2.5. Surface functionalization of the FO‐SPR probes with antibodies

As shown in Figure 2 and with the real‐time sensorgram in Figure 3, COOH SAM functionalized FO‐SPR probes were first immersed in a freshly made mixture of 0.4 M EDC and 0.1 M NHS, dissolved in 50 mM MES buffer at pH 6, for 15 min to activate COOH groups of the SAM. Next, EV specific capture monoclonal antibodies (anti‐CD9, anti‐CD63 and anti‐EpCAM) were initially diluted in 3 different buffers (50 mM MES at pH 6 and 10 mM sodium acetate at pH 5.2 or pH 5.6) at a concentration of 20 μg/ml and were immobilized on the FOR‐SPR probe with gentle shaking at 200 rpm, for 30 min. During this step, the antibodies were bound covalently to the activated COOH groups. Subsequently, the FO‐SPR probes were immersed sequentially in blocking buffers: 1 M ethanolamine at pH 8, Superblock and again in 1 M ethanolamine to minimize the non‐specific binding. Based on the obtained results (see further), 10 mM sodium acetate at pH 5.6 was selected to functionalize the FO‐SPR surface with anti‐CD9, anti‐CD63 and anti‐EpCAM capture antibodies in all the subsequent experiments.

A schematic overview of the EV detection bioassay. Carboxylic acid self‐assembling monolayer (COOH SAM) was first formed on the surface of a gold coated FO‐SPR probe. Capture antibodies (anti‐CD9, anti‐CD63 or anti‐EpCAM) were covalently immobilized on the surface through activated COOH groups. EVs were bound to the capture antibodies in a label‐free manner. The signal was then amplified by using biotinylated detection antibodies (^Banti‐CD9, ^Banti‐CD63 or ^Banti‐CD81) and AuNPs functionalized with anti‐biotin antibody. Stars on detection antibodies depict biotin

FO‐SPR sensorgram depicting each step of the bioassay, as detailed in Section 2.5