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Using Lifetime and Quenching Rate Constant to Determine Optimal Quencher Concentration.

ABSTRACT: Excited state quenching is a key step in photochemical reactions that involve energy or electron transfer. High reaction quantum yields require sufficiently high concentrations of a quencher to ensure efficient quenching. The determination of quencher concentrations is typically done through trial and error. Using kinetic modeling, however, a simple relationship was developed that predicts the concentration of quencher necessary to quench 90% of excited states, using only the photosensitizer lifetime and the rate constant for quenching as inputs. Comparison of the predicted quencher concentrations and quencher concentrations used in photoredox reactions featuring acridinium-based photocatalysts reveals that the majority of reactions used quencher concentrations significantly below the predicted concentration. This suggests that these reactions exhibit low quantum yields, requiring long reaction times and/or intense light sources.

SUBMITTER: Soto XL

PROVIDER: S-EPMC9330265 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Using Lifetime and Quenching Rate Constant to Determine Optimal Quencher Concentration.

Soto Xena L XL Swierk John R JR

ACS omega 20220712 29

Excited state quenching is a key step in photochemical reactions that involve energy or electron transfer. High reaction quantum yields require sufficiently high concentrations of a quencher to ensure efficient quenching. The determination of quencher concentrations is typically done through trial and error. Using kinetic modeling, however, a simple relationship was developed that predicts the concentration of quencher necessary to quench 90% of excited states, using only the photosensitizer lif ...[more]

PMID: 35910131

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Project description:The model-free analysis (MFA) was applied to measure the average rate constant (<k>) for pyrene excimer formation (PEF) in a series of pyrene-labeled dendrons referred to as Pyx-G(N), where x (= 2N) is the number of pyrenyl labels born by a dendron of generation N ranging from 1 to 6. <k> was measured in four different solvents, namely tetrahydrofuran (THF), toluene, N,N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO). <k> was found to increase linearly with increasing local pyrene concentration ([Py]loc), where [Py]loc had been determined mathematically for the Pyx-G(N) dendrons. The slope of each straight line changed with the nature of the solvent and represented kdiff, the bimolecular rate constant for PEF. kdiff depended on the solvent viscosity (η) and the probability (p) for PEF upon encounter between an excited and a ground-state pyrene. In a same solvent, kdiff for the Pyx-G(N) dendrons was about 360 ± 30 times smaller than kdiff obtained for ethyl 4-(1-pyrene)butyrate (PyBE), a pyrene model compound similar to the pyrene derivative used to label the dendrons. The massive decrease in kdiff observed for the Pyx-G(N) samples reflected the massive loss in mobility experienced by the pyrenyl labels after being covalently attached onto a macromolecule compared to freely diffusing PyBE. Interestingly, the kdiff values obtained for the Pyx-G(N) dendrons and the PyBE model compound followed similar trends as a function of solvent, indicating that the difference in behavior between the kdiff values obtained in different solvents were merely due to the changes in the η and p values between the solvents. Normalizing the <k> values obtained with the Pyx-G(N) dendrons by the kdiff values obtained for PyBE in the same solvents accounted for changes in η and p, resulting in a master curve upon plotting <k>/(fdiff × kdiff) as a function of [Py]loc, where fdiff was introduced to account for some pyrene aggregation in the higher generation dendron (Py64-G(6)). This result demonstrates that <k> represents a direct measure of [Py]loc in pyrene-labeled macromolecules.

Project description:Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 μm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Dataset Information

Using Lifetime and Quenching Rate Constant to Determine Optimal Quencher Concentration.

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Using Lifetime and Quenching Rate Constant to Determine Optimal Quencher Concentration.

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