Daniel Zabetakis

Identification of explosives with two-dimensional ultraviolet resonance Raman spectroscopy.

The first two-dimensional (2D) resonance Raman spectra of TNT, RDX, HMX, and PETN are measured with an instrument that sequentially and rapidly switches between laser wavelengths, illuminating these explosives with forty wavelengths between 210 nm and 280 nm. Two-dimensional spectra reflect variations in resonance Raman scatter with illumination wavelength, adding information not available from single or few one-dimensional spectra, thereby increasing the number of variables available for use in identification, which is especially useful in environments with contaminants and interferents. We have recently shown that 2D resonance Raman spectra can identify bacteria. Thus, a single device that identifies the presence of explosives, bacteria, and other chemicals in complex backgrounds may be feasible.

Selection and evaluation of single domain antibodies toward MS2 phage and coat protein.

MS2 phage (MS2 Ø) is a coli phage, non-pathogenic to eukaryotic cells, which has been used as a simulant for viral biothreats, such as those causing smallpox and hemorrhagic fever. MS2 Ø consists of an icosahedral capsid, 28nm in diameter, and a single stranded RNA genome; the viral capsid is composed of 180 copies of coat protein (CP). In this study, we isolated anti-MS2 Ø single domain antibodies (sdAbs) for the sensitive detection of the MS2 Ø. To achieve this, a first immune sdAb library was prepared from llamas immunized with purified coat protein and a second from animals immunized with MS2 Ø. By panning the two libraries against CP, MS2 Ø, or alternating between the two targets, anti-MS2 Ø and anti-CP sdAbs were selected, sequenced, and characterized for their binding affinity. Both direct binding assays and capture sandwich assays were performed on the MAGPIX platform. One of the best anti-MS2 Ø sdAb, Lib2CP12H, could detect MS2 Ø concentrations as low as 1.45ng/mL (∼5.0E+6pfu/mL), providing equivalent detection to conventional antibodies. This sdAb is thermally stable with a melting temperature around 60°C and recovered 80% of its secondary structure after heat denaturation.

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

Production of cumulative jets by ablatively-driven implosion of hollow cones and wedges

Cumulative plasma jets formed by hollow cones imploded via laser ablation of their outer surfaces were observed. The velocity, shape, and density of the jets are measured with monochromatic 0.65keV x-ray imaging. Depending on cone geometry, cumulative jets with ion density ∼2×1020cm−3 and propagation velocities >10km∕s are formed. Similar results are observed when jets are formed by imploding wedges. Such jets can be used to simulate hydrodynamics of astrophysical jets interacting with stellar or interstellar matter.

Integrating Paper Chromatography with Electrochemical Detection for the Trace Analysis of TNT in Soil

We report on the development of an electrochemical probe for the trace analysis of 2,4,6-trinitrotoluene (TNT) in soil samples. The probe is a combination of graphite electrodes, filter paper, with ethylene glycol and choline chloride as the solvent/electrolyte. Square wave chromatovoltammograms show the probes have a sensitivity for TNT of 0.75 nA/ng and a limit of detection of 100 ng. In addition, by taking advantage of the inherent paper chromatography step, TNT can be separated in both time and cathodic peak potential from 4-amino-dinitrotolene co-spotted on the probe or in soil samples with the presence of methyl parathion as a possible interferent.

Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better. Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

A Simple and Inexpensive Electrochemical Assay for the Identification of Nitrogen Containing Explosives in the Field

We report a simple and inexpensive electrochemical assay using a custom built hand-held potentiostat for the identification of explosives. The assay is based on a wipe test and is specifically designed for use in the field. The prototype instrument designed to run the assay is capable of performing time-resolved electrochemical measurements including cyclic square wave voltammetry using an embedded microcontroller with parts costing roughly

Plasma-Modified, Epitaxial Fabricated Graphene on SiC for the Electrochemical Detection of TNT

250 USD. We generated an example library of cyclic square wave voltammograms of 12 compounds including 10 nitroaromatics, a nitramine (RDX), and a nitrate ester (nitroglycine), and designed a simple discrimination algorithm based on this library data for identification.

Incorporation of nano-particle sites in polymer matrix by Metal ion imprinting

Using square wave voltammetry, we show an increase in the electrochemical detection of trinitrotoluene (TNT) with a working electrode constructed from plasma modified graphene on a SiC surface vs. unmodified graphene. The graphene surface was chemically modified using electron beam generated plasmas produced in oxygen or nitrogen containing backgrounds to introduce oxygen or nitrogen moieties. The use of this chemical modification route enabled enhancement of the electrochemical signal for TNT, with the oxygen treatment showing a more pronounced detection than the nitrogen treatment. For graphene modified with oxygen, the electrochemical response to TNT can be fit to a two-site Langmuir isotherm suggesting different sites on the graphene surface with different affinities for TNT. We estimate a limit of detection for TNT equal to 20 ppb based on the analytical standard S/N ratio of 3. In addition, this approach to sensor fabrication is inherently a high-throughput, high-volume process amenable to industrial applications. High quality epitaxial graphene is easily grown over large area SiC substrates, while plasma processing is a rapid approach to large area substrate processing. This combination facilitates low cost, mass production of sensors.