Empirical determination of explosive vapor transport efficiencies.

The transport efficiency of 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazinane (RDX) trace vapors through tubing materials that commonly constitute vapor handling infrastructures have been determined for a variety of tubing dimensions and sampling conditions. Using a programmable temperature vaporization inlet coupled with a gas chromatography mass spectrometer (PTV-GC-MS), the explosive vapors were quantified both with and without a length of tubing of a specific material in the sampling flow path. At vapor temperatures of 30 °C and 66 °C, minimal attenuations were observed for 2,4-DNT and TNT vapor concentrations when the tubing material was in-line with the sampling flow path, indicating that the transport is largely unaffected by interactions with the surface of the tubing materials. In contrast, RDX vapors showed large attenuations as a function of both sampling conditions and tubing materials/dimensions. For those experiments where attenuated RDX vapor transport was observed, the mass sequestered by interactions between the flowing vapor and the internal tubing surface was determined to be in the range of tens to hundreds of picograms. Of all the materials examined for RDX transport, fluorinated ethylene propylene (FEP) tubing resulted in the least amount of mass loss to surface interactions, with vapor transport efficiencies (VTEs) between 95-100%. However, for some materials, the combination of tubing dimensions and sampling conditions resulted in no RDX transport, even after sampling more than 250.0 L of vapor through the tubing.

Trace Explosives Vapor Generation and Quantitation at Parts per Quadrillion Concentrations.

The generation of trace 2,4,6-trinitrotoluene (TNT), cyclotrimethylenetrinitramine (RDX), and pentaerythritol tetranitrate (PETN) vapors using a pneumatically modulated liquid delivery system (PMLDS) coupled to a polytetrafluoroethylene (PTFE) total-consumption micronebulizer is presented. The vapor generator operates in a continuous manner with final vapor concentrations proportional to the explosive concentration in aqueous solution delivered through the nebulizer and the diluent air flow rate. For quantitation of concentrations in the parts per billionvolume (ppbv) to parts per trillionvolume (pptrv) range, Tenax-TA thermal desorption tubes were used for vapor collection with subsequent analysis on a thermal-desorption system programmable-temperature vaporization gas chromatograph (TDS-PTV-GC) with a µ-ECD detector. With 30 min sample times and an average sampling rate of 100 mL min(-1), vapor concentrations of 38 pptrv for TNT, 25 pptrv for RDX, and 26 pptrv for PETN were determined. For parts per quadrillionvolume (ppqv) vapor quantitation of TNT and RDX, an online PTV-GC system with a negative-ion chemical ionization mass spectrometer (methane reagent gas) was used for direct sampling and capture of the vapor on the PTV inlet. Vapor concentrations as low as 160 ppqv and 710 ppqv for TNT and RDX were quantified, respectively, with an instrument duty cycle as low as 4 min.

Catalytic activity and thermal stability of horseradish peroxidase encapsulated in self-assembled organic nanotubes.

Horseradish peroxidase (HRP) was encapsulated in self-assembled lithocholic acid (LCA) based organic nanotubes and its catalytic activity before and after thermal treatment was measured for comparison with free HRP. The apparent kcat (kcat/Km) for nanotube encapsulated HRP remained almost the same before and after thermal treatment, reporting an average value of 3.7 ± 0.4 µM(-1) s(-1). The apparent kcat value for free HRP decreased from 14.8 ± 1.3 µM(-1) s(-1) for samples stored at 4 °C to 2.4 ± 0.1 µM(-1) s(-1) after thermal treatment for 8 h at 55 °C. The Michaelis-Menten constants, Km, determined for encapsulated HRP and free HRP were relatively unperturbed by storage conditions at 4 °C or thermally treated at 55 °C for varying time periods from 2-8 h, with encapsulated HRP having a slightly higher Km than free HRP (13.4 ± 0.9 µM versus 11.7 ± 0.4 µM). The amount of HRP encapsulated in LCA nanotubes increased dramatically when the mixture of HRP and LCA nanotubes was brought to an elevated temperature. Within 4 h of thermal treatment at 55 °C, the amount of HRP encapsulated by the LCA nanotubes was more than 4 times the amount of HRP encapsulated when equilibrated at 4 °C for 7 days. Molecular dynamics (MD) simulations show that the higher degree of exposure of hydrophobic residues in HRP at elevated temperatures enhances the hydrophobic interaction between HRP and the nanotube wall, resulting in the increased amount of HRP surface adsorption and, hence, the overall amount of encapsulation inside the nanotubes.

Pico-force optical exchange (pico-FOX): utilizing optical forces applied to an orthogonal electroosmotic flow for particulate enrichment from mixed sample streams.

Results are reported from a combined optical force and electrokinetic microfluidic device that separates individual particulates from molecular components in a mixed sample stream. A pico-Newton optical force was applied to an orthogonal electroosmotic flow carrying a hydrodynamically pinched, mixed sample, resulting in the separation of the various particles from the sample stream. Different combinations of polystyrene, PMMA, and silica particles with a commercially available dye were utilized to test the different separation modes available, from purely optical force to combined optical and electrophoretic forces. The impact of various particle properties on particle separation and separation efficiency were explored, including size (2, 6, 10 µm), refractive index, and electrophoretic mobility. Particle addressability was achieved by moving particles to different outlets on the basis of particle size, refractive index, and electrophoretic differences. Separations of 6 and 10 µm polystyrene particles led to only 3% particle contamination in the original sample stream and interparticle type enrichment levels >80%. The unique addressability of three different particle materials (polystyrene, PMMA, and silica) of the same size (2 µm) led to each being separated into a unique outlet without measurable contamination of the other particle types using optical force and electrophoretic mobility. In addition to particle separation, the device was able to minimize dye diffusion, leading to >95% dye recovery. This combined platform would have applications for noninvasive sample preparation of mixed molecular/particulate systems for mating with traditional analytics as well as efficient removal of harmful, degrading components from complex mixtures.

Orthogonal optical force separation simulation of particle and molecular species mixtures under direct current electroosmotic driven flow for applications in biological sample preparation.

Presented here are the results from numerical simulations applying optical forces orthogonally to electroosmotically induced flow containing both molecular species and particles. Simulations were conducted using COMSOL v4.2a Multiphysics® software including the particle tracking module. The study addresses the application of optical forces to selectively remove particulates from a mixed sample stream that also includes molecular species in a pinched flow microfluidic device. This study explores the optimization of microfluidic cell geometry, magnitude of the applied direct current electric field, EOF rate, diffusion, and magnitude of the applied optical forces. The optimized equilibrium of these various contributing factors aids in the development of experimental conditions and geometry for future experimentation as well as directing experimental expectations, such as diffusional losses, separation resolution, and percent yield. The result of this work generated an optimized geometry with flow conditions leading to negligible diffusional losses of the molecular species while also being able to produce particle removal at near 100% levels. An analytical device, such as the one described herein with the capability to separate particulate and molecular species in a continuous, high-throughput fashion would be valuable by minimizing sample preparation and integrating gross sample collection seamlessly into traditional analytical detection methods.

Trace vapor generator for Explosives and Narcotics (TV-Gen).

The Trace Vapor Generator for Explosives and Narcotics (TV-Gen) is a portable and compact instrument designed to deliver a continuous source of trace-level vapors and vapor mixtures. It provides a tool to assist in the independent validation and verification of new materials and sensors under development for the vapor detection of explosives and narcotics. The design was conceived for use with a broad range of analytes, detection systems, materials, and sensors and to switch easily between the clean and analyte vapor streams. The TV-Gen system utilizes nebulization of aqueous analyte solutions, an oven to promote efficient transport, and a control box that provides dedicated computer control with logging capabilities. Resultant vapor streams are stable over several hours, with the vapor concentration controlled by a combination of aqueous analyte solution concentration, liquid flow rate through the nebulizer, and volume flow rate of air through the TV-Gen manifold.

A fritless, EOF microchip pump for high pressure pumping of aqueous and organic solvents.

A fritless, microchip electroosmotic flow (EOF) pump is microfabricated and demonstrated on a planar soda lime glass substrate to be capable of supplying reasonable flow rates under high back pressures, such as that required for micro-high pressure liquid chromatography (micro-HPLC). The microchip EOF pump is composed of a densely packed microchannel containing 800 nm silica particles and was capable of generating a maximum pressure > 1000 psi ( approximately 7 MPa) and a maximum flow rate of 282 nL/min (aqueous cyclohexylamino alkyl sulfonate (CHES) buffer, 10 mM, pH 9.0, 200 V/cm). Other pumping fluids, such as CHES buffer-acetonitrile mixture (50%, v/v), CHES buffer-methanol mixture (50%, v/v), and pure acetonitrile were also used in a characterization of pump performance that included determinations of the maximum flow rate, maximum pressure, and resulting flow rate against an applied, downstream back pressure. The flow rate under a 200 psi ( approximately 1.4 MPa) back pressure at an applied electric field strength of 250 V/cm ranged from 285 nL/min for aqueous CHES buffer to 44 nL/min for CHES buffer-acetonitrile mixture (50%, v/v), indicating that this EOF pump will meet the future requirements of a micro-HPLC system.

Micellar Electrokinetic Chromatography.

Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis that allows for the separation of neutral molecules in an electric field. Typically, neutral molecules move with electroosmotic flow (EOF) or bulk flow during electrophoretic separations resulting in no temporal resolution between mixtures of neutral analytes. Inclusion of surfactant micelles in the separation buffer allows for the separation of neutral analytes from one another through association with the micelle. Here we outline the implementation of MEKC for the separation of neutral molecules using a mixture of nitroaromatic explosives and their degradation products serving as a test analyte mixture.

Flow-Through Optical Chromatography in Combination with Confocal Raman Microspectroscopy: A Novel Label-Free Approach To Detect Responses of Live Macrophages to Environmental Stimuli.

Flow-through optical chromatography (FT-OC), an advanced mode of optical chromatography, achieved baseline separation of a mixture of silica microparticles (SiO2, 1.00 and 2.50 µm) and a mixture of polystyrene microparticles (PS, 1.00, 2.00, and 3.00 µm) based on particle size. Comparisons made between experimentally determined velocities for the microparticles and theoretically derived velocities from Mie theory and Stokes' law validated the data collection setup and the data analysis for FT-OC. A population shift in live macrophages (cell line IC-21, ATCC TIB-186) responding to environmental stimuli was sensitively detected by FT-OC. The average velocity of macrophages stressed by nutritional deprivation was decreased considerably together with a small but statistically significant increase in cell size. Mie scattering calculations demonstrated that the small increase in cell size of macrophages stressed by nutritional deprivation was not entirely responsible for this decrease. Confocal fluorescence microscopy and atomic force microscopy (AFM) studies revealed morphological changes of macrophages induced by nutritional deprivation, and these changes were more likely responsible for the decrease in average velocity detected by FT-OC. Confocal Raman microspectroscopy was used to shed light upon biochemical transformations of macrophages suffering from nutritional deprivation.

Electroosmotic flow-based pump for liquid chromatography on a planar microchip.

An electroosmotic flow (EOF)-based pump, integrated with a sol-gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 microm x 100 microm) packed with 5-microm porous silica beads. The sol-gel stationary phase was derived from a precursor mixture of methyltrimethoxy- and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 microm x 100 microm. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging.

Simple and rapid extraction, separation, and detection of alkaloids in beverages.

Implementation of an uncomplicated SPE process for the rapid extraction and preconcentration of the alkaloids, colchicine, strychnine, aconitine, and nicotine, from water, apple juice, and nonfat milk samples is presented. When coupled to analysis via micellar EKC (MEKC), the total analysis time per sample was less than 15 min for the water and juice samples and less than 20 min for the milk. The SPE process allowed for anywhere from a three to a fourteen-fold improvement in the LOD for each alkaloid when compared to detecting the alkaloids in a nontreated water sample matrix. Following SPE, the LODs for colchicine, strychnine, and nicotine were sufficient to meet levels from 150 to 5000 times more dilute than the LD(50) for a 50 kg individual drinking 12 oz of a contaminated beverage. Aconitine, on the other hand, was detected at approximately the LD(50) level. The percent recoveries for the SPE ranged from 37% to as high as 99%. Nicotine attained the highest recovery efficiencies, followed by colchicine, and finally, aconitine and strychnine, which were nearly identical. The greatest recovery efficiencies were achieved from apple juice and water, whereas nonfat milk yielded the lowest.

Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies.

A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-linkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody-gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose-response curve is 1 ng/mL, corresponding to 5 pg in a 5-microL sample. Coupling the specificity of immunoassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices.

Microchip-based electrochemical enzyme immunoassays.

In this chapter a microchip-based electrochemical enzyme immunoassay is developed and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. Such a direct homogeneous immunoassay requires the integration of electrokinetic mixing of alkaline phosphatase (ALP)-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl phosphate (p-APP) substrate (S) and downstream amperometric detection of p-aminophenol (p-AP) product. Factors influencing the reaction, injection, separation, and detection processes are optimized. We have characterized the microchip-based immunoassay protocol. The resulting attractive analytical performance, along with distinct miniaturization and portability advantages of the electrochemical microsystem, offer considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics.

Trace explosives sensor testbed (TESTbed).

A novel vapor delivery testbed, referred to as the Trace Explosives Sensor Testbed, or TESTbed, is demonstrated that is amenable to both high- and low-volatility explosives vapors including nitromethane, nitroglycerine, ethylene glycol dinitrate, triacetone triperoxide, 2,4,6-trinitrotoluene, pentaerythritol tetranitrate, and hexahydro-1,3,5-trinitro-1,3,5-triazine. The TESTbed incorporates a six-port dual-line manifold system allowing for rapid actuation between a dedicated clean air source and a trace explosives vapor source. Explosives and explosives-related vapors can be sourced through a number of means including gas cylinders, permeation tube ovens, dynamic headspace chambers, and a Pneumatically Modulated Liquid Delivery System coupled to a perfluoroalkoxy total-consumption microflow nebulizer. Key features of the TESTbed include continuous and pulseless control of trace vapor concentrations with wide dynamic range of concentration generation, six sampling ports with reproducible vapor profile outputs, limited low-volatility explosives adsorption to the manifold surface, temperature and humidity control of the vapor stream, and a graphical user interface for system operation and testing protocol implementation.

Factors affecting the intramolecular decomposition of hexamethylene triperoxide diamine and implications for detection.

Hexamethylene triperoxide diamine (HMTD) is an easily synthesized and highly sensitive organic peroxide frequently used as a primary explosive. The vapor pressure of HMTD is very low, impeding vapor detection, especially when compared to other peroxide explosives, such as triacetone triperoxide (TATP) or diacetone diperoxide (DADP). Despite this fact, HMTD has a perceptible odor that could be utilized in the indirect detection of HMTD vapor. Headspace measurements above solid HMTD samples confirm that HMTD readily decomposes under ambient conditions to form highly volatile products that include formic acid, ammonia, trimethylamine and formamides. The presence and quantity of these compounds are affected by storage condition, time, and synthetic method, with synthetic method having the most significant effect on the content of the headspace. A kinetic study of HMTD decomposition in solution indicated a correlation between degradation rate and the presence of decomposition species identified in the headspace, and provided further insight into the mechanism of decomposition. The study provided evidence for a proton assisted decomposition reaction with water, as well as an intramolecular decomposition process facilitated by the presence of water.

Analysis of ammonium nitrate headspace by on-fiber solid phase microextraction derivatization with gas chromatography mass spectrometry.

A novel analytical method has been developed for the quantitation of trace levels of ammonia in the headspace of ammonium nitrate (AN) using derivatized solid phase microextraction (SPME) fibers with gas chromatography mass spectrometry (GC-MS). Ammonia is difficult to detect via direct injection into a GC-MS because of its low molecular weight and extreme polarity. To circumvent this issue, ammonia was derivatized directly onto a SPME fiber by the reaction of butyl chloroformate coated fibers with the ammonia to form butyl carbamate. A derivatized externally sampled internal standard (dESIS) method based upon the reactivity of diethylamine with unreacted butyl chloroformate on the SPME fiber to form butyl diethylcarbamate was established for the reproducible quantification of ammonia concentration. Both of these compounds are easily detectable and separable via GC-MS. The optimized method was then used to quantitate the vapor concentration of ammonia in the headspace of two commonly used improvised explosive device (IED) materials, ammonium nitrate fuel oil (ANFO) and ammonium nitrate aluminum powder (Ammonal), as well as identify the presence of additional fuel components within the headspace.

Impact of confinement on proteins concentrated in lithocholic acid based organic nanotubes.

Organic nanotubes form in aqueous solution near physiological pH by self-assembly of lithocholic acid (LCA) with inner diameters of 20-40nm. The encapsulation of enhanced green fluorescent protein (eGFP) and resultant confinement effect for eGFP within these nanotubes is studied via confocal microscopy. Timed release rate studies of eGFP encapsulated in LCA nanotubes and fluorescence recovery after photobleaching (FRAP) indicate that the diffusive transport of eGFP out of and/or within the nanotubes is very slow, in contrast to the rapid introduction of eGFP into the nanotubes. By encapsulating two fluorescent proteins in LCA nanotubes, eGFP and mCherry, as a fluorescence resonance energy transfer (FRET) pair, the FRET efficiencies are determined using FRET imaging microscopy at three different protein concentrations with a fixed donor-to-acceptor ratio of 1:1. Förster theory reveals that the proteins are spatially separated by 4.8-7.2nm in distance inside these nanotubes. The biomimetic nanochannels of LCA nanotubes not only afford a confining effect on eGFP that results in enhanced chemical and thermal stability under conditions of high denaturant concentration and temperature, but also function as protein concentrators for enriching protein in the nanochannels from a diluted protein solution by up to two orders of magnitude.

Compact, high voltage power supply for the lab-on-a-chip.

The design, construction and operation of a simple, inexpensive and compact high voltage power supply (HVPS) for use in conjunction with a simple cross capillary electrophoresis microchip is presented. The microchip HVPS utilizes a single high voltage power supply (15 kV), a voltage-divider network, to give the voltages necessary to operate a gated injection valve, and two high voltage relays for switching between the open and closed gate sequences of the injection. In order to accommodate the application of different simple cross microchip dimensions, a set of equations for defining the resistor network and ensuring proper gate performance are presented.

Nonaqueous based microchip separation of toxic metal ions using 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol.

The colorimetric metal chelating agent, 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (5-Br-PAPS), was demonstrated on a capillary electrophoresis microchip in the separation and detection of six metal ions of environmental concern, Cd2+, Pb2+, Cu2+, Co2+, Ni2+, and Hg2+. The inclusion of methanol in the buffer was found to improve both the separation efficiency and sensitivity, in addition to making the technique directly amenable to the application of solid-phase extraction. The combination of metal chelation with solid-phase extraction on a C18 silica gel microcolumn gave several hundred fold improvements in detection limits for the CE microchip measurements of toxic metal ions in water and extracted from a solid Plexiglas surface.

Microscale solid-phase extraction system for explosives.

A simple, semi-automated, microcolumn solid-phase extraction (SPE) system is optimized for the extraction, preconcentration and HPLC analysis of seven different explosives and explosive derivatives contaminating seawater, river water and well water samples. The microcolumns were constructed from 1/16 in. O.D. PTFE tubing (1 in.=2.54 cm) packed with 0.5-1.5 mg of SPE material. LiChrolut EN or Porapak R. The extraction system consisted of two syringe pumps and several solenoid valves. Optimal detection limits were realized when the sample water flow-rate was maximally increased within the limits of the pump, 5-10 ml/min (despite exceeding the breakthrough threshold of the SPE microcolumn), and when the eluate volume collected from the column was minimized, <5 microl (despite very low recovery percentages).