Counting Single Redox Turnovers: Fluorogenic Antioxidant Conversion and Mass Transport Visualization via Single Molecule Spectroelectrochemistry

We report herein the use of single molecule spectroelectrochemistry (SMS-EC) to optically detect electrochemically activated individual molecules of the redox-active, tocopherol-like fluorogenic probe H₂B-PMHC. Single molecule detection combined with a fluorescence burst frequency analysis is shown to provide a most sensitive method to monitor subnanomolar concentrations of the electrochemically activated probe. By measuring changes in activated probe concentration over time and distance from a planar electrode, mass transport characteristics of a prototype infinite planar electrode system are retrieved as a showcase study. We show that fluorescence activation may be achieved under both oxidative (direct oxidation of H₂B-PMHC) and reductive (via O₂^•‑ production) conditions. Our results conducted in organic solvents broaden the scope of electrochemical reactions that can be studied by SMS-EC. The methodology we describe may potentially aid the study of mass transport in systems with complex electrode geometries or hampered diffusion

Redox-Based Photostabilizing Agents in Fluorescence Imaging: The Hidden Role of Intersystem Crossing in Geminate Radical Ion Pairs

Here we report transient absorption studies on the ground-state recovery dynamics of the single-molecule fluorophore Cy3B in the presence of four different photostabilizing agents, namely β-mercaptoethanol (β-ME), Trolox (TX), <i>n</i>-propyl gallate (<i>n</i>-PG), and ascorbic acid (AA). These are triplet-state quenchers that operate via photoinduced electron transfer (PeT). While quantitative geminate recombination was recorded following PeT for β-ME (∼100%), for Trolox, <i>n</i>-propyl gallate, and ascorbic acid the extent of geminate recombination was >48%, >27%, and >13%, respectively. The results are rationalized in terms of the rates of intersystem crossing (ISC) in the newly formed geminate radical ion pairs (GRIPs). Rapid spin relaxation in the radicals formed accounts for quantitative geminate recombination with β-ME and efficient geminate recombination with TX. Our results illustrate how the interplay of PeT quenching efficiency and geminate recombination dynamics may lead to improved photostabilization strategies, critical for single-molecule fluorescence and super-resolution imaging

Cy3 Photoprotection Mediated by Ni²⁺ for Extended Single-Molecule Imaging: Old Tricks for New Techniques

The photostability of reporter fluorophores in single-molecule fluorescence imaging is of paramount importance, as it dictates the amount of relevant information that may be acquired before photobleaching occurs. Quenchers of triplet excited states are thus required to minimize blinking and sensitization of singlet oxygen. Through a combination of single-molecule studies and ensemble mechanistic studies including laser flash photolysis and time-resolved fluorescence, we demonstrate herein that Ni²⁺ provides a much desired physical route (chemically inert) to quench the triplet excited state of Cy3, the most ubiquitous green emissive dye utilized in single-molecule studies

Improving the Photostability of Red- and Green-Emissive Single-Molecule Fluorophores via Ni²⁺ Mediated Excited Triplet-State Quenching

Methods to improve the photostability/photon output of fluorophores without compromising their signal stability are of paramount importance in single-molecule fluorescence (SMF) imaging applications. We show herein that Ni²⁺ provides a suitable photostabilizing agent for three green-emissive (Cy3, ATTO532, Alexa532) and three red-emissive (Cy5, Alexa647, ATTO647N) fluorophores, four of which are regularly utilized in SMF studies. Ni²⁺ works via photophysical quenching of the triplet excited state eliminating the potential for reactive intermediates being formed. Measurements of survival time, average intensity, and mean number of photons collected for the six fluorophores show that Ni²⁺ increased their photostability 10- to 45-fold, comparable to photochemically based systems, without compromising the signal intensity or stability. Comparative studies with existing photostabilizing strategies enabled us to score different photochemical and photophysical stabilizing systems, based on their intended application. The realization that Ni²⁺ allowed achieving a significant increase in photon output both for green- and red-emissive fluorophores positions Ni²⁺ as a widely applicable tool to mitigate photobleaching, most suitable for multicolor single-molecule fluorescence studies

How Lipid Unsaturation, Peroxyl Radical Partitioning, and Chromanol Lipophilic Tail Affect the Antioxidant Activity of α-Tocopherol: Direct Visualization via High-Throughput Fluorescence Studies Conducted with Fluorogenic α-Tocopherol Analogues

The preparation of two highly sensitive fluorogenic α-tocopherol (TOH) analogues which undergo >30-fold fluorescence intensity enhancement upon reaction with peroxyl radicals is reported. The probes consist of a chromanol moiety coupled to the <i>meso</i> position of a BODIPY fluorophore, where the use of a methylene linker (BODIPY-2,2,5,7,8-pentamethyl-6-hydroxy-chroman adduct, H₂B-PMHC) vs an ester linker (<i>meso</i>-methanoyl BODIPY-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, H₂B-TOH) enables tuning their reactivity toward H-atom abstraction by peroxyl radicals. The development of a high-throughput fluorescence assay for monitoring kinetics of peroxyl radical reactions in liposomes is subsequently described where the evolution of the fluorescence intensity over time provides a rapid, facile method to conduct competitive kinetic studies in the presence of TOH and its analogues. A quantitative treatment is formulated for the temporal evolution of the intensity in terms of relative rate constants of H-atom abstraction (<i>k</i>_inh) from the various tocopherol analogues. Combined, the new probes, the fluorescence assay, and the data analysis provide a new method to obtain, in a rapid, parallel format, relative antioxidant activities in phospholipid membranes. The method is exemplified with four chromanol-based antioxidant compounds differing in their aliphatic tails (TOH, PMHC, H₂B-PMHC, and H₂B-TOH). Studies were conducted in six different liposome solutions prepared from poly- and mono-unsaturated and saturated (fluid vs gel phase) lipids in the presence of either hydrophilic or lipophilic peroxyl radicals. A number of key insights into the chemistry of the TOH antioxidants in lipid membranes are provided: (1) The relative antioxidant activities of chromanols in homogeneous solution, arising from their inherent chemical reactivity, readily translate to the microheterogeneous environment at the water/lipid interface; thus similar values for <i>k</i>_inh^H₂B‑PMHC/<i>k</i>_inh^H₂B‑TOH in the range of 2–3 are recorded both in homogeneous solution and in liposome suspensions with hydrophilic or lipophilic peroxyl radicals. (2) The relative antioxidant activity between tocopherol analogues with the same inherent chemical reactivity but bearing short (PMHC) or long (TOH) aliphatic tails, <i>k</i>_inh^PMHC/<i>k</i>_inh^TOH, is ∼8 in the presence of hydrophilic peroxyl radicals, regardless of the nature of the lipid membrane into which they are embedded. (3) Antioxidants embedded in saturated lipids do not efficiently scavenge hydrophilic peroxyl radicals; under these conditions wastage reactions among peroxyl radicals become important, and this translates into larger times for antioxidant consumption. (4) Lipophilic peroxyl radicals show reduced discrimination between antioxidants bearing long and short aliphatic tails, with <i>k</i>_inh^PMHC/<i>k</i>_inh^TOH in the range of 3–4 for most lipid membranes. (5) Lipophilic peroxyl radicals are scavenged with the same efficiency by all four antioxidants studied, regardless of the nature of their aliphatic tail or the lipid membrane into which they are embedded. These data underpin the key role the lipid environment plays in modulating the rate of reaction of antioxidants characterized by similar inherent chemical reactivity (arising from a conserved chromanol moiety) but differing in their membrane mobility (structural differences in the lipophilic tail). Altogether, a novel, facile method of study, new insights, and a quantitative understanding on the critical role of lipid diversity in modulating antioxidant activity in the lipid milieu are reported

Conformational Changes Spanning Angstroms to Nanometers via a Combined Protein-Induced Fluorescence Enhancement–Förster Resonance Energy Transfer Method

Förster resonance energy transfer (FRET)-based single-molecule techniques have revolutionized our understanding of conformational dynamics in biomolecular systems. Recently, a new single-molecule technique based on protein-induced fluorescence enhancement (PIFE) has aided studies in which minimal (<3 nm) displacements occur. Concerns have been raised regarding whether donor fluorophore intensity (and correspondingly fluorescence quantum yield Φ_f) fluctuations, intrinsic to PIFE methods, may adversely affect FRET studies when retrieving the donor–acceptor dye distance. Here, we initially show through revisions of Förster’s original equation that distances may be calculated in FRET experiments regardless of protein-induced intensity (and Φ_f) fluctuations occurring in the donor fluorophore. We additionally demonstrate by an analysis of the recorded emission intensity and competing decay pathways that PIFE and FRET methods may be conveniently combined, providing parallel complementary information in a single experiment. Single-molecule studies conducted with Cy3- and ATTO647N-labeled RNA structures and the HCV-NS5B polymerase protein undergoing binding dynamics along the RNA backbone provide a case study to validate the results. The analysis behind the proposed method enables for PIFE and FRET changes to be disentangled when both FRET and PIFE fluctuate over time following protein arrival and, for example, sliding. A new method, intensity-FRET, is thus proposed to monitor conformational changes spanning from angstroms to nanometers

Rate of Lipid Peroxyl Radical Production during Cellular Homeostasis Unraveled via Fluorescence Imaging

Reactive oxygen species (ROS) and their associated byproducts have been traditionally associated with a range of pathologies. It is now believed, however, that at basal levels these molecules also have a beneficial cellular function in the form of cell signaling and redox regulation. Critical to elucidating their physiological role is the opportunity to visualize and quantify the production of ROS with spatiotemporal accuracy. Armed with a newly developed, extremely sensitive fluorogenic α-tocopherol analogue (H₄BPMHC), we report herein the observation of steady concentrations of lipid peroxyl radicals produced in live cell imaging conditions. Imaging studies with H₄BPMHC indicate that the rate of production of lipid peroxyl radicals in HeLa cells under basal conditions is 33 nM/h within the cell. Our work further provides indisputable evidence on the antioxidant role of Vitamin E, as lipid peroxidation was suppressed in HeLa cells both under basal conditions and in the presence of Haber–Weiss chemistry, generated by the presence of cumyl hydroperoxide and Cu²⁺ in solution, when supplemented with the α-tocopherol surrogate, PMHC (2,2,5,7,8-pentamethyl-6-hydroxy-chromanol, an α-tocopherol analogue lacking the phytyl tail). H₄BPMHC has the sensitivity needed to detect trace changes in oxidative status within the lipid membrane, underscoring the opportunity to illuminate the physiological relevance of lipid peroxyl radical production during cell homeostasis and disease

Fluorogenic α‑Tocopherol Analogue for Monitoring the Antioxidant Status within the Inner Mitochondrial Membrane of Live Cells

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain autoxidation. Mito-Bodipy-TOH enables monitoring of the antioxidant status, i.e., the antioxidant load and ability to prevent lipid chain autoxidation, within the inner mitochondrial membrane of live cells. The new probe consists of 3 segments: a receptor, a reporter, and a mitochondria-targeting element, constructed, respectively, from an α-tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion (and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology

Exploiting Conjugated Polyelectrolyte Photophysics toward Monitoring Real-Time Lipid Membrane-Surface Interaction Dynamics at the Single-Particle Level

Herein we report the real-time observation of the interaction dynamics between cationic liposomes flowing in solution and a surface-immobilized charged scaffolding formed by the deposition of conjugated polyanion poly­[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) onto 100-nm-diameter SiO₂ nanoparticles (NPs). Contact of the freely floating liposomes with the polymer-coated surfaces led to the formation of supported lipid bilayers (SLBs). The interaction of the incoming liposomes with MPS-PPV adsorbed on individual SiO₂ nanoparticles promoted the deaggregation of the polymer conformation and led to large emission intensity enhancements. Single-particle total internal reflection fluorescence microscopy studies exploited this phenomenon as a way to monitor the deformation dynamics of liposomes on surface-immobilized NPs. The MPS-PPV emission enhancement (up to 25-fold) reflected on the extent of membrane contact with the surface of the NP and was correlated with the size of the incoming liposome. The time required for the MPS-PPV emission to reach a maximum (ranging from 400 to 1000 ms) revealed the dynamics of membrane deformation and was also correlated with the liposome size. Cryo-TEM experiments complemented these results by yielding a structural view of the process. Immediately following the mixing of liposomes and NPs the majority of NPs had one or more adsorbed liposomes, yet the presence of a fully formed SLB was rare. Prolonged incubation of liposomes and NPs showed completely formed SLBs on all of the NPs, confirming that the liposomes eventually ruptured to form SLBs. We foresee that the single-particle studies we report herein may be readily extended to study membrane dynamics of other lipids including cellular membranes in live cell studies and to monitor the formation of polymer-cushioned SLBs

Enhancing the Emissive Properties of Poly(<i>p</i>-phenylenevinylene)-Conjugated Polyelectrolyte-Coated SiO₂ Nanoparticles

Here we describe single-particle imaging studies conducted on the conjugated polyelecrolyte poly­[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) supported on SiO₂ nanoparticles. The particles are subjected to a time-programmed sequence involving addition and removal of different additives, including excited-triplet-state quenchers and scavengers of singlet oxygen as well as ground-state oxygen. Our studies show that these additives enhance the emission intensity and photostability of the nanoparticles and may further repair photodamaged conjugated polymer. The ability to monitor the emission from individual particles along multiple cycles under a range of conditions provides a mechanistic insight into the action of these additives