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We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL. (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 °C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 °C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required.

Photochemical cyclobutane dimerization of adjacent thymines generates the major lesion in DNA caused by exposure to sunlight. Not all nucleotide sequences and structures are equally susceptible to this reaction or its potential to create mutations. Photostationary levels of the cyclobutane thymine dimer have now been quantified in homogenous samples of DNA reconstituted into nucleosome core particles to examine the basis for previous observations that such structures could induce a periodicity in dimer yield when libraries of heterogeneous sequences were used. Initial rate studies did not reveal a similar periodicity when a homogenous core particle was analyzed, but this approach examined only formation of this photochemically reversible cyclobutane dimer. Photostationary levels result from competition between dimerization and reversion and, as described in this study, still express none of the periodicity within two alternative core particles that was evident in heterogeneous samples. Such periodicity likely arises from only a limited set of sequences and structural environments that are not present in the homogeneous and well-characterized assemblies available to date.

Biological systems have evolved the exquisite ability to spatially combine many weak, non-covalent chemical interactions to direct the molecular recognition and self-assembly of incredibly complex materials. The ability to control assembly at the molecular level has led to an interest in harnessing nature’s building blocks (e.g., polypeptides, DNA, etc.) to bind inorganic or synthetic compounds for multi-scale fabrication (nano-to macro) of advanced materials. The utility of this approach is evidenced by the large and growing body of research reports highlighting peptides generated through biopanning of surface display peptide libraries.1–5 Examples include a wide range of peptide binders to pure metals,6–10 metal oxides,11–13 metal alloys,14 metal salts,15 and semiconductors,16–18 as well as hydroxyapatite—the inorganic component of teeth and bone.19 Inorganic binding peptides, no matter the source, are widely recognized for their specificity and design control, and present a remarkable opportunity for advanced materials development.20 However, the rules governing this type of peptide binding are not fully understood.18, 20, 21 A variety of factors have been implicated in playing a role in peptide-inorganic surface interactions, including conformational effects,22–25 electrostatic effects,26, 27 relative residue placement in the sequence,28, 29 acid-base chemistry,30 and hydrogen bond formation.14, 21

We report on peptide‐based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide‐alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti‐VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half‐life (T 1/2) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti‐PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti‐PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

In the present work, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described. DNA-Surfactant complex formation renders the DNA soluble in organic solvents offering an exciting way to bridge the transition of DNA origami materials electronics applications. The DNA retains its structural features, and these unique geometries provide an interesting candidate for future electronics and nanofabrication applications with potential for new properties. The DNA architectures were first assembled under aqueous conditions, and then characterized in solution (using circular dichroism (CD) spectroscopy) and on the surface (using atomic force microscopy (AFM)). Following aqueous assembly, the DNA nanostructures were transitioned to a non-aqueous environment, where butanol was chosen for optical compatibility and thermal properties. The retention of DNA hierarchical structure and thermal stability in non-aqueous conditions were confirmed via CD spectroscopy. The formation and characterization of these higher order DNA-surfactant complexes is described in this paper.

A series of 1,5-diaminonaphthalene derivatives were synthesized and characterized to provide ground- and excited-state electron donors of similar structure but varying potential. Electrochemical and spectroscopic properties of the series are reported and together illustrate two opposing consequences of alkyl substitution on the aryl amines. Inductive effects of methylation are evident from the decrease in ground-state oxidation potential for derivatives containing monomethylamino substituents. In contrast, steric effects seem to dominate the increase in the ground-state oxidation potential of derivatives containing dimethylamino substituents since the conformational constraints created by dimethylation suppress delocalization of the nonbonding electrons. Absorption and emission properties also respond to increasing levels of N-methylation, and the excited-state oxidation potentials of the parent 1,5-diaminonaphthalene and its monomethylamine derivatives (ca. -3.2 V) are approximately 200 mV lower than the corresponding dimethylamino derivatives (-3.0 V).

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

Biopanning by bacterial display has many advantages over yeast and phage display, including the speed to discovery of affinity reagents and direct amplification of bound cells without the need to elute and reinfect. However, widespread use is limited, in part due to poor performance achieved using manual Magnetic-Activated Cell Sorting (MACS) methods, and an absence of widely-available, low cost, high-performance sorting alternatives. Here, we have developed a methodology for bacterial cell sorting using the semi-automated autoMACS® Pro Separator for the first time, and have produced a complete method for sorting of bacteria displaying 15-mer peptides on their cell surface using this device, including downstream bioinformatic analysis of candidates for binding to a target of interest. Two autoMACS® programs designed for isolation of target cells with low frequency were evaluated and adapted to bacterial biopanning, using protective antigen (PA) of Bacillus anthracis as the model system. In contrast to manual MACS, the bacterial display library was preferentially enriched by autoMACS® sorting, yielding several promising candidates after only three rounds of biopanning and bioinformatic analysis. Individual candidates were evaluated for relative binding to fluorescently-labeled PA target or streptavidin negative control using Fluorescence-Activated Cell Sorting (FACS). The top thirteen peptide candidates from the autoMACS® sort demonstrate binding to PA with low cross-reactivity to streptavidin, while only two of eighteen candidates from the manual sort showed binding to PA, and both demonstrated greater cross-reactivity to streptavidin. Overall, the autoMACS® platform quickly harvested higher affinity peptide candidates with demonstrated specificity to the PA target. Peptide candidates produced with this method contained the previously reported PA consensus WXCFTC, further validating this method and the commercially available autoMACS® platform as the first low cost, semi-automated biopanning approach for bacterial display that is widely accessible and more reliable than the MACS/FACS standard protocol.

Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

The ability to conveniently and immediately test and diagnose in a diverse and rapidly changing environment is critical for field diagnostics. Smart biomedical sensors employ many different diagnostic/therapeutic methodologies; however, an ideal system would include the ability for results to be shared instantaneously with all members of the team through a software interface. We discuss our efforts towards the development and use of a robust, mobile platform (Android-based smart phone) that incorporates stable molecular recognition elements in sensor development. The inexpensive, compact, robust, archival, and portable design is ideal for rapid field diagnostics.

Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

Reliable, robust, and portable technologies are needed for the rapid identification and detection of chemical, biological, and explosive (CBE) materials. A key to addressing the persistent threat to U.S. troops in the current war on terror is the rapid detection and identification of the precursor materials used in development of improvised explosive devices, homemade explosives, and bio-warfare agents. However, a universal methodology for detection and prevention of CBE materials in the use of these devices has proven difficult. Herein, we discuss our efforts towards the development of a modular, robust, inexpensive, pervasive, archival, and compact platform (android based smart phone) enabling the rapid detection of these materials.

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

A methodology that allows for the coupling of biology and electronic materials is presented, where double stranded DNA will ultimately serve as a template for electronic material growth. Self-assembled DNA structures allow for a variety of patterns to be achieved on the nanometer size scale that is difficult to achieve using conventional patterning techniques. DNA self assembly under non-aqueous conditions has yet to be presented in literature, and is necessary if unwanted oxidation of certain electronic substrates is to be avoided. Solubilization of the DNA in non-aqueous solvents is achieved by replacing charge stabilizing salts with the surfactant cetyl trimethyl ammonium chloride (CTAC). Herein, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described, and these structures are subsequently deposited (drop cast, spin cast, and physically adsorbed) onto freshly cleaved mica or silicon wafers. The DNA architectures are characterized either in solution (circular dichroism spectroscopy (CD)) or on the surface (AFM). These studies illustrate the retention of DNA hierarchical structure under both conditions and this data will be presented by observing the structures using AFM imaging and CD spectroscopic studies© (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

A methodology that allows for the coupling of biology and electronic materials is presented, where double stranded DNA serves as a template for electronic material growth. Self-assembled DNA structures allow for a variety of patterns to be achieved on the nanometer size scale. These DNA architectures allow for feature sizes that are difficult to achieve using conventional patterning techniques. Herein, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described, and these structures are subsequently deposited onto substrates of interest. DNA self assembly under non-aqueous conditions has yet to be presented in literature, and is necessary if unwanted oxidation of certain electronic substrates is to be avoided. Solubilization of the DNA in non-aqueous solvents is achieved by replacing charge stabilizing salts with surfactants. Retention of DNA hierarchical structure under both conditions will be presented by observing the structures using AFM imaging and circular dichroism spectroscopic studies.

In today s Army, there is a pressing need to improve the quality of the electronics used in the field. Some requirements include the fabrication of electronic devices that are cheaper, smaller, and lighter, all the while maintaining or even expanding their technical capabilities. An example of this research is the use of deoxyribonucleic acid (DNA) to create thin-film membranes that could be used in electronics. For example, these thin films have been successfully used in light-emitting diodes (LEDs) (1). However, this work focuses on the optimization of membrane fabrication parameters, such as when the sample volume should be added to the spin-coater, spin-coat speed, and solvent type. This can be accomplished by mixing a surfactant, cetyltrimethylammonium chloride (CTAC), with the DNA, causing DNA-cetyltrimethylammonium (DNA-CTMA) to precipitate out of solution. Then, the DNA-CTMA is redissolved in an organic solvent and spin-coated onto a silicon wafer to create the thin film. We demonstrated that certain conditions yield DNA-CTMA films that were thinner and more uniform. We envision that this work could be used in several electronic, photonic, and electro-optic applications.

Microbial fuel cells (MFCs) were used to monitor metabolism changes in Clostridium acetobutylicum fermentations. When MFCs were inoculated with C. acetobutylicum, they generated a unique voltage output pattern where two distinct voltage peaks occurred over a weeklong period. This result was markedly different to previously studied organisms which usually generate one sustained voltage peak. Analysis of the fermentation products indicated that the dual voltage peaks correlated with glucose metabolism. The first voltage peak correlated with acidogenic metabolism (acetate and butyrate production) and the second peak with solventogenic metabolism (acetone and butanol production). This demonstrates that MFCs can be applied as a novel tool to monitor the shift from acid production to solvent production in C. acetobutylicum.