Comparison Of Various Quantification Methods For Protein Adsorption

The extent of protein adsorption is an important consideration for the biocompatibility of biomaterials. Accurate quantification of non-specific protein adsorption on biomaterial surfaces is essential for evaluation of their antifouling properties. In order to obtain the nanogram range of protein adsorption on biomaterial surface or compare subtle differences in protein adsorption between various surfaces, high requirement are put forward to the accuracy of quantification methods for protein adsorption. Various experimental methods, such as radiolabeling, surface plasma resonance(SPR) and quartz crystal micro balances(QCM) can be used to determine the quantity of protein adsorbed, but the results usually differ. In addition, the majority of quantification methods obtain physical signal instead of the adsorbed mass. The applicability of equations or models to convert physical signals to the adsorbed mass may also influence the final results of detection, which will lead to erroneous evaluation on the bio-functional properties. Therefore, it is necessary to compare the difference among various methods to discuss the applicability of quantification methods for protein adsorption on typical model surface systematically.In the present thesis, SPR and QCM, widely used quantification methods were compared to discuss applicability of them for protein adsorption on typical model surface systematically.1. Self-assembled monolayers(SAMs) were used to prepare a series of model gold surfaces varying systematically in water wettability, from hydrophilic to hydrophobic. Three commonly used methods, namely, surface plasmon resonance(SPR), quartz crystal microbalance with dissipation(QCM-D) and 125I-radiolabeling were employed to quantify fibrinogen(Fg) adsorption on these surfaces. This approach allows a direct comparison of the mass of Fg adsorbed using these three techniques. The results from all three methods showed that protein adsorption increases with increasing surface hydrophobicity. The increase in the mass of Fg adsorbed with increasing surface hydrophobicity in the SPR data was parallel to that from 125I-radiolabeling, but the absolute values were different and there does not seem to be a “universally congruent” Stenberg relationship(1 RU·mm2/pg) between the two methods for surfaces with varying wettability. For QCM-D, the variation in protein adsorption with wettability was different from that for SPR and radiolabeling. On the more hydrophobic surfaces, QCM-D gave an adsorbed mass much higher than from the two other methods, possibly because QCM-D measures both the adsorbed Fg and its associated water. However, on the more hydrophilic surfaces, the adsorbed mass from QCM-D was slightly greater than that from SPR, and both were smaller than from 125I-radiolabeling; this was true no matter whether the Sauerbrey equation or the Voigt model was used to convert QCM-D data to adsorbed mass. Furthermore, the trend of QCM-D was quiet different to that from SPR and radiolabeling atrributed to the viarous water relative content of the adsorbed proein layer because of the difference in surface properties.2. Accurate quantification of non-specific protein adsorption on biomaterial surfaces is essential for evaluation of their antifouling properties. QCM-D is one of the most widely practiced methods for the measurement of protein adsorption. Although it is highly sensitive, it does have performance limitations. In the case of polymer brush surfaces, factors such as the thickness and viscoelastic properties of the brush may bring such limitations into play. Three types of antifouling biomaterials were used to explore the applicability of QCM-D for the evaluation of the protein resistance of hydrophilic polymer brush surfaces. Adsorption was also measured by surface plasmon resonance as a reference standard. In order to discuss the factors which should be paid attention to when using QCM to measure the protein adsorption on polymer brush surface, we focused on the situation apparent adsorption can be detected by SPR, while QCM measurement was nearly zero. It is shown that, for thicker or rigid layers and where adsorption is primarily on top of the brush as expected for larger proteins like fibrinogen, the adsorbed protein may be not detected. The decrease in the accuracy may be attributed to the influence of thickness and viscoelastic properties of brush and the extent of dissipation due to polymer brush layer.