Showing papers on "Substrate (chemistry) published in 2018"

Use of Fluorogenic Model Substrates for Extracellular Enzyme Activity (EEA) Measurement of Bacteria

Abstract: "ectoenzymes" and the latter "extracellular" enzymes. Substrates for hydrolysis are generally proteins, carbohydrates, fats and organic P- or S- compounds. Mechanisms of decomposition of individual compounds within these groups may be studied by in vitro experiments. However, aquatic microbial ecologists require, in many cases, a more general measurement of the in situ hydrolytic capacity of the prevailing bacterial community. This has led to the adaptation of biochemical methods for determination of overall bacterial extracellular enzyme activities (peptidases, a- and P-glucosidases, chi- tinases, etc.) in natural waters (Table 1). These methods enable us to study the impact of extracellular enzyme activity (EEA) on bacterial substrate uptake, bacterial growth, and water chemistry. The quantitative estimates of total bacterial extracellular enzyme activity are completed by rapid and sensitive tests for the detection of enzymatic properties of bacterial isolates. The methods used for these purposes are based on the application of fluorogenic model substrates. These substrates have some characteristics in common: (1) they contain an artificial fluorescent molecule and one or more natural molecules (e.g., glucose, amino acids), linked by a specific binding (e.g., peptide binding, ester binding); fluorescence is observed after enzymatic splitting of the complex molecule (Figure 1); (2) the hydrolysis of model substrates is competitively inhibited by a variety of natural compounds with the same structural characteristics; (3) hydrolysis of model substrates follows first order enzyme kinetics; and (4) application of those model substrates allows enzyme activity measurements under natural (in situ) conditions within short incubation periods. The latter is highly im- portant for microbial ecologists, because the process of enzymatic hydrolysis is fully

Substrate-driven chemotactic assembly in an enzyme cascade.

Abstract: Enzymes that form a metabolic pathway in which the product of one enzyme is the substrate for the next have now been shown to associate through a process of sequential, directed chemotactic movement. The extent of enzyme migration is proportional to the exposure time to the substrate gradient.

New insights into the inhibition mechanism of betulinic acid on α-glucosidase

Abstract: Betulinic acid (BA), an important pentacyclic triterpene widely distributed in many foods, possesses high antidiabetic activity. In this study, BA was found to exhibit stronger inhibition of α-glucosidase than acarbose with an IC50 value of (1.06 ± 0.02) × 10–5 mol L–1 in a mixed-type manner. BA bound with α-glucosidase to form a BA−α-glucosidase complex, resulting in a more compact structure of the enzyme. The obtained concentrations and spectra profiles of the components resolved by the multivariate-curve resolution–alternating least-squares confirmed the formation of the BA−α-glucosidase complex. Molecular docking showed that BA tightly bound to the active cavity of α-glucosidase, which might hinder the entrance of the substrate leading to a decline in enzyme activity. The chemical modification of α-glucosidase verified the results of the computer simulation that the order of importance of the four amino acid residues in the binding process was His > Tyr > Lys > Arg.

Vertical Alignment of Liquid Crystals Over a Functionalized Flexible Substrate.

Abstract: A simple and effective approach for vertical alignment of liquid crystals (LCs) over a functionalized transparent flexible substrate is described. Surface characterization of this commercially available plastic substrate through X-ray photoelectron spectroscopy (XPS) and attenuated total reflection infrared spectroscopy (ATR-IR) indicated that cellulose acetate is main component of the transparent substrate. This substrate was chemically functionalized with a suitable LC compound. A trimethoxysilane terminated new rod-shaped mesogen is synthesized and covalently attached to the pre-treated film through silane condensation reaction. LC functionalization of the polymer film is confirmed through contact angle (CA), atomic force microscopy (AFM), XPS and ATR-IR spectroscopy studies. Versatility of the LC modified flexible substrates for the alignment of bulk LC sample at substrate-LC interface was assessed for nematic (N) and smectic A (SmA) phases. Remarkably, LC functionalized cellulose acetate films were found to be highly efficient in assisting a perfect homeotropic alignment of LCs (for both, a room temperature N and a high temperature SmA phase) over the entire area of the LC sample under observation indicating their superior aligning ability in comparison to their unmodified and octadecyltrimethoxysilane (OTS) modified counterparts. The demonstrated method of surface modification of flexible polymer film is easy, surface modified substrates are stable for several months, retained their aligning ability intact and more importantly they are reusable with maximum delivery.

Intrinsic peroxidase-like activity of rhodium nanoparticles, and their application to the colorimetric determination of hydrogen peroxide and glucose

Abstract: The intrinsic peroxidase-like activity of rhodium nanoparticles (RhNPs) and their use as catalytic labels for sensitive colorimetric assays is presented. RhNPs catalyze the oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue reaction product with a maximum absorbance at 652 nm. Kinetic studies show catalysis to follow Michaelis-Menten kinetics and a “ping-pong” mechanism. The calculated kinetic parameters indicate high affinity of RhNPs for both the substrate TMB and H2O2. In fact, they are better than other peroxidase mimicking nanomaterials and even the natural enzyme horseradish peroxidase. On the other hand, RhNPs exhibit no reactivity towards saccharides, thiols, amino acids and ascorbic acid. Based on these findings, a sensitive and selective colorimetric method was worked out for the determination of H2O2 in real samples with a linear response in the 1–100 μM concentration range. By employing glucose oxidase, the glucose assay has a linear range that covers the 5 to 125 μM glucose concentration range. The detection limits are <0.75 μM for both species. The methods were applied to the determination of H2O2 in spiked pharmaceutical formulations, and of glucose in soft drinks and blood plasma. Figures of merit include (a) good accuracy (with errors of <6%), (b) high recoveries (96.5–103.7%), and (c) satisfactory reproducibility (<6.3%).

A Synthetic Light-Driven Substrate Channeling System for Precise Regulation of Enzyme Cascade Activity Based on DNA Origami.

Abstract: Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By ...

NiFe layered double hydroxide nanosheets as an efficiently mimic enzyme for colorimetric determination of glucose and H2O2

Abstract: The ultrathin NiFe-layered double hydroxide nanosheets (NiFe-LDHNS) were prepared by exfoliating bulk LDH in l -asparagine aqueous medium. The transmission electron microscopy and atomic force microscopy results reveal that NiFe-LDHNS have about 200–300 nm lateral size and less than 2 nm thickness with the broken edges rather than the original hexagon of hydrotalcite. The peroxidase-like activity of the 2D NiFe-LDHNS was investigated by using oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine in presence of H2O2. The 2D NiFe-LDHNS exhibited the superior enzyme mimic activity to the bulk NiFe-LDH, which was attributed to the more exposed active sites and more accessible inner surface of single or several layers. Thus, a simple and sensitive non-enzyme biosensor was developed for colorimetric detection of H2O2 and glucose. The proposed method was successfully applied to glucose detection in real samples with satisfactory results.

Fabrication and Characterization of Glucose Biosensors by Using Hydrothermally Grown ZnO Nanorods

Abstract: Highly oriented ZnO nanorod (NR) arrays were fabricated on a seeded substrate through a hydrothermal route. The prepared ZnO nanorods were used as an amperometric enzyme electrode, in which glucose oxidase (GOx) was immobilised through physical adsorption. The modified electrode was designated as Nafion/GOx/ZnO NRs/ITO. The morphology and structural properties of the fabricated ZnO nanorods were analysed using field-emission scanning electron microscope and X-ray diffractometer. The electrochemical properties of the fabricated biosensor were studied by cyclic voltammetry and amperometry. Electrolyte pH, electrolyte temperature and enzyme concentration used for immobilisation were the examined parameters influencing enzyme activity and biosensor performance. The immobilised enzyme electrode showed good GOx retention activity. The amount of electroactive GOx was 7.82 × 10−8 mol/cm2, which was relatively higher than previously reported values. The Nafion/GOx/ZnO NRs/ITO electrode also displayed a linear response to glucose ranging from 0.05 mM to 1 mM, with a sensitivity of 48.75 µA/mM and a low Michaelis–Menten constant of 0.34 mM. Thus, the modified electrode can be used as a highly sensitive third-generation glucose biosensor with high resistance against interfering species, such as ascorbic acid, uric acid and L-cysteine. The applicability of the modified electrode was tested using human blood samples. Results were comparable with those obtained using a standard glucometer, indicating the excellent performance of the modified electrode.

Phoresis and Enhanced Diffusion Compete in Enzyme Chemotaxis.

Abstract: Chemotaxis of enzymes in response to gradients in the concentration of their substrate has been widely reported in recent experiments, but a basic understanding of the process is still lacking. Here, we develop a microscopic theory for chemotaxis that is valid for enzymes and other small molecules. Our theory includes both nonspecific interactions between enzyme and substrate as well as complex formation through specific binding between the enzyme and the substrate. We find that two distinct mechanisms contribute to enzyme chemotaxis: a diffusiophoretic mechanism due to the nonspecific interactions and a new type of mechanism due to binding-induced changes in the diffusion coefficient of the enzyme. The latter chemotactic mechanism points toward lower substrate concentration if the substrate enhances enzyme diffusion and toward higher substrate concentration if the substrate inhibits enzyme diffusion. For a typical enzyme, attractive phoresis and binding-induced enhanced diffusion will compete against each other. We find that phoresis dominates above a critical substrate concentration, whereas binding-induced enhanced diffusion dominates for low substrate concentration. Our results resolve an apparent contradiction regarding the direction of urease chemotaxis observed in experiments and, in general, clarify the relation between the enhanced diffusion and the chemotaxis of enzymes. Finally, we show that the competition between the two distinct chemotactic mechanisms may be used to engineer nanomachines that move toward or away from regions with a specific substrate concentration.

Substrate Directed Asymmetric Reactions.

Abstract: Historically, reagent controlled reactions (mechanism controlled reactions) have played a significant role in the asymmetric synthesis of complex structures. In contrast, today’s asymmetric synthesis is greatly dependent on substrate directed approaches. In this approach, a polar functional group, namely, a “directing group”, in the vicinity of the reactive site inside the substrate has been documented to preassociate with the chiral catalyst, which exerts stereodirecting influence by directing the reacting partner toward one of the enantiotopic faces of the reaction center. Those reactions usually proceed through exceptionally ordered transition states and result in extraordinary levels of stereoselection. Within the last four decades, the substrate directed approach has become an indispensible tool for the preparation of complex chiral frameworks starting directly from relatively simple achiral substrate molecules via asymmetric induction or various resolution techniques or both. Likewise, the substrate...

Polysilane-Immobilized Rh-Pt Bimetallic Nanoparticles as Powerful Arene Hydrogenation Catalysts: Synthesis, Reactions under Batch and Flow Conditions and Reaction Mechanism.

Abstract: Hydrogenation of arenes is an important reaction not only for hydrogen storage and transport but also for the synthesis of functional molecules such as pharmaceuticals and biologically active compounds. Here, we describe the development of heterogeneous Rh–Pt bimetallic nanoparticle catalysts for the hydrogenation of arenes with inexpensive polysilane as support. The catalysts could be used in both batch and continuous-flow systems with high performance under mild conditions and showed wide substrate generality. In the continuous-flow system, the product could be obtained by simply passing the substrate and 1 atm H2 through a column packed with the catalyst. Remarkably, much higher catalytic performance was observed in the flow system than in the batch system, and extremely strong durability under continuous-flow conditions was demonstrated (>50 days continuous run; turnover number >3.4 × 105). Furthermore, details of the reaction mechanisms and the origin of different kinetics in batch and flow were stud...

Enzyme-like Click Catalysis by a Copper-Containing Single-Chain Nanoparticle.

Abstract: A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the micromolar range. We report that copper cross-linked single-chain nanoparticles (SCNPs) are able to significantly increase the efficiency of copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC) reactions at low substrate concentration in aqueous buffer by promoting substrate binding. Using a fluorogenic click reaction and dye uptake experiments, a structure–activity study is performed with SCNPs of different size and copper content and substrates of varying charge and hydrophobicity. The high catalytic efficiency and selectivity are attributed to a mechanism that involves an enzyme-like substrate binding process. Saturation-transfer difference (STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentrations, and computational simulations are consistent with a Michaelis–Menten, two-substrate, random-sequential enzyme-like kinetic profile. This gener...

Enhanced High-Solids Fed-Batch Enzymatic Hydrolysis of Sugar Cane Bagasse with Accessory Enzymes and Additives at Low Cellulase Loading

Abstract: High cellulase loading is still a major impediment in the production of fermentative sugars from high-solids enzymatic hydrolysis of lignocellulosic substrates in the enzyme-based “biorefinery” industry. This study attempted a high-solids (20%) enzymatic hydrolysis of lignocellulosic substrate at a very low cellulase loading with mixed use of additives and accessory enzymes by fed-batch mode. To avoid the high initial biomass viscosity, the high-solids enzymatic hydrolysis of lignocellulosic substrates was initiated with a solids content of 8%. Thereafter, 4% of the additional substrates were consecutively fed into the hydrolysis system after 6, 12, and 18 h to reach a final solids content of 20%. Some additive mixtures (40 mg/g substrateTween 80 + 10 mg/g substrate tea saponin +20 mg/g substrate BSA) were observed to enable this fed-batch hydrolysis to increase 30% of the glucose yield after the 48 h. The combination of these additives and accessory enzymes (2.4 mg/g substrate xylanase and 1 mg/g substra...

LaMnO3-based perovskite with in-situ exsolved Ni nanoparticles: a highly active, performance stable and coking resistant catalyst for CO2 dry reforming of CH4

Abstract: Wet impregnation method has been widely adopted to synthesize the oxide substrate supported catalysts for CO2 dry reforming of CH4. However, their particle size and distribution are not stable and uniform, causing rapid particle growth and carbon deposition. Here, we show that these problems can be overcome by using a LaMnO3 (LM)-based perovskite in which 20 mol% Ni is doped (La0.9Mn0.8Ni0.2O3, LMN) and then exsolved (R-LMN) by in-situ reduction in 5%H2-N2 stream at 800 °C. The perfomance of such catalyst is compared with that of the Ni-impregnated LM (NLM) reduced under the same conditions (R-NLM). The SEM and TEM analyses reveal that the exsolved Ni nanoparticles in R-LMN are uniform in size, evenly distributed and partially embedded into and hence bonded strongly with the substrate; but the Ni particles in R-NLM are variable in size and weakly bonded with the substrate. Therefore, the particle growth (coalescence) is largely suppressed for the exsolved Ni nanoparticles in R-LMN but not for the Ni nanoparticles in R-NLM. It is also comfirmed that the Ni particles in R-NLM are lifted off from the substrate by carbon fibers formed during test. Consequently, R-LMN demonstrates high and stable conversion (above 80%) and selectivity (above 90%) with a strong resistance to carbon deposition at 700 °C for 24 h. But, a large amount of carbon fiber is formed with a Ni particle at the tip in tested R-NLM, resulting in rapid performance degradation. The high performance of R-LMN for CO2 dry reforming of CH4 is attributed to the stable nanosized Ni particles and LMN substrate that provides more oxygen vacancies for CO2 activation and in turn carbon oxidation.

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings.

Abstract: Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

Towards High‐Mobility Heteroepitaxial β‐Ga2O3 on Sapphire − Dependence on The Substrate Off‐Axis Angle

Abstract: This paper presents the heteroepitaxial growth of β-Ga2O3 thin films on off-axis (0001) c-sapphire substrates by low pressure chemical vapor deposition (LPCVD). ( 201) oriented β-Ga2O3 thin films are grown using high purity metallic gallium (Ga) and oxygen (O2) as the precursors. N-type conductivity in silicon doped β-Ga2O3 thin films is demonstrated. It is found that the film crystalline quality, surface morphology, and electrical conductivity are remarkably sensitive to the off-axis angles. X-ray phi-scan measurements of the β-Ga2O3 film grown on on-axis c-sapphire indicate the presence of six in-plane rotational domains due to the substrate symmetry. With the increase of off-axis angle toward <11–20> of sapphire, one of the in-plane orientations is strongly favored. The use of off-axis substrate also reduced the X-ray rocking curve full width at half maximum and increased the intensities of the Raman peaks. The best electrical properties of the β-Ga2O3 film are exhibited by the film grown on 6 off-axis c-sapphire. The room temperature electron Hall mobility was 106.6 cmV 1 s 1 with an n-type carrier concentration of 4.83 10cm . The results from this study demonstrate high electrical quality β-Ga2O3 thin films grown on off-axis c-sapphire substrates, which are promising for high power electronic and short wavelength optoelectronic device applications.

Diffusion Measurements of Swimming Enzymes with Fluorescence Correlation Spectroscopy

Abstract: Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors' active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature's nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme's diffusion due to active self-propulsion must be observed on top of the enzyme's passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme's substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme's diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes' catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.

Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase

Abstract: The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis.

Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

Abstract: Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

Fe-Porphyrin-Based Covalent Organic Framework As a Novel Peroxidase Mimic for a One-Pot Glucose Colorimetric Assay

Abstract: Covalent organic frameworks (COFs) have recently emerged as very fascinating porous polymers due to their attractive design synthesis and various applications. However, the catalytic application of COF materials as enzymatic mimics remains largely unexplored. In this work, the Fe-porphyrin-based covalent organic framework (Fe-COF) has been successfully synthesized through a facile postsynthetic strategy for the first time. In the presence of hydrogen peroxide (H2O2), the Fe-COF can catalyze a chromogenic substrate (3,3′,5,5′-tetramethylbenzidine (TMB)) to produce color, and this just goes to show that it has an inner peroxidase-like activity. Moreover, the kinetic studies indicate that the Fe-COF nanomaterial has a higher affinity toward both the substrate H2O2 and TMB than the natural enzyme, horseradish peroxidase (HRP). Under the optimized conditions, the Fe-COF nanomaterial was applied in a colorimetric sensor for the sensitive detection of H2O2. The detection range was from 7 to 500 μM, and the detec...

Non-labeled selective virus detection with novel SERS-active porous silver nanofilms fabricated by Electron Beam Physical Vapor Deposition

Abstract: Virus detection is often performed using antibody-based and polymerase chain reaction-based techniques. Such methods have major deficiencies, caused by time-consuming and labor-intensive incubation and purification steps. In this contribution, a novel SERS substrate for qualitative virus detection was developed and described. The substrate is composed of a thin silver film with folded surface structure containing pore-like nanoscale cavities and indentations, deposited on mica substrate by electron beam physical vapor deposition method. Pore-like structures are semi-regularly arrayed, with a rough surface in between, allowing for SERS activity, and their size and periodicity can be manipulated in the manufacturing process. It was speculated that viral particles could be trapped in these structures and would generate easily detectable enhanced Raman signals. The SERS substrate was tested against detection of four virus species – rabbit myxomatosis virus, canine distemper virus, tobacco mosaic virus and potato virus X. Specific spectra were obtained and analyzed for each virus. Data analysis demonstrated successful differentiation between tested species. The reported results demonstrate that novel SERS substrate is suitable for detection and identification of viral particles.

A Theory of Enzyme Chemotaxis: From Experiments to Modeling

Abstract: Enzymes show two distinct transport behaviors in the presence of their substrates in solution. First, their diffusivity enhances with an increasing substrate concentration. In addition, enzymes perform directional motion toward regions with a high substrate concentration, termed as chemotaxis. While a variety of enzymes has been shown to undergo chemotaxis, there remains a lack of quantitative understanding of the phenomenon. Here, we derive a general expression for the active movement of an enzyme in a concentration gradient of its substrate. The proposed model takes into account both the substrate-binding and catalytic turnover step, as well as the enhanced diffusion of the enzyme. We have experimentally measured the chemotaxis of a fast and a slow enzyme: urease under catalytic conditions and hexokinase for both full catalysis and for simple noncatalytic substrate binding. There is good agreement between the proposed model and the experiments. The model is general, has no adjustable parameters, and onl...

Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells’ morphology and proliferation

Abstract: The effect of substrate stiffness on the cellular morphology, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) has been extensively researched and well established. However, the majority of these studies are done with a low seeding density where cell to cell interactions do not play a significant role. While these conditions permit an analysis of cell-substratum interactions at the single cell level, such a model system fails to capture a critical aspect of the cellular micro-environment in vivo, i.e. the cell-cell interaction via matrix deformation (i.e., strain). To address this question, we seeded hMSCs on soft poly-acrylamide (PAA) gels, at a seeding density that permits cells to be mechanically interacting via the underlying substrate. We found that as the intercellular distance decreases with the increasing seeding density, cellular sensitivity towards the substrate rigidity becomes significantly diminished. With the increasing seeding density, the cell spread area increased on a soft substrate (500 Pa) but reduced on an even slightly stiffer substrate (2 kPa) as well as on glass making them indistinguishable at a high seeding density. Not only in terms of cell spread area but also at a high seeding density, cells formed mature focal adhesions and prominent stress fibres on a soft substrate similar to that of the cells being cultured on a stiff substrate. The decreased intercellular distance also influenced the proliferation rate of the cells: higher seeding density on the soft substrate showed cell cycle progression similar to that of the cells on glass substrates. In summary, this paper demonstrates how the effect of substrate rigidity on the cell morphology and fate is a function of inter-cellular distance when seeded on a soft substrate. Our AFM data suggest that such changes happen due to local strain stiffening of the soft PAA gel, an effect that has been rarely reported in the literature so far.

Higher Substrate Ratios of Ethanol to Acetate Steered Chain Elongation Toward n-Caprylate in a Bioreactor With Product Extraction

Abstract: Syngas fermentation to ethanol and acetate has recently been coupled to microbial chain elongation to produce medium-chain carboxylates, including n-caproate and n-caprylate. These medium-chain carboxylates are relatively hydrophobic, and thus easier to extract from solution than miscible ethanol. Here, we examined the effect of 11 different ethanol-to-acetate substrate ratios (ranging from 1.8 to 275 g COD g COD-1 [1.2 to 183 mol mol-1]) on directing chain elongation toward n-caprylate in a 0.7-L upflow anaerobic filter with product extraction. During an eight-month operating period, we monitored the performance and characterized the microbiome composition of this chain-elongating bioreactor. We also developed a thermodynamic model to predict the favorability of n-caprylate production at different substrate ratios. As predicted by our model, higher ethanol-to-acetate substrate ratios fed to our bioreactor led to higher specificities for n-caprylate production. We observed that feeding primarily ethanol to the bioreactor (i.e., ethanol-to-acetate substrate ratio of 275 g COD g COD-1) resulted in the highest specificity for n-caprylate, but the n-caprylate production rate decreased at this high ratio, resulting in lower conversion efficiencies. Thus, care should be taken not to overload the system with primarily ethanol as the substrate and to lower the organic loading rate.