Fluorescamine-based assay

Fluorescamine-based assay

1. A series of silane-PEG_5k-NH₂ PB buffered solutions with known concentrations (0.125, 0.25, 0.5, 0.75, 1, 1.25 μM) are prepared.
2. Fluorescamine solution (2 μM) is added into silane-PEG_5k-NH₂ PB buffered solution (3 ml).
3. The mixture reacts in the dark at room temperature for 15 min.
4. Fluorescent intensity is recorded at 480 nm (λ_ex: 390 nm) and a calibration curve is obtained by plotting the fluorescent intensity as a function of the concentration of silane-PEG_5k-NH₂.
5. For quantification of surface PEG density, the unreacted silane-PEG_5k-NH₂ is first calculated. After the PEGylation of MSN, the PEGylated MSNs are harvested by centrifugation, and the supernatant solution is collected.
6. Unreacted silane-PEG_5k-NH₂ is detected using the same procedure by replacing the silane-PEG_5k-NH₂ PB buffered solution with supernatant solution, which is also diluted into 3 ml of PB buffer.
7. The surface PEG density of MSN (chains/nm2) can be calculated according to Equation 1 and 2.

Solutions

1. Phosphate buffer (PB buffer, pH = 10).

2. Fluorescamine solution (2 μM) in acetone.

   
   

The calibration curve for silane-PEG_5k-NH₂ of fluorescamine-based assay at pH =10, showing a direct linear correlation between the fluorescence intensity and the concentration of silane-PEG_5k-NH₂. (R² = 0.9897)

Notes

1. The method of quantification of surface PEG density by fluorescamine-based assay is according to a previous report (1).
2. The analyses require to concentrate the dilute reaction supernatant.
3. The absolute PEG density is presented in the number of PEG molecules packed per square nanometer of apparent surface area (not BET surface area since the critical parameter that affects the conformation should be the PEG density based on the projected area that ignored the influence of pores).

References

1. X. H. Xia, M. X. Yang, Y. C. Wang, Y. Q. Zheng, Q. G. Li, J. Y. Chen, Y. N. Xia, Quantifying the Coverage Density of Poly(ethylene glycol) Chains on the Surface of Gold Nanostructures. ACS nano 6, 512-522 (2012).

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