Table 1.

Conspiring Factors that Influence Measurement of Protein Adsorption

Experimental Variable	Factor	Impact	Comment
Protein	Molecular weight	Size matters	Adsorbent capacity is a function of MW [31, 33, 34, 101] and larger proteins can occupy multiple layers in the adsorbed state [31, 101].
	Solution concentration	Kinetics and subsequent protein- surface interactions	Solution concentration should be compared to that which saturates adsorbent surface which is seldom reported in literature. Adsorption from concentrated solution is rapid compared to dilute solution. Protein denaturation is relatively rapid on sub-saturated solution with decreasing rate of denaturation with increasing solution concentration [38].
	Source	Molecular shape/volume	Blood proteins are oblate spheroids in solution [102-107]. Other proteins (such as environmental or food proteins) may not share this commonality. Degree of glycosylation or de-lipidization.
	Number of proteins in solution	Adsorption competition	Adsorption from binary solution is vastly more complex than adsorption from purified protein solutions [13, 35].
Adsorbent	Hydrophilic v. hydrophobic	Terminology	Little agreement among investigators on terminology [71]. Broad categorization of surfaces can ignore important differences in surface chemistry.
	Surface characterization	Chemistry matters	Surface functional groups have different Lewis acid/base strength that affects interaction with water and proteins [110]. Hydrophilic surface functionalities strong Lewis acid/base strength (electric field) can adsorb protein by ion-exchange not available to surface functionalities such as hydroxyl, carboxyl, ether, etc. [34].
	ADsorption v. ABsorption	On v. In	Protein entrapped IN the matrix of porous or water-swollen surfaces can appear to be adsorbed. Adsorption and absorption can be difficult to differentiate and can frequently occur in hydrophilic materials [17, 110].
Aqueous Phase	Media ionic strength	Electrostatic screening	Electrostatic interactions of proteins with adsorbents is shielded in high ionic strength media unless surface functional groups exhibit very high Lewis acid/base properties [34].
	Surface hydration	Role of water	High vacuum spectroscopies do not account for hydration reactions.
Protocol	Adsorbent rinsing	Perturbation of the interfacial region	“Dip-rinse-measure” protocol destroys integrity of the interphase [14] and removes loosely-bound protein that interacts with more strongly bound protein [96], underestimating total amount adsorbed [85, 218]. Efficiency of rinsing at interfacial dimensions is unclear and untested.
	Protein labeling	Experimental artifacts	Radio [85-93] and fluorescent [94-96] protein labels significantly affect protein structure and adsorption properties.
	Adsorption Isotherm	Scaling: moles v. weight concentration	Complete characterization of adsorption requires measurement of a full adsorption isotherm. One or a few arbitrarily-selected solution concentrations is usually an inadequate basis for general conclusions. Should adsorption be compared on a molar or mass basis?
	Gravimetry and Spectroscopy	Surface Sensitivity and Selectivity	Gravimetric methods measure the same mass? Evanescent wave methods must capture entire interphase depth and resolve bulk solution contribution.