Plasma and serum samples (BioIVT, Hicksville NY) were diluted 1:5 in a dilution buffer composed of TE buffer (10 mM Tris, 1 mM disodium EDTA, 150 mM KCl) with 0.05% CHAPS. NP powder was reconstituted by sonicating for 10 min in DI water followed by vortexing for 2–3 sec. To form the protein corona, 100 µL of NP suspension (SP-003, 5 mg/ml; SP-007, 2.5 mg/ml; SP-011, 10 mg/ml) was mixed with 100 µL of diluted biological samples in microtiter plates. The plates were sealed and incubated at 37 °C for 1 h with shaking at 300 rpm. After incubation, the plate was placed on top of a magnetic collection device for 5 min to draw down the NPs. Unbound proteins in supernatant were pipetted out. The protein corona was further washed with 200 µL of dilution buffer three times with magnetic separation.
For the 10-NP screen, the five additional assay conditions evaluated were identical to those described above, with one of the following exceptions. First, a low concentration of NPs was evaluated that was 50% the original concentration (ranging from 2.5–15 mg/ml for each NP, depending on expected peptide yield). For the second and third assay variations, both low and high NP concentrations were run using an undiluted, neat plasma rather than diluting the plasma in buffer. For the fourth and fifth assay variations, both low and high NP concentrations were run using a pH 5 citrate buffer for both dilution and rinse.
To digest the proteins bound onto NPs, a trypsin digestion kit (iST 96×, PreOmics, Germany) was used according to protocols provided. Briefly, 50 µL of Lyse buffer was added to each well and heated at 95 °C for 10 min with agitation. After cooling the plates to room temperature, trypsin digestion buffer was added, and the plate incubated at 37 °C for 3 h with shaking. The digestion process was stopped with a stop buffer. The supernatant was separated from the NPs by a magnetic collector and further cleaned up by a peptide cleanup cartridge included in the kit. The peptide was eluted with 75 µL of elution buffer twice and combined. Peptide concentration was measured by a quantitative colorimetric peptide assay kit from Thermo Fisher Scientific (Waltham, MA).
For the 10-NP screen, the five additional assay conditions evaluated were identical to those described above, with one of the following exceptions. First, a low concentration of NPs was evaluated that was 50% the original concentration (ranging from 2.5–15 mg/ml for each NP, depending on expected peptide yield). For the second and third assay variations, both low and high NP concentrations were run using an undiluted, neat plasma rather than diluting the plasma in buffer. For the fourth and fifth assay variations, both low and high NP concentrations were run using a pH 5 citrate buffer for both dilution and rinse.
To digest the proteins bound onto NPs, a trypsin digestion kit (iST 96×, PreOmics, Germany) was used according to protocols provided. Briefly, 50 µL of Lyse buffer was added to each well and heated at 95 °C for 10 min with agitation. After cooling the plates to room temperature, trypsin digestion buffer was added, and the plate incubated at 37 °C for 3 h with shaking. The digestion process was stopped with a stop buffer. The supernatant was separated from the NPs by a magnetic collector and further cleaned up by a peptide cleanup cartridge included in the kit. The peptide was eluted with 75 µL of elution buffer twice and combined. Peptide concentration was measured by a quantitative colorimetric peptide assay kit from Thermo Fisher Scientific (Waltham, MA).
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