Defluorination of Organofluorine Compounds Using Dehalogenase Enzymes from Delftia acidovorans (D4B).

Organofluorine compounds have been widely used as pharmaceuticals, agricultural pesticides, and water-resistant coatings for decades; however, these compounds are recognized as environmental pollutants. The capability of microorganisms and enzymes to defluorinate organofluorine compounds is both rare and highly desirable to facilitate environmental remediation efforts. Recently, a strain of Delftia acidovorans (D4B) was identified with potential biodegradation activity toward perfluoroalkyl substances (PFAS) and other organofluorine compounds. Genomic analysis found haloacid and fluoroacetate dehalogenases as enzymes associated with Delftia acidovorans. Here, defluorination activity of these enzymes toward different fluorinated substrates was investigated after their recombinant expression and purification from E. coli. Using an electrochemical fluoride probe, 19F NMR, and mass spectrometry to monitor defluorination, we identified two dehalogenases, DeHa2 (a haloacid dehalogenase) and DeHa4 (a fluoroacetate dehalogenase), with activity toward mono- and difluoroacetate. Of the two dehalogenases, DeHa4 demonstrated a low pH optimum compared to DeHa2, which lost catalytic activity under acidic conditions. DeHa2 and DeHa4 are relatively small proteins, operate under aerobic conditions, and remain active for days in the presence of substrates. Significantly, while there have been many reports on dehalogenation of monofluoroacetate by dehalogenases, this study adds to the relatively small list of enzymes reported to carry out enzymatic defluorination of the more recalcitrant disubstituted carbon in an organofluorine compound. Thus, DeHa2 and DeHa4 represent organofluorine dehalogenases that may be used in the future to design and engineer robust defluorination agents for environmental remediation efforts.

Programming Fluorogenic DNA Probes for Rapid Detection of Steroids.

The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 µM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate-reader), and low sample volumes (10 µL).

In Silico Discovery and Validation of Neuropeptide-Y-Binding Peptides for Sensors.

Wearable sensors for human health, performance, and state monitoring, which have a linear response to the binding of biomarkers found in sweat, saliva, or urine, are of current interest for many applications. A critical part of any device is a biological recognition element (BRE) that is able to bind a biomarker at the surface of a sensor with a high affinity and selectivity to produce a measurable signal response. In this study, we discover and compare 12-mer peptides that bind to neuropeptide Y (NPY), a stress and human health biomarker, using independent and complimentary experimental and computational approaches. The affinities of the NPY-binding peptides discovered by both methods are equivalent and below the micromolar level, which makes them suitable for application in sensors. The in silico design protocol for peptide-based BREs is low cost, highly efficient, and simple, suggesting its utility for discovering peptide binders to a variety of biomarker targets.

Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Advancing Peptide-Based Biorecognition Elements for Biosensors Using in-Silico Evolution.

Sensors for human health and performance monitoring require biological recognition elements (BREs) at device interfaces for the detection of key molecular biomarkers that are measurable biological state indicators. BREs, including peptides, antibodies, and nucleic acids, bind to biomarkers in the vicinity of the sensor surface to create a signal proportional to the biomarker concentration. The discovery of BREs with the required sensitivity and selectivity to bind biomarkers at low concentrations remains a fundamental challenge. In this study, we describe an in-silico approach to evolve higher sensitivity peptide-based BREs for the detection of cardiac event marker protein troponin I (cTnI) from a previously identified BRE as the parental affinity peptide. The P2 affinity peptide, evolved using our in-silico method, was found to have ∼16-fold higher affinity compared to the parent BRE and ∼10 fM (0.23 pg/mL) limit of detection. The approach described here can be applied towards designing BREs for other biomarkers for human health monitoring.

Structured DNA Aptamer Interactions with Gold Nanoparticles.

DNA aptamers that bind biomolecular targets are of interest as the recognition element in colorimetric sensors based on gold nanoparticles (AuNP), where sensor functionality is related to changes in AuNP colloidal stability upon target binding. In order to understand the role of target binding on DNA-AuNP colloidal stability, we have used high-resolution NMR to characterize the interactions of the 36 nucleotide cocaine-binding aptamer (MN4) and related aptamers with AuNPs, cocaine, and cocaine metabolites. Changes in the aptamer imino proton NMR spectra with low (20 nM) concentrations of AuNP show that the aptamers undergo fast-exchange adsorption on the nanoparticle surface. An analysis of the spectral changes and the comparison with modified MN4 aptamers shows that the AuNP binding domain is localized on stem two of the three-stemmed aptamer. The identification of an AuNP recognition domain allows for the incorporation of AuNP binding functionality into a wide variety of aptamers. AuNP-induced spectral changes are not observed for the aptamer-AuNP mixtures in the presence of cocaine, demonstrating that aptamer absorption on the AuNP surface is modulated by aptamer-target interactions. The data also show that the DNA-AuNP interactions and sensor functionality are critically dependent on aptamer folding.

Single-Round Patterned DNA Library Microarray Aptamer Lead Identification.

A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint.

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.

Stability, surface features, and atom leaching of palladium nanoparticles: toward prediction of catalytic functionality.

Surfactant-stabilized metal nanoparticles have shown promise as catalysts although specific surface features and their influence on catalytic performance have not been well understood. We quantify the thermodynamic stability, the facet composition of the surface, and distinct atom types that affect rates of atom leaching for a series of twenty near-spherical Pd nanoparticles of 1.8 to 3.1 nm size using computational models. Cohesive energies indicate higher stability of certain particles that feature an approximate 60/20/20 ratio of {111}, {100}, and {110} facets while less stable particles exhibit widely variable facet composition. Unique patterns of atom types on the surface cause apparent differences in binding energies and changes in reactivity. Estimates of the relative rate of atom leaching as a function of particle size were obtained by the summation of Boltzmann-weighted binding energies over all surface atoms. Computed leaching rates are in good qualitative correlation with the measured catalytic activity of peptide-stabilized Pd nanoparticles of the same shape and size in Stille coupling reactions. The agreement supports rate-controlling contributions by atom leaching in the presence of reactive substrates. The computational approach provides a pathway to estimate the catalytic activity of metal nanostructures of engineered shape and size, and possible further refinements are described.

Keratin-based antimicrobial textiles, films, and nanofibers.

The combination of appealing structural properties, biocompatibility, and the availability of renewable and inexpensive raw materials, make keratin-based materials attractive for a variety of applications. In this paper, we report on the antimicrobial functionalization of keratin-based materials, including wool cloth and regenerated cellulose/keratin composite films and nanofibers. The functionalization of these materials was accomplished utilizing a facile chlorination reaction that converts the nitrogen-bearing moieties of keratin into halamine compounds. Halamine-charged wool cloth exhibited rapid and potent bactericidal activity against several species of bacteria and induced up to a 5.3 log (i.e., 99.9995%) reduction in the colony forming units of Bacillus thuringiensis spores within 10 min. Keratin-containing composites were prepared by the spin coating and coaxial electrospinning of extracted/oxidized alpha-keratin and cellulose acetate (CA) solubilized in formic acid, followed by CA deacetylation. Regenerated cellulose/keratin materials chlorinated to display halamines were also effective in killing Escherichia coli and Staphylococcus aureus bacteria. Electrospun core/shell nanofibers engineered to maximize keratin-Cl surface area displayed higher activity against S. aureus than films composed of the same materials. The halamine-based antimicrobial functionalization methods demonstrated for keratin-based materials in this paper are anticipated to translate to other protein biopolymers of interest to the biomaterials community.

Sporicidal/bactericidal textiles via the chlorination of silk.

Bacterial spores, such as those of the Bacillus genus, are extremely resilient, being able to germinate into metabolically active cells after withstanding harsh environmental conditions or aggressive chemical treatments. The toughness of the bacterial spore in combination with the use of spores, such as those of Bacillus anthracis, as a biological warfare agent necessitates the development of new antimicrobial textiles. In this work, a route to the production of fabrics that kill bacterial spores and cells within minutes of exposure is described. Utilizing this facile process, unmodified silk cloth is reacted with a diluted bleach solution, rinsed with water, and dried. The chlorination of silk was explored under basic (pH 11) and slightly acidic (pH 5) conditions. Chloramine-silk textiles prepared in acidified bleach solutions were found to have superior breaking strength and higher oxidative Cl contents than those prepared under caustic conditions. Silk cloth chlorinated for ≥1 h at pH 5 was determined to induce >99.99996% reduction in the colony forming units of Escherichia coli, as well as Bacillus thuringiensis Al Hakam (B. anthracis simulant) spores and cells within 10 min of contact. The processing conditions presented for silk fabric in this study are highly expeditionary, allowing for the on-site production of protein-based antimicrobial materials from a variety of agriculturally produced feed-stocks.

Structure of peptides on metal oxide surfaces probed by NMR.

Peptides that bind inorganic surfaces and template the formation of nanometer-sized inorganic particles are of great interest for the self- or directed assembly of nanomaterials for sensors and diagnostic applications. These surface-recognizing peptides can be identified from combinatorial phage-display peptide libraries, but little experimental information is available for understanding the relationship between the peptide sequence, structure at the nanoparticle surface, and function. We have developed NMR methods to determine the structures of peptides bound to inorganic nanoparticles and report on the structure of three peptides bound to silica and titania surfaces. Samples were prepared under conditions leading to rapid peptide exchange at the surface such that solution-based nuclear Overhauser experiments can be used to determine the three-dimensional structure of the bound peptide. The binding motif is defined by a compact "C"-shaped structure for the first six amino acids in the 12-mer. The orientation of the peptide on the nanoparticle surface was determined by magnetization transfer from the nanoparticle surface to the nearby peptide protons. These methods can be applied to a wide variety of abiotic interfaces to provide an insight into the relationship between the primary sequence of peptides and their functionality at the interface.

Canopy dynamics in nanoscale ionic materials.

Nanoscale ionic materials (NIMS) are organic-inorganic hybrids in which a core nanostructure is functionalized with a covalently attached corona and an ionically tethered organic canopy. NIMS are engineered to be liquids under ambient conditions in the absence of solvent and are of interest for a variety of applications. We have used nuclear magnetic resonance (NMR) relaxation and pulse-field gradient (PFG) diffusion experiments to measure the canopy dynamics of NIMS prepared from 18-nm silica cores modified by an alkylsilane monolayer possessing terminal sulfonic acid functionality, paired with an amine-terminated ethylene oxide/propylene oxide block copolymer canopy. Carbon NMR studies show that the block copolymer canopy is mobile both in the bulk and in the NIMS and that the fast (ns) dynamics are insensitive to the presence of the silica nanoparticles. Canopy diffusion in the NIMS is slowed relative to the neat canopy, but not to the degree predicted from the diffusion of hard-sphere particles. Canopy diffusion is not restricted to the surface of the nanoparticles and shows unexpected behavior upon addition of excess canopy. Taken together, these data indicate that the liquid-like behavior in NIMS is due to rapid exchange of the block copolymer canopy between the ionically modified nanoparticles.

Structure and dynamics of surfactant interfaces in organically modified clays.

Organic modification of clays with surfactants is required for the preparation of polymer-clay nanocomposites for a variety of applications. We have studied the structure and dynamics of interfaces in synthetic clays modified with phosphonium surfactants. The chemical shifts, line widths, and relaxation times measured by 31P, 13C, and 1H NMR and the relaxation times measured by impedance spectroscopy allow us to monitor the dynamics over a wide range of time scales. The results show that the phosphonium headgroup is most restricted and that the mobility increases with increasing separation from the clay surface. The carbon chemical shifts show that the 16-carbon and 12-carbon surfactant tails of hexadecyltributyl phosphonium and dodecytriphenyl phosphonium are disordered at the interface and experience mobility over a range of time scales. The dynamics depend most strongly on the structure of the surfactant headgroup, and tributylphosphoniums are more mobile than the triphenylphosphoniums. Two dimensional chemical shift anisotropy spin exchange experiments show that the phosphorus atoms in the triphenylphosphonium surfactant are immobile on the clay surface on a 1 s time scale. The dynamics measured by impedance spectroscopy show a similar dependence on headgroup structure, even though the processes occur on very different time scales and length scales. The relationship between the structure and dynamics of the interface and the properties of composites are considered.