We report the fabrication of an oriented bioelectrode of photosystem I (PSI) on single-layer graphene (SLG). This bioelectrode demonstrates improved photocurrent generation, which can be directly attributed to the molecular conductive interface formed by cytochrome c553 (cyt c553) promoting the uniform orientation of PSI with its donor side towards the electrode. The conductive interface between PSI-cyt c553 and SLG is facilitated by a monolayer composed of π–π-stacked pyrene functionalized with the Ni-NTA moiety, which binds the His6-tagged cyt c553. The surface uniformity of the PSI protein orientation in the electrode structure is evidenced by cross-sectional scanning electron microscopy and fluorescence microscopy, with the latter also proving the efficient electronic coupling between majority of the PSI complexes and graphene. With the uniform organization of the biological photoactive layer, photocurrents are generated at the open circuit potential, which can be further increased when a negative potential is applied. Indeed, at the highest applied negative potential (−0.3 V), over 5-fold increase in the cathodic photocurrent for the PSI complexes conjugated via cyt c553 to the SLG substrate is observed compared with that obtained for the randomly oriented structure where PSI is physisorbed on graphene. These results indicate the key role of a strictly defined orientation of photoactive proteins on electrodes for proper electron transfer and substantial improvement in photocurrent generation in the present or similar bioelectrode architectures.

We theoretically study the electrokinetic problem of a pressure-induced liquid flow through a narrow long channel with charged walls, going beyond the classical Helmholtz-Schmolukowski picture by considering the surprisingly strong combined effect of (i) Stern-layer conductance and (ii) dynamic charge-regulating rather than fixed surface charges. We find that the water flow induces, apart from the well-known streaming potential, also a strongly heterogeneous surface charge and zeta potential on chemically homogeneous channel walls. Moreover, we identify a novel steady state with a nontrivial 3D electric flux with 2D surface charges acting as sources and sinks. For a pulsed pressure drop our findings also provide a first-principles explanation for ill-understood experiments on the effect of flow on interfacial chemistry [D. Lis et al., Science 344, 1138 (2014)].

Charged surfaces in contact with liquids containing ions are accompanied in equilibrium by an electric double layer consisting of a layer of electric charge on the surface that is screened by a diffuse ion cloud in the bulk fluid. This screening cloud determines not only the interactions between charged colloidal particles or polyelectrolytes and their self-assembly into ordered structures, but it is also pivotal in understanding energy storage devices, such as electrochemical cells and supercapacitors. However, little is known to what spatial complexity the electric double layers can be designed. Here, we show that electric double layers of non-trivial topology and geometry -including tori, multi-tori and knots- can be realised in charged topological colloidal particles, using numerical modelling within a mean-field Poisson-Boltzmann theory. We show that the complexity of double layers -including geometry and topology- can be tuned by changing the Debye screening length of the medium, or by changing the shape and topology of the (colloidal) particle. More generally, this work is an attempt to introduce concepts of topology in the field of charged colloids, which could lead to novel exciting material design paradigms.

One of the main players in the process of mitochondrial fragmentation is dynamin-related protein 1 (Drp1), which assembles into a helical ring-like structure on the mitochondria and facilitates fission. The fission mechanism is still poorly understood and detailed information concerning oligomeric form of Drp1, its cellular distribution and the size of the fission complex is missing. To estimate oligomeric forms of Drp1 in the cytoplasm and on the mitochondria, we performed a quantitative analysis of Drp1 diffusion and distribution in gene-edited HeLa cell lines. This paper provides an insight into the fission mechanism based on the quantitative description of Drp1 cellular distribution. We found that approximately half of the endogenous GFP-Drp1 pool remained in the cytoplasm, predominantly in a tetrameric form, at a concentration of 28 ± 9 nM. The Drp1 mitochondrial pool included many different oligomeric states with equilibrium distributions that could be described by isodesmic supramolecular polymerization with a K_d of 31 ± 10 nM. We estimated the average number of Drp1 molecules forming the functional fission complex to be approximately 100, representing not more than 14% of all Drp1 oligomers. We showed that the upregulated fission induced by niclosamide is accompanied by an increase in the number of large Drp1 oligomers.

Fast and reliable bacteria detection is crucial for lowering the socioeconomic burden related to bacterial infections (e.g., in healthcare, industry or security). Bacteriophages (i.e., viruses with bacterial hosts) pose advantages such as great specificity, robustness, toughness and cheap preparation, making them popular biorecognition elements in biosensors and other assays for bacteria detection. There are several possible designs of bacteriophage-based biosensors. Here, we focus on developments based on whole virions as recognition agents. We divide the review into sections dealing with phage lysis as an analytical signal, phages as capturing elements in assays and phage-based sensing layers, putting the main focus on development reported within the past three years but without omitting the fundamentals.

The monomeric photosystem I-light-harvesting antenna complex I (PSI-LHCI) supercomplex from the extremophilic red alga Cyanidioschyzon merolae represents an intermediate evolutionary link between the cyanobacterial PSI reaction center and its green algal/higher plant counterpart. We show that the C. merolae PSI-LHCI supercomplex is characterized by robustness in various extreme conditions. By a combination of biochemical, spectroscopic, mass spectrometry, and electron microscopy/single particle analyses, we dissected three molecular mechanisms underlying the inherent robustness of the C. merolae PSI-LHCI supercomplex: (1) the accumulation of photoprotective zeaxanthin in the LHCI antenna and the PSI reaction center; (2) structural remodeling of the LHCI antenna and adjustment of the effective absorption cross section; and (3) dynamic readjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK core subunit. We show that the largest low light-treated C. merolae PSI-LHCI supercomplex can bind up to eight Lhcr antenna subunits, which are organized as two rows on the PsaF/PsaJ side of the core complex. Under our experimental conditions, we found no evidence of functional coupling of the phycobilisomes with the PSI-LHCI supercomplex purified from various light conditions, suggesting that the putative association of this antenna with the PSI supercomplex is absent or may be lost during the purification procedure.

We theoretically and experimentally investigate colloid–oil–water-interface interactions of charged, sterically stabilized, poly(methyl-methacrylate) colloidal particles dispersed in a low-polar oil (dielectric constant ε = 5–10) that is in contact with an adjacent water phase. In this model system, the colloidal particles cannot penetrate the oil–water interface due to repulsive van der Waals forces with the interface whereas the multiple salts that are dissolved in the oil are free to partition into the water phase. The sign and magnitude of the Donnan potential and/or the particle charge is affected by these salt concentrations such that the effective interaction potential can be highly tuned. Both the equilibrium effective colloid–interface interactions and the ion dynamics are explored within a Poisson–Nernst–Planck theory, and compared to experimental observations.

Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are difficult to quantify, especially in the locally confined, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with fluorescence correlation spectroscopy (FCS). STED reduces the length-scale of observation to tens of nanometres (2D)/attoliters (3D) and the time-scale to microseconds, with direct, gradual control. This allows one to distinguish diffusional and binding processes of complex-formation, even at reaction rates higher by an order of magnitude than in confocal FCS. We provide analytical autocorrelation formulas for probes undergoing diffusion-reaction processes under STED condition. We support the theoretical analysis of experimental STED-FCS data on a model system of dye–micelle, where we retrieve the equilibrium and rates constants. Our work paves a promising way toward quantitative characterization of molecular interactions in vivo.

This work, based on in vivo and in vitro measurements, as well as in silico simulations, provides a consistent analysis of diffusion of polydisperse nanoparticles in the cytoplasm of living cells. Using the example of fluorescence correlation spectroscopy (FCS), we show the effect of polydispersity of probes on the experimental results. Although individual probes undergo normal diffusion, in the ensemble of probes, an effective broadening of the distribution of diffusion times occurs—similar to anomalous diffusion. We introduced fluorescently labeled dextrans into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic drag, exponentially dependent on probe size, extraordinarily broadens the distribution of diffusion times across the focal volume. As a result, the in vivo FCS data were effectively fitted with the anomalous subdiffusion model while for a monodisperse probe the normal diffusion model was most suitable. Diffusion time obtained from the anomalous diffusion model corresponds to a probe whose size is determined by the weight-average molecular weight of the polymer. The apparent anomaly exponent decreases with increasing polydispersity of the probes. Our results and methodology can be applied in intracellular studies of the mobility of nanoparticles, polymers, or oligomerizing proteins.

Transport of heat to the surface of a liquid is a limiting step in the evaporation of liquids into an inert gas. Molecular dynamics (MD) simulations of a two component Lennard-Jones (LJ) fluid revealed two modes of energy transport from a vapour to an interface of an evaporating droplet of liquid. Heat is transported according to the equation of temperature diffusion, far from the droplet of radius R. The heat flux, in this region, is proportional to temperature gradient and heat conductivity in the vapour. However at some distance from the interface, Aλ, (where λ is the mean free path in the gas), the temperature has a discontinuity and heat is transported ballistically i.e. by direct individual collisions of gas molecules with the interface. This ballistic transport reduces the heat flux (and consequently the mass flux) by the factor R/(R + Aλ) in comparison to the flux obtained from temperature diffusion. Thus it slows down the evaporation of droplets of sizes R ∼ Aλ and smaller (practically for sizes from 10³ nm down to 1 nm). We analyzed parameter A as a function of interactions between molecules and their masses. The rescaled parameter, A(k_BT_b/ε₁₁)^1/2, is a linear function of the ratio of the molecular mass of the liquid molecules to the molecular mass of the gas molecules, m₁/m₂ (for a series of chemically similar compounds). Here ε₁₁ is the interaction parameter between molecules in the liquid (proportional to the enthalpy of evaporation) and T_b is the temperature of the gas in the bulk. We tested the predictions of MD simulations in experiments performed on droplets of ethylene glycol, diethylene glycol, triethylene glycol and tetraethylene glycol. They were suspended in an electrodynamic trap and evaporated into dry nitrogen gas. A changes from ∼1 (for ethylene glycol) to approximately 10 (for tetraethylene glycol) and has the same dependence on molecular parameters as obtained for the LJ fluid in MD simulations. The value of x = A(k_BT_b/ε₁₁)^1/2 is of the order of 1 (for water x = 1.8, glycerol x = 1, ethylene glycol x = 0.4, tetraethylene glycol x = 2.1 evaporating into dry nitrogen at room temperature and for Lennard-Jones fluids x = 2 for m₁/m₂ = 1 and low temperature).

Superresolution fluorescence microscopy is becoming a widely available, standard tool in biophysical research. The leading deterministic approach, stimulated emission depletion (STED), can enhance the capabilities of various fluorescence techniques, including fluorescence correlation spectroscopy (FCS). Until now, STED-FCS has been successfully applied to diffusion studies in 2D systems such as membranes. Severe deficiencies, including overestimation of the detected number of probes as well as underestimation of their diffusion coefficients (both parameters differing from the expected values by up to an order of magnitude) impeded STED-FCS studies in solutions. Here, we introduce a realistic 3D model of the detection volume for STED-FCS and use it to resolve the apparent inconsistencies. To validate the model, we show a range of STED-FCS experimental data on free diffusion of probes in solutions, covering a broad range of diffusion coefficients and STED power levels. We define the limitations of STED-FCS in 3D and provide simple guidelines for experiment design and data analysis. The proposed approach should prove useful for particle mobility and reaction kinetics studies in polymer solutions as well as in bulk biomimetic and biological systems, especially when reactant concentrations exceeding 100 nM are required.

Faster and more sensitive environmental monitoring should be developed to face the worldwide problem of bacterial infections. To remedy this issue, we demonstrate a bacteria-sensing element that utilizes dense and ordered layers of bacteriophages specific to the given bacteria strain. We combine (1) the chemical modification of a surface to increase the surface coverage of bacteriophages (2) with an alternating electric field to greatly increase the number of properly oriented bacteriophages at the surface. Usually, in sensing elements, a random orientation of bacteriophages results in steric hindrance, which results in no more than a few percent of all receptors being available. An increased number of properly ordered phages results in the optimal performance of phage receptors, manifesting in up to a 64-fold increase in sensitivity and a limit of detection as low as 100 CFU mL^–1. Our sensing elements can be applied for selective, sensitive, and fast (15 min) bacterial detection. A well-studied pair T4 bacteriophage—bacteria Escherichia coli, was used as a model; however, the method could be adapted to prepare bacteriophage-based sensors for detection of a variety of bacterial strains.

We measured macroscopic viscosity as well as nanoviscosity experienced by molecular probes diffusing in solutions containing two polymer species vastly differing in the molecular weight. On this basis we postulated a scaling equation for viscosity of complex liquids characterized by two distinct length-scales. As an experimental model, we used aqueous solutions of low-polydispersity poly(ethylene glycol) and poly(ethylene oxide) with molecular weight ranging from 6 to 1000 kg/mol, polymer concentrations from 0.25% up to 50%, and viscosity up to 500 mPa·s. The proposed model distinguishes between the contributions to the total viscosity stemming from the mesoscopic structure of the complex liquid and from the magnitude of interactions dictated by the chemical nature of its constituents. It allows to predict diffusion rates of nanoscaled probes in polymer solution mixtures and can be adapted to various multilength-scale complex systems.

We showed that the Taylor Dispersion Analysis (TDA) is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS). A series of measurements at constant protein concentration (for transferrin was 1.9 x 10⁻⁵ M, for β- lactoglobulin was 7.6 x 10⁻⁵ M, and for insulin was 1.2 x 10⁻⁴ M) and varying SDS concentrations were carried out in the phosphate-buffered saline (PBS). The structural changes were analyzed based on the diffusion coefficients of the complexes formed at various surfactant concentrations. The concentration of surfactant was varied in the range from 1.2 x 10⁻⁴ M to 8.7 x 10⁻² M. We determined the minimum concentration of the surfactant necessary to change the native conformation of the proteins. The minimal concentration of SDS for β-lactoglobulin and transferrin was 4.3 x 10⁻⁴ M and for insulin 2.3 x 10⁻⁴ M. To evaluate the TDA as a novel method for studying denaturation of proteins we also applied other methods i.e. electronic circular dichroism (ECD) and dynamic light scattering (DLS) to study the same phenomenon. The results obtained using these methods were in agreement with the results from TDA.

Fluorescence correlation spectroscopy (FCS) can provide information about diffusion coefficients and rate constants of chemical reactions in small systems of interacting molecules. However, the interpretation of FCS experiments depends crucially on the model of the autocorrelation function for the fluorescence intensity fluctuations. In this theoretical work, we consider a system of fluorescent molecules that diffuse and interact with massive particles, e.g. surfactant micelles. Using the general formalism of FCS, we derive a new analytical approximation of the autocorrelation function for systems in which both diffusion and a binary reaction occur. This approximation provides a smooth interpolation between the limit of fast reaction (much faster than diffusion), and the opposite limit of slow reaction. Our studies of noncovalent interactions of micelles with dyes by FCS provided an experimental case to which the approximate autocorrelation function was successfully applied [X. Zhang, A. Poniewierski, A. Jelińska, A. Zagożdżon, A. Wisniewska, S. Hou and R. Hołyst, Soft Matter, 2016, 12, 8186–8194].