Label-Free Optical Method for Quantifying Molecular Transport Across Cellular Membranes In Vitro

We demonstrate a nonlinear optical method for the label-free quantification of membrane transport rates of small/medium size molecules in living cells. Specifically, second-harmonic generation (SHG) laser scattering permits surface-specific characterization of transport across membranes. Unfortunately, most biologically relevant molecules are SHG-inactive. In the interest of extending this methodology for characterizing transport of any molecule, we monitor the SHG produced from an SHG-active reference molecule, in the presence of an SHG-inactive target molecule-of-interest as both molecules compete to cross a membrane. Of significance, the SHG-inactive target transport rate can be deduced as a perturbation in the measured transport rate of the reference. As proof-of-principle, we examine competitive transport of the strongly SHG-active cation, malachite green (MG), in the presence of a weakly SHG-active dication, propidium (Pro), across the outer-membrane protein channels in living bacteria. Comparison of the extracted and directly measured Pro transport rates validates the effectiveness of the method

Azithromycin-Induced Changes to Bacterial Membrane Properties Monitored <i>in Vitro</i> by Second-Harmonic Light Scattering

We present a nonlinear light scattering method for monitoring, with real-time resolution and membrane specificity, changes in molecular adsorption, and transport at bacterial membranes induced by an antimicrobial compound. Specifically, time-resolved second-harmonic light scattering (SHS) is used to quantify azithromycin-induced changes to bacterial membrane permeability in colloidal suspensions of living <i>Escherichia coli</i>. Variations in membrane properties are monitored through changes in the adsorption and transport rates of malachite green, a hydrophobic cation that gives SHS signal. Regardless of concentration, instantaneous treatment with azithromycin showed no significant changes in membrane permeability. However, 1 h pretreatment with subminimum inhibitory concentrations of azithromycin induced an order-of-magnitude enhancement in the permeability of both the outer membrane and, through facilitation of a new transport mechanism, the cytoplasmic membrane of the bacteria as well. This study illustrates SHS as a novel tool for monitoring antimicrobial-induced changes to membrane properties in living bacteria

Mitigation of Thin-Film Composite Membrane Biofouling via Immobilizing Nano-Sized Biocidal Reservoirs in the Membrane Active Layer

This work investigates the use of a silver-based metal–organic framework (MOF) for mitigating biofouling in forward-osmosis thin-film composite (TFC) membranes. This is the first study of the use of MOFs for biofouling control in membranes. MOF nanocrystals were immobilized in the active layer of the membranes via dispersion in the organic solution used for interfacial polymerization. Field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) characterization results showed the presence of the MOF nanocrystals in the active layer of the membranes. The immobilization improved the membrane active layer in terms of hydrophilicity and transport properties without adversely affecting the selectivity. It imparted antibacterial activity to the membranes; the number of live bacteria attached to the membrane surface was over 90% less than that of control membranes. Additionally, the MOF nanocrystals provided biocidal activity that lasted for 6 months. The immobilization improved biofouling resistance in the membranes, whose flux had a decline of 8% after 24 h of operation in biofouling experiments, while that of the control membranes had a greater decline of ∼21%. The better biofouling resistance is due to simultaneous improvement of antiadhesive and antimicrobial properties of the membranes. Fluorescence microscopy and FE-SEM indicated simultaneous improvement in antiadhesive and antimicrobial properties of the TFN membranes, resulting in limited biofilm formation