Colorimetric Method for the Determination of Proteins Using Immobilized Microbial Protease and a Smartphone Camera

Abstract

The determination of proteins is very important in industry, medicine, analytical chemistry, and biotechnology. A novel colorimetric method using magnetic microparticles as a carrier of immobilized microbial protease on surface was developed and smartphone camera was employed for detection. Folin and Ciocalteu’s phenol reagent was selected as the chromogen and casein from bovine milk was used as the substrate. Casein was cleaved by immobilized enzyme to tyrosine, which together with chromogen spontaneously generated a blue color measurable by a spectrophotometer or photographic detection by a smartphone camera. The photographs were processed by a computer and selected points were transformed into RGB numeric values. A reference spectrophotometric method provided results that provide a Michaelis–Menten dependence with the Michaelis–Menten constant equal to 0.11 mmol/l and a limit of detection equal to 41 µg/ml. The novel method using spectrophotometric detection shows the same Michaelis–Menten dependence with a Michaelis–Menten constant equal to 0.11 mmol/l with a limit of detection of 4.27 µg/ml. The RGB method using a smartphone for the determination of proteins was demonstrated to be an alternative to the standard spectrophotometer. The limit of detection was 242 µg/ml, which is sufficient for protein determination in real samples. Easy processing, good analytical results, and low demands on equipment make this method a suitable tool for a wide range of applications in industry, analytical chemistry, medicine, biotechnology, and biosensors.