Table 1.

Various Analytical Techniques Used to Evaluate Nanoparticles and Plasma Proteins Interactions

Analytic Method	Type of Evaluation and Comments
1D-polyacrylamide gel electrophoresis (PAGE) and 2D PAGE	Identification of proteins in corona on nanoparticles. The order of protein adsorbed onto the surface of nanoparticles can be determined. With the help of densitometry, the half-max protein adsorption and hill slope for protein. Highly laborious and not helpful/useful for quick kinetic adsorption evaluations.
BSA Quantification	Half-max protein adsorption. Hill slope for protein adsorption.
UV-Vis extinction spectroscopy	Half-max protein adsorption. Hill slope for protein adsorption. Apparent thickness, orientation and refractive index of the adsorbed proteins. Simultaneous protein adsorption measurements.
Gel permeation chromatography	Determination of molecular weight of plasma proteins bound to nanoparticles. Comparative evaluation of bound proteins on nanoparticles. Requires larger volumes of samples for analysis.
LC-MS/MS	Efficient separation of bound plasma proteins. Identification of protein/peptide sequence. Accurate molecular weight distribution. Requires larger volumes of samples for analysis.
Capillary electrophoresis	Analysis of protein–nanoparticle interaction with high degree of resolution. Efficient separation of plasma proteins and very small sample volumes are required for analysis. Determination of the fate of nanoparticles in vivo including delivery to tumors due to their interactions with specific plasma proteins. Stable nanoparticle-protein complexes and transient complexes were resolved by capillary zone electrophoresis and affinity capillary electrophoresis. This technique is also used in combination with other analytical methods to evaluate each type and percentage of various bound proteins on nanoparticles.
Atomic force microscope	Measure of change of indention before and after protein corona in situ modification. Determined the work of adhesion. Evaluation of surface free energy during protein corona formation. Force of adhesion involved in protein corona formation.
Fluorescence correlation spectroscopy	Used to quantitatively monitor protein binding or adsorption onto fluorescent nanoparticles. Correlation analysis of the fluorescence emission time traces yields a characteristic time scale of diffusion from the hydrodynamic radius. Size of the nanoparticles after protein deposition on their surfaces can be monitored. Nanomolar concentrations of the nanoparticles in microlitre-sized volumes required.
Particle Size and Distribution (TEM and DLS)	Plasma protein corona structure examination. Particle’s dispersibility before and after protein corona formation on nanoparticles. Particle’s zeta potential before and after protein corona formation on nanoparticles.