Direct functionalization of AFM probes (DNP-10 D tips, Bruker, Coventry, UK) was performed by rinsing the probes in deionised H2O x5, before immersion in HAPTES buffer (0.1 % v/v, pH 7.0; Sigma Aldrich, Gillingham, UK) for 7 mins at 25 °C. The sialianized probes were rinsed x5 in deionised H2O before immersion in glutaraldehyde (0.5 % w/v pH 7.0) for 7 mins at 25 °C. The probes were further rinsed in deionised H2O before being immersed in either 200 µg/ml mouse VU4H5 anti-MUC1 antibody or 200 µg/ml mouse anti-GAPDH antibody (Santa Cruz Biotechnology, Heidelberg, Germany) for 15 mins at 25 °C. The AFM probes were then rinsed x5 in Tris HCl (5 % w/v pH 7.0) before immersion and storage in Tris HCl. Linker functionalisation of AFM probes with L-selectin and associated negative control followed a two-step chemical procedure [28]. The AFM probes (DNP-10; Bruker-nano, Coventry, UK) were briefly washed in acetone for 5 mins before immersion in piranha solution (H2SO4:H2O2; 3:1; v/v) for 30 mins. The cantilevers were then incubated in 1 mL of APTES (0.1 % w/v, pH 7.2) for 10 mins to create an amino-terminated tip surface. The probes were then rinsed with PBS (x5) followed by rinsing with water (x5) before incubation in LC-SPDP (succinimidyl 6-(3(2-pyrifyldithio)propionamido)hexanoate) for 45 mins to obtain a reactive pyridyl-disulfide surface. The LC-SPDP functionalised probes were then rinsed again in PBS (x5) and water (x5). L-selectin (200 µg/mL; Randox Laboratories, County Antrim, UK) was modified by reaction with SATP (N-succinimidyl-S-acetylthiopropionate) for 30 mins in order to produce a free sulfhydryl group, before a series of purification steps through a dextran salting column (5.0k MWCO; Pierce, Thermo Fisher, UK) were performed. LC-SPDP functionalized probes were then incubated in the presence of thiolated-activated L-selectin, forming an L-selectin functionalised probe through a disulfide exchange reaction with SPDP-activated protein [55]. The AFM probes were functionalised fresh before every experiment and kept submerged in a solution of 20 mM sodium phosphate, 0.15 M NaCl and 10 mM EDTA at pH 7.2. ATPES was freshly made and adjusted to pH 7 before the functionalisation to prevent hydrolyses of ATPES which could interfere with sialinization reaction with the AFM tip.

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