Phosphates form spectroscopically dark state assemblies in common aqueous solutions.

Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically "dark." Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate's native state in biological solutions.

Spectroscopically dark phosphate features revealed by chemical exchange saturation transfer.

Phosphate is an essential anion in the human body, comprising approximately 1% of the total body weight, and playing a vital role in metabolism, cell membranes, and bone formation. We have recently provided spectroscopic, microscopic, and computational evidence indicating that phosphates can aggregate much more readily in solution than previously thought. This prior work provided indirect evidence through the observation of unusual 31 P NMR relaxation and line-broadening effects with increasing temperature. Here, we show that, under conditions of slow exchange and selective RF saturation, additional features become visible in chemical exchange saturation transfer (CEST) experiments, which appear to be related to the previously reported phosphate clustering. In particular, CEST shows pronounced dips several ppm upfield of the main phosphate resonance at low temperatures, while direct 31 P spectroscopy does not produce any signals in that range. We study the pH dependence of these new spectroscopic features and present exchange and spectroscopic parameters based on fitting the CEST data. These findings could be of importance in the investigation of phosphate dynamics, especially in the biological milieu.

Entangled dark state mediated by a dielectric cavity within epsilon-near-zero materials.

Two emitters can be entangled by manipulating them through optical fields within a photonic cavity. However, maintaining entanglement for a long time is challenging due to the decoherence of the entangled qubits, primarily caused by cavity loss and atomic decay. Here, we found the entangled dark state between two emitters mediated by a dielectric cavity within epsilon-near-zero (ENZ) materials, ensuring entanglement maintenance over an extended period. To obtain the entangled dark state, we derived an effective model with degenerate mode modulation. In the dielectric cavities within ENZ materials, the decay rate of emitters can be regarded as 0, which is the key to achieving the entangled dark state. Meanwhile, the dark state immune to cavity loss exists when two emitters are in symmetric positions in the dielectric cavity. Additionally, by adjusting the emitters to specific asymmetric positions, it is possible to achieve transient entanglement with higher concurrence. By overcoming the decoherence of the entangled qubits, this study demonstrates stable, long-term entanglement with ENZ materials, holding significant importance for applications such as nanodevice design for quantum communication and quantum information processing.

Population and Energy Transfer Dynamics in an Open Excitonic Quantum Battery.

In a previous study, we proposed an open quantum network model of a quantum battery (QB) that possesses dark states owing to its structural exchange symmetries. While in a dark state, the QB is capable of storing an exciton without any environment-induced population losses. However, when the structural exchange symmetry is broken, the QB begins to discharge the exciton towards its exit site. In this article, we start by demonstrating that this QB is not only loss-free with respect to exciton population during the storage phase, but also with respect to the QB energy. We then explore the exciton population and energy transfer dynamics of the QB during the discharge phase over a wide range of site energies, bath temperatures, and bath reorganization energies. Our results shed light on how to optimize the QB's population and energy transfer dynamics for different purposes.

Tetrazine-Isonitrile Bioorthogonal Fluorogenic Reactions Enable Multiplex Labeling and Wash-Free Bioimaging of Live Cells.

Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine-functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post-reacted probes displayed remarkable fluorescence turn-on ratios, up to 3184-fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659 nm. Quantum chemical calculations further elucidate the turn-on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.

Fluorinated graphene grating metasurface for terahertz dark state excitation.

We propose an original technique for the grating metasurfaces fabrication by low-power ultraviolet laser treatment of fluorinated graphene (FG) films with the focus on terahertz applications. The laser treatment reduces dielectric FG to its conductive counterparts, increasing DC conductivity to 170 S·m-1for treated areas. The electromagnetic response of the grating metasurfaces studied by THz time-domain spectroscopy in the 100 GHz-1 THz frequency range demonstrates enhanced resonant transmittance through metasurfaces. The intensity and position of transmittance peak could be tuned by changing the metasurface geometry, i.e. the period of the structure and width of the reduced and unreduced areas. In particular, the decrease of the reduced FG area width from 400 to 170µm leads to the shift of the resonance peak from 0.45 THz to the higher frequencies, 0.85 THz. Theoretical description based on the multipole theory supported by finite element numerical calculations confirms the excitation of the dark state in the metasurface unit cells comprising reduced and unreduced FG areas at resonance frequency determined by the structure geometrical features. Fabricated metasurfaces have been proved to be efficient narrowband polarizers being rotated by 50° about the incident THz field vector.

Creation of Single-Photon Emitters in WSe2 Monolayers Using Nanometer-Sized Gold Tips.

Due to their tunable bandgaps and strong spin-valley locking, transition metal dichalcogenides constitute a unique platform for hosting single-photon emitters. Here, we present a versatile approach for creating bright single-photon emitters in WSe2 monolayers by the deposition of gold nanostars. Our molecular dynamics simulations reveal that the formation of the quantum emitters is likely caused by the highly localized strain fields created by the sharp tips of the gold nanostars. The surface plasmon modes supported by the gold nanostars can change the local electromagnetic fields in the vicinity of the quantum emitters, leading to their enhanced emission intensities. Moreover, by correlating the emission energies and intensities of the quantum emitters, we are able to associate them with two types of strain fields and derive the existence of a low-lying dark state in their electronic structures. Our findings are highly relevant for the development and understanding of single-photon emitters in transition metal dichalcogenide materials.

Exchange saturation transfer and associated NMR techniques for studies of protein interactions involving high-molecular-weight systems.

A brief overview of theoretical and experimental aspects of the Dark state Exchange Saturation Transfer (DEST) and lifetime line broadening ([Formula: see text]) NMR methodologies is presented from a physico-chemical perspective. We describe how the field-dependence of [Formula: see text] can be used for determining the exchange regime on the transverse spin relaxation time-scale. Some limitations of DEST/[Formula: see text] methodology in applications to molecular systems with intermediate molecular weights are discussed, and the means of overcoming these limitations via the use of closely related exchange NMR techniques is presented. Finally, several applications of DEST/[Formula: see text] methodology are described from a methodological viewpoint, with an emphasis on providing examples of how kinetic and relaxation parameters of exchange can be reliably extracted from the experimental data in each particular case.

Composition-Dependent Energy Splitting between Bright and Dark Excitons in Lead Halide Perovskite Nanocrystals.

Perovskite semiconductor nanocrystals with different compositions have shown promise for applications in light-emitting devices. Dark excitonic states may suppress light emission from such nanocrystals by providing an additional nonradiative recombination channel. Here, we study the composition dependence of dark exciton dynamics in nanocrystals of lead halides by time-resolved photoluminescence spectroscopy at cryogenic temperatures. The presence of a spin-related dark state is revealed by magneto-optical spectroscopy. The energy splitting between bright and dark states is found to be highly sensitive to both halide elements and organic cations, which is explained by considering the effects of size confinement and charge screening, respectively, on the exchange interaction. These findings suggest the possibility of manipulating dark exciton dynamics in perovskite semiconductor nanocrystals by composition engineering, which will be instrumental in the design of highly efficient light-emitting devices.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from (15)N, (1)HN, and (13)Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

Sensing mechanism of the benzo-bodipy based fluorescent probe for Hypochlorous acid detection: Invalidity of photoinduced electron transfer.

In vivo real-time detection of hypochlorous acid (HClO) in biological systems plays a crucial role in diagnosing immune-related diseases. Experimentally, a benzo-bodipy probe based on the photo-induced electron transfer (PeT) sensing mechanism has been developed for live fluorescence imaging. However, there have been no theoretical studies conducted to substantiate the precision of the sensing mechanism. This paper employs density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods to investigate the fluorescence detection mechanism of benzo-bodipy derivatives (BBy-T and BBy-TO), proposing a detection approach based on dark nπ* state quenching. The study reveals that the fluorescence quenching mechanism of BBy-T is primarily regulated by a thiomorpholine moiety, involving a dark nπ* state transition non-radiatively. Furthermore, this paper explains the fluorescence enhancement observed in BBy-TO. Theoretical investigations demonstrate, based on frontier molecular orbitals (FMOs) and hole-electron analysis, that the fluorescence enhancement for BBy-TO is not governed by the previously proposed intramolecular charge transfer (ICT) mechanism in experiments but rather follows a locally excited (LE) ππ* pattern. This work offers new insights for the design of novel fluorescence probes based on bodipy and benzo derivatives, expanding the understanding of their fluorescence properties.

Cavity-Induced Optical Nonreciprocity Based on Degenerate Two-Level Atoms.

We developed and experimentally realized a scheme of optical nonreciprocity (ONR) by using degenerate two-level atoms embedded in an optical ring cavity. For the degenerate transition Fg = 4 ↔ Fe = 3, we first studied the cavity-transmission property in different coupling field configurations and verified that under the strong-coupling regime, the single-dark-state peak formed by electromagnetically induced transparency (EIT) showed ONR. The stable ground-state Zeeman coherence for Λ-chains involved in the degenerate two-level system was found to be important in the formation of intracavity EIT. However, different from the three-level atom-cavity system, in the degenerate two-level system, the ONR effect based on intracavity EIT occurred only at a low probe intensity, because the cavity-atom coupling strength was weakened in the counter-propagating probe and coupling field configuration. Furthermore, ONR transmission with a high contrast and linewidth-narrowing was experimentally demonstrated.

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain.

The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions. Here, conformational equilibria between a major and two minor states were highlighted exclusively in ZK2, by using CPMG and CEST NMR experiments. These minor states appear to be temperature dependent, and their populations are highest at physiological temperature. These minor states are present both in NCp7, the mature form of NCd, and in NCp9 and NCp15, the precursor forms of NCd, with increased populations. The role of these minor states in the targeting of NCd by drugs and its binding properties is discussed.

NMR methods for exploring 'dark' states in ligand binding and protein-protein interactions.

A survey, primarily based on work in the authors' laboratory during the last 10 years, is provided of recent developments in NMR studies of exchange processes involving protein-ligand and protein-protein interactions. We start with a brief overview of the theoretical background of Dark state Exchange Saturation Transfer (DEST) and lifetime line-broadening (ΔR2) NMR methodology. Some limitations of the DEST/ΔR2 methodology in applications to molecular systems with intermediate molecular weights are discussed, along with the means of overcoming these limitations with the help of closely related exchange NMR techniques, such as the measurements of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, exchange-induced chemical shifts or rapidly-relaxing components of relaxation decays. Some theoretical underpinnings of the quantitative description of global dynamics of proteins on the surface of very high molecular weight particles (nanoparticles) are discussed. Subsequently, several applications of DEST/ΔR2 methodology are described from a methodological perspective with an emphasis on providing examples of how kinetic and relaxation parameters for exchanging systems can be reliably extracted from NMR data for each particular model of exchange. Among exchanging systems that are not associated with high molecular weight species, we describe several exchange NMR-based studies that focus on kinetic modelling of transient pre-nucleation oligomerization of huntingtin peptides that precedes aggregation and fibril formation.

Competition between Factors Determining Bright versus Dark Atomic States within a Laser Mode.

We observe bimodal fluorescence patterns from atoms in a fast atomic beam when the laser excitation occurs in the presence of a magnetic field and the atoms sample only a portion of the laser profile. The behavior is well explained by competition between the local intensity of the laser, which tends to generate a coherent-population-trapping (CPT) dark state in the J = 1 to J' = 0 system, and the strength of an applied magnetic field that can frustrate the CPT process. This work is relevant for understanding and optimizing the detection process for clocks or other coherent systems utilizing these transitions and could be applicable to in situ calibration of the laser-atom interaction, such as the strength of the magnetic field or laser intensity at a specific location.

Molecular Origins of Heteroatom Engineering on the Emission Wavelength Tuning, Quantum Yield Variations and Fluorogenicity of NBD-like SCOTfluors.

Molecular engineering of fluorophore scaffolds, especially heteroatom replacement, is a promising method to yield novel fluorophores with tailored properties for various applications. Yet, molecular origins of the distinct fluorescent properties in newly developed SCOTfluors, i. e., varied emission wavelengths, distinct quantum yields, and fluorogenicity, remain elusive. Such understanding, however, is critical for the rational molecular engineering of high-performance fluorophores. Herein, we employed quantum chemical calculations to understand the structure-property relationships of nitrobenzoxadiazole (NBD)-like SCOTfluors. Our findings are important not only for the rational deployment of SCOTfluors, but also for the effective modifications of other fluorophore scaffolds, for satisfying the increasingly diversified requirements of bioimaging and biosensing applications.

Interactions of IDPs with Membranes Using Dark-State Exchange NMR Spectroscopy.

Membrane interactions of proteins play a role in essential cellular processes in both physiological and disease states. The structural flexibility of intrinsically disordered proteins (IDPs) allows for interactions with multiple partners, including membranes. However, determining conformational states of IDPs when interacting with membranes can be challenging. Here we describe the use of nuclear magnetic resonance (NMR), including dark-state exchange saturation transfer (DEST), to probe IDP-membrane interactions in order to determine whether there is an interaction, which residues participate, and the extent/nature of the interaction between the protein and the membrane. Using α-synuclein and tau as typical examples, we provide protocols for how the membrane interactions of IDPs can be probed, including details of how the samples should be prepared and guidelines on how to interpret the results.

The origin of the dark S1 state in carotenoids: a comprehensive model.

In carotenoids, by analogy to polyenes, the symmetry of the π-electron system is often invoked to explain their peculiar electronic features, in particular the inactivity of the S0 → S1 transition in one-photon excitation. In this review, we verify whether the molecular symmetry of carotenoids and symmetry of their π-electron system are supported in experimental and computational studies. We focus on spectroscopic techniques which are sensitive to the electron density distribution, including the X-ray crystallography, electronic absorption, two-photon techniques, circular dichroism, nuclear magnetic resonance, Stark and vibrational spectroscopies, and on this basis we seek for the origin of inactivity of the S1 state. We come across no experimental and computational evidence for the symmetry effects and the existence of symmetry restrictions on the electronic states of carotenoids. They do not possess an inversion centre and the C2h symmetry approximation of carotenoid structure is by no means justified. In effect, the application of symmetry rules (and notification) to the electronic states of carotenoids in this symmetry group may lead to a wrong interpretation of experimental data. This conclusion together with the results summarized in the review allows us to advance a consistent model that explains the inactivity of the S0 → S1 transition. Within this model, S1 is never accessible from S0 due to the negative synergy of (i) the contributions of double excitations of very low probability, which elevate S1 energy, and (ii) a non-verticality of the S0 → S1 transition, due to the breaking of Born-Oppenheimer approximation. Certainly, our simple model requires a further experimental and theoretical verification.