Improving capture efficiency of human cancer cell derived exosomes with nanostructured metal organic framework functionalized beads

Affibody Functionalized Beads for the Highly Sensitive Detection of Cancer Cell-Derived Exosomes

“…The minimum number of microbeads needed to achieve a detectable fluorescence signal was found to be 50,000. The optimal concentration of antibody for the complete saturation of 10 µm polystyrene beads was calculated from our previous study [ 34 ]; 1.5 µg antibody was needed for the saturation of 1 mg of 10 µm polystyrene microbeads, which contained 1.83 × 10 6 microbeads according to the manufacturer’s datasheet (Bangs Laboratories Inc.). To compare the capturing efficiency of exosomes for the Affibody and the antibody probes, 10-fold molar excess of affibody (1.5 µg, ~0.1 nmole) compared to antibody (1.5 µg, ~0.01 nmole) is used to saturate the same amount of microbeads, as Affibody (13.9 kDa, mostly in dimeric form) is around 10 times smaller than antibody (150 kDa).…”

“…The amount of fluorescence detector including both anti-EGFR-FITC Affibody and anti-EGFR-Alexa Fluor 488 was adjusted to maximize the signal-to-noise ratio performance when tested with a flow cytometer. The maximum number of exosomes captured per 10 µm bead was calculated based upon the average diameter of the exosome at 150 nm using an equation described in our previous study [ 34 ]. In brief, a 10 μm polystyrene bead could attach to a maximum of 18,470 exosomes, as in our flow cytometry experiment, we used 50,000 beads for each run, a sample with 9.2 × 10 8 exosomes would saturate the beads.…”

“…Media containing released EVs was collected and subjected to multiple centrifugation and ultracentrifuge steps, as was comprehensively described in our previous work [ 34 ]. In brief, the cells, dead cells, and cell debris were removed using a three-step centrifuge including a 300× g for 10 min and 2000× g for 10 min, followed by 10,000× g (R18A rotor, HITACHI CR22N, Minato, Tokyo 108-6020 Japan) for 30 min.…”

“…The dehydration process was performed using 50%, 60%, 70%, 80%, 90%, and 100% ethanol for 20 min after the addition of hexamethyldisilazane (HDMS, Sigma, Darmstadt, Germany) at a ratio of 1:2 with 100% ethanol into fixed samples for 20 min. This was followed by critical point drying as described in our previous paper [ 34 ]. The fixed samples were sputter-coated with a 20 nm Au/Pd coating in a vacuum.…”

“…After two times washing with MES buffer (pH = 5.7), 1 mg of functionalized polystyrene microbeads were co-incubated separately with 1.5 µg of anti-human EGFR Affibody (10.1886.01.0001, Sweden) and anti-human EGFR antibody (352,902, BioLegend, USA) dissolved in 250 µL of PBS buffer (pH = 7.4) and incubated at room temperature for 4 h with constant mixing. The optimum time of Affibody/antibody incubation with the functionalized beads and the concentration of Affibody/antibody needed for the saturation of beads, and consequently achieving the signal in flow cytometry, was characterized in our previous study [ 34 ]. In the final step, washed beads were resuspended in 1 mL of quenching solution containing 40 mM glycine and 0.05–1% ( w / v ) BSA.…”