Enzyme-activity assays are used, for example, for screening enzyme inhibitors and activators to discover novel drug candidates.[1] 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. [2,3] 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), [4] 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), [5] 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. [6] 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 1 a).Benzonase endonuclease was chosen as a model enzyme for the upconversion FRET-based assay, because it efficiently degrades oligonucleotides to shorter fragments. The substrate oligonucleotide had three modifications: an Alexa Fluor 680 (AF680) fluorophore and biotin at the 5' end and a BlackBerry Quencher 650 quencher (BBQ650) at the 3' end. The quenching efficiency of BBQ650 in the double-labeled substrate was found to be very good (> 96 %). First, the enzyme reaction was carried out and this was followed by a second step in which all of the biotinylated oligonucleotide substrates (intact or cleaved) were collected on the streptavidin-coated UCPs. The energy-transfer-excited emission of the AF680 was directly proportional to the extent of substrate digestion until all of the substrates were...

Upconverting phosphors (UCPs) are lanthanide-doped sub-micrometer-sized particles, which produce multiple narrow and well-separated anti-Stokes emission bands at visible wavelengths under infrared excitation (980 nm). The advantageous features of UCPs were utilized to construct a dual-parameter, homogeneous sandwich hybridization assay based on a UCP donor and lanthanide resonance energy transfer (LRET). UCPs with two emission bands (540 nm and 653 nm) were exploited together with two appropriate fluorophores as acceptors. The energy transfer excited emissions of the acceptors were measured at 600 nm and 740 nm without any significant interference from each other. The autofluorescence limitation associated with conventional fluorescence was totally avoided as the measurements were carried out at shorter wavelength relative to the excitation. In the sandwich hybridization assay two different single-stranded target-oligonucleotide sequences were detected simultaneously and quantitatively with a dynamic range from 0.03 to 0.4 pmol (corresponding 0.35-5.4 nM). The UCPs enable multiplexed homogeneous LRET-based assay requiring only a single excitation wavelength, which simplifies the detection and extends the applicability of upconversion in bioanalytical measurements.

Enzyme-activity assays are used, for example, for screening enzyme inhibitors and activators to discover novel drug candidates.[1] 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. [2,3] 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), [4] 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), [5] 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. [6] 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 1 a).Benzonase endonuclease was chosen as a model enzyme for the upconversion FRET-based assay, because it efficiently degrades oligonucleotides to shorter fragments. The substrate oligonucleotide had three modifications: an Alexa Fluor 680 (AF680) fluorophore and biotin at the 5' end and a BlackBerry Quencher 650 quencher (BBQ650) at the 3' end. The quenching efficiency of BBQ650 in the double-labeled substrate was found to be very good (> 96 %). First, the enzyme reaction was carried out and this was followed by a second step in which all of the biotinylated oligonucleotide substrates (intact or cleaved) were collected on the streptavidin-coated UCPs. The energy-transfer-excited emission of the AF680 was directly proportional to the extent of substrate digestion until all of the substrates were...