Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins.

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

Effect of Gd3+, La3+, Lu3+ Co-Doping on the Morphology and Luminescent Properties of NaYF4:Sm3+ Phosphors.

The series of luminescent NaYF4:Sm3+ nano- and microcrystalline materials co-doped by La3+, Gd3+, and Lu3+ ions were synthesized by hydrothermal method using rare earth chlorides as the precursors and citric acid as a stabilizing agent. The phase composition of synthesized compounds was studied by PXRD. All synthesized materials except ones with high La3+ content (where LaF3 is formed) have a ß-NaYF4 crystalline phase. SEM images demonstrate that all particles have shape of hexagonal prisms. The type and content of doping REE significantly effect on the particle size. Upon 400 nm excitation, phosphors exhibit distinct emission peaks in visible part of the spectrum attributed to 4G5/2→6HJ transitions (J = 5/2-11/2) of Sm3+ ion. Increasing the samarium (III) content results in concentration quenching by dipole-dipole interactions, the optimum Sm3+concentration is found to be of 2%. Co-doping by non-luminescent La3+, Gd3+ and Lu3+ ions leads to an increase in emission intensity. This effect was explained from the Sm3+ local symmetry point of view.

Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values.

Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.

Copper-Ruthenium Composite as Perspective Material for Bioelectrodes: Laser-Assisted Synthesis, Biocompatibility Study, and an Impedance-Based Cellular Biosensor as Proof of Concept.

Copper is an inexpensive material that has found wide application in electronics due to its remarkable electric properties. However, the high toxicity of both copper and copper oxide imposes restrictions on the application of this metal as a material for bioelectronics. One way to increase the biocompatibility of pure copper while keeping its remarkable properties is to use copper-based composites. In the present study, we explored a new copper-ruthenium composite as a potential biocompatible material for bioelectrodes. Sample electrodes were obtained by subsequent laser deposition of copper and ruthenium on glass plates from a solution containing salts of these metals. The fabricated Cu-Ru electrodes exhibit high effective area and their impedance properties can be described by simple R-CPE equivalent circuits that make them perspective for sensing applications. Finally, we designed a simple impedance cell-based biosensor using this material that allows us to distinguish between dead and alive HeLa cells.

Laser-assisted surface activation for fabrication of flexible non-enzymatic Cu-based sensors.

A rapid and effective technique has been develped for the fabrication of sensor-active copper-based materials on the surface of such flexible polymers as terephthalate, polyethylene naphthalate, and polyimide using the method of laser surface modification. For this purpose, we optimized the polymer surface activation parameters using laser sources with a picosecond pulse duration for subsequent selective metallization within the activated region. Furthermore, the fabricated copper structures were modified with gold nanostructures and by electrochemical passivation to produce copper-gold and oxide-containing copper species, respectively. As a result, in comparison with pure copper electrodes, these composite materials exhibit much better electrocatalytic performance concerning the non-enzymatic identification of biologically important disease markers such as glucose, hydrogen peroxide, and dopamine.

4,5-Bis(arylethynyl)-1,2,3-triazoles-A New Class of Fluorescent Labels: Synthesis and Applications.

Cu-catalyzed 1,3-dipolar cycloaddition of ethyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Sonogashira cross-coupling were used to synthesize a large series of new triazole-based push-pull chromophores: 4,5-bis(arylethynyl)-1H-1,2,3-triazoles. The study of their optical properties revealed that all molecules have fluorescence properties, the Stokes shift values of which exceed 150 nm. The fluorescent properties of triazoles are easily adjustable depending on the nature of the substituents attached to aryl rings of the arylethynyl moieties at the C4 and C5 atoms of the triazole core. The possibility of 4,5-bis(arylethynyl)-1,2,3-triazoles' application for labeling was demonstrated using proteins and the HEK293 cell line. The results of an MTT test on two distinct cell lines, HEK293 and HeLa, revealed the low cytotoxicity of 4,5-bis(arylethynyl)triazoles, which makes them promising fluorescent tags for labeling and tracking biomolecules.

Azobenzene/Tetraethyl Ammonium Photochromic Potassium Channel Blockers: Scope and Limitations for Design of Para-Substituted Derivatives with Specific Absorption Band Maxima and Thermal Isomerization Rate.

Azobenzene/tetraethyl ammonium photochromic ligands (ATPLs) are photoactive compounds with a large variety of photopharmacological applications such as nociception control or vision restoration. Absorption band maximum and lifetime of the less stable isomer are important characteristics that determine the applicability of ATPLs. Substituents allow to adjust these characteristics in a range limited by the azobenzene/tetraethyl ammonium scaffold. The aim of the current study is to find the scope and limitations for the design of ATPLs with specific spectral and kinetic properties by introducing para substituents with different electronic effects. To perform this task we synthesized ATPLs with various electron acceptor and electron donor functional groups and studied their spectral and kinetic properties using flash photolysis and conventional spectroscopy techniques as well as quantum chemical modeling. As a result, we obtained diagrams that describe correlations between spectral and kinetic properties of ATPLs (absorption maxima of E and Z isomers of ATPLs, the thermal lifetime of their Z form) and both the electronic effect of substituents described by Hammett constants and structural parameters obtained from quantum chemical calculations. The provided results can be used for the design of ATPLs with properties that are optimal for photopharmacological applications.

Ultrasound-Assisted Synthesis of Luminescent Micro- and Nanocrystalline Eu-Based MOFs as Luminescent Probes for Heavy Metal Ions.

The luminescent coarse-, micro- and nanocrystalline europium(III) terephthalate tetrahydrate (Eu2bdc3·4H2O) metal-organic frameworks were synthesized by the ultrasound-assisted wet-chemical method. Electron micrographs show that the europium(III) terephthalate microparticles are 7 µm long leaf-like plates. According to the dynamic light scattering technique, the average size of the Eu2bdc3·4H2O nanoparticles is equal to about 8 ± 2 nm. Thereby, the reported Eu2bdc3·4H2O nanoparticles are the smallest nanosized rare-earth-based MOF crystals, to the best of our knowledge. The synthesized materials demonstrate red emission due to the 5D0-7FJ transitions of Eu3+ upon 250 nm excitation into 1ππ* state of the terephthalate ion. Size reduction results in broadened emission bands, an increase in the non-radiative rate constants and a decrease in both the quantum efficiency of the 5D0 level and Eu3+ and the luminescence quantum yields. Cu2+, Cr3+, and Fe3+ ions efficiently and selectively quench the luminescence of nanocrystalline europium(III) terephthalate, which makes it a prospective material for luminescent probes to monitor these ions in waste and drinking water.

Laser-Assisted Surface Modification of Ni Microstructures with Au and Pt toward Cell Biocompatibility and High Enzyme-Free Glucose Sensing.

We investigated the influence of morphology of Ni microstructures modified with Au and Pt on their cell biocompatibility and electrocatalytic activity toward non-enzymatic glucose detection. Synthesis and modification were carried out using a simple and inexpensive approach based on the method of laser-induced deposition of metal microstructures from a solution on the surface of various dielectrics. Morphological analysis of the fabricated materials demonstrated that the surface of the Ni electrode has a hierarchical structure with large-scale 10 µm pores and small-scale 10 nm irregularities. In turn, the Ni-Pt surface has large-scale cavities, small-scale pores (1-1.5 µm), and a few tens of nanometer particles opposite to Ni-Au that reveals no obvious hierarchical structure. These observations were supported by impedance spectroscopy confirming the hierarchy of the surface topography of Ni and Ni-Pt structures. We tested the biocompatibility of the fabricated Ni-based electrodes with the HeLa cells. It was shown that the Ni-Au electrode has a much better cell adhesion than Ni-Pt with a more complex morphology. On the contrary, porous Ni and Ni-Pt electrodes with a more developed surface area than that of Ni-Au have better catalytic performance toward enzymeless glucose sensing, revealing greater sensitivity, selectivity, and stability. In this regard, modification of Ni with Pt led to the most prominent results providing rather good glucose detection limits (0.14 and 0.19 µA) and linear ranges (10-300 and 300-1500 µA) as well as the highest sensitivities of 18,570 and 2929 µA mM-1 cm-2. We also proposed some ideas to clarify the observed behavior and explain the influence of morphology of the fabricated electrodes on their electrocatalytic activity and biocompatibility.

Ultrafast Excited-State Dynamics of CuBr3- Complex Studied with Sub-20 fs Resolution.

Ultrafast excited-state dynamics of CuBr3- complex was studied in acetonitrile and dichloromethane solutions using femtosecond transient absorption spectroscopy with 18 fs temporal resolution and quantum-chemical DFT calculations. Upon 640 nm excitation, the CuBr3- complex is promoted to the ligand-to-metal charge transfer (LMCT) state, which then shortly undergoes internal conversion into the vibrationally hot ligand field (LF) excited state with time constants of 30 and 40 fs in acetonitrile and dichloromethane, respectively. The LF state nonradiatively relaxes into the ground state in 2.6 and 7.3 ps in acetonitrile and dichloromethane, respectively. Internal conversion of the LF state is accompanied by vibrational relaxation that occurs on the same time scale. Based on the analysis of coherent oscillations and quantum-chemical calculations, the predominant forms of the CuBr3- complex in acetonitrile and dichloromethane solutions were revealed. In acetonitrile, the CuBr3- complex exists as [CuBr3(CH3CN)2]-, whereas three forms of this complex, [CuBr3CH2Cl2]-, [CuBr3(CH2Cl2)2]-, and [CuBr3(CH2Cl2)3]-, are present in equilibrium in dichloromethane.

Copper and Nickel Microsensors Produced by Selective Laser Reductive Sintering for Non-Enzymatic Glucose Detection.

In this work, the method of selective laser reductive sintering was used to fabricate the sensor-active copper and nickel microstructures on the surface of glass-ceramics suitable for non-enzymatic detection of glucose. The calculated sensitivities for these microsensors are 1110 and 2080 µA mM-1·cm-2 for copper and nickel, respectively. Linear regime of enzymeless glucose sensing is provided between 0.003 and 3 mM for copper and between 0.01 and 3 mM for nickel. Limits of glucose detection for these manufactured micropatterns are equal to 0.91 and 2.1 µM for copper and nickel, respectively. In addition, the fabricated materials demonstrate rather good selectivity, long-term stability and reproducibility.

Simple Models to Study Spectral Properties of Microbial and Animal Rhodopsins: Evaluation of the Electrostatic Effect of Charged and Polar Residues on the First Absorption Band Maxima.

A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.

High rate fabrication of copper and copper-gold electrodes by laser-induced selective electroless plating for enzyme-free glucose sensing.

In the current study, the method of Selective Surface Activation Induced by Laser (SSAIL) was used for the fabrication of metallic and bimetallic structures based on copper and gold on the surface of glass and glass-ceramics. It was shown that the fabricated electrodes are suitable for non-enzymatic detection of biologically essential analytes such as glucose. The implemented approach allows performing high-rate metallization of various dielectrics. Voltammetric methods were applied to evaluate the electrocatalytic activity of the obtained structures, which were used as working electrodes. The most promising results were revealed by copper-gold electrode structures manufactured on glass-ceramics. For these structures, sensitivity towards glucose sensing was 3060 µA mM-1 cm-2. The linear range of glucose detection varied between 0.3 and 1000 µM. Besides, the manufactured electrodes exhibited high selectivity and long-term stability.

In Situ Laser-Induced Fabrication of a Ruthenium-Based Microelectrode for Non-Enzymatic Dopamine Sensing.

In this paper, we propose a fast and simple approach for the fabrication of the electrocatalytically active ruthenium-containing microstructures using a laser-induced metal deposition technique. The results of scanning electron microscopy and electrical impedance spectroscopy (EIS) demonstrate that the fabricated ruthenium-based microelectrode had a highly developed surface composed of 10 µm pores and 10 nm zigzag cracks. The fabricated material exhibited excellent electrochemical properties toward non-enzymatic dopamine sensing, including high sensitivity (858.5 and 509.1 µA mM-1 cm-2), a low detection limit (0.13 and 0.15 µM), as well as good selectivity and stability.

An assessment of water placement algorithms in quantum mechanics/molecular mechanics modeling: the case of rhodopsins' first spectral absorption band maxima.

Quantum mechanics/molecular mechanics (QM/MM) models are a widely used tool to obtain detailed insight into the properties and functioning of proteins. The outcome of QM/MM studies heavily depends on the quality of the applied QM/MM model. Prediction and right placement of internal water molecules in protein cavities is one of the critical parts of any QM/MM model construction. Herein, we performed a systematic study of four protein hydration algorithms. We tested these algorithms for their ability to predict X-ray-resolved water molecules for a set of membrane photosensitive rhodopsin proteins, as well as the influence of the applied water placement algorithms on the QM/MM calculated absorption maxima (λmax) of these proteins. We used 49 rhodopsins and their intermediates with available X-ray structures as the test set. We found that a proper choice of hydration algorithms and setups is needed to predict functionally important water molecules in the chromophore-binding cavity of rhodopsins, such as the water cluster in the N-H region of bacteriorhodopsin or two water molecules in the binding pocket of bovine visual rhodopsin. The QM/MM calculated λmax of rhodopsins is also quite sensitive to the applied protein hydration protocols. The best methodology allows obtaining an 18.0 nm average value for the absolute deviation of the calculated λmax from the experimental λmax. Although the major effect of water molecules on λmax originates from the water molecules located in the binding pocket, the water molecules outside the binding pocket also affect the calculated λmax mainly by causing a reorganization of the protein structure. The results reported in this study can be used for the evaluation and further development of hydration methodologies, in general, and rhodopsin QM/MM models, in particular.

In situ laser-induced codeposition of copper and different metals for fabrication of microcomposite sensor-active materials.

We report one-step in situ laser-induced synthesis of the conductive copper microstructures doped with iron, zinc, nickel, and cobalt with highly developed surface area. It was observed that the presence of chlorides of the aforementioned metals in the solutions used in our experiments increases the deposition rate and the amount of copper in the resulting deposits; it also leads to the deposit miniaturization. The laser deposition from solutions containing cobalt (II) chloride of concentration more than 0.003 M results in fabrication of copper microelectrode with better electrochemical properties than those deposited from solutions containing chlorides of other metals of the same concentration. Moreover, copper microelectrode doped with cobalt has demonstrated good reproducibility and long-run stability as well as sensitivity and selectivity towards determination of hydrogen peroxide (limit of detection-0.2 µM) and d-glucose (limit of detection-2.2 µM). Thus, in this article we have shown the opportunity to manufacture two-phase microcomposite materials with good electrical conductivity and electrochemical characteristics using in situ laser-induced metal deposition technique. These materials might be quite useful in development of new perspective sensors for non-enzymatic detection of such important analytes as hydrogen peroxide and glucose.

Ultrafast Excited-State Dynamics of Ligand-Field and Ligand-to-Metal Charge-Transfer States of CuCl42- in Solution: A Detailed Transient Absorption Study.

Ultrafast excited-state dynamics of CuCl42- in acetonitrile is studied by femtosecond broadband transient absorption spectroscopy following excitation of the complex into all ligand-field (LF or d-d) states and into the two ligand-to-metal charge transfer (LMCT) states corresponding to the most intense steady-state absorption bands. The LF excited states are found to be nonreactive. The lowest-lying 2E LF excited state has a lifetime less than 150 fs, and the lifetimes of the second (2B1) and the third (2A1) LF excited states are 1 and 5 ps, respectively. All three LF states decay directly into the ground 2B2 state. Such significant differences in excited-state decay time constants were rationalized computationally through time-dependent density functional theory (TD-DFT) computations. TD-DFT mapping of the relaxation pathway along the symmetric Cl-Cu-Cl umbrella bending vibration gives evidence for a conical intersection between the 2E excited state and the ground 2B2 state. The LMCT states decay within 200 fs with the primary deactivation mode consistent to be Cu-Cl stretch. A fraction of the CuCl42- complexes excited into the LMCT states undergoes ionic dissociation to form products that survive longer than 1 ns. The remaining fraction undergoes internal conversion, which can be viewed as back electron transfer, populating the lower vibrationally hot LF states. The LF states populated from the LMCT states exhibit the same lifetimes as the Franck-Condon LF states and likewise decay directly into the ground state.

A Comparative Study of Modern Homology Modeling Algorithms for Rhodopsin Structure Prediction.

Rhodopsins are seven α-helical membrane proteins that are of great importance in chemistry, biology, and modern biotechnology. Any in silico study on rhodopsin properties and functioning requires a high-quality three-dimensional structure. Due to particular difficulties with obtaining membrane protein structures from the experiment, in silico prediction of the three-dimensional rhodopsin structure based only on its primary sequence is an especially important task. For the last few years, significant progress was made in the field of protein structure prediction, especially for methods based on comparative modeling. However, the majority of this progress was made for soluble proteins and further investigations are needed to achieve similar progress for membrane proteins. In this paper, we evaluate the performance of modern protein structure prediction methodologies (implemented in the Medeller, I-TASSER, and Rosetta packages) for their ability to predict rhodopsin structures. Three widely used methodologies were considered: two general methodologies that are commonly applied to soluble proteins and a methodology that uses constraints that are specific for membrane proteins. The test pool consisted of 36 target-template pairs with different sequence similarities that was constructed on the basis of 24 experimental rhodopsin structures taken from the RCSB database. As a result, we showed that all three considered methodologies allow obtaining rhodopsin structures with the quality that is close to the crystallographic one (root mean square deviation (RMSD) of the predicted structure from the corresponding X-ray structure up to 1.5 Å) if the target-template sequence identity is higher than 40%. Moreover, all considered methodologies provided structures of average quality (RMSD < 4.0 Å) if the target-template sequence identity is higher than 20%. Such structures can be subsequently used for further investigation of molecular mechanisms of protein functioning and for the development of modern protein-based biotechnologies.

A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling.

It was demonstrated in recent studies that some rhodopsins can be used in optogenetics as fluorescent indicators of membrane voltage. One of the promising candidates for these applications is archaerhodopsin-3. While it has already shown encouraging results, there is still a large room for improvement. One of possible directions is increasing the intensity of the protein's fluorescent signal. Rational design of mutants with an improved signal is an important task, which requires both experimental and theoretical studies. Herein, we used a homology-based computational approach to predict the three-dimensional structure of archaerhodopsin-3, and a Quantum Mechanics/Molecular Mechanics (QM/MM) hybrid approach with high-level multireference ab initio methodology (SORCI+Q/AMBER) to model optical properties of this protein. We demonstrated that this methodology allows for reliable prediction of structure and spectral properties of archaerhodopsin-3. The results of this study can be utilized for computational molecular design of efficient fluorescent indicators of membrane voltage for modern optogenetics on the basis of archaerhodopsin-3.

Solvent Effects on Nonradiative Relaxation Dynamics of Low-Energy Ligand-Field Excited States: A CuCl42- Complex.

Nonradiative relaxation dynamics of CuCl42- complexes photoexcited into the highest-energy ligand-field electronic state (2A1) is studied in acetonitrile, dichloromethane, and chloroform solvents, as well as in acetonitrile-water and in acetonitrile-deuterated water mixtures. Due to ultrafast internal conversion, this excited state directly converts to the electronic ground state in dichloromethane and chloroform. The nonradiative relaxation constant is similar in anhydrous acetonitrile. Addition of water to acetonitrile solutions efficiently quenches the excited ligand-field 2A1 state. The quenching is proposed to be due to the diffusion-controlled formation of an electronically excited pentacoordinated [CuCl4H2O]2- encounter complex or a short-lived exciplex of similar structure, in which the electronic excitation energy transfers into the O-H stretch of the coordinated H2O molecule. This is followed by the dissociation of the pentacoordinated species, resulting in the reformation of the ground-state CuCl42- and free H2O molecules.