Automatic spot preparation and image processing of paper microzone-based assays for analysis of bioactive compounds in plant extracts

•The colorimetric assay on a paper microzone plates (PμZPs) was developed.•We demonstrate that PμZPs can be prepared in large numbers by using robotic spotter.•Automatic image processing software has been developed.•The test is a green analytical chemistry assay. The colorimetric determination of the concentration of phytochemicals in plant extract samples using a spotting automatic system, mobile phone camera and a computer with developed software for quantification is described. Method automation was achieved by using a robotic system for spotting. The instrument was set to disperse the appropriate aliquots of the reagents and sample on a Whatman paper sheet. Spots were photographed and analysed by ImageJ software or by applying the developed MatLab based algorithm. The developed assay was found to be effective, with a linear response at the concentration range of 0.03–0.25g/L for polyphenols. The detection limit of the proposed method is sub 0.03g/L. The paper microzone-based assays for flavonoids and amino acids/peptides were also developed and evaluated as applicable. Comparing the results with conventional PμZP methods demonstrates that both methods yield similar results. At the same time, the proposed method has an attractive advantage in analysis time and repeatability/reproducibility.