Probing phosphatase activity using redox active nanoparticles: A novel colorimetric approach for the detection of enzyme activity

• A rapid single step colorimetric assay for alkaline phosphatase (ALP) activity is demonstrated.
• The assay is based on the color change of nanoceria particles after interaction with ALP generated products.
• The assay allows detection of hydrolytic products of more stable substrates that could previously not be used in ALP assays.
• The method is easy-to-use, robust and cost effective and can be easily integrated in affinity bioassays.

A new colorimetric assay for the detection of alkaline phosphatase (ALP) activity is reported based on the surface reactivity and optical properties of redox active nanoparticles of cerium oxide, or nanoceria. The method takes advantage of nanoceria color changes after interaction with products of the ALP catalyzed reaction, resulting in charge transfer complexes with very strong absorption characteristics. The developed assay is easy-to-use, robust and cost effective and does not involve labeled reagents, secondary enzymes or soluble dyes. Hydrolytic products of more stable substrates (catechol monophosphate, ascorbic 2-phosphate and hydroquinone diphosphate) that could previously not be used in ALP assays can be conveniently colorimetrically detected with this assay. A detection limit of 0.04 U/L ALP with a linear range up to 2 U/L was obtained with ascorbic 2-phosphate substrate. The proposed assay can eliminate multistep procedures and minimize problems associated with the poor stability of substrates and enzyme labels of conventional ALP assays. The assay has been adapted to a paper platform and has demonstrated functionality for ALP detection in human serum. This sensing concept can find wide applications as a general approach for improving sensitivity and simplifying detection schemes of colorimetric bioassays, e.g. enzyme, gene, immuno and aptamer assays and related affinity sensing methods.