Immobilization of living micro-organisms on predefined areas of substrates is a prerequisite for their characterizations by atomic force microscopy (AFM) in culture media. It remains challenging since micro-organisms should not be denatured but attached strongly enough to be scanned with an AFM tip, in a liquid phase. In this work, a novel approach is proposed to electrostatically assemble biological objects of interest on 2 nm thick polyethylenimine (PEI) patterns fabricated by nanoxerography. This nanoxerography process involves electrostatic trapping of PEI chains on negatively charged patterns written on electret thin films by AFM or electrical microcontact printing. The capability of this approach is demonstrated using a common biological system, Pseudomonas aeruginosa bacteria. These negatively charged bacteria are selectively assembled on large scale arrays of PEI patterns. In contrast to other PEI continuous films commonly used for cell anchoring, these ultrathin PEI patterns strongly attached on the surface do not cause any denaturation of the assembled Pseudomonas aeruginosa bacteria. AFM characterizations of large populations of individual living bacteria in culture media can thus be easily performed through this approach, providing the opportunity to perform representative statistical data analysis. Interestingly, this process may be extended to any negatively charged micro-organism in solution.

A new strategy for AFM (atomic force microscopy) tip functionalization based on the use of aldehyde-phosphorus dendrimers for the immobilization of biomolecules such as proteins (e.g. antibodies) is presented. Firstly functionalized with amino groups, the tips are reacted with dendrimers leading to dendrimer-activated tips (so-called dendritips). Free aldehyde functions on the dendrimer are therefore available to react with amino-functions present on every protein and many biomolecules. Using biofunctionalized-dendritip, single molecule force interactions between glutathione-S-transferase (GST) and its cognate antibody immobilized on dendritips (67±11pN for single interaction) were probed by AFM spectroscopy. The specificity of our measurements was demonstrated by performing blocking tests resulting in the loss of interactions.

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.