Biomedical subjects

Greg E Collins

Publications and source records attributed to Greg E Collins.

16 recordsLinked to original sources

PubMed2007

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.

Antibodies↗

PubMed2006

Bead-assisted displacement immunoassay for staphylococcal enterotoxin B on a microchip.

A microchip-based, displacement immunoassay for the sensitive laser-induced fluorescence detection of staphylococcal enterotoxin B is presented. The glass microchip device consists of a microchannel that contains a double weir structure for supporting antibody-functionalized microbeads. After a 30-min sample preparation step, the displacement assay was performed without user intervention and produced quantitative results in an additional 20 min. Linear detection responses were observed over 6 orders of magnitude and provided detection limits down to 1 fM (28.5 fg/mL). The surprisingly low detection limits are hypothesized to arise from field-based enrichment analogous to field-amplified stacking, chromatographic effects, and limited diffusion lengths in the microbead bed. The assay was challenged with bovine serum albumin, casein, and milk sample matrixes. This system has the potential to provide highly sensitive detection capabilities for target biomolecules.

Animals↗

PubMed2006

Aptamer-based detection and quantitative analysis of ricin using affinity probe capillary electrophoresis.

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (approximately 14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.

Aptamers, Nucleotide↗

PubMed2006

Micellar electrokinetic chromatography and capillary electrochromatography of nitroaromatic explosives in seawater.

The ability to separate nitroaromatic and nitramine explosives in seawater sample matrices is demonstrated using both MEKC and CEC. While several capillary-based separations exist for explosives, none address direct sampling from seawater, a sample matrix of particular interest in the detection of undersea mines. Direct comparisons are made between MEKC and CEC in terms of sensitivity and separation efficiency for the analysis of 14 explosives and explosive degradation products in seawater and diluted seawater. The use of high-salt stacking with MEKC results, on average, in a three-fold increase in the number of theoretical plates, and nearly double resolution for samples prepared in 25% seawater. By taking advantage of long injection times in conjunction with stacking, detection limits down to sub mg/L levels are attainable; however, resolution is sacrificed. CEC of explosive mixtures using sol-gels prepared from methyltrimethoxysilane does not perform as well as MEKC in terms of resolving power, but does permit extended injection times for concentrating analyte onto the head of the separation column with little or no subsequent loss in resolution. Electrokinetic injections of 8 min at high voltage allow for detection limits of explosives below 100 microg/L.

Borates↗

PubMed2006

Microchip-based CEC of nitroaromatic and nitramine explosives using silica-based sol-gel stationary phases from methyl- and ethyl-trimethoxysilane precursors.

Microchip-based CEC of nitroaromatic and nitramine explosives with UV absorbance detection is described. The stationary phase was deposited in the microchip using the sol-gel process. Unique to this work, is the exclusive use of alkylated-trimethoxysilane precursors in the gel solution. Using alkylated precursors allows for the synthesis of a hydrophobic stationary phase in a single step. Three sol-gel formulations of increasing hydrophobicity and suitable for the separation of explosives are established from methyl- and ethyl-trimethoxysilane precursors. Increasing the alkyl-chain length improved the resolution significantly, allowing for the separation of up to seven analytes. Direct injection onto the head of the stationary phase for long injection times, results in sub-mg/L detection limits with little effect on separation efficiency.

Aniline Compounds↗

PubMed2005

Integrated microfluidic device for solid-phase extraction coupled to micellar electrokinetic chromatography separation.

An integrated microdevice was utilized for the autonomous coupling of solid-phase extraction (SPE) to micellar electrokinetic chromatography (MEKC). Porous plugs of polymethacrylate polymer approximately 200 microm in length) were fabricated by ultraviolet irradiation in microchannels. Microcolumns of hydrophobic beads packed against the polymethacrylate plugs were utilized for the quantitative extraction of rhodamine B, yielding preconcentration factors over 200 for a 90-s extraction. The calculated detection limit for this dye was 60 fM. A sample of coumarin dyes were concentrated by SPE, eluted in a nonaqueous solvent from a separate on-chip reservoir, and injected by a gated valve onto a separate column for MEKC analysis. Using the integrated device, a completely automated sequence of extraction, elution, injection, separation, and detection were performed in less than 5 min. Observed separation efficiencies were high, with plate heights below 2 microm. The analysis was at least 3 times faster than semiautomated, conventional, solid-phase extraction, while requiring no user intervention. The design, fabrication, and autonomous operation of the device is discussed.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2004

Electrochemical coding for multiplexed immunoassays of proteins.

An electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies. Each biorecognition event yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of beta(2)-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity.

Animals↗

PubMed2004

Microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of V-type nerve agents.

A microchip protocol for the capillary electrophoresis separation and electrochemical detection of thiol-containing degradation products of V-type nerve agents is described. The microchip assay relies on the derivatization reaction of 2-(dimethylamino)ethanethiol (DMAET), 2-(diethylamino)ethanethiol (DEAET), and 2-mercaptoethanol (ME) with o-phthaldialdehyde in the presence of the amino acid valine along with amperometric monitoring of the isoindole derivatives. Both off-chip and on-chip derivatization reactions have led to highly sensitive and rapid detection of the thiol degradation products. Various parameters influencing the derivatization, separation, and detection processes were examined and optimized. These include the amino acid co-reagent, reagent-mixing ratio, reaction time, injection time, separation voltage, and detection potential. The chip microsystem offers a rapid (<4 min) simultaneous detection of micromolar concentrations of DMAET, DEAET, and ME. Linear calibration plots were observed for the V-type nerve agent thiol degradation products, along with good stability and reproducibility (RSD < 8.0%). Detection limits of 5 and 8 microM were obtained for the off-chip reaction of DMAET and DEAET, respectively, following a 2-s injection. The suitability for assays of environmental matrixes was demonstrated for the determination of DMAET and DEAET in untreated tap and river water samples. The favorable analytical performance makes the new microfluidic device attractive for addressing the needs of various security scenarios.

Biodegradation, Environmental↗

PubMed2004

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.

Electric Power Supplies↗

PubMed2004

Vapor and liquid phase detection of cyanide on a microchip.

A capillary electrophoresis microchip is used to selectively and sensitively monitor cyanide levels in both vapor (HCN((g))) and aqueous (NaCN in drinking water) phases. Laser-induced fluorescence detection is applied using a violet diode laser to monitor the fluorescent isoindole derivative formed by the reaction of cyanide with 2,3-naphthalenedicarboxaldehyde (NDA) and taurine. Air sampling of hydrogen cyanide is achieved using a miniature impinger (2 mL), giving collection efficiencies as high as 79% for a sampling rate of 1.0 L/min and a 10 s sampling time (relative standard deviation RSD: 2.7% for n = 5). Following the addition of NDA and taurine to either the vapor phase impinger sample or an aqueous drinking water sample, the NDA/cyanide derivative can be detected in just over 40 s on the microchip, giving a detection limit of 0.56 microg/L and a linear dynamic range from 0.56 microg/L-2.4 mg/L. The detection limit for hydrogen cyanide in air was determined to be 2.3 ppb (mole%). On-chip derivatization of cyanide by NDA was successful, although a 50% decrease in signal intensity was observed due to insufficient time for completion of the reaction on the microchip. A number of different interferents were examined, and only iron(II) and chlorine showed any interference due to their capability for masking the presence of cyanide by reacting with free cyanide in solution.

Air Pollution↗

PubMed2004

Separation of thiol and cyanide hydrolysis products of chemical warfare agents by capillary electrophoresis.

The fluorescence derivatizing agent, o-phthalaldehyde (OPA), has been applied to the separation and detection of cyanide and several structurally similar thiols by capillary electrophoresis (CE)-laser induced fluorescence (LIF). Of particular interest to this investigation was the separation of 2-dimethylaminoethanethiol, 2-diethylaminoethanethiol, and cyanide, each of which are hydrolysis products or hydrolysis product simulants of the chemical warfare (CW) agents O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), O-isobutyl S-2-diethylaminoethyl methylphosphonothiolate (R-VX), and tabun (GA). Other structurally similar thiols simultaneously resolved by this method include 1-pentanethiol and 2-mercaptoethanol. Instrumental parameters were probed and optimum values for capillary length (50 cm) and inner diameter (75 microm), injection time (30 s) and field strength (15 kV) were determined. Sample stacking methods enabled detection limits of 9.3 microg/L for cyanide, 1.8 microg/L for 2-diethylaminoethanethiol, 35 microg/L for 2-dimethylaminoethanethiol, 15 microg/L for 2-mercaptoethanol, and 89 microg/L for 1-pentanethiol. The linearity of the method was verified over an order of magnitude and the reproducibility was found to be 3.0%.

Chemical Warfare Agents↗

PubMed2003

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).

Automation↗

PubMed2003

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.

Azo Compounds↗

PubMed2003

Analysis of inorganic and small organic ions with the capillary electrophoresis microchip.

Capillary electrophoresis microchip devices are receiving considerable attention due to their versatility, portability, and sample handling capabilities. This article is a comprehensive review of the analysis of inorganic and small, charged organic species on microchip platforms. The application of conductivity, amperometry, laser-induced fluorescence, absorbance, and chemiluminescence detection methods are discussed. The potential utilization of these devices for miniaturized analytical systems is described.

Electrochemistry↗

PubMed2003

Electrophoretic microchip with dual-opposite injection for simultaneous measurements of anions and cations.

A novel dual-injection poly(methylmethacrylate) (PMMA) microchip electrophoretic system has been designed and fabricated for simultaneous measurements of anions and cations using a single channel and detection device. It consists of two sample reservoirs, on both sides of a common separation channel. Anions and cations can be simultaneously electrokinetically injected into both ends of the separation channel. Due to lower electroosmotic flow in polymer channels compared to glass ones, the cations and anions migrate in opposite directions and can be separated from each other and detected using a movable contactless conductivity detector (MCCD) positioned around the center of the separation channel. The effects of the detector position and of the separation voltage on the response and resolution have been studied and optimized for simultaneous determination of six low-energy explosive-related ions, including ammonium, methyl ammonium, sodium, chloride, nitrate, and perchlorate in a single analytical run (of ca. 3 min). Simultaneous detection of nerve-agent degradation products along with explosive-related anions and cations is also demonstrated. The versatile system can also be used for separately measuring anions or cations. The attractive behavior of the dual-opposite injection microchip offers great promise for a wide range of applications, including "total ion analysis" of various samples.

Anions↗

PubMed2002

Measurements of chemical warfare agent degradation products using an electrophoresis microchip with contactless conductivity detector.

This paper reports on a microfluidic device for the screening of organophosphonate nerve agent degradation products. The miniaturized system relies on an efficient chip-based separation of alkyl methylphosphonic acids (breakdown products of Sarin, Soman, and VX nerve agents) followed by their sensitive contactless conductivity detection. Experimental parameters relevant to the separation and detection processes have been optimized to yield high sensitivity (with 48-86 microg L(-1) detection limits), fast response (50 s for a three alkyl methylphosphonic acid mixture), high precision (RSD = 3.8-5.0%), and good linearity (over the 0.3-100 mg L(-1) range). Applicability to natural (river) water samples is demonstrated. The new microsystem offers promise for monitoring degradation products of chemical warfare agents, with advantages of speed/warning, efficiency, portability, sample size, and cost compared to conventional ion chromatography or capillary electrophoresis systems.

Chemical Warfare Agents↗