6 research outputs found

    Photophysics and Release Kinetics of Enzyme-Activatable Optical Probes Based on H‑Dimerized Fluorophores on Self-Immolative Linkers

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    A series of activatable optical probes for the model enzyme penicillin G amidase based on intramolecular formation of non-fluorescent H-dimer between two identical dyes were synthesized. The probes are based on a self-immolative linker, which allows positioning both dyes in close spatial proximity to ensure efficient quenching of probes with absorption and fluorescence emission in the near-infrared (NIR) range. A detailed photophysical investigation of the novel optical probes led to a revision of a previously anticipated quenching mechanism and revealed their potential for optimizing the performance of activatable probes based on H-dimer formation. A kinetic analysis indicated that the fluorescence progress curves can be used to qualitatively extract enzyme kinetic parameters

    Chiral, J‑Aggregate-Forming Dyes for Alternative Signal Modulation Mechanisms in Self-Immolative Enzyme-Activatable Optical Probes

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    Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored. We report herein newly synthesized probes based on self-immolative linkers containing chiral J-aggregate-forming dyes. Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye–dye interactions not observed for the free dyes in solution as well as dye–protein interactions with the enzyme. This suggested that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes

    Stimuli-Responsive Polyguanidino-Oxanorbornene Membrane Transporters as Multicomponent Sensors in Complex Matrices

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    We introduce guanidinium-containing synthetic polymers based on polyguanidino-oxanorbornenes (PGONs) as anion transporters in lipid bilayers that can be activated and inactivated by chemical stimulation. According to fluorogenic anion export experiments with vesicles, PGON transporters are most active in neutral bilayers near their phase transition, with EC<sub>50</sub>’s in the nanomolar range. Six times higher effective transporter concentrations were measured with aminonaphthalene-1,3,6-trisulfonate than with 5(6)-carboxyfluorescein, demonstrating the importance of anion binding for transport and excluding nonspecific efflux. Negative surface potentials efficiently annihilate transport activity, while inside-negative membrane potentials slightly increase it. These trends demonstrate the functional importance of counterions to hinder the binding of hydrophilic counterions and to minimize the global positive charge of the transporter−counterion complexes. Strong, nonlinear increases in activity with polymer length reveal a significant polymer effect. Overall, the characteristics of PGONs do not match those of similar systems (for example, polyarginine) and hint toward an interesting mode of action, clearly different from nonspecific leakage caused by detergents. The activity of PGONs increases in the presence of amphiphilic anions such as pyrenebutyrate (EC<sub>50</sub> = 70 μM), while several other amphiphilic anions tested were inactive. PGONs are efficiently inactivated by numerous hydrophilic anions including ATP (IC<sub>50</sub> = 150 μM), ADP (IC<sub>50</sub> = 460 μM), heparin (IC<sub>50</sub> = 1.0 μM), phytate (IC<sub>50</sub> = 0.4 μM), and CB hydrazide (IC<sub>50</sub> = 26 μM). The compatibility of this broad responsiveness with multicomponent sensing in complex matrices is discussed and illustrated with lactate sensing in sour milk. The PGON lactate sensor operates together with lactate oxidase as a specific signal generator and CB hydrazide as an amplifier for covalent capture of the pyruvate product as CB hydrazone (IC<sub>50</sub> = 1.5 μM)

    Nanomolar Binding of Steroids to Cucurbit[<i>n</i>]urils: Selectivity and Applications

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    Cucurbit­[<i>n</i>]­urils (CB<i>n</i>, <i>n</i> = 7, 8) serve as artificial receptors for steroids (21 tested), including the hormones testosterone and estradiol as well as steroidal drugs. Fluorescence displacement titrations and isothermal titration calorimetry (ITC) provided up to nanomolar binding affinities in aqueous solution for these hydrophobic target molecules, exceeding the values of known synthetic receptors. Remarkable binding selectivities, even for homologous steroid pairs, were investigated in detail by NMR, X-ray crystal diffraction, ITC, and quantum chemical calculations. Notably, the CB<i>n</i>•steroid complexes are stable in water and buffers, in artificial gastric acid, and even in blood serum. Numerous applications have been demonstrated, which range from the solubility enhancement of the steroids in the presence of the macrocycles (up to 100 times, for drug delivery) and the principal component analysis of the fluorescence responses of different CB<i>n</i>•reporter dye combinations (for differential sensing of steroids) to the real-time monitoring of chemical conversions of steroids as substrates (for enzyme assays)

    Nanomolar Binding of Steroids to Cucurbit[<i>n</i>]urils: Selectivity and Applications

    No full text
    Cucurbit­[<i>n</i>]­urils (CB<i>n</i>, <i>n</i> = 7, 8) serve as artificial receptors for steroids (21 tested), including the hormones testosterone and estradiol as well as steroidal drugs. Fluorescence displacement titrations and isothermal titration calorimetry (ITC) provided up to nanomolar binding affinities in aqueous solution for these hydrophobic target molecules, exceeding the values of known synthetic receptors. Remarkable binding selectivities, even for homologous steroid pairs, were investigated in detail by NMR, X-ray crystal diffraction, ITC, and quantum chemical calculations. Notably, the CB<i>n</i>•steroid complexes are stable in water and buffers, in artificial gastric acid, and even in blood serum. Numerous applications have been demonstrated, which range from the solubility enhancement of the steroids in the presence of the macrocycles (up to 100 times, for drug delivery) and the principal component analysis of the fluorescence responses of different CB<i>n</i>•reporter dye combinations (for differential sensing of steroids) to the real-time monitoring of chemical conversions of steroids as substrates (for enzyme assays)

    Scope and Limitations of Surface Functional Group Quantification Methods: Exploratory Study with Poly(acrylic acid)-Grafted Micro- and Nanoparticles

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    The amount of grafted poly­(acrylic acid) on poly­(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, <sup>13</sup>C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]­uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly­(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network
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