# Development of Three Alternative Strategies for the Binding of Cells to Functionalized DeepTipTM AFM Probes

## TL;DR

This paper presents three new methods to bind cells to AFM probes, improving the study of cell-material interactions for biohybrid material design.

## Contribution

Three versatile and robust cell-binding strategies for AFM probes are developed and compared for non-adherent and adherent cells.

## Key findings

- The streptavidin/biotin, sulfhydryl, and click chemistry methods enable cell binding to AFM probes.
- Specific antibody binding showed the strongest interaction, followed by concanavalin A and azide-derivatized proteins.
- The methods work for both CD4+ T-lymphocytes and mesenchymal stem cells.

## Abstract

The efficient design of biohybrid materials requires controlling the interaction between the cell and the material for a wide range of possible combinations. Single cell force spectroscopy (SCFS), an atomic force microscopy (AFM) experimental procedure based on the binding of an individual cell to an AFM cantilever and the assessment of the adhesion force between the cell and a target substrate, represents one of the most promising alternatives to characterize the interaction between cell and material. However, SCFS relies on the efficient binding of the cell to the AFM in order to avoid drawbacks, such as the detachment of the cell. In this work, three different versatile and robust procedures are presented that allow for the binding of either non-adherent (CD4+ T-lymphocytes) or adherent (mesenchymal stem cells, MSC) cells to the AFM probe. The three crosslinking strategies comprise (1) the streptavidin/biotin system, (2) sulfhydryl group-based crosslinkers, and (3) “click” (bioorthogonal) chemistry. Additionally, three decoration schemes of the functionalized AFM probes are explored: a specific antibody, concanavalin A, and direct binding of the cell through azide-derivatized membrane proteins. Differences are observed between these alternatives and it is found that the strength of the interaction (in decreasing order) is as follows: specific antibody, concanavalin A, and binding through azide-derivatized proteins.