Surface Tethering Research Articles

Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides

AbstractEnzyme cascades are promising for multistep biocatalytic synthesis, but their effective use beyond the proof‐of‐concept stage is challenging. Strategies to recycle the individual enzymes are critical for the applicability of such cascades. Immobilization on solid support is well developed for single enzymes but remains difficult for enzyme ensembles. Here, we show a controlled co‐immobilization of three glycoside phosphorylases to establish a highly active and recyclable biocatalyst for the conversion of sucrose and glucose into soluble (short‐chain) cello−oligosaccharides. We use protein fusion with the binding module Zbasic2 to enable non‐covalent surface tethering of all enzymes according to a uniform principle and in a programmable fashion. We thus achieve loading of the phosphorylases in an activity ratio optimal for the overall conversion and for controlling the cello−oligosaccharide chain length (≤6), hence the solubility, in the reaction. We demonstrate efficient production of ∼12 g/L cello−oligosaccharides with integrated enzyme re‐use, retaining ∼85 % of the overall initial activity after five reaction cycles. This study presents a major advance toward the practical use of systems bio‐catalysis on solid support.

Bacterial activation of surface-tethered antimicrobial peptides for the facile construction of a surface with self-defense

In this study, a charge-conversion mechanism was introduced into the design of a bacteria-responsive antibacterial surface. The antibacterial surface was facilely constructed via surface tethering of antimicrobial peptides (AMPs) to solid supports and subsequently integrating charge-conversion moieties into the peptide structure. The resulting zwitterionic structure of the AMPs rendered the antibacterial surface biocompatible under normal physiological conditions. Importantly, the surface exhibited self-defense properties specifically against pathogenic bacteria; the toxicity of AMPs was activated in bacterially induced acidic environments. Unlike previously described self-defense systems that utilize an antimicrobial release mechanism, this antibacterial surface exerts its bactericidal effects by diminishing the nonbactericidal masking moieties and activating the surface-tethered AMPs to avoid the undesirable toxic effects caused by planktonic AMPs. In contrast to the prevailing passive switchable antibacterial surfaces, our surface uses a unique self-adaptive zwitterionic-to-cationic transition mechanism to inhibit the development of bacterial infections.

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

Surface tethering of stem cells with H2O2-responsive anti-oxidizing colloidal particles for protection against oxidation-induced death

Mesenchymal stem cells are the new generation of medicine for treating numerous vascular diseases and tissue defects because of their ability to secrete therapeutic factors. Poor cellular survival in an oxidative diseased tissue, however, hinders the therapeutic efficacy. To this end, we hypothesized that tethering the surface of stem cells with colloidal particles capable of discharging antioxidant cargos in response to elevated levels of hydrogen peroxide (H2O2) would maintain survival and therapeutic activity of the stem cells. We examined this hypothesis by encapsulating epigallocatechin gallate (EGCG) and manganese oxide (MnO2) nanocatalysts into particles comprising poly(d,l-lactide-co-glycolide)-block-hyaluronic acid. The MnO2 nanocatalysts catalyzed the decomposition of H2O2 into oxygen gas, which increased the internal pressure of particles and accelerated the release of EGCG by 1.5-fold. Consequently, stem cells exhibited 1.2-fold higher metabolic activity and 2.8-fold higher secretion level of pro-angiogenic factor in sub-lethal H2O2 concentrations. These stem cells, in turn, performed a greater angiogenic potential with doubled number of newly formed mature blood vessels. We envisage that the results of this study will contribute to improving the therapeutic efficacy of a wide array of stem cells.

Stable Carboxylate-Terminated Gold Surfaces Produced by Spontaneous Grafting of an Alkyltin Compound.

Self-assembled monolayers formed by chemisorption of thiolated molecules on gold surfaces are widely applied for biosensing. Moreover, and due to the low stability of thiol-gold chemistry, contributions to the functionalisation of gold substrates with linkers that provide a more stable platform for the immobilisation of electroactive or biological molecules are highly appreciated. Herein, it is demonstrated that a carboxylated organotin compound can be successfully grafted onto gold substrates to form a highly stable organic layer with reactivity for subsequent binding to an aminated molecule. A battery of techniques were used to characterise the surface chemistry. The grafted layer was used to anchor aminoferrocene and subjected to both thermostability tests and long-term stability studies over a period of one year, demonstrating thermostability up to 90 °C and storage stability for at least 12 months at 4 °C protected from light. The stable surface tethering of molecules on gold substrates can be exploited in a plethora of applications, including molecular techniques, such as solid-phase amplification and solid-phase melting curve analysis, that require elevated temperature stability, as well as biosensors, which require long-term storage stability.

Wide-Field Monitoring of Single Fluorescent Molecules and Nanoparticles without Immobilization

Surface-based wide-field imaging (“surface-WIFI”) of single fluorescent molecules has transformed our understanding of many biological mechanisms by directly accessing molecular heterogeneity and real-time reaction trajectories. By monitoring the function of hundreds of immobilised molecules simultaneously, surface-WIFI achieves high throughput, outperforming its confocal microscopy counterpart that relies on point detection. However, surface-WIFI relies on surface tethering of molecules, which requires the preparation of modified surfaces and insertion of biomolecule modifiers; further, the molecules of interest (e.g., proteins and their complexes) may stick to surfaces non-specifically and lose their function. These issues significantly limit the appeal of surface-WIFI. To address these challenges, we introduce solution-based wide-field imaging (“solution-WIFI”), a method that combines wide-field ratiometric imaging and tracking of diffusing fluorescent molecules. Solution-WIFI requires no surface attachment, is immune to surface interactions, and also reports on molecular size. To implement solution-WIFI, we used oblique illumination and ratiometric imaging of diffusing molecules on a sCMOS camera with a temporal resolution down to 2.5 ms. We used custom software to localize and track particles, extract their intensities and associated ratios (e.g., Förster energy resonance transfer (FRET), spectral signatures, molecular stoichiometries), and characterize thousands of individual molecules per minute of imaging. For validation, we tracked doubly labelled double-stranded DNA that serve as FRET standards, and recovered FRET distributions similar to those for surface measurements. Our system was capable of tracking tiny fluorescent molecules, such as single Cy3B dyes (with characteristic size of ∼1 nm and diffusion coefficients reaching 50 µm2/s in 50% glycerol). We also determined the size distribution of fluorescently labelled nanodiamonds, as well as the fraction of nanodiamond population that contain fluorescent nitrogen vacancy (NV) centers. Solution-WIFI is a general method for high-throughput analysis of structure, dynamics, and diffusion of many biomolecules without immobilization.

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Frontispiece: Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

Author(s): Britton, Joshua; Dyer, Rebekah P; Majumdar, Sudipta; Raston, Colin L; Weiss, Gregory A

Frontispiz: Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

Frontispiz: Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

AbstractNature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous‐flow, vortex fluidic device (VFD). Fused Hisn‐tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi‐aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this “reaction‐ready” system demonstrated excellent stability during five days of continuous‐flow processing. Towards multi‐step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous‐flow biosynthesis.

Anti-adhesive antimicrobial peptide coating prevents catheter associated infection in a mouse urinary infection model.

Catheter-associated urinary tract infections (CAUTIs) represent one of the most common hospital acquired infections with significant economic consequences and increased patient morbidity. CAUTIs often start with pathogen adhesion and colonization on the catheter surface followed by biofilm formation. Current strategies to prevent CAUTIs are insufficiently effective and antimicrobial coatings based on antimicrobial peptides (AMPs) hold promise in curbing CAUTIs. Here we report an effective surface tethering strategy to prepare AMP coatings on polyurethane (PU), a common biomedical plastic used for catheter manufacture, by using an anti-adhesive hydrophilic polymer coating. An optimized surface active AMP, labeled with cysteine at the C-terminus (RRWRIVVIRVRRC), was used. The coated PU surface was characterized using ATR-FTIR, XPS and atomic force microscopy analyses. The tethered peptides on the PU catheter surface displayed broad spectrum antimicrobial activity and showed long term activity invitro. The surface coating prevented bacterial adhesion by up to 99.9% for both Gram-positive and -negative bacteria, and inhibited planktonic bacterial growth by up to 70%. Invivo, the coating was tested in a mouse urinary catheter infection model; the AMP-coated PU catheter was able to prevent infection with high efficiency by reducing the bacteria adhesion on catheter surface by more than 4 logs (from 1.2×106CFU/mL to 5×101CFU/mL) compared to the uncoated catheter surface, and inhibit planktonic bacterial growth in the urine by nearly 3 logs (1.1×107CFU/mL to 1.47×104CFU/mL). The AMP-brush coating also showed good biocompatibility with bladder epithelial cells and fibroblast cells in cell culture. The new coating might find clinical applications in preventing CAUTIs.

Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament

Actin filaments have key roles in cell motility but are generally claimed to be passive interaction partners in actin-myosin-based motion generation. Here, we present evidence against this static view based on an altered myosin-induced actin filament gliding pattern in an in vitro motility assay at varied [MgATP]. The statistics that characterize the degree of meandering of the actin filament paths suggest that for [MgATP] ≥ 0.25 mM, the flexural rigidity of heavy meromyosin (HMM)-propelled actin filaments is similar (without phalloidin) or slightly lower (with phalloidin) than that of HMM-free filaments observed in solution without surface tethering. When [MgATP] was reduced to ≤0.1 mM, the actin filament paths in the in vitro motility assay became appreciably more winding in both the presence and absence of phalloidin. This effect of lowered [MgATP] was qualitatively different from that seen when HMM was mixed with ATP-insensitive, N-ethylmaleimide-treated HMM (NEM-HMM; 25–30%). In particular, the addition of NEM-HMM increased a non-Gaussian tail in the path curvature distribution as well as the number of events in which different parts of an actin filament followed different paths. These effects were the opposite of those observed with reduced [MgATP]. Theoretical modeling suggests a 30–40% lowered flexural rigidity of the actin filaments at [MgATP] ≤ 0.1 mM and local bending of the filament front upon each myosin head attachment. Overall, the results fit with appreciable structural changes in the actin filament during actomyosin-based motion generation, and modulation of the actin filament mechanical properties by the dominating chemomechanical actomyosin state.

ATR-IR Investigation of Solvent Interactions with Surface-Bound Polymers.

Solvent interactions with bulk and surface-bound polymer brushes are crucial for functionalities such as controlled friction and thermoresponsive adhesion. To study such interactions, the temperature-induced solvent-quality changes and the effect of surface tethering on the mechanical and tribological properties of poly(dodecyl methacrylate) (P12MA) brushes have been investigated by means of attenuated total reflection infrared spectroscopy (ATR-IR), as well as atomic force microscopy (AFM) and lateral force microscopy (LFM). These results have been compared with temperature-dependent UV–visible spectrophotometry (UV–vis) data for the corresponding bulk polymer solutions. The ATR-IR results clearly show that increasing temperature enhances ethanol uptake in P12MA, which results in film swelling. This is accompanied by a marked increase in both adhesion and friction. We have also shown that a combination of solvents, such as toluene and ethanol, can lead to a temperature-dependent solvent partitioning within the polymer brush. To our knowledge this is the first time preferential solvent uptake in a grafted-from brush has been monitored via in situ ATR-IR. Moreover, we have observed remarkably different behavior for polymer chains in solution compared to the behavior of similar chains bound to a surface. The presented findings on the temperature-dependent solvent interactions of surface-grafted P12MA reveal previously unknown solvation phenomena and open up a range of possible applications in the area of stimuli-responsive materials.

Mesothelin's minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane.

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies.

Anchoring sulfonic acid on silica surface through Si[sbnd]C bond for immobilization of catalyst for polyketone synthesis

Sulfonic acid groups were anchored on a silica surface through robust SiC bonds. The successive treatment of dehydroxylated silica with benzylmagnesium chloride and H2SO4 resulted in the surface tethering of CH2C6H4SO3H groups at a high coverage rate (0.50-CH2C6H4SO3H/nm2). The pore structure of the silica remained unchanged during this surface-modification process. Next, the CH2C6H4SO3H groups on the surface were successfully used for preparing a supported catalyst for CO/ethylene copolymerization; the ≡SiCH2C6H4SO3H groups on the surface were reacted with [1,3-bis(di(2-methoxyphenyl)phosphino)propane]Pd(OAc)2 to generate dicationic palladium species, which were anchored on the silica surface through ionic interactions with the sulfonate anions generated on the surface. The supported catalyst prepared in this way exhibited a high activity (up to 43kg/g-Pd or 0.61kg/g-cat) with respect to CO/ethylene copolymerization. The morphology of the obtained polymer particles replicated that of the silica particles. Thus, a polymer powder that exhibited a high bulk density (0.30g/mL) could be obtained while causing minimal reactor fouling.

Surface functionalisation of carbon for low cost fabrication of highly stable electrochemical DNA sensors.

An alternative strategy for surface tethering of DNA probes, where highly reactive glassy carbon (GC) substrates are prepared via electrochemical hydrogenation and electrochemical/chemical chlorination is reported. Thiolated DNA probes and alkanethiols were stably immobilised on the halogenated carbon, with electrochemical chlorination being milder, thus producing less damage to the surface. Electrochemical DNA sensors prepared using this surface chemistry on carbon with electrochemical chlorination providing an improved performance, producing a highly ordered surface and the use of lateral spacers to improve steric accessibility to immobilised probes was not required.

Surface-Free Single-Molecule Force Spectroscopy

Mechanical forces play key roles throughout biology. Most existing methods for probing forces in/between molecules and cells, such as the AFM, optical tweezers, magnetic tweezers, traction force microscopy, etc., require molecules to be tethered to the surfaces of cover glasses or beads. However, surface tethering has a number of drawbacks. First, the conjugation chemistry for tethering can be challenging. Second, the spatial confinement and the nonspecific attraction from the surface can induce artifacts to the dynamics of the molecule. For semi-rigid objects, such as actin filaments, surface attachment could also induce bending and twisting of the filament, complicating the applied force profile. To overcome these issues, we present a surface-free force spectroscopy method that uses a high-speed cross-slot hydrodynamic trap, capable of stretching molecules and cells with hydrodynamic drag. The trap is based on a glass microfluidic cross-slot flow chamber. Buffer flows in from two opposite directions and exits via the two orthogonal outlets to create an elongational flow field with a stagnation point in the center. As a result, objects near the stagnation point are stretched by the viscous drag from the flow. In addition, the pressure in one of the outlet reservoirs is electronically controlled with a high-speed feedback algorithm to stabilize the object at the stagnation point. Thanks to the high-speed feedback, we can apply much higher flow rate and therefore much higher stretching force on the trapped object than with the cross slot alone. We demonstrate tension-dependent actin severing, extension of von Willebrand Factor (a key protein in haemostasis that changes conformation in response to hydrodynamic stress in blood stream), overstretching of double strand DNA, and stretching deformation of red blood cells. In summary, the high-speed cross-slot hydrodynamic trap can be a powerful, surface-free alternative to more commonly used force spectroscopy methods.

Virus-based surface patterning of biological molecules, probes, and inorganic materials

An essential requirement for continued technological advancement in many areas of biology, physics, chemistry, and materials science is the growing need to generate custom patterned materials. Building from recent achievements in the site-specific modification of virus for covalent surface tethering, we show in this work that stable 2D virus patterns can be generated in custom geometries over large area glass surfaces to yield templates of biological, biochemical, and inorganic materials in high density. As a nanomaterial building block, filamentous viruses have been extensively used in recent years to produce materials with interesting properties, owing to their ease of genetic and chemical modification. By utilizing un-natural amino acids generated at specific locations on the filamentous fd bacteriophage protein coat, surface immobilization is carried out on APTES patterned glass resulting in precise geometries of covalently linked virus material. This technique facilitated the surface display of a high density of virus that were labeled with biomolecules, fluorescent probes, and gold nanoparticles, thereby opening the possibility of integrating virus as functional components for surface engineering.

Site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens.

Bacterial colonization of urinary catheters is a common problem leading to Catheter Associated Urinary Tract Infections (CAUTIs) in patients, which result in high treatment costs and associated complications. Due to the advantages of antimicrobial peptides (AMPs) compared to most other antimicrobial molecules, an increasing number of AMP-coated surfaces is being developed but their efficacy is hindered by suboptimal coating methods and loss of peptide activity upon surface tethering. This study aims to address this issue by employing a methodic approach that combines a simple selective chemical immobilization platform developed on a silicone catheter with the choice of a potent AMP, Lasioglossin-III (Lasio-III), to allow site specific immobilization of Lasio-III at an effective surface concentration. The Lasio-III peptide was chemically modified at the N-terminal with a cysteine residue to facilitate cysteine-directed immobilization of the peptide onto a commercial silicone catheter surface via a combination of an allyl glycidyl ether (AGE) brush and polyethylene glycol (PEG) based chemical coupling. The amount of immobilized peptide was determined to be 6.59 ± 0.89 μg cm-2 by Sulfo-SDTB assay. The AMP-coated catheter showed good antimicrobial activity against both Gram positive and negative bacteria. The antimicrobial properties of the AMP-coated catheter were sustained for at least 4 days post-incubation in a physiologically relevant environment and artificial urine and prevented the biofilm growth of E. coli and E. faecalis. Adenosine tri-phosphate leakage and propidium iodide fluorescence studies further confirmed the membranolytic mode of action of the immobilized peptide. To the best of our knowledge, this is the first proof-of-concept study that reports the efficacy of AMP immobilization by sulfhydryl coupling on a real catheter surface.