Water-soluble conjugated polymers for continuous and sensitive fluorescence assays for phosphatase and peptidase

A new method has been developed for continuous and real-time assays for enzymes using complexes of conjugated polymers/enzymatic substrates by electrostatic interactions, where the products are the substrate fragments and the free chains of conjugated polymers. The charged substrates and enzymes as a proof of concept are anionic adenosine triphosphate (ATP) and alkaline phosphatase (ALP), as well as cationic polyarginine peptide (Arg(6)) and trypsin. The charged water-soluble polyfluorenes containing 2,1,3-benzothiadiazole (BT) units demonstrate intramolecular energy transfer from the fluorene units to the BT sites when oppositely charged substrates (ATP or Arg6) are added, following by a shift in emission color from blue to green. When the substrates are cleaved into fragments, the relatively weak electrostatic interactions of substrate fragments with conjugated polymers keep their main chains separated and fluorescence resonance energy transfer (FRET) from the fluorene units to the BT sites is inefficient, and the conjugated polymers emit blue fluorescence. By triggering the shift in emission color and the change of emission intensity of conjugated polymers, it is possible to assay the enzyme activity and screen drugs based on the inhibition of enzymes. This protocol combines the fluorescence resonance energy transfer and light harvesting properties of conjugated polymers. The assay technique has two significant characteristics offered by the conjugated polymers: (i) the use of water-soluble conjugated polymers imparts the sensor high sensitivity; (ii) this method does not require fluorescent labels on the substrates, which should significantly reduce the cost.