Recent advances in bacteriophage-based methods for bacteria detection

Ł. Richter, M. Janczuk-Richter, J. Niedziółka-Jönsson, J. Paczesny, R. Hołyst

Drug Discovery Today, 2018, 23, 2, 448-455

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.

Colloid–oil–water-interface interactions in the presence of multiple salts: charge regulation and dynamics

J. C. Everts, S. Samin, N. A. Elbers, J. E. S. van der Hoeven, A. van Blaaderen and R. van Roij

Phys. Chem. Chem. Phys., 2017, 19, 14345-14357

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.

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level

X. Zhang, E. Sisamakis, K. Sozanski and R. Holyst

J. Phys. Chem. Lett. 2017, 8, 23, 5785–5791

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.

Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes’ Polydispersity

T. Kalwarczyk, K. Kwapiszewska, K. Szczepanski, K. Sozanski, J. Szymanski, B. Michalska, P. Patalas-Krawczyk, J. Duszynski, and R. Hołyst

J. Phys. Chem. B 2017, 121, 42, 9831–9837

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.

Evaporation of liquid droplets of nano- and micro-meter size as a function of molecular mass and intermolecular interactions: experiments and molecular dynamics simulations

R. Hołyst, M. Litniewski and D. Jakubczyk

Soft Matter, 2017, 13, 5858-5864

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, , (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 + ) in comparison to the flux obtained from temperature diffusion. Thus it slows down the evaporation of droplets of sizes R ∼  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(kBTb/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 Tb 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(kBTb/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).

Quantitative fluorescence correlation spectroscopy in three-dimensional systems under stimulated emission depletion conditions

K. Sozanski, E. Sisamakis, X. Zhang and R. Hołyst

Optica, 2017, 4, 8, 982-988

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.

Dense Layer of Bacteriophages Ordered in Alternating Electric Field and Immobilized by Surface Chemical Modification as Sensing Element for Bacteria Detection

Ł.Richter, K. Bielec, A. Leśniewski, M. Łoś, J. Paczesny and Robert Hołyst

ACS Appl. Mater. Interfaces 2017, 9, 23, 19622–19629

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.

Scaling Equation for Viscosity of Polymer Mixtures in Solutions with Application to Diffusion of Molecular Probes

A. Wisniewska, K. Sozanski, T. Kalwarczyk, K. Kedra-Krolik, and R. Holyst

Macromolecules 2017, 50, 11, 4555–4561

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.

Denaturation of proteins by surfactants studied by the Taylor dispersion analysis

A. Jelińska, A. Zagożdżon, M. Górecki, A. Wisniewska, J. Frelek and R. Holyst

PLOS ONE, 2017

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.

Analytical form of the autocorrelation function for the fluorescence correlation spectroscopy

R. Hołyst, A. Poniewierski and X. Zhang

Soft Matter, 2017, 13, 1267-1275

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].

Bacteriophage-Based Bioconjugates as a Flow Cytometry Probe for Fast Bacteria Detection

M. Janczuk, Ł. Richter, G. Hoser, J. Kawiak, M. Łoś, J. Niedziółka-Jönsson, J. Paczesny, and R. Hołyst

Bioconjugate Chem. 2017, 28, 2, 419–425

Robust detection of bacteria can significantly reduce risks of nosocomial infections, which are a serious problem even in developed countries (4.1 million cases each year in Europe). Here we demonstrate utilization of novel multifunctional bioconjugates as specific probes for bacteria detection. Bifunctional magnetic-fluorescent microparticles are coupled with bacteriophages. The T4 bacteriophage, due to its natural affinity to bacterial receptors, namely, OmpC and LPS, enables specific and efficient detection of Escherichia coli bacteria. Prepared probes are cheap, accessible (even in nonbiological laboratories), as well as versatile and easily tunable for different bacteria species. The magnetic properties of the bioconjugates facilitate the separation of captured target bacteria from other components of complex samples and other bacteria strains. Fluorescence enables simple analysis. We chose flow cytometry as the detection method as it is fast and widely used for biotests. The capture efficiency of the prepared bioconjugates is close to 100% in the range of bacteria concentrations from tens to around 105 CFU/mL. The limit of detection is restricted by flow cytometry capabilities and in our case was around 104 CFU/mL.

Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry

E. Witkowska, D. Korsak, A. Kowalska, M. Księżopolska-Gocalska, J. Niedziółka-Jönsson, E. Roźniecka, W. Michałowicz, P. Albrycht, M. Podrażka, R. Hołyst, J. Waluk and A.Kamińska

Analytical and Bioanalytical Chemistry, 2017, 409, 1555–1567

We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290–1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures.

Preparation of Biocompatible, Luminescent-Plasmonic Core/Shell Nanomaterials Based on Lanthanide and Gold Nanoparticles Exhibiting SERS Effects

M. Runowski, S. Goderski, J. Paczesny, M. Księżopolska-Gocalska, A. Ekner-Grzyb, T. Grzyb, J. D. Rybka, M. Giersig, and S. Lis

J. Phys. Chem. C 2016, 120, 41, 23788–23798

Multifunctional core/shell type nanomaterials composed of nanocrystalline, lanthanide doped fluorides and gold nanoparticles (Au NPs) were successfully prepared. The products were synthesized to combine luminescence properties of the core NPs, i.e., LnF3/SiO2–NH2 and KLn3F10/SiO2–NH2, and plasmonic activity of the shell Au NPs within a single nanomaterial. The luminescent lanthanide NPs (10 or 150–200 nm) were separated from the gold NPs (6–30 nm) using an amine modified silica shell (thickness ≈30 nm). The synthesized products exhibited bright green (Tb3+) and red (Eu3+) emission under UV light irradiation. Surface modification with Au NPs influenced the product emission and luminescence decay characteristics. The luminescent-plasmonic nanomaterials were used as platforms for surface enhanced Raman scattering (SERS) measurements. 4-Mercaptobenzoic acid, choline, and T4 bacteriophages were utilized as SERS probes. For all synthesized nanomaterials, the SERS spectra for all probes studied exhibited higher intensity in comparison with the spectra measured using a commercial SERS substrate. Cytotoxicity of the products was evaluated in fibroblast cells. The results obtained showed biocompatibility of the synthesized nanomaterials in a dose-dependent manner.

Density-induced reentrant melting of colloidal Wigner crystals

J. C. Everts, N. Boon and R. van Roij

Phys. Chem. Chem. Phys., 2016, 18, 5211-5218

Electrostatic repulsions can drive crystallization in many-particle systems. For charged colloidal systems, the phase boundaries as well as crystal structure are highly tunable by experimental parameters such as salt concentration and pH. By using projections of the colloid–ion mixture to a system of (soft) repulsive spheres and the one-component plasma (OCP), we study the hitherto unexplained experimentally observed reentrant melting of electrostatically repelling colloids upon increasing the colloid density. Our study shows that the surface chemistry should involve a competition between adsorption of cations and anions to explain the observed density-induced reentrant melting.

A Landau-de Gennes theory for hard colloidal rods: Defects and tactoids

J. C. Everts, . T. J. J. M. Punter, S. Samin, P. van der Schoot and R. van Roij

J. Chem. Phys. 2016, 144, 194901

We construct a phenomenological Landau-de Gennes theory for hard colloidal rods by performing an order parameter expansion of the chemical-potential dependent grand potential. By fitting the coefficients to known results of Onsager theory, we are not only able to describe the isotropic-nematic phase transition as function of density, including the well-known density jump, but also the isotropic-nematic planar interface. The resulting theory is applied in calculations of the isotropic core size in a radial hedgehog defect, the density dependence of linear defects of hard rods in square confinement, and the formation of a nematic droplet in an isotropic background.

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