Stella H. North

Interpenetrating networks based on gelatin methacrylamide and PEG formed using concurrent thiol click chemistries for hydrogel tissue engineering scaffolds.

The integration of biological extracellular matrix (ECM) components and synthetic materials is a promising pathway to fabricate the next generation of hydrogel-based tissue scaffolds that more accurately emulate the microscale heterogeneity of natural ECM. We report the development of a bio/synthetic interpenetrating network (BioSINx), containing gelatin methacrylamide (GelMA) polymerized within a poly(ethylene glycol) (PEG) framework to form a mechanically robust network capable of supporting both internal cell encapsulation and surface cell adherence. The covalently crosslinked PEG network was formed by thiol-yne coupling, while the bioactive GelMA was integrated using a concurrent thiol-ene coupling reaction. The physical properties (i.e. swelling, modulus) of BioSINx were compared to both PEG networks with physically-incorporated gelatin (BioSINP) and homogenous hydrogels. BioSINx displayed superior physical properties and significantly lower gelatin dissolution. These benefits led to enhanced cytocompatibility for both cell adhesion and encapsulation; furthermore, the increased physical strength provided for the generation of a micro-engineered tissue scaffold. Endothelial cells showed extensive cytoplasmic spreading and the formation of cellular adhesion sites when cultured onto BioSINx; moreover, both encapsulated and adherent cells showed sustained viability and proliferation.

Plasma-Based Surface Modification of Polystyrene Microtiter Plates for Covalent Immobilization of Biomolecules

In recent years, polymer surfaces have become increasingly popular for biomolecule attachment because of their relatively low cost and desirable bulk physicochemical characteristics. However, the chemical inertness of some polymer surfaces poses an obstacle to more expansive implementation of polymer materials in bioanalytical applications. We describe use of argon plasma to generate reactive hydroxyl moieties at the surface of polystyrene microtiter plates. The plates are then selectively functionalized with silanes and cross-linkers suitable for the covalent immobilization of biomolecules. This plasma-based method for microtiter plate functionalization was evaluated after each step by X-ray photoelectron spectroscopy, water contact angle analysis, atomic force microscopy, and bioimmobilization efficacy. We further demonstrate that the plasma treatment followed by silane derivatization supports direct, covalent immobilization of biomolecules on microtiter plates and thus overcomes challenging issues typically associated with simple physisorption. Importantly, biomolecules covalently immobilized onto microtiter plates using this plasma-based method retained functionality and demonstrated attachment efficiency comparable to commercial preactivated microtiter plates.

Critical aspects of biointerface design and their impact on biosensor development

AbstractThe stable integration of a biological recognition element on a transducing substrate surface is the single most important step in the creation of a high-functioning sensor surface. The key factors affecting biotic and abiotic functionalities at the biointerface are both chemical and physical. Understanding the interactions between biomolecules and surfaces, and their emergent complexity, is critical for biointerface implementation for sensing applications. In this overview, we highlight materials and methods typically used for biosensor development. Particular emphasis has been given to the experimental evaluation of biointerfacial properties and functionality. Promising research directions for application of biointerfaces to biosensing are suggested. FigureCorrelation between nanoscale roughness, surface elemental composition, and functional bio-immobilization.

Effect of Physicochemical Anomalies of Soda-Lime Silicate Slides on Biomolecule Immobilization

Glass microscope slides are considered by many as the substrate of choice for microarray manufacturing due to their amenability to various surface chemistry modifications. The use of silanes to attach various functional groups onto glass slides has provided a versatile tool for the covalent immobilization of many diverse biomolecules of interest. We recently noted a dramatic reduction in biomolecule immobilization efficiency on standard microscope slides prepared using a well-characterized silanization method. A survey of commercial soda-lime slides yielded the surprising result that slides purchased prior to 2008 had superior immobilization efficiencies when compared to those purchased after 2008. Characterization of the slides by X-ray photoelectron spectroscopy (XPS), contact angle measurements, and atomic force microscopy (AFM), revealed a significant correlation (R > 0.9) between magnesium content, surface roughness, and bioimmobilization efficiency. High performance slides had higher magnesium content and higher root-mean-square (rms) roughness (P < 0.005) than slides with lower bioimmobilization efficiencies. Although the exact mechanism of how magnesium content and surface roughness affect silane deposition has not yet been defined, we show that recent changes in the chemical and physical properties of commercial soda-lime slides affect the ability of these slides to be covalently modified.

Rapid Analytical Methods for On-Site Triage for Traumatic Brain Injury

Traumatic brain injury (TBI) results from an event that causes rapid acceleration and deceleration of the brain or penetration of the skull with an object. Responses to stimuli and questions, loss of consciousness, and altered behavior are symptoms currently used to justify brain imaging for diagnosis and therapeutic guidance. Tests based on such symptoms are susceptible to false-positive and false-negative results due to stress, fatigue, and medications. Biochemical markers of neuronal damage and the physiological response to that damage are being identified. Biosensors capable of rapid measurement of such markers in the circulation offer a solution for on-site triage, as long as three criteria are met: (a) Recognition reagents can be identified that are sufficiently sensitive and specific, (b) the biosensor can provide quantitative assessment of multiple markers rapidly and simultaneously, and (c) both the sensor and reagents are designed for use outside the laboratory.

Surface immobilization chemistry influences peptide-based detection of lipopolysaccharide and lipoteichoic acid

Antimicrobial peptides (AMPs) have recently gained attention as potentially valuable diagnostic and therapeutic agents. The utilization of these peptides for diagnostic purposes relies on the ability to immobilize them on the surface of a detection platform in a predictable and reliable manner that facilitates target binding. The method for attachment of peptides to a solid support is guided by peptide length, amino acid composition, secondary structure, and the nature of the underlying substrate. While immobilization methods that target amine groups of amino acid sequences are widely used, they can result in heterogeneous conjugation at multiple sites on a peptide and have direct implications for peptide presentation and function. Using two types of commercial amine‐reactive microtiter plates, we described the effects of analogous immobilization chemistries on the surface attachment of AMPs and their differential binding interaction with Gram‐specific bacterial biomarkers, lipopolysaccharide and lipoteichoic acid. As might be expected, differences in overall binding affinities were noted when comparing AMPs immobilized on the two types of plates. However, the two‐amine‐targeted linking chemistries also affected the specificity of the attached peptides; lipopolysaccharide generally demonstrated a preference for peptides immobilized on one type of plate, while (when observed at all) lipoteichoic acid bound preferentially to AMPs immobilized on the other type of plate. These results demonstrate the potential for tuning not only the binding affinities but also the specificities of immobilized AMPs by simple alterations in linking strategy. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite

BackgroundA complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.MethodsThe main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions.ResultsWe report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.ConclusionsThe expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Bradys reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

Application of Circular Dichroism for Structural Analysis of Surface-Immobilized Cecropin A Interacting with Lipoteichoic Acid

The development of biomaterials integrating antimicrobial peptides (AMPs) for improved pathogen detection or use as therapeutic agents requires an understanding of how a peptide may behave once immobilized. Here, we use a combination of circular dichroism and capture assays to assess the structure-function relationship of the cationic amphipathic AMP, cecropin A (cecA), upon interaction with Gram-positive lipoteichoic acids (LTAs). In solution, cecA peptides underwent a change from a largely unstructured conformation in water to structures with significant α-helical content in the presence of both Bacillus subtilis and Staphylococcus aureus LTAs. After surface immobilization, cecA peptides attached by either C- or N-terminus were able to capture both LTAs as well as to undergo conformational changes in the presence of SDS similar to those observed in solution. However, in spite of demonstrated LTA binding activity and the ability to undergo conformational changes (i.e., with SDS), no structural changes were observed when cecA immobilized by its N-terminus was treated with either LTA preparation. On the other hand, cecA immobilized by its C-terminus underwent a conformational change in the presence of S. aureus, but not B. subtilis, LTA. These results indicate that after immobilization recognition of different targets by cationic AMPs may occur by mechanisms quite different from those in solution and that selectivity of these mechanisms is further dependent on the orientation of the immobilized peptide.

Antimicrobial Peptides for Detection and Diagnostic Assays

Considered gold standards for biodetection, immunoassays and nucleic acid-based assays are sensitive, highly selective, and well characterized. However, they are capable of detecting only those targets for which specific reagents (such as antibodies or nucleic acid primers or probes) have been developed. Furthermore, new, emerging, and unexpected pathogens may not be detected. To address the challenge of detecting both known and unknown microbes, assays utilizing antimicrobial peptides (AMPs) are being developed for integration into both biosensors and high-throughput platforms. AMP-based detection represents a new paradigm in sensing—namely, the ability to screen a sample for the presence of many different microbes without target-specific reagents, and to provide broad classification information on the species detected.