Abstract

Cell adhesion constitutes a cornerstone of fundamental biological processes, including tissue morphogenesis, immune surveillance and tumour progression, yet its initial stages remain poorly characterised due to methodological constraints. Conventional techniques either lack real-time capability, rely on indirect readouts or are limited in throughput. The Single Cell Adhesion Dot Array (SCADA) platform, previously developed in our group, enables the evaluation of cell adhesion to protein matrices using simple optical microscopy, thereby affording real time measurements with single cell resolution. Here, our group advances the SCADA platform by implementing photopatterning assisted by UV-light (254 nm) for its fabrication. This strategy enables rapid, reproducible and cost-effective fabrication of protein micropatterns eliminating the need for microcontact printing previously required. Building on this improved fabrication method, we applied SCADA to explore biologically relevant questions. The primary objectives of this study were to investigate the affinity of HeLa cells toward specific adhesion proteins and, simultaneously, to evaluate the effectiveness of SCADA in conducting cellular affinity studies, obtaining comparative and statistical analysis of the results. To this end, SCADA platforms were utilised to assess the adhesion of HeLa cells to individual extracellular matrix components: fibronectin (FN), laminin (LAM), collagen IV (COL IV); anti-adhesive substrates bovine serum albumin (BSA); molecular mixtures and Matrigel (MG). HeLa cells exhibited the highest affinity for the protein mixture FN:LAM:COL IV (ratio 1:1:1), followed by the mixture LAM:COL IV (ratio 1:1) and MG, whereas BSA showed minimal affinity. These findings confirm that SCADA reliably discriminates between pro- and anti- adhesive environments and reveals synergistic effects among extracellular matrix components on cellular adhesion, thereby establishing it as a robust platform for quantitative analysis of early adhesion events with single-cell resolution, high reproducibility and reduced reagent consumption.