Fluorescence‐Quenching‐Based Enzyme‐Activity Assay by Using Photon Upconversion

Abstract

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...

Quenching of the Luminescence of Upconverting Luminescent Nanoparticles by Heavy Metal Ions

“…Moreover, NIR light is safe to tissue which it can penetrate to a depth of up to several mm. [33,34] UCLNPs have found applications in chemical sensing, [35] labeling of biomolecules, [36][37][38][39] in enzymatic and affinity assays, [40,41] and in intracellular sensing of temperature [42] because of their brightness and other attractive features. Despite many studies on the spectral properties of UCLNPs, it is rather surprising that the quenching of their luminescence by heavy metal (HM) ions has not been investigated so far.…”

Photonen aufkonvertierende Nanopartikel zur optischen Codierung und zum Multiplexing von Zellen, Biomolekülen und Mikrosphären

“…[224] Deshalb sind kleine UCNPs, die gut in Wasser dispergierbar sind, vorteilhaft für die Verwendung in homogenen Assays. Die Effizienz, mit der die Emission organischer Farbstoffe gelçscht werden kann, liegt jedoch typischerweise unterhalb von 50 %, auch wenn die Lçscheffizienz erst kürzlich auf 82 % erhçht wurde.…”

“…[225] Um die geringe Lçscheffizienz des Aufkonversions-LRET zu umgehen, wurde ein Nucleaseassay entwickelt, der auf einem sequenziellen Energietransfer beruht (Abbildung 11). [224] Abbildung 10. a) Schema eines auf LRET basierenden Assays für Avidin. Biotinylierte UCNPs wirken als Energiedonoren und biotinylierte Goldnanopartikel als Energieakzeptoren.…”

“…Da der fluoreszierende Donorfarbstoff durch ein UCNP ersetzt werden kann, ist es unkompliziert, die LRET-Aufkonversionstechnik auf vorhandene FRETSubstrate zu übertragen. Dieses Detektionssystem wurde eingesetzt, um Assays für Endonucleasen, [224] Caspasen, [234] Thrombin [218] oder Metalloproteinasen [219] zu entwickeln.…”

Upconverting Nanoparticles

“…In 2008, Rantanen et al have published a sensitive enzyme activity assay where they used upconverting donors (average size 340 nm) and a Black-Berry 650 quencher [51]. Again, the nanoparticles used were comparatively large.…”