before and after 2 h of EGTA exposure (b).

Figure 4

Illustration of the working mechanism of the polymeric ion selective sensors exposed at the established electrochemical protocol: membrane impregnation (a); pre-concentration step (b); anodic stripping voltammetry (c).

Figure 5

Electrochemical characterization of ion sensors. Square wave voltammograms of Cu²⁺ at different concentrations (1 × 10⁻¹¹, 1 × 10⁻¹⁰, 1 × 10⁻⁹, 1 × 10⁻⁷, 1 × 10⁻⁶ M) in PBS (a); plot of CuCl₂ peak currents vs. log Cu²⁺ concentrations (b). Square wave voltammograms of Zn²⁺ at different concentrations (1 × 10⁻¹¹, 1 × 10⁻¹⁰, 1 × 10⁻⁹, 1 × 10⁻⁷, 1 × 10⁻⁶ M) in PBS (c); plot of ZnCl₂ peak currents vs. log Zn²⁺ concentrations (d).

Figure 6

Square-wave anodic stripping voltammograms of Cu²⁺ and Zn²⁺ in Transwell™ cell culture systems. SWASV for copper detection in apical compartment (a) and basolateral side (b) of transwell Caco-2 culture system at different time points before (T0) and after 1 h, 2 h of EGTA treatment. SWASV for zinc detection in apical compartment (c) and basolateral side (d) of transwell Caco-2 culture system at different time points before and after EGTA exposure.

Figure 7

Fluorescent microscopy images of Caco-2 epithelial cell layer. Caco-2 monolayer, 21 days after seeding, before (a) and 2 h after (b) EGTA exposure. Cells were stained for E-cadherin (anti-E-cadherin Alexa Fluor 647, red) and nuclei (DAPI, blue).