Martin H. Moore

Development of Antiricin Single Domain Antibodies Toward Detection and Therapeutic Reagents

Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricins A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricins biological activity. One of the sdAb (C8) was particularly effective and blocked ricins biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

Magnetic directed assembly of molecular junctions

We present a technique for fabricating molecular junctions for molecular electronic devices. Silica microspheres are rendered magnetically susceptible and electrically conductive by the sequential deposition of nickel and gold films. The metallized microspheres undergo directed assembly into lithographically defined magnetic arrays functionalized with self-assembled monolayers of prototypical molecular wire candidates. We characterize the resulting junctions by scanning electron microscopy and measure their current-voltage characteristics. Magnetic directed assembly provides a wafer-level route for the fabrication of molecular junctions and opens up the potential for hybrid complementary metal-oxide semiconductor∕molecular electronic applications.

Multiplexed Detection of Mycotoxins in Foods with a Regenerable Array

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Biotemplating rod-like viruses for the synthesis of copper nanorods and nanowires

BackgroundIn the past decade spherical and rod-like viruses have been used for the design and synthesis of new kind of nanomaterials with unique chemical positioning, shape, and dimensions in the nanosize regime. Wild type and genetic engineered viruses have served as excellent templates and scaffolds for the synthesis of hybrid materials with unique properties imparted by the incorporation of biological and organic moieties and inorganic nanoparticles. Although great advances have been accomplished, still there is a broad interest in developing reaction conditions suitable for biological templates while not limiting the material property of the product.ResultsWe demonstrate the controlled synthesis of copper nanorods and nanowires by electroless deposition of Cu on three types of Pd-activated rod-like viruses. Our aqueous solution-based method is scalable and versatile for biotemplating, resulting in Cu-nanorods 24–46 nm in diameter as measured by transmission electron microscopy. Cu2+ was chemically reduced onto Pd activated tobacco mosaic virus, fd and M13 bacteriophages to produce a complete and uniform Cu coverage. The Cu coating was a combination of Cu0 and Cu2O as determined by X- ray photoelectron spectroscopy analysis. A capping agent, synthesized in house, was used to disperse Cu-nanorods in aqueous and organic solvents. Likewise, reactions were developed to produce Cu-nanowires by metallization of polyaniline-coated tobacco mosaic virus.ConclusionsSynthesis conditions described in the current work are scalable and amenable for biological templates. The synthesized structures preserve the dimensions and shape of the rod-like viruses utilized during the study. The current work opens the possibility of generating a variety of nanorods and nanowires of different lengths ranging from 300 nm to micron sizes. Such biological-based materials may find ample use in nanoelectronics, sensing, and cancer therapy.

Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30?nm viral particle. These quantum dot?protein assemblies were studied in detail. The IgG?QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV?QD complexes have a local concentration of quantum dots greater than 3000?nmol?ml?1, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

Molecular electronics based nanosensors on a viral scaffold

Assembling and interconnecting the building blocks of nanoscale devices and being able to electronically address or measure responses at the molecular level remains an important challenge for nanotechnology. Here we show the usefulness of bottom-up self-assembly for building electronic nanosensors from multiple components that have been designed to interact in a controlled manner. Cowpea mosaic virus was used as a scaffold to control the positions of gold nanoparticles. The nanoparticles were then interconnected using thiol-terminated conjugated organic molecules, resulting in a three-dimensional conductive network. Biotin molecules were attached to the virus scaffold using linkers to act as molecular receptors. We demonstrated that binding avidin to the biotin receptors on the self-assembled nanosensors causes a significant change in the network conductance that is dependent on the charge of the avidin protein.

Quantum Dot Fluorescence as a Function of Alkyl Chain Length in Aqueous Environments

In this work, we examine the dependence of the fluorescence quantum yield of water-soluble CdSe/ZnS quantum dots on the local environment. The hydrophobicity of the local environment was modified by using different alkyl chain lengths in a set of oligo-ethylene glycols. Our results show that the quantum yield of CdSe/ZnS quantum dots is highest for the longest alkyl chain length, suggesting that a more hydrophobic environment is beneficial for generating bright, water-soluble quantum dots.

Using click chemistry toward novel 1,2,3-triazole-linked dopamine D3 receptor ligands

The dopamine D3 receptor (D3R) is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, Parkinsons disease, restless leg syndrome, and drug addiction. A common molecular template used in the development of D3R-selective antagonists and partial agonists incorporates a butylamide linker between two pharmacophores, a phenylpiperazine moiety and an extended aryl ring system. The series of compounds described herein incorporates a change to that chemical template, replacing the amide functional group in the linker chain with a 1,2,3-triazole group. Although the amide linker in the 4-phenylpiperazine class of D3R ligands has been previously deemed critical for high D3R affinity and selectivity, the 1,2,3-triazole moiety serves as a suitable bioisosteric replacement and maintains desired D3R-binding functionality of the compounds. Additionally, using mouse liver microsomes to evaluate CYP450-mediated phase I metabolism, we determined that novel 1,2,3-triazole-containing compounds modestly improves metabolic stability compared to amide-containing analogues. The 1,2,3-triazole moiety allows for the modular attachment of chemical subunit libraries using copper-catalyzed azide-alkyne cycloaddition click chemistry, increasing the range of chemical entities that can be designed, synthesized, and developed toward D3R-selective therapeutic agents.

Accelerating the initial rate of hydrolysis of methyl parathion with laser excitation using monolayer protected 10 nm Au nanoparticles capped with a Cu(bpy) catalyst

Using a low power green laser, we have demonstrated a rate acceleration of ~2-fold for the hydrolysis of methyl parathion by irradiating the plasmon absorption band of Au nanoparticles capped with a Cu(bpy) catalyst.

Molecular sensing: modulating molecular conduction through intermolecular interactions.

We observe changes in the molecular conductivity of individual oligophenylene-vinylene (OPV) molecules due to interactions with small aromatic molecules. Fluorescence experiments were correlated with scanning tunneling microscopy measurements in order to determine the origin of the observed effect. Both nitrobenzene and 1,4-dinitrobenzene decreased fluorescence intensity and molecular conductivity, while toluene had no effect. The observed changes in the fluorescence and conduction of OPV correlate well with the electron withdrawing ability of the interacting aromatic molecules. These results demonstrate the potential usefulness of OPV as a sensor for aromatic compounds containing electron withdrawing groups.