Investigation of the Efficacy of a Listeria monocytogenes Biosensor Using Chicken Broth Samples.

Foodborne pathogens are microbes present in food that cause serious illness when the contaminated food is consumed. Among these pathogens, Listeria monocytogenes is one of the most serious bacterial pathogens, and causes severe illness. The techniques currently used for L. monocytogenes detection are based on common molecular biology tools that are not easy to implement for field use in food production and distribution facilities. This work focuses on the efficacy of an electrochemical biosensor in detecting L. monocytogenes in chicken broth. The sensor is based on a nanostructured electrode modified with a bacteriophage as a bioreceptor which selectively detects L. monocytogenes using electrochemical impedance spectroscopy. The biosensing platform was able to reach a limit of detection of 55 CFU/mL in 1× PBS buffer and 10 CFU/mL in 1% diluted chicken broth. The biosensor demonstrated 83-98% recovery rates in buffer and 87-96% in chicken broth.

Comparative Study of the Biological Degradation of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Microbeads in Municipal Wastewater in Environmental and Controlled Laboratory Conditions.

From April to June 2019, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3(HA)) microbead samples were exposed to an operational wastewater reclamation facility (WWRF) in an aerobic aeration basin in Athens, Georgia. Samples were withdrawn from the facility over a 13-week timeframe, and the particles were examined by Raman microscopy and thermogravimetric analysis/mass spectroscopy (TGA/MS) coupled with differential scanning calorimetry (DSC). The activated sludge from this facility was also used as an inoculum to examine carbon mineralization under controlled respirometry experiments to corroborate biological degradation rates determined from both the environmental and laboratory approach. Respirometry, Raman microscopy, and TGA/MS-DSC methods all measured similar biodegradation timelines for microbeads bound to an epoxy substrate, indicating that the three methods are temporally comparable and may be used to measure material biological degradation. Samples of epoxy-bound P3(HA) microbeads, free microbeads, the P3(HA) film, and poly(lactic acid) (PLA) film demonstrated carbon mineralization of 90.0, 89.4, 95.0, and 8.15%, respectively, relative to the cellulose positive control. Using a modified Gompertz growth model, the biological degradation rate coefficients (Rm) were determined for cellulose, P3(HA) film, epoxy-bound P3(HA) microbeads, and free P3(HA) microbeads and found to be 31.6, 30.2, 17.5, and 18.7 mL CO2·g-1·day-1, respectively. Moreover, P3(HA) microbeads can efficiently mineralize in WWRF infrastructure at a rate comparable to cellulose.

Distinct Mycoplasma pneumoniae Interactions with Sulfated and Sialylated Receptors.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the conducting airways, causing bronchitis and atypical or "walking" pneumonia in humans. M. pneumoniae recognizes sialylated and sulfated oligosaccharide receptors to colonize the respiratory tract, but the contribution of the latter is particularly unclear. We used chamber slides coated with sulfatide (3-O-sulfogalactosylceramide) to provide a baseline for M. pneumoniae binding and gliding motility. As expected, M. pneumoniae bound to surfaces coated with sulfatide in a manner that was dependent on sulfatide concentration and incubation temperature and inhibited by competing dextran sulfate. However, mycoplasmas bound to sulfatide exhibited no gliding motility, regardless of receptor density. M. pneumoniae also bound lactose 3'-sulfate ligated to an inert polymer scaffold, and binding was inhibited by competing dextran sulfate. The major adhesin protein P1 mediates adherence to terminal sialic acids linked α-2,3, but P1-specific antibodies that blocked M. pneumoniae hemadsorption (HA) and binding to the sialylated glycoprotein laminin by 95% failed to inhibit mycoplasma binding to sulfatide, suggesting that P1 does not mediate binding to sulfated galactose. Consistent with this conclusion, the M. pneumoniae HA-negative mutant II-3 failed to bind to sialylated receptors but adhered to sulfatide in a temperature-dependent manner.

Sialylated Receptor Setting Influences Mycoplasma pneumoniae Attachment and Gliding Motility.

Mycoplasma pneumoniae is a common cause of human respiratory tract infections, including bronchitis and atypical pneumonia. M. pneumoniae binds glycoprotein receptors having terminal sialic acid residues via the P1 adhesin protein. Here, we explored the impact of sialic acid presentation on M. pneumoniae adherence and gliding on surfaces coated with sialylated glycoproteins, or chemically functionalized with α-2,3- and α-2,6-sialyllactose ligated individually or in combination to a polymer scaffold in precisely controlled densities. In both models, gliding required a higher receptor density threshold than adherence, and receptor density influenced gliding frequency but not gliding speed. However, very high densities of α-2,3-sialyllactose actually reduced gliding frequency over peak levels observed at lower densities. Both α-2,3- and α-2,6-sialyllactose supported M. pneumoniae adherence, but gliding was only observed on the former. Finally, gliding on α-2,3-sialyllactose was inhibited on surfaces also conjugated with α-2,6-sialyllactose, suggesting that both moieties bind P1 despite the inability of the latter to support gliding. Our results indicate that the nature and density of host receptor moieties profoundly influences M. pneumoniae gliding, which could affect pathogenesis and infection outcome. Furthermore, precise functionalization of polymer scaffolds shows great promise for further analysis of sialic acid presentation and M. pneumoniae adherence and gliding.

Ingested Micronizing Plastic Particle Compositions and Size Distributions within Stranded Post-Hatchling Sea Turtles.

From July 2015 to November 2016, 96 post-hatchling sea turtles were collected from 118 km of the Atlantic coastline in Florida, USA, including loggerhead, green, and hawksbill sea turtle species. Forty-five of the recovered turtles were rehabilitated and released, but the remaining 52 died and were frozen. At necropsy, the gastrointestinal tracts of most the turtles contained visible plastic, and collected particles of 27 individuals were chemically characterized by Raman microscopy as polyethylene, polypropylene, polyethylene terephthalate, and polystyrene. Mesoparticle plastic fragments 1.0-8.7 mm, microparticle fragments 20-1000 µm, and nanoparticles 5-169 nm were identified in the turtles. Polyethylene and polypropylene were the most common plastics ingested from specimens representing 54.1 and 23.7% of the total observed mesoparticles and 11.7 and 21.0% of the total observed microparticles, respectively. A plastic-to-body mass ratio of 2.07 mg/g was determined for this group. The authors suggest that ingestion of micronizing plastic by post-hatchling sea turtles is likely a substantial risk to survival of these endangered and threatened species. This study also provides some of the first evidence for the formation of nanoscopic plastic particles that we theorize forms in the post-hatchling and juvenile environment and are present post-ingestion.

Biodegradation of Poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) Plastic under Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbial Diversity.

Poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (poly(3HB- co-3HHx)) thermoplastics are a promising biodegradable alternative to traditional plastics for many consumer applications. Biodegradation measured by gaseous carbon loss of several types of poly(3HB- co-3HHx) plastic was investigated under anaerobic conditions and aerobic seawater environments. Under anaerobic conditions, the biodegradation levels of a manufactured sheet of poly(3HB- co-3HHx) and cellulose powder were not significantly different from one another over 85 days with 77.1 ± 6.1 and 62.9 ± 19.7% of the carbon converted to gas, respectively. However, the sheet of poly(3HB- co-3HHx) had significantly higher methane yield ( p ≤ 0.05), 483.8 ± 35.2 mL·g-1 volatile solid (VS), compared to cellulose controls, 290.1 ± 92.7 mL·g-1 VS, which is attributed to a greater total carbon content. Under aerobic seawater conditions (148-195 days at room temperature), poly(3HB- co-3HHx) sheets were statistically similar to cellulose for biodegradation as gaseous carbon loss (up to 83% loss in about 6 months), although the degradation rate was lower than that for cellulose. The microbial diversity was investigated in both experiments to explore the dominant bacteria associated with biodegradation of poly(3HB- co-3HHx) plastic. For poly(3HB- co-3HHx) treatments, Cloacamonales and Thermotogales were enriched under anaerobic sludge conditions, while Clostridiales, Gemmatales, Phycisphaerales, and Chlamydiales were the most enriched under aerobic seawater conditions.

Covalent Grafting of Antifouling Phosphorylcholine-Based Copolymers with Antimicrobial Nitric Oxide Releasing Polymers to Enhance Infection-Resistant Properties of Medical Device Coatings.

Medical device coatings that resist protein adhesion and bacterial contamination are highly desirable in the healthcare industry. In this work, an antifouling zwitterionic terpolymer, 2-methacryloyloxyethyl phosphorylcholine-co-butyl methacrylate-co-benzophenone (BPMPC), is covalently grafted to a nitric oxide (NO) releasing antimicrobial biomedical grade copolymer of silicone-polycarbonate-urethane, CarboSil, to significantly enhance the biocompatibility, nonspecific protein repulsion and infection-resistant properties. The NO donor embedded into CarboSil is S-nitroso-N-acetylpenicillamine (SNAP) and covalent grafting of the BPMPC is achieved through rapid UV-cross-linking, providing a stable, hydrophilic coating that has excellent durability over a period of several weeks under physiological conditions. The protein adsorption test results indicate a significant reduction (∼84-93%) of protein adhesion on the test samples compared to the control samples. Bacteria tests were also performed using the common nosocomial pathogen, Staphylococcus aureus. Test samples containing both NO donor and BPMPC show a 99.91 ± 0.06% reduction of viable bacteria when compared to control samples. This work demonstrates a synergistic combination of both antimicrobial and antifouling properties in medical devices using NO donors and zwitterionic copolymers that can be covalently grafted to any polymer surface.

Versatile Methodology for Glycosurfaces: Direct Ligation of Nonderivatized Reducing Saccharides to Poly(pentafluorophenyl acrylate) Grafted Surfaces via Hydrazide Conjugation.

In this work, we report a convenient and versatile strategy for surface-grafted glycopolymer constructs with the goal of surface modification that controls the chemical presentation and grafting density of carbohydrate side chains. This approach employs a difunctional hydrazine linker, chemically modified to an active ester containing poly(pentafluorophenyl acrylate) grafted scaffold, to conjugate a variety of saccharides through the reducing end. The successive conjugation steps are carried out under mild conditions and yield high surface densities of sugars, as high as 4.8 nmol·cm-2, capable of multivalency, with an intact structure and retained bioactivity. We also demonstrate that this glycosylated surface can bind specific lectins according to the structure of its pendant carbohydrate. To demonstrate bioactivity, this surface platform is used to study the binding events of a human respiratory tract pathogen, Mycoplasma pneumoniae, on surfaces conjugated with sialylated sugars.

SuFEx Click: New Materials from SOx F and Silyl Ethers.

New forms of click chemistry present new opportunities in materials science. Sulfur(VI) fluoride exchange (SuFEx) is a recently discovered click reaction between molecules containing SOx F groups and silyl ethers, two functionalities that are orthogonal to all other known click chemistries, that generates sulfate or sulfonate connections upon the addition of certain organobases or fluoride sources. SuFEx also has several important advantages over other click reactions in that it is insensitive to ambient oxygen and water, and its precursor materials, especially SOx F, are chemically, UV, and thermally inert. This Concept article focuses on the unique reactivity of SuFEx and its relation to building high molecular weight polymers and surface coatings, both of which make it a powerful new tool for materials science.

Multifunctional Surface Manipulation Using Orthogonal Click Chemistry.

Polymer brushes are excellent substrates for the covalent immobilization of a wide variety of molecules due to their unique physicochemical properties and high functional group density. By using reactive microcapillary printing, poly(pentafluorophenyl acrylate) brushes with rapid kinetic rates toward aminolysis can be partially patterned with other click functionalities such as strained cyclooctyne derivatives and sulfonyl fluorides. This trireactive surface can then react locally and selectively in a one pot reaction via three orthogonal chemistries at room temperature: activated ester aminolysis, strain promoted azide-alkyne cycloaddition, and sulfur(VI) fluoride exchange, all of which are tolerant of ambient moisture and oxygen. Furthermore, we demonstrate that these reactions can also be used to create areas of morphologically distinct surface features on the nanoscale, by inducing buckling instabilities in the films and the grafting of nanoparticles. This approach is modular, and allows for the development of highly complex surface motifs patterned with different chemistry and morphology.

A dynamic duo: pairing click chemistry and postpolymerization modification to design complex surfaces.

Advances in key 21st century technologies such as biosensors, biomedical implants, and organic light-emitting diodes rely heavily on our ability to imagine, design, and understand spatially complex interfaces. Polymer-based thin films provide many advantages in this regard, but the direct synthesis of polymers with incompatible functional groups is extremely difficult. Using postpolymerization modification in conjunction with click chemistry can circumvent this limitation and result in multicomponent surfaces that are otherwise unattainable. The two methods used to form polymer thin films include physisorption and chemisorption. Physisorbed polymers suffer from instability because of the weak intermolecular forces between the film and the substrate, which can lead to dewetting, delamination, desorption, or displacement. Covalent immobilization of polymers to surfaces through either a "grafting to" or "grafting from" approach provides thin films that are more robust and less prone to degradation. The grafting to technique consists of adsorbing a polymer containing at least one reactive group along the backbone to form a covalent bond with a complementary surface functionality. Grafting from involves polymerization directly from the surface, in which the polymer chains deviate from their native conformation in solution and stretch away from the surface because of the high density of chains. Postpolymerization modification (PPM) is a strategy used by our groups over the past several years to immobilize two or more different chemical functionalities onto substrates that contain covalently grafted polymer films. PPM exploits monomers with reactive pendant groups that are stable under the polymerization conditions but are readily modified via covalent attachment of the desired functionality. "Click-like" reactions are the most common type of reactions used for PPM because they are orthogonal, high-yielding, and rapid. Some of these reactions include thiol-based additions, activated ester coupling, azide-alkyne cycloadditions, some Diels-Alder reactions, and non-aldol carbonyl chemistry such as oxime, hydrazone, and amide formation. In this Account, we highlight our research combining PPM and click chemistry to generate complexity in polymer thin films. For the purpose of this Account, we define a complex coating as a polymer film grafted to a planar surface that acts as a template for the patterning of two or more discrete chemical functionalities using PPM. After a brief introduction to grafting, the rest of the review is arranged in terms of the sequence in which PPM is performed. First, we describe sequential functionalization using iterations of the same click-type reaction. Next, we discuss the use of two or more different click-like reactions performed consecutively, and we conclude with examples of self-sorting reactions involving orthogonal chemistries used for one-pot surface patterning.

Magnetic-Field-Assisted Fabrication and Manipulation of Nonspherical Polymer Particles in Ferrofluid-Based Droplet Microfluidics.

We report a novel magnetic-field-assisted method for the fabrication and manipulation of nonspherical polymer particles within a ferrofluid-based droplet microfluidic device. Shape control and chain assembly of droplets with tunable lengths have been achieved.

Degradable Polycaprolactone and Polylactide Homopolymer and Block Copolymer Brushes Prepared by Surface-Initiated Polymerization with Triazabicyclodecene and Zirconium Catalysts.

Surface-initiated ring-opening polymerization (SI-ROP) of polycaprolactone (PCL) and polylactide (PLA) polymer brushes with controlled degradation rates were prepared on oxide substrates. PCL brushes were polymerized from hydroxyl-terminated monolayers utilizing triazabicyclodecene (TBD) as the polymerization catalyst. A consistent brush thickness of 40 nm could be achieved with a reproducible unique crystalline morphology. The organocatalyzed PCL brushes were chain extended using lactide in the presence of zirconium n-butoxide to successfully grow PCL/PLA block copolymer (PCL-b-PLA) brushes with a final thickness of 55 nm. The degradation properties of "grafted from" PCL brush and the PCL-b-PLA brush were compared to "grafted to" PCL brushes, and we observed that the brush density plays a major role in degradation kinetics. Solutions of methanol/water at pH 14 were used to better solvate the brushes and increase the kinetics of degradation. This framework enables a control of degradation that allows for the precise removal of these coatings.

SuFEx on the Surface: A Flexible Platform for Postpolymerization Modification of Polymer Brushes.

Polymer brushes present a unique architecture for tailoring surface functionalities due to their distinctive physicochemical properties. However, the polymerization chemistries used to grow brushes place limitations on the monomers that can be grown directly from the surface. Several forms of click chemistry have previously been used to modify polymer brushes by postpolymerization modification with high efficiency, however, it is usually difficult to include the unprotected moieties in the original monomer. We present the use of a new form of click chemistry known as SuFEx (sulfur(VI) fluoride exchange), which allows a silyl ether to be rapidly and quantitatively clicked to a polymer brush grown by free-radical polymerization containing native -SO2F groups with rapid pseudo-first-order rates as high as 0.04 s(-1). Furthermore, we demonstrate the use of SuFEx to facilely add a variety of other chemical functional groups to brush substrates that have highly useful and orthogonal reactivity, including alkynes, thiols, and dienes.

Rapid electrochemical reduction of Ni(II) generates reactive monolayers for conjugated polymer brushes in one step.

This article reports the development of a robust, one-step electrochemical technique to generate surface-bound conjugated polymers. The electrochemical reduction of arene diazonium salts at the surface of a gold electrode is used to generate tethered bromobenzene monolayers quickly. The oxidative addition of reactive Ni(0) across the aryl halide bond is achieved in situ through a concerted electrochemical reduction of Ni(dppp)Cl2. This technique limits the diffusion of Ni(0) species away from the surface and overcomes the need for solution deposition techniques which often require multiple steps that result in a loss of surface coverage. With this electrochemical technique, the formation of the reactive monolayer resulted in a surface coverage of 1.29 × 10(14) molecules/cm(2), which is a 6-fold increase over previously reported results using solution deposition techniques.

π-Complexation in nickel-catalyzed cross-coupling reactions.

The kinetic isotope effect (KIE) is used to experimentally elucidate the first irreversible step in oxidative addition reactions of a zerovalent nickel catalyst to a set of haloarene substrates. Halogenated o-methylbenzene, dimethoxybenzene, and thiophene derivatives undergo intramolecular oxidative addition through irreversible π-complexation. Density functional theory computations at the B3LYP-D3/TZ2P-LANL2TZ(f)-LANL08d level predict η(2)-bound π-complexes are generally stable relative to a solvated catalyst plus free substrate and that ring-walking of the Ni(0) catalyst and intramolecular oxidative addition are facile in these intermediates.

Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Experimentally Determined Hansen Solubility Parameters of Biobased and Biodegradable Polyesters.

Hansen solubility parameters (HSP) of 15 commercially relevant biobased and biodegradable polyesters were experimentally determined by applying a novel approach to the classic solubility study method. In this approach, the extent of swelling in polymer films was determined using a simple equation based on the mass difference between swollen and nonswollen film samples to obtain normalized solvent uptake (N). Using N and HSPiP software, highly accurate HSP values were obtained for all 15 polyesters. Qualitative evaluation of the HSP values was conducted by predicting the miscibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB-co-HHx, 7 mol % HHx) and poly(lactic acid) (PLA) with a novel lignin-based plasticizer (ethyl 3-(4-ethoxy-3-methoxyphenyl)propanoate, EP) with a relative energy difference (RED) less than 0.4. Additionally, an HSP-predicted plasticizer (di(2-ethylhexyl) adipate, DA) with a larger RED (>0.7) was used to demonstrate the effects of less-miscible additives. Plasticized samples were analyzed by differential scanning calorimetry and polarized optical microscopy (POM) to determine the Tg depression, with EP showing linear Tg depression up to 50% plasticizer loading, whereas DA shows minimal Tg depression past 10% loading. Further analysis by POM reveals that the DA phase separates from both polymers at loadings as low as 2.5% (PHB-co-HHx, 7 mol % HHx) and 5% (PLA), while the EP phase separates at a much higher loading of 50% (PHB-co-HHx, 7 mol% HHx) and 30% (PLA).

Exact ligand cone angles.

Many properties of transition-metal complexes depend on the steric bulk of bound ligands, usually quantified by the Tolman (θ) and solid (Θ) cone angles, which have proven utility but suffer from various limitations and coarse approximations. Here, we present an improved, mathematically rigorous method to determine an exact cone angle (θ°) by solving for the most acute right circular cone that contains the entire ligand. The procedure is applicable to any ligand, planar or nonplanar, monodentate or polydentate, bound to any metal center in any environment, and it is ideal for analyzing structures from quantum chemical computations as well as X-ray crystallography experiments. Exact cone angles were evaluated for a wide array of phosphine and amine ligands bound to palladium, nickel, or platinum by optimizing structures using B3LYP/6-31G* density functional theory with effective core potentials for the transition metals. The mean absolute deviations of the standard θ and Θ parameters from the exact cone angles were 15-25°, mostly caused by distortions from the assumed idealized structures.

Self-sorting click reactions that generate spatially controlled chemical functionality on surfaces.

This Article describes the generation of a patterned surface that can be postpolymerization modified to incorporate fragile macromolecules or delicate biomolecules without the need for special equipment. Two monomers that undergo different click reactions, pentafluorophenyl acrylate (PFPA) and 4-(trimethylsilyl) ethynylstyrene (TMSES), were sequentially polymerized from a silicon surface in the presence of a shadowmask with UV light, generating 12.5 and 62 µm pitch patterns. Two different dyes, 1-aminomethylpyrene (AMP) and 5-azidofluorescein (AF), were covalently attached to the polymer brushes through aminolysis and dual desilylation/copper(I)-catalyzed alkyne/azide cycloaddition (CuAAC) in one pot. Unlike most CuAAC reactions, the terminal alkyne of TMSES was not deprotected prior to functionalization. Although a 2 nm thickness increase was observed for poly(PFPA) brushes after polymerization of TMSES, cross-contamination was not visible through fluorescence microscopy after functionalization.