Development of a rapid, high-sensitivity, low-cost fluorescence method for protein surface hydrophobicity determination using a Nanodrop fluorospectrometer

•A novel method for protein surface hydrophobicity determination was developed.•Linearity, precision, accuracy, and robustness were within the guideline’s values.•Selectivity, linear ranges, and sensitivity are dependent upon the target protein.•Limits of detection and quantification should be considered and preferably reported. A microvolumetric method for surface hydrophobicity (H0) determination of proteins using a Nanodrop fluorospectrometer was developed. This method reduces the protein and fluorophore quantities that are necessary for sample preparations and readings by two and three orders of magnitude, respectively, compared to conventional methods. In addition, readings can be obtained in just 2–6 s. Bovine serum albumin (BSA) and 1-anilino 8-naphthalene sulfonic acid (ANS) were used for the first optimization of appropriate fluorophore-protein conditions for H0 determination (20 μM ANS, 0.5–4 μM BSA, pH 5). Based on validation guidelines, the novel method shows linear behavior, good intraday precision, accuracy, and sensitivity. This method was robust against several factors, as determined by a Youden-Steiner test. Additional surface hydrophobicity determinations using several proteins demonstrate suitable method applicability. The present microvolumetric method provides a reliable technique to determine the H0 of proteins for pharmaceutical, biotechnological, and food applications.