Birgitta Knudsen and colleagues exploited the fact that the enzymes investigated (Flp, Cre and topoisomerase I) can transform a single-stranded linear DNA molecule into a closed circle shape. This circle than then be "magnified" using a technique called "rolling circle amplification" and the resulting product visualized at the single molecule level using fluorescent probes.

Flp and Cre are currently used to integrate genes and control gene expression in animal models systems and cell lines, explains Knudsen. Scientists are also investigating these enzymes for use as tools in gene therapy – in particular, for integrating therapeutic genes in the human genome.

"Our multiplexed enzyme detection system holds great promise for developing enzyme activity chips in the future," Knudsen told nanotechweb.org. "These chips could allow us to detect multiple clinical targets in small tissue or cell samples at the same time."

Detecting topoisomerase I could be important for cancer prognosis. This is because cells with high levels of topoisomerase activity would be very sensitive to cancer-treating drugs, like camptothecins, that target this enzyme.

The team, which includes researchers from Aarhus University Hospital and the departments of molecular biology and pathology at Aarhus University, is now setting up a single-molecule multiplexed detection system that can sense enzymes from various parasites and disease-causing bacteria. "We expect to present a modified version of our current set-up that offers highly sensitive, quick, easy and inexpensive diagnosis of various infectious diseases, such as malaria and tuberculosis, in the near future," said Knudsen.

The work was reported in ACS Nano.