Cw Electron Paramagnetic Resonance Research Articles

Frequency multiplexing enables parallel multi-sample EPR

Electron paramagnetic resonance (EPR) spectroscopy stands out as a powerful analytical technique with extensive applications in the fields of biology, chemistry, physics, and material sciences. It proves invaluable for investigating the molecular structure and reaction mechanisms of substances containing unpaired electrons, such as metal complexes, organic and inorganic radicals, and intermediate states in chemical reactions. However, despite their remarkable capabilities, EPR systems face significant limitations in terms of sample throughput, as current commercial systems only target the analysis of one sample at a time. Here we introduce a novel scheme for conducting ultra-high frequency continuous-wave EPR (CW EPR) targeting the EPR spectroscopy of multiple microliter volume samples in parallel. Our proof-of-principle prototype involves two decoupled detection cells equipped with high qualty factor Q=104\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$Q=104$$\\end{document} solenoidal coils tuned to 488 and 589 MHz, ensuring a significant frequency gap for effective radio frequency (RF) decoupling between the channels. To further enhance electromagnetic decoupling, an orthogonal alignment of the coils was adopted. The paper further presents an innovative radiofrequency circuit concept that utilizes a single physical RF channel to simultaneously conduct parallel EPR on up to eight cells. Parallel EPR experiments on two BDPA samples, each with a sample volume of 18.3 μL, registered signal-to-noise ratios of 255 and 252 for the two EPR measurement cells, with no observable coupling. The showcased prototype, built using cost-effective commercially available fabrication technology, is readily scalable and represents an initial step with promising potential for advancing sample screening with high-throughput parallel EPR.

The CW-EPR Capabilities of a Dual DNP/EPR Spectrometer Operating at 14 and 7 T

High-field electron paramagnetic resonance (EPR) measurements are indispensable for a better understanding of dynamic nuclear polarization (DNP), which relies on polarization transfer between electron and nuclear spins. DNP experiments are typically performed at high > 7 T magnetic fields and low ≤ 100 K temperatures, while EPR instrumentation capable of EPR measurements under these conditions is scarce. In this paper, we describe the CW EPR capabilities of a dual DNP/EPR spectrometer that is designed to carry out EPR experiments under “DNP conditions” at 14 and 7 T. In the first part, we present the design of this instrument, highlighting the choices made to allow for both DNP and EPR operations. The spectrometer uses a sweepable cryogen-free magnet with NMR-grade homogeneity, a closed-cycle cooling system, a quasi-optical induction mode bridge, and a superheterodyne receiver system. The probe design is optimized for low heat load and fast sample exchange under cryogenic conditions. The spectrometer can operate in frequency and field sweep modes, including wide field sweeps using the main coil of the magnet. In the second part, we present EPR spectra acquired over a wide range of samples and operating conditions, illustrating the CW EPR capabilities of the instrument.

Conformational investigation of the asymmetric periplasmic domains of E. coli LptB2FGC using SDSL CW EPR spectroscopy.

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptB2FG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptB2FGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.

Conventional and high-field pulsed EPR experimental studies on Bazhenov oil formation under the influence of 50 Hz electromagnetic field

Heavy and extra-heavy oil are generating considerable interest in terms of replacing light oil role in the next few years. Nowadays, various enhanced oil recovery (EOR) methods are under development for an effective exploration and oil refining of unconventional oil sources. Electrical methods are among the most commonly discussed types of EOR techniques. Despite this interest, no one as far as we know has studied the effectiveness and mechanisms of electromagnetic (EM) heating influence on heavy oil conversion. It is common knowledge that heavy oils often contain stable paramagnetic centers, which potentially can serve as at least signaling species to follow the EOR upgrading degree. For the first time, to the best of our knowledge, pulsed and high-frequency (high-field, with 3.4 T detection magnetic field) electron paramagnetic resonance (EPR) techniques were exploited to investigate the EM heating influence on Bazhenov formation heavy oil by subjecting it to 50 Hz (0.75 T) electromagnetic field in laboratory conditions. The obtained samples were analyzed by a set of physico-chemical methods including SARA analysis (S: Saturates, A: Aromatics, R: Resins, A: Asphaltenes), gas chromatography–mass spectrometry (GCMS) in addition to pulsed and conventional (cw) EPR spectroscopy. SARA analysis has shown an increase in the content of asphaltenes from 1.2 ± 0.3 wt% in the initial oil to 1.4 ± 0.4, 1.4 ± 0.3, 1.3 ± 0.2, 1.3 ± 0.5 wt% in the samples obtained at 10, 20, 30 and 40 min respectively. Interestingly, this content has drastically dropped to 0.2 ± 0.05 and 0.3 ± 0.02 wt% in the samples obtained at 50 and 60 min of EM heating. Analogically for resins content change which has firstly increased from 1.5 ± 0.5 wt% in the initial oil to 1.9 ± 0.5, 2.6 ± 0.6, 3.0 ± 0.2, 3.0 ± 0.6 wt% in the samples obtained at 10, 20, 30 and 40 min respectively. Then this content has decreased to 0.5 ± 0.05 and 1.0 ± 0.04 wt% in the samples obtained at 50 and 60 min of EM heating. The content of saturated and aromatic compounds has been found to reach 99.3 ± 0.6 wt% in the sample obtained at 50 min comparing to 97.3 ± 1.1 wt% in the initial oil. Moreover, the GCMS data have showed a significant increase in the content of normal alkanes, which resulted from the processes of oil high molecular compounds fractions conversion as a consequence of alkyl substituents elimination. Our data points towards the idea that the investigated oil sample EPR signal intensity and their spectroscopic characteristics do not change with the EM heating either in the X- or W-band frequencies. g|| and g⊥ were found equal to 2.0024 and 2.0015 respectively at 3357 and 3358 mT for the initial sample and the obtained sample after 60 min of EM heating. Similar results (g||=2.0004 and g⊥=1.9991) were obtained for these samples at 3360 and 3362 mT. The findings of this study can help for better understanding of the mechanisms ruling the impact of electromagnetic heating on enhancing oil recovery.

Dynamic Nuclear Polarization with Vanadium(IV) Metal Centers

Summary Dynamic nuclear polarization (DNP) harnesses the large polarization of electron spins to dramatically increase nuclear magnetic resonance (NMR) sensitivity. This study expands the scope of DNP beyond its traditional focus on hyperpolarizing the solvent network using exogenous polarizing agents (PAs). We introduce 1H DNP with endogenous V4+ centers positioned in a set of vanadyl complexes with tunable V4+-1H distances. We traced the polarization transfer from V4+ to 1H spins, specifically differentiating between direct V4+-1Hs polarization transfer and the 1H spin-diffusion-mediated bulk solvent 1H polarization buildup and illuminated the effect of the V4+-1H distance on these processes. These results deepen our understanding of polarization pathways and expand the catalog of PAs to broad-line transition metals. This study establishes crucial first steps toward employing strategically positioned endogenous paramagnetic metal centers for DNP and the conceptual framework of hyperfine DNP spectroscopy that merges both spatial and chemical diagnosis of target nuclear spins.

CW EPR Investigation of Red‐Emitting CaS:Eu Phosphors: Rationalization of Local Electronic Structure

AbstractA series of commercial and prepared CaS:Eu2+ rare earth activated phosphors are investigated following different post‐synthetic treatments. A number of species directly related to the function of the material are characterized using electron paramagnetic resonance (EPR) spectroscopy. Isolated Eu2+ sites are identified and associated with the substitutional doping for Ca2+ in the lattice which are responsible for the 645 nm emission of interest. Another inactive Eu2+ site based within a “EuO” type phase aggregated on the surface of the material is also identified, as well as competitive F+ center defects that are known to reduce emission from the Eu2+ sites. Intrinsic Mn2+ impurities are identified and used as local order probes to determine changes in ordering and symmetry upon cryo‐milling and heating treatments of the samples. X‐ray photoelectron (XPS) and photoluminescence (PL) spectroscopies are also conducted to complement the local structure observations. The reported data is useful in understanding how the nature of the lattice affects ground state electronic structure of functional defective sites, for the development of efficient and selective materials.

Unusual 31P Hyperfine Strain Effects in a Conformationally Flexible Cu(II) Complex Revealed by Two-Dimensional Pulse EPR Spectroscopy.

Strain effects on g and metal hyperfine coupling tensors, A, are often manifested in Electron Paramagnetic Resonance (EPR) spectra of transition metal complexes, as a result of their intrinsic and/or solvent-mediated structural variations. Although distributions of these tensors are quite common and well understood in continuous-wave (cw) EPR spectroscopy, reported strain effects on ligand hyperfine coupling constants are rather scarce. Here we explore the case of a conformationally flexible Cu(II) complex, [Cu{Ph2P(O)NP(O)Ph2-κ2O,O'}2], bearing P atoms in its second coordination sphere and exhibiting two structurally distinct CuO4 coordination spheres, namely a square planar and a tetrahedrally distorted one, as revealed by X-ray crystallography. The Hyperfine Sublevel Correlation (HYSCORE) spectra of this complex exhibit 31P correlation ridges that have unusual inverse or so-called "boomerang" shapes and features that cannot be reproduced by standard simulation procedures assuming only one set of magnetic parameters. Our work shows that a distribution of isotropic hyperfine coupling constants (hfc) spanning a range between negative and positive values is necessary in order to describe in detail the unusual shapes of HYSCORE spectra. By employing DFT calculations we show that these hfc correspond to molecules showing variable distortions from square planar to tetrahedral geometry, and we demonstrate that line shape analysis of such HYSCORE spectra provides new insight into the conformation-dependent spectroscopic response of the spin system under investigation.

Methane Activation on H-ZSM-5 Zeolite with Low Copper Loading. The Nature of Active Sites and Intermediates Identified with the Combination of Spectroscopic Methods.

Cu-modified zeolites have enormous potential as the catalysts facilitating the conversion of methane to methanol. It becomes important to investigate the active sites and the reaction mechanisms involved. In this paper, several spectroscopic methods such as UV-vis diffuse reflectance spectroscopy (UV-visDRS), pulse electron paramagnetic resonance (EPR), diffuse reflectance Fourier transform infrared spectroscopy, and solid-state (13C MAS) NMR have been employed to characterize the state of the Cu sites and the intermediates formed during the catalyst activation and methane-to-methanol transformation on Cu/H-ZSM-5 zeolite with low (0.10 wt %) Cu content. UV-vis DRS and EPR data imply the presence of two types of Cu2+ cations bound to the zeolite framework Si-O--Al sites (Z). One of them is a species of the type Z[Cu(II)O] or Z[Cu(II)(OH)] with extra-framework O- or OH- ligands. The other one refers to Z2Cu(II) species without extra-framework O-containing ligands. CW EPR studies reveal that the Z2Cu(II) species are the major part of the Cu(II) sites present in the zeolite. 1H HYSCORE and DRIFTS data are supportive of the formation of a molecular complex of methane and Z2Cu(II) species, with a strongly polarized C-H bond and a 3.3 Å separation between the hydrogen atom of methane and Cu. 13C MAS NMR provides evidence for the formation of both the surface methoxy intermediate and physisorbed methanol. It is suggested that experimentally identified Z[Cu(II)O] or Z[Cu(II)(OH)] are those sites that provide a homolytic cleavage of the methane C-H bond to yield surface bound methoxy species and/or methanol molecule, the possibility that has been recently justified with density functional theory ( Kulkarni et al. Catal. Sci. Technol. 2018 , 8 , 114 ). The comparison of the amount of the surface methoxy intermediates formed and the number of different Cu(II) sites present in the zeolite allowed us to conclude the involvement of Z2Cu(II) sites in methane C-H bond activation. The mechanism of methane activation on Z2Cu(II) sites has been proposed. It includes two steps: (1) the formation of the molecular complex of methane with Z2Cu(II); (2) heterolytic dissociation of the polarized C-H bond affording surface copper(II) hydride and methoxy species, both bound to zeolite framework Si-O--Al sites.

An EPR characterisation of stable and transient reactive oxygen species formed under radiative and non-radiative conditions

Electron paramagnetic resonance (EPR) spectroscopy is the ideal method of choice when detecting and studying the wide variety of paramagnetic oxygen-centred radicals. For simple diatomic radicals, such as the superoxide (O2−) or peroxy ({text{ROO}}^{bullet}) species, the CW EPR profile (in particular the g-values) of these species can appear similar and indeed indistinguishable in some cases. Experiments using 17O-enriched oxygen, revealing a rich 17O hyperfine pattern, are therefore essential to distinguish between the two species. However, in many cases, particularly involving TiO2 photocatalysis, the peroxy-type ({text{ROO}}^{bullet}) radicals or other intermediate species such as the [O2−…organic]-type adducts can be transient in nature and once again can produce similar g-values. In general terms, these reactive oxygen species (ROS) are formed and detected at low-temperature conditions. Hence, the application of EPR spectroscopy to studies of surface-stabilised oxygen-centred radicals must be performed under carefully selected conditions in order to confidently distinguish between the differing types of diatomic radicals, such as O2−, {text{ROO}}^{bullet} or [O2−…organic].

A radical containing injectable in-situ-oleogel and emulgel for prolonged in-vivo oxygen measurements with CW EPR

Molecular oxygen, reactive oxygen species and free radicals derived from oxygen play important roles in a broad spectrum of physiological and pathological processes. The quantitative measurement of molecular oxygen in tissues by electron paramagnetic resonance (EPR) has great potential for understanding and diagnosing a number of diseases, and for developing and guiding therapies. This requires improvements in the free radical probe systems that sense and report molecular oxygen levels in vivo. We report on the encapsulation of existing free radical probes in lipophilic gel implants: an in-situ-oleogel and an emulgel, based only on well-known, safe excipients for the incorporation of lipophilic and hydrophilic radicals, respectively. The EPR signals of encapsulated radicals were not altered compared to dissolved radicals. The high solubility of oxygen in lipophilic solvents enhanced oxygen sensitivity. The gels extended the lifetime of the radicals in tissues from tens of minutes to many days, simplifying studies with extended series of measurements. The encapsulated radicals showed a good in vivo response to changes in oxygen supply and seem to circumvent concerns from toxicity of the radical probes. These gels simplify the development of new oxygen-sensitive free radical probes for EPR oximetry by making their in vivo stability, persistence and toxicity a function of the encapsulating gel and not a set of additional requirements for the free radical probe.

Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced EPR Techniques

An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron‐electron resonance (DEER) is emerging as a powerful technique that is complementary to other well established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often by a site‐specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. The most common spin‐labeling methodology conjugates (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site‐specifically, all native surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions.Support or Funding InformationThe work is funded by the Pennsylvania State University startup fund of Alexey Silakov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ENDOR-Induced EPR of Disordered Systems: Application to X-Irradiated Alanine

The electron paramagnetic resonance (EPR) spectra of radiation-induced radicals in organic solids are generally composed of multiple components that largely overlap due to their similar weak g anisotropy and a large number of hyperfine (HF) interactions. Such properties make these systems difficult to study using standard cw EPR spectroscopy even in single crystals. Electron-nuclear double-resonance (ENDOR) spectroscopy is a powerful and widely used complementary technique. In particular, ENDOR-induced EPR (EIE) experiments are useful for separating the overlapping contributions. In the present work, these techniques were employed to study the EPR spectrum of stable radicals in X-irradiated alanine, which is widely used in dosimetric applications. The principal values of all major proton HF interactions of the dominant radicals were determined by analyzing the magnetic field dependence of the ENDOR spectrum at 50 K, where the rotation of methyl groups is frozen. Accurate simulations of the EPR spectrum were performed after the major components were separated using an EIE analysis. As a result, new evidence in favor of the model of the second dominant radical was obtained.

Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5).

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21−49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.

ChemInform Abstract: Defects in Nanodiamonds: Application of High‐Frequency CW and Pulse EPR, ODMR

AbstractReview: 45 refs.

Physiological concentrations of albumin favor drug binding.

The ability to track drug binding and release makes electron paramagnetic resonance (EPR) spectroscopy well suited for drug delivery studies. Using the continuous wave (cw) EPR technique to extract information about the dynamics of the spin labeled drugs we can simultaneously determine the bound and unbound drugs. Here, spin labeled salicylic acid (SLSA) binding to and release from bovine serum albumin (BSA) is investigated, as a model for drug-transport protein interaction. We studied SLSA-BSA binding in a wide concentration range and found that the stoichiometry of the drug-protein increases significantly when the physiological range of BSA concentration is reached. Our EPR results explicitly reveal that up to ∼7 SLSA can bind to one albumin at the physiological concentration, whereas at lower BSA concentrations (<0.125 mM) the SLSA-BSA stoichiometry is maximum 2. Moreover, we studied drug release and showed that the ratio of bound to unbound SLSA concentrations remains relatively stable during dialysis. This indicates that the binding equilibrium of SLSA is not altered through the process of dialysis. This study demonstrates that cw EPR spectroscopy in combination with spin labeled drugs is an effective technique for binding and release studies and stoichiometric analysis of drug-protein interactions.

Combining Single Crystal Electron Paramagnetic Resonance and X-Ray Crystallography to Study the Orientation and Dynamics of MTSSL Spin Labels in T4 Lysozyme

The line shape of cw electron paramagnetic resonance (EPR) spectra of spin labeled proteins has been used extensively to analyze local dynamics, backbone fluctuations and structural rearrangements of these systems. The commonly used paramagnetic spin label MTSSL, a methanethiosulfonate derivative, attached to the protein via covalent disulfide coupling on a designated cysteine residue serves as a reporter of these properties. Much effort has been made to extract structural and dynamic information as encoded in the observed line shapes.In this study we use single crystals of MTSSL labeled T4 lysozyme as a prototype example of an α-helical protein to investigate the structure and dynamics of the paramagnetic side chain in detail by a combination of X-ray crystallography and EPR spectroscopy. X-ray diffraction is used to determine the orientation of the unit cell with respect to the EPR sample tube, which in turn allows to determine the orientation of the spin labels for every EPR spectrum of an angular dependent series given that the structure and orientation of the protein in the unit cell is known. To this end high-resolution crystal structures (0.9 - 1.3 A) of spin labeled T4 lysozyme were taken at 100 K and are compared to data deposited in the protein data bank.On this poster we will discuss the angular dependent line shape of the EPR spectra taken at room temperature with respect to the orientation of the spin label as deduced from X-ray crystallography at 100 K. Particular emphasize will be on the ability of currently available models to describe the observed dynamics of the spin label and the implications of this for the line shape analysis of disordered samples.

Demystifying EPR: A Rookie Guide to the Application of Electron Paramagnetic Resonance Spectroscopy on Biomolecules

Electron Paramagnetic Resonance (EPR) spectroscopy, also known as Electron Spin Resonance(ESR) especially among physicists, is a strong and versatile spectroscopic method forinvestigation of paramagnetic systems, i.e. systems like free radicals and most transition metalions, which have unpaired electrons. The sensitivity and selectivity of EPR are notable andintriguing as compared to other spectroscopic methods and approaches. As a qualitative method,EPR can detect species down to the nanomolar range. On the other hand, the specificity of themethod stems from spectral features which directly depend on the types, distances, and relativeorientations of the atoms in the neighborhood of the electron spin centers. In addition tostructural information, EPR can be used to elucidate time dependent behavior of the studiedsystem and it is applicable to systems of different size, ranging from small molecules tomacromolecules. The following short review of general EPR methods is intended for anaudience with little prior knowledge about EPR. It includes examples of suitable representativesystems, techniques for the study of short lived paramagnetic species and even diamagneticmolecules, and introduces the reader to tools necessary for making sense of the spectra. Thispaper focuses only on continuous wave (cw) EPR and does not elaborate on the more advancedpulsed EPR methods.

Defects in Nanodiamonds: Application of High-Frequency cw and Pulse EPR, ODMR

Different aspects of applications of electron paramagnetic resonance (EPR) based techniques including high frequency (HF) electron spin echo (ESE), electron-nuclear double resonance (ENDOR) and optically detected magnetic resonance (ODMR) approaches to study diamond nanostructures are examined.

Structure and Dynamics of Spin-Labeled Insulin Entrapped in a Silica Matrix by the Sol–Gel Method

The structure and conformational dynamics of insulin entrapped into a silica matrix was monitored during the sol to maturated-gel transition by electron paramagnetic resonance (EPR) spectroscopy. Insulin was successfully spin-labeled with iodoacetamide and the bifunctional nitroxide reagent HO-1944. Room temperature continuous wave (cw) EPR spectra of insulin were recorded to assess the mobility of the attached spin labels. Insulin conformation and its distribution within the silica matrix were studied using double electron-electron resonance (DEER) and low-temperature cw-EPR. A porous oxide matrix seems to form around insulin molecules with pore diameters in the order of a few nanometers. Secondary structure of the encapsulated insulin investigated by Fourier transform infrared spectroscopy proved a high structural integrity of insulin even in the dried silica matrix. The results show that silica encapsulation can be used as a powerful tool to effectively isolate and functionally preserve biomolecules during preparation, storage, and release.

Spin probing of supramolecular structures in 1-butyl-3-methyl-imidazolium tetrafluoroborate/water mixtures

We have systematically studied micro-heterogeneous mixtures of the room temperature ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim+][BF4−]) and water using continuous wave electron paramagnetic resonance (CW EPR) and the spin-probing methodology. Using cryo TEM, a mesoscopic picture of the micro-heterogeneous mixtures could be revealed. Six spin-probes differing in polarity, charge and Lewis basicity have been used to map the dependence of the micro-polarity and rotation motion of the probe on the ionic liquid (IL) concentration. The electron paramagnetic resonance (EPR) spectra of all probe molecules have been found to be unimodal. The critical aggregation concentration has been determined and the local water concentration sensed by the probes extracted from the Mukerjee hydrophilicity index. Surprisingly, four probes of the piperidinoxyl type were found to sense very similar local water concentrations irrespective of the substituent, monovalent ionic or H-bonding, at the 4-position. Using a simple geometrical model of the primary IL aggregates observed in the micro-heterogeneous concentration range, we show that these probes cannot be statically located within the aggregates on the EPR timescale. Instead, diffusive trajectories of the probe molecules extend into the aqueous phase, i.e. aqueous and [bmim+][BF4−]-rich phases are sampled by the probes in swift succession. No details of the internal structure of the [bmim+][BF4−] aggregates can in general be elucidated by the spin-probing methodology under these conditions. On the other hand, the dianionic Fremy's salt binds to the surface of the IL aggregates thereby sampling predominantly the aqueous phase. At large IL concentrations, a micro-viscosity drastically smaller than the macroscopic viscosity is typically observed.Supplementary Materials (Figures S1–S5) for this paper are available online on the journal website.