Fluorescence-quenching-based enzyme-activity assay by using photon upconversion.

Enzyme-activity assays are used, for example, for screening enzyme inhibitors and activators to discover novel drug candidates. A homogeneous assay principle for hydrolyzing enzymes based on a double-labeled fluorogenic substrate is commonly employed and is suitable for high-throughput screening. This separation-free assay concept relies on the strong distance dependency of fluorescence resonance energy transfer (FRET), which takes place only at distances below 10 nm. A synthetic internally quenched substrate for the enzyme is labeled with a fluorophore at one end and a quencher at the other end of the molecule. When the enzyme digests the substrate, the two labels are separated and fluorescence is recovered. The performance of fluorescence-quenching-based homogeneous assays is still limited due to the autofluorescence originating from biological materials. This problem can be solved by a novel label technology based on upconverting phosphors (UCPs), which have the unique property of photoluminescence emission at visible wavelengths under near-infrared (NIR) excitation. No autofluorescence is detected at shorter wavelengths, because the upconversion phenomenon requires sequential multiphoton absorption not observed in nature. Due to the NIR excitation, UCP technology is also applicable to strongly colored samples (for example, whole blood), which absorb at ultraviolet and visible wavelengths, a process that interferes with other fluorescence technologies. The aim of this study was to combine the advantageous features of UCP donors and fluorescence-quenching assays to construct a sensitive enzyme-activity assay. Wang et al. have quenched around 70% of the emission of nanosized UCPs by using gold particles. More efficient quenching, however, is required for a practical assay as described above since the best theoretical signal-to-background ratio with these components would be as poor as 3:1. It is not possible to entirely quench the anti-Stokes photoluminescence originating from multiple dopant ions within submicrometer-sized UCPs because only those emitter ions located near the surface of UCP can be quenched. We have now solved this problem with a sequential energy-transfer-based assay concept (Figure 1a).