Multi-parametric Surface Plasmon Resonance Research Articles

Properties of APTES-Modified CNC Films.

Sulfated cellulose nanocrystals' (CNCs') facile aqueous dispersibility enables producing films, fibers, and other materials using only water as a solvent but prevents using sulfated CNCs in applications that require water immersion. We report that modifying CNCs with 3-aminopropyl-triethoxysilane (APTES) via a simple, single-pot reaction scheme dramatically improves the hydrolytic stability of CNC films. The effects of APTES modification on CNCs' properties were studied using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force and optical microscopy, thermogravimetric analysis, dynamic light scattering, and ultimate analysis. Substituting a mere 12.6% of the CNCs' available hydroxyl groups with APTES dramatically increased the hydrolytic stability of shear cast films while only having minor impacts on their mechanical properties. In addition, quartz crystal microbalance with dissipation monitoring (QCMD) and multiparametric surface plasmon resonance (MP-SPR) studies showed that the CNC-APTES films also had a greater irreversible binding with carbofuran, a pesticide and emerging contaminant. These results highlight that APTES modification is a promising method for increasing the utility of sulfated CNCs in sensors, adsorbents, and other applications requiring water immersion.

Monitoring silica core@shell nanoparticle‐bacterial film interactions using the multi‐parametric surface plasmon resonance technique

AbstractIn a healthcare setting, biofilms are a major source of infection and difficult to eradicate once formed. Nanoparticles (NPs) can be designed to effectively penetrate biofilms to more efficiently either deliver antibiotic drugs throughout the biofilm matrix or elicit inherent antibiofilm activity. Antibacterial cerium oxide (CeO2) NPs were employed as core material and coated with a mesoporous silica shell (MSN) to generate cerium oxide coated mesoporous silica NPs (CeO2@MSN). Detailed studies of NP‐biofilm interactions are required to rationally develop NP platforms to prevent biofilm‐related infections. This work developed and implemented a unique label‐free analysis platform for the real‐time monitoring of bacterial biofilm formation and then assessed the interactions of antibacterial NPs. An analysis platform which allows bacterial biofilms to grow and develop in situ in flow within the multi‐parametric surface plasmon resonance (MP‐SPR) instrument was established. This enabled simultaneous monitoring and detection of biofilm growth phases, structure, and interactions between differentially charged CeO2@MSNs and bacterial biofilms. Positively charged antibacterial NPs (polyethyleneimine functionalized CeO2@MSNs) were found to be the most efficient to penetrate the biofilm. The MP‐SPR analysis platform was shown to be a powerful tool for monitoring biofilm development in real‐time and to analyze biofilm properties and NP‐biofilm interactions.

A stable physisorbed layer of packed capture antibodies for high-performance sensing applications.

Antibody physisorption at a solid interface is a very interesting phenomenon that has important effects on applications such as the development of novel biomaterials and the rational design and fabrication of high-performance biosensors. The strategy selected to immobilize biorecognition elements can determine the performance level of a device and one of the simplest approaches is physical adsorption, which is cost-effective, fast, and compatible with printing techniques as well as with green-chemistry processes. Despite its huge advantages, physisorption is very seldom adopted, as there is an ingrained belief that it does not lead to high performance because of its lack of uniformity and long-term stability, which, however, have never been systematically investigated, particularly for bilayers of capture antibodies. Herein, the homogeneity and stability of an antibody layer against SARS-CoV-2-Spike1 (S1) protein physisorbed onto a gold surface have been investigated by means of multi-parametric surface plasmon resonance (MP-SPR). A surface coverage density of capture antibodies as high as (1.50 ± 0.06) × 1012 molecules per cm-2 is measured, corresponding to a thickness of 12 ± 1 nm. This value is compatible with a single monolayer of homogeneously deposited antibodies. The effect of the ionic strength (is) of the antibody solution in controlling physisorption of the protein was thoroughly investigated, demonstrating an enhancement in surface coverage at lower ionic strength. An atomic force microscopy (AFM) investigation shows a globular structure attributed to is-related aggregations of antibodies. The long-term stability over two weeks of the physisorbed proteins was also assessed. High-performance sensing was proven by evaluating figures of merit, such as the limit of detection (2 nM) and the selectivity ratio between a negative control and the sensing experiment (0.04), which is the best reported performance for an SPR S1 protein assay. These figures of merit outmatch those measured with more sophisticated biofunctionalization procedures involving chemical bonding of the capture antibodies to the gold surface. The present study opens up interesting new pathways toward the achievement of a cost-effective and scalable biofunctionalization protocol, which could guarantee the prolonged stability of the biolayer and easy handling of the biosensing system.

Mechanisms of Influenza Virus HA2 Peptide Interaction with Liposomes Studied by Dual-Wavelength MP-SPR.

A phospholipid-based liposome layer was used as an effective biomimetic membrane model to study the binding of the pH-dependent fusogenic peptide (E4-GGYC) from the influenza virus hemagglutinin HA2 subunit. To this end, a multiparameter surface plasmon resonance approach (MP-SPR) was used for monitoring peptide-liposome interactions at two pH values (4.5 and 8) by means of recording sensorgrams in real time without the need for labeling. Biotinylated liposomes were first immobilized as a monolayer onto the surface of an SPR gold chip coated with a streptavidin layer. Multiple sets of sensorgrams with different HA2 peptide concentrations were generated at both pHs. Dual-wavelength Fresnel layer modeling was applied to calculate the thickness (d) and the refractive index (n) of the liposome layer to monitor the change in its optical parameters upon interaction with the peptide. At acidic pH, the peptide, in its α helix form, entered the lipid bilayer of liposomes, inducing vesicle swelling and increasing membrane robustness. Conversely, a contraction of liposomes was observed at pH 8, associated with noninsertion of the peptide in the double layer of phospholipids. The equilibrium dissociation constant KD = 4.7 × 10-7 M of the peptide/liposome interaction at pH 4.5 was determined by fitting the "OneToOne" model to the experimental sensorgrams using Trace Drawer software. Our experimental approach showed that the HA2 peptide at a concentration up to 100 μM produced no disruption of liposomes at pH 4.5.

Multi‐parametric surface plasmon resonance‐based intake quantification of label‐free light‐activated nanoparticles by therapeutic limbal stem cells for corneal blindness

AbstractAdvanced therapies with combined approaches of cell and nanomedicine‐based interventions are emerging. Corneal blindness with a severe form of limbal stem cell (LSC) deficiency is an example of defect with unmet clinical need. Towards cell‐based therapy, advanced enabling technologies are needed for efficient intracellular delivery of biomolecules both for in vitro disease modeling and for in vivo interventions. For this nanomedicine creates novel possibilities as light activatable polymeric nanoparticles (NPs) hold potential for controlled on‐demand cargo delivery with control of light. In this study, we used multi‐parametric surface plasmon resonance (MP‐SPR) technique in vitro for measuring NP intake in real‐time by therapeutic LSCs. Although a variety of NPs has been described, many challenges remain, and one is related to cell uptake of the formulations. Here, three different NP formulations were analyzed, one demonstrating clearly the highest cellular intake by LSCs. Importantly, data demonstrate that the labeling of NPs significantly reduce the cellular intake highlighting the importance of label‐free administration and quantification. The MP‐SPR based approach hold high potential as a powerful non‐invasive platform to be implemented in manufacturing of therapeutic cell‐ and cargo‐based interventions. For any therapy applications, validated non‐invasive and label independent NP intake measurement systems would be important.

Refractive index and temperature dual parameter sensor based on a twin-core photonic crystal fiber

A twin-core photonic crystal fiber sensor is proposed for measuring liquid refractive index (RI) and temperature simultaneously. The air holes of the sensor are arranged in a hexagonal pattern, and two planes are introduced by polishing in the cladding. On one side of the plane, the gold film is deposited for RI measurement, and on the other side, the gold film and polydimethylsiloxane are deposited for temperature measurement. We analyzed its sensing characteristics by using the finite element method. The numerical results show that the two channels for measuring RI and temperature have no mutual interference and the arrangement reduces the complexity of the sensing measurement. The maximum spectral sensitivity of the sensor is 20 000 nm/RIU and 9.2 nm °C−1, respectively, when the liquid RI is in the range of 1.36–1.42 and the temperature is in the range of 0 °C–50 °C. The results also show the sensing accuracy was not very sensitive to the change of structural parameters, which makes the sensor very easy to fabricate. Our work is very helpful for implementation of a high sensitivity, easy fabrication and real-time multi-parameter surface plasmon resonance sensor.

Polystyrene sulfonate is effective for enhancing biomass enzymatic saccharification under green liquor pretreatment in bioenergy poplar

BackgroundWater-soluble lignin (particularly lignosulfonate, LS) has been well documented for its significance on enzymatic saccharification of lignocellulose, though the promotion mechanism has not been fully understood. Much attention has been paid to natural lignin or its derivatives. The disadvantage of using natural lignin-based polymers as promoting agents lies in the difficulty in tailor-incorporating functional groups due to their complex 3D structures. To further improve our understanding on the promotion mechanism of water-soluble lignin in the bioconversion of lignocellulose and to pursue better alternatives with different skeleton structures other than natural lignin, herein we reported a synthetic soluble linear aromatic polymer, sodium polystyrene sulfonate (PSS), to mimic LS for enhancing the efficiency of enzymatic saccharification.ResultsThe role of PSS in enzymatic saccharification of pure cellulose and green liquor-pretreated poplar (GL-P) was explored by analyzing substrate enzymatic digestibility (SED) under different addition dosages and various pH media, along with LS for comparison. At the cellulase loading of 13.3 FPU/g-glucan, the glucose yield of GL-P increased from 53% for the control to 81.5% with PSS addition of 0.1 g/g-substrate. It outperformed LS with the addition of 0.2 g/g-substrate by 6.3%. In the pH range from 4.5 to 6, PSS showed a positive effect on lignocellulose saccharification with the optimum pH at 4.8, where the most pronounced SED of GL-P was achieved. The underlying mechanism was unveiled by measuring zeta potential and using Quartz Crystal Microbalance (QCM) and Multi-parametric Surface Plasmon Resonance (MP-SPR). The results confirmed that the complexes of cellulase and PSS were conjugated and the negatively supercharged complexes reduced non-productive binding effectively along with the improved saccharification efficiency. The thickness of PSS required to block the binding sites of cellulase film was less than half of that of LS, and the PSS adlayer on cellulase film is also more hydrated and with a much lower shear modulus than LS adlayer.ConclusionsPSS as LS analogue is effective for enhancing the biomass enzymatic saccharification of GL-pretreated poplar. PSS exhibited a severer inhibition on the enzymatic saccharification of pure cellulose, while a more positive effect on bioconversion of lignocellulose (GL-P) than LS. In addition, a much lower dosage is required by PSS. The dynamic enzymatic hydrolysis indicated PSS could prolong the processive activity of cellulase. The valid data stemmed from QCM and SPR expressed that PSS bound to cellulases and the as-formed complexes reduced the non-productive adsorption of cellulase onto substrate lignin more efficiently than LS due to its flexible skeleton and highly hydrated structure. Therefore, PSS is a promising alternative promoting agent for lignocellulose saccharification. From another perspective, the synthetic lignin mimics with controllable structures enable us to reach an in-depth understanding of the promotion mechanism of soluble lignins on enzymatic saccharification.

Characterization of protein corona formation on nanoparticles via the analysis of dynamic interfacial properties: Bovine serum albumin - silica particle interaction

Protein corona adsorption layers on nanoparticle surfaces, dispersed in biological fluids, can significantly change the interfacial interactions, reactivity, and mobility of the original nanoparticles designed as a drug carrier or existing as bioaerosols, bacteria, or viruses. The evaluation of the level of interactions (hard/soft corona), dispersion stability, and the ratio of the attached proteins per nanoparticle are essential parameters for the characterization of the protein corona formation on nanoparticle (PCN). In spite of development of several experimental techniques for this purpose, still more powerful, economical and fast measuring techniques are needed to work in-situ at original solution samples (near the physiological conditions), without requirement of additional sample preparation which can change the quality/quantity of the original interactions. In this study, a novel experimental protocol based on the analysis of dynamic interfacial properties (ADIP) is developed for in-situ evaluation of the protein–nanoparticle interactions under original conditions. For this purpose, dynamic surface tension and interfacial elasticity values of the bovine serum albumin (BSA) solutions alone and in mixed solutions with silica nanofluids are measured using drop profile analysis tensiometry. A considerable difference between the dynamic surface tension of BSA and protein corona solutions (BSA + SiO2 NPs complexes) demonstrates significant adsorption of the protein molecules at nanoparticles, confirmed by Fourier Transform Infrared Spectroscopy (FTIR) analysis. PCN complexes illustrate much slower kinetics of adsorption due to a smaller diffusion coefficient according to their larger size. The free proteins available in the solution were estimated considering early-time values of the dynamic surface tension, used for estimation of the adsorbed proteins per unit area of the silica surface (mol/cm2), considering the initial protein concentration in the bulk. The results show very good agreement with others’ results provided by AFM, DLS, UV–vis Spectroscopy, Multi-Parametric Surface Plasmon Resonance (MP-SPR), and Quartz Crystal Microbalance (QCM). Our novel experimental protocols and data analysis, demonstrates promising results for differentiation of the hard and soft corona layers, and unique for soft corona layer recognition, which is difficult by other available techniques, due to quick disturbances of weak protein-protein interaction in soft corona. The measured interfacial elasticity values confirm PCN formation and provides additional information for better differentiations of the corona layers.

Label-free methods of multiparametric surface plasmon resonance and MPQ-cytometry for quantitative real-time measurements of targeted magnetic nanoparticles complexation with living cancer cells

Most of the methods used today for investigation of nanoparticle binding to living cells imply a quantitative or qualitative analysis and do not provide information on the binding dynamics. To meet the challenge, we show here for the first time that the interaction of metal nanoparticles with eukaryotic cells in the flow system in dynamics can be studied by the method of multiparametric surface plasmon resonance. To this aim, we obtained a spectrum of colloidally stable targeted magnetite nanoparticles modified by phytolectins (SBA, WGA, ConA) of different specificity to monosaccharides (GalNAc, GlcNAc, and Man, respectively), and studied the interaction of these conjugates with human epidermoid carcinoma A431 cells. We showed that multiparametric surface plasmon resonance can be effectively used as a label-free method to study the process of dynamic mass transfer in the nanoparticle*cell system in the fluid cell. We show that not only the commonly used parameter in such systems, the angle of the minimum peak, θSPR, at the full angular spectrum, but also the intensity, I, of this peak, can be used to study the binding of targeted nanoparticles with living cells in dynamics. This is due to the contribution of metal nanoparticles to resonant absorption of incident electromagnetic radiation by free electrons at the interface between media with different refractive indices. To get the most relevant quantitative data on nanoparticle binding to the cell surface, we combined this assay with our original MPQ-cytometry method (Magnetic Particle Quantification based cytometry) precisely quantifying the nanoparticles in the static cell sample. By the combination of label-free plasmonic and magnetometry-based MPQ-cytometry methods, we showed that nanoparticles modified with agglutinin from soybeans, SBA, most efficiently (up to 4.2 ± 0.1 pg/cell) bind to epidermoid carcinoma cells with achieving a saturation at 12 min

Lipid Cubic Systems for Sustained and Controlled Delivery of Antihistamine Drugs.

Antihistamines are capable of blocking mediator responses in allergic reactions including allergic rhinitis and dermatological reactions. By incorporating various H1 receptor antagonists into a lipid cubic phase network, these active ingredients can be delivered locally over an extended period of time owing to the mucoadhesive nature of the system. Local delivery can avoid inducing unwanted side effects, often observed after systematic delivery. Lipid-based antihistamine delivery systems are shown here to exhibit prolonged release capabilities. In vitro drug dissolution studies investigated the extent and release rate of two model first-generation and two model second-generation H1 antagonist antihistamine drugs from two monoacyglycerol-derived lipid models. To optimize the formulation approach, the systems were characterized macroscopically and microscopically by small-angle X-ray scattering and polarized light to ascertain the mesophase accessed upon an incorporation of antihistamines of varying solubilities and size. The impact of encapsulating the antihistamine molecules on the degree of mucoadhesivity of the lipid cubic systems was investigated using multiparametric surface plasmon resonance. With the ultimate goal of developing therapies for the treatment of allergic reactions, the ability of the formulations to inhibit mediator release utilizing RBL-2H3 mast cells with the propensity to release histamine upon induction was explored, demonstrating no interference from the lipid excipient on the effectiveness of the antihistamine molecules.

Surface Plasmon Resonance Assay for Label‐Free and Selective Detection of Xylella Fastidiosa

Xylella fastidiosa is among the most dangerous plant bacteria worldwide causing a variety of diseases, with huge economic impact on agriculture and environment. A surveillance tool, ensuring the highest possible sensitivity enabling the early detection of X. fastidiosa outbreaks, would be of paramount importance. So far, a variety of plant pathogen biomarkers are studied by means of surface plasmon resonance (SPR). Herein, multiparameter SPR (MP‐SPR) is used for the first time to develop a reliable and label‐free detection method for X. fastidiosa. The real‐time monitoring of the bioaffinity reactions is provided as well. Selectivity is guaranteed by biofunctionalizing the gold transducing interface with polyclonal antibodies for X. fastidiosa and it is assessed by means of a negative control experiment involving the nonbinding Paraburkholderia phytofirmans bacterium strain PsJN. Limit of detection of 105 CFU mL−1 is achieved by transducing the direct interaction between the bacterium and its affinity antibody. Moreover, the binding affinity between polyclonal antibodies and X. fastidiosa bacteria is also evaluated, returning an affinity constant of 3.5 × 107 m−1, comparable with those given in the literature for bacteria detection against affinity antibodies.

Surface Plasmon Resonance Monitoring of Mono-Rhamnolipid Interaction with Phospholipid-Based Liposomes.

The interactions of mono-rhamnolipids (mono-RLs) with model membranes were investigated through a biomimetic approach using phospholipid-based liposomes immobilized on a gold substrate and also by the multiparametric surface plasmon resonance (MP-SPR) technique. Biotinylated liposomes were bound onto an SPR gold chip surface coated with a streptavidin layer. The resulting MP-SPR signal proved the efficient binding of the liposomes. The thickness of the liposome layer calculated by modeling the MP-SPR signal was about 80 nm, which matched the average diameter of the liposomes. The mono-RL binding to the film of the phospholipid liposomes was monitored by SPR and the morphological changes of the liposome layer were assessed by modeling the SPR signal. We demonstrated the capacity of the MP-SPR technique to characterize the different steps of the liposome architecture evolution, i.e., from a monolayer of phospholipid liposomes to a single phospholipid bilayer induced by the interaction with mono-RLs. Further washing treatment with Triton X-100 detergent left a monolayer of phospholipid on the surface. As a possible practical application, our method based on a biomimetic membrane coupled to an SPR measurement proved to be a robust and sensitive analytical tool for the detection of mono-RLs with a limit of detection of 2 μg mL-1.

Adsorption and Conformation Behavior of Lysozyme on a Gold Surface Determined by QCM-D, MP-SPR, and FTIR.

The physicochemical properties of protein layers at the solid–liquid interface are essential in many biological processes. This study aimed to link the structural analysis of adsorbed lysozyme at the water/gold surface at pH 7.5 in a wide range of concentrations. Particular attention was paid to the protein’s structural stability and the hydration of the protein layers formed at the interface. Complementary methods such as multi-parameter surface plasmon resonance (MP-SPR), quartz crystal microbalance with energy dissipation (QCM-D), and infrared spectroscopy (FTIR) were used for this purpose. The MP-SPR and QCM-D studies showed that, during the formation of a monolayer on the gold surface, the molecules’ orientation changes from side-on to end-on. In addition, bilayer formation is observed when adsorbing in the high-volume concentration range >500 ppm. The degree of hydration of the monolayer and bilayer varies depending on the degree of surface coverage. The hydration of the system decreases with filling the layer in both the monolayer and the bilayer. Hydration for the monolayer varies in the range of 50–70%, because the bilayer is much higher than 80%. The degree of hydration of the adsorption layer has a crucial influence on the protein layers’ viscoelastic properties. In general, an increase in the filling of a layer is characterized by a rise in its rigidity. The use of infrared spectroscopy allowed us to determine the changes taking place in the secondary structure of lysozyme due to its interaction with the gold surface. Upon adsorption, the content of II-structures corresponding to β-turn and random lysozyme structures increases, with a simultaneous decrease in the content of the β-sheet. The increase in the range of β-turn in the structure determines the lysozyme structure’s stability and prevents its aggregation.

Interactions and Dissociation Constants of Galactomannan Rendered Cellulose Films with Concavalin A by SPR Spectroscopy.

Interactions of biomolecules at interfaces are important for a variety of physiological processes. Among these, interactions of lectins with monosaccharides have been investigated extensively in the past, while polysaccharide-lectin interactions have scarcely been investigated. Here, we explore the adsorption of galactomannans (GM) extracted from Prosopis affinis on cellulose thin films determined by a combination of multi-parameter surface plasmon resonance spectroscopy (MP-SPR) and atomic force microscopy (AFM). The galactomannan adsorbs spontaneously on the cellulose surfaces forming monolayer type coverage (0.60 ± 0.20 mg·m−2). The interaction of a lectin, Concavalin A (ConA), with these GM rendered cellulose surfaces using MP-SPR has been investigated and the dissociation constant KD (2.1 ± 0.8 × 10−8 M) was determined in a range from 3.4 to 27.3 nM. The experiments revealed that the galactose side chains as well as the mannose reducing end of the GM are weakly interacting with the active sites of the lectins, whereas these interactions are potentially amplified by hydrophobic effects between the non-ionic GM and the lectins, thereby leading to an irreversible adsorption.

Gamma estimator of Jarzynski equality for recovering binding energies from noisy dynamic data sets

A fundamental problem in thermodynamics is the recovery of macroscopic equilibrated interaction energies from experimentally measured single-molecular interactions. The Jarzynski equality forms a theoretical basis in recovering the free energy difference between two states from exponentially averaged work performed to switch the states. In practice, the exponentially averaged work value is estimated as the mean of finite samples. Numerical simulations have shown that samples having thousands of measurements are not large enough for the mean to converge when the fluctuation of external work is above 4 kBT, which is easily observable in biomolecular interactions. We report the first example of a statistical gamma work distribution applied to single molecule pulling experiments. The Gibbs free energy of surface adsorption can be accurately evaluated even for a small sample size. The values obtained are comparable to those derived from multi-parametric surface plasmon resonance measurements and molecular dynamics simulations.

In-situ and real-time probing cellulase biosensor formation and its interaction with lignosulfonate in varied media

It is a challenge to interpret the signals obtained by Quartz Crystal Microbalance (QCM) and Surface Plasmon Resonance (SPR) accompanied with in the significant changes of the density and/or refractivity of media. The work presented herein focuses on demonstrating a universal in-situ and real-time monitoring methodology for ultrathin film formation on gold chips in varied media. Biomimetic model cellulase/enzyme film was immobilized in-situ on gold surface of sensors, followed by revealing its interaction with lignosulphonate in the media changed from ethanol-based to aqueous ones. Combining the techniques of Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and Multi-parametric Surface Plasmon Resonance (MP-SPR) along with respective software and models enabled us to precisely characterize the thickness, viscous and shear elastic moduli, and the coupled solvent for individual layer formed over the entire process, which are unachievable with conventional methods. The methodology provides real-time monitoring of filming process and offers us an in-situ qualify control approach to the biomimetic model films formed on different substrates including gold chip.

Effect of laminin, polylysine and cell medium components on the attachment of human hepatocellular carcinoma cells to cellulose nanofibrils analyzed by surface plasmon resonance

The development of in vitro cell models that mimic cell behavior in organs and tissues is an approach that may have remarkable impact on drug testing and tissue engineering applications in the future. Plant-based, chemically unmodified cellulose nanofibrils (CNF) hydrogel is a natural, abundant, and biocompatible material that has attracted great attention for biomedical applications, in particular for three-dimensional cell cultures. However, the mechanisms of cell-CNF interactions and factors that affect these interactions are not yet fully understood. In this work, multi-parametric surface plasmon resonance (SPR) was used to study how the adsorption of human hepatocellular carcinoma (HepG2) cells on CNF films is affected by the different proteins and components of the cell medium. Both human recombinant laminin-521 (LN-521, a natural protein of the extracellular matrix) and poly-l-lysine (PLL) adsorbed on CNF films and enhanced the attachment of HepG2 cells. Cell medium components (glucose and amino acids) and serum proteins (fetal bovine serum, FBS) also adsorbed on both bare CNF and on protein-coated CNF substrates. However, the adsorption of FBS hindered the attachment of HepG2 cells to LN-521- and PLL-coated CNF substrates, suggesting that serum proteins blocked the formation of laminin-integrin bonds and decreased favorable PLL-cell electrostatic interactions. This work sheds light on the effect of different factors on cell attachment to CNF, paving the way for the utilization and optimization of CNF-based materials for different tissue engineering applications.

It takes two to tango - The case of thebaine 6-O-demethylase

Thebaine 6-O-demethylase (T6ODM) is an Fe(II)/2-oxoglutarate-dependent dioxygenase catalysing two oxidative O-demethylation reactions in morphine biosynthesis. Its crystal structure revealed a large active site pocket which is at least two times larger than necessary to accommodate a substrate (thebaine or oripavine) molecule. Since so far no crystal structures have been obtained for enzyme-substrate complex, which is necessary to explain the enzyme regiospecificity towards the C6-bound methoxy group, in this work we used computational methods and multi-parametric surface plasmon resonance measurements to elucidate the most likely structure of this complex and the reaction mechanism starting therefrom. Results of simulations and experiments unanimously indicate that the enzyme-substrate complex of T6ODM has a 1:2 stoichiometry. The key residues responsible for substrate binding are: Val-128, Glu-133, Met-150 and Agr-219 for the substrate in the distal position, and Asp-144, Leu-235 and Leu-353 for the proximal substrate molecule. QM/MM and DFT calculations show that the oxo ligand is bound trans to His-295 and the enzyme catalyzes hydroxylation of the C6-bound methoxy group according to the established rebound mechanism. The final stage of the demethylation reaction, which includes deformylation and enol-keton tautomerization steps, is most likely catalysed by water molecules and takes place in the solvent.

Partitioning of Catechol Derivatives in Lipid Membranes: Implications for Substrate Specificity to Catechol-O-methyltransferase.

We have utilized multiparametric surface plasmon resonance and impendance-based quartz crystal microbalance instruments to study the distribution coefficients of catechol derivatives in cell model membranes. Our findings verify that the octanol–water partitioning coefficient is a poor descriptor of the total lipid affinity for small molecules which show limited lipophilicity in the octanol–water system. Notably, 3-methoxytyramine, the methylated derivative of the neurotransmitter dopamine, showed substantial affinity to the lipids despite its nonlipophilic nature predicted by octanol–water partitioning. The average ratio of distribution coefficients between 3-methoxytyramine and dopamine was 8.0. We also found that the interactions between the catechols and the membranes modeling the cell membrane outer leaflet are very weak, suggesting a mechanism other than the membrane-mediated mechanism of action for the neurotransmitters at the postsynaptic site. The average distribution coefficient for these membranes was one-third of the average value for pure phosphatidylcholine membranes, calculated using all compounds. In the context of our previous work, we further theorize that membrane-bound enzymes can utilize membrane headgroup partitioning to find their substrates. This could explain the differences in enzyme affinity between soluble and membrane-bound isoforms of catechol-O-methyltransferase, an essential enzyme in catechol metabolism.

Multilayer Density Analysis of Cellulose Thin Films.

An approach for the multilayer density analysis of polysaccharide thin films at the example of cellulose is presented. In detail, a model was developed for the evaluation of the density in different layers across the thickness direction of the film. The cellulose thin film was split into a so called “roughness layer” present at the surface and a “bulk layer” attached to the substrate surface. For this approach, a combination of multi-parameter surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM) was employed to detect changes in the properties, such as cellulose content and density, thickness and refractive index, of the surface near layer and the bulk layer. The surface region of the films featured a much lower density than the bulk. Further, these results correlate to X-ray reflectivity studies, indicating a similar layered structure with reduced density at the surface near regions. The proposed method provides an approach to analyse density variations in thin films which can be used to study material properties and swelling behavior in different layers of the films. Limitations and challenges of the multilayer model evaluation method of cellulose thin films were discussed. This particularly involves the selection of the starting values for iteration of the layer thickness of the top layer, which was overcome by incorporation of AFM data in this study.