2.4. Assessment of Surface Protein Levels

The concentration of residual protein on healing abutment was determined using a Micro BCA assay (Pierce Biotechnology, Rockford, Illinois).

The MicroBCA assay is based on the colorimetric change due to bicinchonic acid (BCA) chelating the copper ion (Cu¹⁺) resulting from the reduction of copper ion (Cu²⁺) in the presence of protein and in an alkaline environment [35,36]. The colorimetric change goes from green to purple as the amount of protein in solution increases (Figure 3).

Colorimetric variation based on reduction of Cu²⁺ in the presence of protein and in an alkaline environment and subsequent chelation reaction between BCA and Cu¹⁺ resulting from the first reaction.

The color change is detected in the visible light spectrum using a spectrophotometer with an optical density (OD) of 562 nm.

In spectrophotometry, OD is the basis of quantitative chemical analysis and is linearly related to the concentration of the sample.

The steps applied to the HAs of each group were as follows:

Sterile tube containing a HA in BCA solution.

Sterile tubes free of HAs and containing only BCS.

Aliquots of 150 µL were added to 96-well plates in triplicate. All tubes retain the green coloring as the reaction to test for surface proteins has not yet been performed. The three sterile tubes free of HAs and containing only BCS are placed apart on the left to distinguish them from the sample under examination.

BSA standard curve.