A sensitive smartphone-based method for measuring phosphate in biological samples and ATPase activities

The quantification of orthophosphate is essential for applications like water quality assessment, soil fertility analysis, metabolic monitoring and enzyme activity evaluation. Chemical quantification methods include the reaction between orthophosphate and molybdate under acidic conditions to form 12-molybdophosphoric acid units, which auto-assembles forming nanometer size particles. The adsorption of malachite green to these nanoparticles allows their spectrophotometric detection constituting one of the most widely used methods to quantify phosphate. Here we introduce a straightforward procedure for precise quantification of the 12-molybdophosphoric-acid-malachite green complex. This technique utilizes a digital image of a 96-well plate, captured with a smartphone, followed by color intensity analysis using open-source image processing software. This approach provides a reliable, reproducible alternative to traditional spectrophotometric methods, offering a broad dynamic range for phosphate quantification while maintaining versatility, high sensitivity and accuracy. Applicability of this method was validated by quantifying phosphate in river water, soil, human serum, as well as by measuring the ATPase activity of a thermophilic protein.