Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples

We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we “count” the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity–based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester–hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.