Automation of the Bradford Method Using Digital Image Analysis for Estimating Proteins in Milk by Spot Test

Food analysis is essential for ensuring food quality and safety, with protein content being one of the most relevant compositional parameters from both nutritional and technological perspectives. Although the Kjeldahl method is the reference technique for protein quantitation, reversed-phase high-performance liquid chromatography (RP-HPLC) provides high specificity for individual proteins and was used in this study as an independent comparative method. Unlike nitrogen-based methods, RP-HPLC enables direct quantitation of defined protein standards. This work proposes the determination of protein concentration using the Bradford assay combined with digital image analysis (DIA), replacing conventional spectrophotometric detection. A glass slide was evaluated as a sample support, requiring only 14 µL of sample, substantially less than conventional microplates. Images were acquired with a smartphone, and the complete analytical workflow, from image acquisition to protein concentration calculation, was semi-automated using MATLAB software. The method was evaluated using bovine serum albumin (BSA) and casein standards and applied to food samples. Protein concentrations obtained by the Bradford-DIA method showed good agreement with RP-HPLC, with errors ranging from 1.7% to 17% for standards and from 1.1% to 23% compared with labelled protein values. The proposed approach provides a rapid, low-cost, and miniaturised alternative for protein determination.