Diffusion and flow in complex liquids

K. Makuch, R. Hołys, T. Kalwarczyk, P. Garstecki and J. F. Brady

Soft Matter, 2020, 16, 114-124

Thermal motion of particles and molecules in liquids underlies many chemical and biological processes. Liquids, especially in biology, are complex due to structure at multiple relevant length scales. While diffusion in homogeneous simple liquids is well understood through the Stokes–Einstein relation, this equation fails completely in describing diffusion in complex media. Modeling, understanding, engineering and controlling processes at the nanoscale, most importantly inside living cells, requires a theoretical framework for the description of viscous response to allow predictions of diffusion rates in complex fluids. Here we use a general framework with the viscosity η(k) described by a function of wave vector in reciprocal space. We introduce a formulation that allows one to relate the rotational and translational diffusion coefficients and determine the viscosity η(k) directly from experiments. We apply our theory to provide a database for rotational diffusion coefficients of proteins/protein complexes in the bacterium E. coli. We also provide a database for the diffusion coefficient of proteins sliding along major grooves of DNA in E. coli. These parameters allow predictions of rate constants for association of proteins. In addition to constituting a theoretical framework for description of diffusion of probes and viscosity in complex fluids, the formulation that we propose should decrease substantially the cost of numerical simulations of transport in complex media by replacing the simulation of individual crowding particles with a continuous medium characterized by a wave-length dependent viscosity η(k).

Transport of nanoprobes in multicellular spheroids (August, 2020)

Pilz, Marta and Kwapiszewska, Karina and Kalwarczyk, Tomasz and Bubak, Grzegorz and Nowis, Dominika and Holyst, Robertv

The Royal Society of Chemistry, Nanoscale, 2020,12, 19880-19887

The efficient delivery of drugs to cells depends on their diffusion through the extracellular matrix (ECM) of tissues. Here we present a study of diffusion of nanoprobes of radius from 1 nm to over 100 nm in the ECM of spheroids of three cell types (HeLa, MCF-7 and fibroblasts). We quantified nanoparticle transport in spheroids’ proliferating zone. We determined the size-dependent viscosity of the ECM. We revealed that nanoobjects up to 10 nm in radius exhibited unobstructed diffusion in the ECM, regardless of the spheroid type. Presented length-scale dependent viscosity profiles for spheroids pave the way for advanced modelling of drug administration through tissue.
Journal: Nanoscale
Publisher: The Royal Society of Chemistry

Features and advantages of flexible silicon nanowires for SERS applications

H. Gebavi, V. Gašparić, D. Risović, P. Albrycht, M. Ivanda

Beilstein Journal of Nanotechnology, 2019, 10, pp. 725–734

The paper reports on the features and advantages of horizontally oriented flexible silicon nanowires (SiNWs) substrates for surface-enhanced Raman spectroscopy (SERS) applications. The novel SERS substrates are described in detail considering three main aspects. First, the key synthesis parameters for the flexible nanostructure SERS substrates were optimized. It is shown that fabrication temperature and metal-plating duration significantly influence the flexibility of the SiNWs and, consequently, determine the SERS enhancement. Second, it is demonstrated how the immersion in a liquid followed by drying results in the formation of SiNWs bundles influencing the surface morphology. The morphology changes were described by fractal dimension and lacunar analyses and correlated with the duration of Ag plating and SERS measurements. SERS examination showed the optimal intensity values for SiNWs thickness values of 60–100 nm. That is, when the flexibility of the self-assembly SiNWs allowed hot spots occurrence. Finally, the test with 4-mercaptophenylboronic acid showed excellent SERS performance of the flexible, horizontally oriented SiNWs in comparison with several other commercially available substrates.

Stability of cytoplasmic nanoviscosity during cell cycle of HeLa cells synchronized with Aphidicolin

K. Szczepański, K. Kwapiszewska and R. Hołyst

Scientific Reports, 2019, 9, 16486

Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and – as a consequence – rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.

Dissolving without mixing

R. Hołyst and P. Garstecki

Nature, 2019, 574, 181-182

Microfluidic devices have revolutionized biological assays, but complex set-ups are required to prevent the unwanted mixing of reagents in the liquid samples being analysed. A simpler solution has just been found.

TMA, A Forgotten Uremic Toxin, but Not TMAO, Is Involved in Cardiovascular Pathology

K. Jaworska, D. Hering , G. Mosieniak, A. Bielak-Zmijewska, M. Pilz, M. Konwerski, A. Gasecka, A. Kapłon-Cieślicka, K. Filipiak, E. Sikora, R. Hołyst and M. Ufnal

Toxins 2019, 11(9), 490

Trimethylamine-N-oxide (TMAO) has been suggested as a marker and mediator of cardiovascular diseases. However, data are contradictory, and the mechanisms are obscure. Strikingly, the role of the TMAO precursor trimethylamine (TMA) has not drawn attention in cardiovascular studies even though toxic effects of TMA were proposed several decades ago. We assessed plasma TMA and TMAO levels in healthy humans (HH) and cardiovascular patients qualified for aortic valve replacement (CP). The cytotoxicity of TMA and TMAO in rat cardiomyocytes was evaluated using an MTT test. The effects of TMA and TMAO on albumin and lactate dehydrogenase (LDH) were assessed using fluorescence correlation spectroscopy. In comparison to HH, CP had a two-fold higher plasma TMA (p < 0.001) and a trend towards higher plasma TMAO (p = 0.07). In CP plasma, TMA was inversely correlated with an estimated glomerular filtration rate (eGFR, p = 0.002). TMA but not TMAO reduced cardiomyocytes viability. Incubation with TMA but not TMAO resulted in the degradation of the protein structure of LDH and albumin. In conclusion, CP show increased plasma TMA, which is inversely correlated with eGFR. TMA but not TMAO exerts negative effects on cardiomyocytes, likely due to its disturbing effect on proteins. Therefore, TMA but not TMAO may be a toxin and a marker of cardiovascular risk.

Phenotypic plasticity of Escherichia coli upon exposure to physical stress induced by ZnO nanorods

K. Matuła, Ł. Richter, M. Janczuk-Richter, W. Nogala, M. Grzeszkowiak, B. Peplińska, S. Jurga, E. Wyroba, Sz. Suski, H. Bilski, A. Silesian, H. A. R. Bluyssen, N. Derebecka, J. Wesoły, J. M. Łoś, M. Łoś, P. Decewicz, Ł. Dziewit, J. Paczesny and R. Hołyst

Scientific Reports, 2019, 9, 8575

Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed Ecoli to a large number of collisions of around 105 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria’s shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such Ecoli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in β-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Flux and storage of energy in nonequilibrium stationary states

R. Holyst, A. Maciołek, Y. Zhang, M. Litniewski, P. Knychała, M. Kasprzak and M. Banaszak

Phys. Rev. E, 2019, 99, 042118

Systems kept out of equilibrium in stationary states by an external source of energy store an energy ΔU=UU0U0 is the internal energy at equilibrium state, obtained after the shutdown of energy input. We determine ΔU for two model systems: ideal gas and a Lennard-Jones fluid. ΔU depends not only on the total energy flux, JU, but also on the mode of energy transfer into the system. We use three different modes of energy transfer where the energy flux per unit volume is (i) constant, (ii) proportional to the local temperature, and (iii) proportional to the local density. We show that ΔU/JU=τ is minimized in the stationary states formed in these systems, irrespective of the mode of energy transfer. τ is the characteristic timescale of energy outflow from the system immediately after the shutdown of energy flux. We prove that τ is minimized in stable states of the Rayleigh-Benard cell.

Kinetics and equilibrium constants of oligonucleotides at low concentrations. Hybridization and melting study.

K. Bielec, K. Sozanski, M. Seynen, Z. Dziekan, P. Rein ten Wolde and R. Holyst

Phys. Chem. Chem. Phys., 2019,21, 10798-10807

Although DNA hybridization/melting is one of the most important biochemical reactions, the non-trivial kinetics of the process is not yet fully understood. In this work, we use Förster resonance energy transfer (FRET) to investigate the influence of temperature, ionic strength, and oligonucleotide length on the kinetic and equilibrium constants of DNA oligonucleotide binding and dissociation. We show that at low reagent concentrations and ionic strength, the time needed to establish equilibrium between single and double strand forms may be of the order of days, even for simple oligonucleotides of a length of 20 base pairs or less. We also identify and discuss the possible artifacts related to fluorescence-based experiments conducted in extremely dilute solutions. The results should prove useful for the judicious design of technologies based on DNA-matching, including sensors, DNA multiplication, sequencing, and gene manipulation.

Determination of oligomerization state of Drp1 protein in living cells at nanomolar concentrations

K. Kwapiszewska, T. Kalwarczyk, B. Michalska, K. Szczepański, J. Szymański, P. Patalas-Krawczyk, T. Andryszewski, M. Iwan, J. Duszyński and R. Hołyst

Scientific Reports, 2019, 9, 5906

Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo, thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, KD) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe’s size, and provide a model for its determination for different protein sizes (1–70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that KD for tetramers was 0.7 ± 0.5 μM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of KD for other protein-protein complexes, or drug-target interactions.

Fluorescence correlation spectroscopy for multiple-site equilibrium binding: a case of doxorubicin–DNA interaction

X. Zhang, A. Poniewierski, K. Sozański, Y. Zhou, A. Brzozowska-Elliott and R. Holyst

Phys. Chem. Chem. Phys., 2019, 21, 1572-1577

Quantitative description of the interaction between doxorubicin (DOX), a broadly used anticancer drug, and DNA is the key to understand the action mechanism and side effects of its clinical use. However, the reported equilibrium constants of DOX–DNA interaction obtained using a range of different analytical methods vary even by several orders of magnitude. Herein, we propose a novel application of a single-molecule technique – fluorescence correlation spectroscopy (FCS) – to probe the interaction between DOX and two types of DNA (pUC19 and calf thymus DNA), taking advantage of intrinsic self-fluorescence of DOX. We provide an analytical formula for autocorrelation analysis to determine the equilibrium constant of DOX–DNA complex-formation, where binding of multiple DOX molecules to a DNA chain is included in the reaction–diffusion model. Our FCS-based method not only quantitatively revealed the values of equilibrium constant, but also implied that the stability of DOX–DNA complex is related to the types of base pair rather than the length or structure of the DNA. This work opens a promising pathway toward quantitative determination of molecular interactions in complex systems such as living cells or organisms at single-molecule level.

Propagation of Oscillating Chemical Signals through Reaction Networks

R. Roszak, M. D. Bajczyk, E. P. Gajewska, R. Hołyst and B. A. Grzybowski

Angewandte Chemie International Edition, 2019, 58, 14, 4520-4525

Akin to electronic systems that can tune to and process signals of select frequencies, systems/networks of chemical reactions also “propagate” time‐varying concentration inputs in a frequency‐dependent manner. Whereas signals of low frequencies are transmitted, higher frequency inputs are dampened and converted into steady‐concentration outputs. Such behavior is observed in both idealized reaction chains as well as realistic signaling cascades, in the latter case explaining the experimentally observed responses of such cascades to input calcium oscillations. These and other results are supported by numerical simulations within the freely available Kinetix web application we developed to study chemical systems of arbitrary architectures, reaction kinetics, and boundary conditions.

Flow-Induced Surface Charge Heterogeneity in Electrokinetics due to Stern-Layer Conductance Coupled to Reaction Kinetics

B. L. Werkhoven, J. C. Everts, S. Samin and R. van Roij

Phys. Rev. Lett. 2018, 120, 264502

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)].

Complex electric double layers in charged topological colloids

J. C. Everts and M. Ravnik

Scientific Reports 2018, 8, 14119

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.

Insight into the fission mechanism by quantitative characterization of Drp1 protein distribution in the living cell

B. M. Michalska, K. Kwapiszewska, J. Szczepanowska, T. Kalwarczyk, P. Patalas-Krawczyk, K. Szczepański, R. Hołyst, J.Duszyński and J. Szymański

Scientific Reports, 2018, 8, 8122

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 Kd 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.

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