Nanoparticle-based amplification for sensitive detection of β-galactosidase activity in fruits

Development of fast and sensitive assays for enzyme activity detection has received a great deal of attention because of the wide spread applications in measurements of numerous clinical, food and environmental processes. Herein, a novel amplification approach to enhance the sensitivity of colorimetric assays for detection of β-galactosidase (β-Gal) activity is proposed. β-Gal detection is important in biomedical applications and in food industry, where it is associated with the ripening process of fruits. The method is based on the use of multivalent cerium oxide nanoparticles (CeNPs) which catalyze the oxidation of 4-aminophenol (4-AP) produced in the hydrolysis process of the 4-aminophenyl-β-d-galactopyranoside substrate (4-APG) by β-Gal, thus enhancing detection sensitivity of β-Gal in the visible range. The developed assay is highly sensitive and easy to use. Using the optimized procedure, a limit of detection of 0.06 mU/mL was obtained with a linearity range up to 2.0 mU/mL. The feasibility of the method was demonstrated for detection of β-Gal activity in fruits and the results were compared with the conventional assay, providing over a 30-fold amplification as compared to a commercially available β-Gal protocol. The advantage of the presented assay is its biocatalytic event amplified by a secondary reaction, which enables much more sensitive detection of the enzymatic product. The sensing platform can be applied broadly to a variety of applications that rely on β-Gal activity measurements.