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1.
PLoS One ; 14(9): e0221831, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31490969

RESUMO

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22-40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.


Assuntos
Bactérias/efeitos dos fármacos , Polissacarídeos/farmacologia , Preservação Biológica/métodos , Bactérias/citologia , Bactérias/metabolismo , Cisteína/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Polissacarídeos Bacterianos/farmacologia , Proteômica , Temperatura
2.
Langmuir ; 33(11): 2790-2798, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28248516

RESUMO

Azides on the periphery of nanodiamond materials (ND) are of great utility because they have been shown to undergo Cu-catalyzed and Cu-free cycloaddition reactions with structurally diverse alkynes, affording particles tailored for applications in biology and materials science. However, current methods employed to access ND featuring azide groups typically require either harsh pretreatment procedures or multiple synthesis steps and use surface linking groups that may be susceptible to undesirable cleavage. Here we demonstrate an alternative single-step approach to producing linker-free, azide-functionalized ND. Our method was applied to low-cost, detonation-derived ND powders where surface carbonyl groups undergo silver-mediated decarboxylation and radical substitution with azide. ND with directly grafted azide groups were then treated with a variety of aliphatic, aromatic, and fluorescent alkynes to afford 1-(ND)-4-substituted-1,2,3-triazole materials under standard copper-catalyzed cycloaddition conditions. Surface modification steps were verified by characteristic infrared absorptions and elemental analyses. High loadings of triazole surface groups (up to 0.85 mmol g-1) were obtained as determined from thermogravimetric analysis. The azidation procedure disclosed is envisioned to become a valuable initial transformation in numerous future applications of ND.

3.
Chem Commun (Camb) ; 52(57): 8802-5, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27292161

RESUMO

Glutardiamidoxime, a structural motif on sorbents used in uranium extraction from seawater, was discovered to cyclize in situ at room temperature to 2,6-diimino-piperidin-1-ol in the presence of uranyl nitrate. The new diimino motif was also generated when exposed to competing transition metals Cu(ii) and Ni(ii). Multinuclear µ-O bridged U(vi), Cu(ii), and Ni(ii) complexes featuring bound diimino ligands were isolated.

4.
Dalton Trans ; 45(28): 11312-25, 2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27184739

RESUMO

The ability to collect uranium from seawater offers the potential for a nearly limitless fuel supply for nuclear energy. We evaluated the use of functionalized nanostructured sorbents for the collection and recovery of uranium from seawater. Extraction of trace minerals from seawater and brines is challenging due to the high ionic strength of seawater, low mineral concentrations, and fouling of surfaces over time. We demonstrate that rationally assembled sorbent materials that integrate high affinity surface chemistry and high surface area nanostructures into an application relevant micro/macro structure enables collection performance that far exceeds typical sorbent materials. High surface area nanostructured silica with surface chemistries composed of phosphonic acid, phosphonates, 3,4 hydroxypyridinone, and EDTA showed superior performance for uranium collection. A few phosphorous-based commercial resins, specifically Diphonix and Ln Resin, also performed well. We demonstrate an effective and environmentally benign method of stripping the uranium from the high affinity sorbents using inexpensive nontoxic carbonate solutions. The cyclic use of preferred sorbents and acidic reconditioning of materials was shown to improve performance. Composite thin films composed of the nanostructured sorbents and a porous polymer binder are shown to have excellent kinetics and good capacity while providing an effective processing configuration for trace mineral recovery from solutions. Initial work using the composite thin films shows significant improvements in processing capacity over the previously reported sorbent materials.

5.
RSC Adv ; 6(107): 105239-105251, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-31354950

RESUMO

Magnetic nanoparticles are well known to possess chemically active surfaces and large surface areas that can be employed to extract a range of ions from aqueous solutions. Additionally, their superparamagnetic properties provide a convenient means for bulk collection of the material from solution after the targeted ions have been adsorbed. Herein, two nanoscale amphoteric metal oxides, each possessing useful magnetic attributes, were evaluated for their ability to collect trace levels of a chemically diverse range of alpha emitting radioactive isotopes (polonium (Po), radium (Ra), uranium (U), and americium (Am)) from a wide range of aqueous solutions. The nanomaterials include commercially available magnetite (Fe3O4) and magnetite modified to incorporate manganese (Mn) into the crystal structure. The chemical stability of these nanomaterials was evaluated in Hanford Site, WA ground water between the natural pH (~8) and pH 1. Whereas the magnetite was observed to have good stability over the pH range, the Mn-doped material was observed to leach Mn at low pH. The materials were evaluated in parallel to characterize their uptake performance of the alpha-emitting radionuclide spikes from ground water across a range of pH (from ~8 down to 2). In addition, radiotracer uptake experiments were performed on Columbia River water, seawater, and human urine at their natural pH and at pH 2. Despite the observed leaching of Mn from the Mn-doped nanomaterial in the lower pH range, it exhibited generally superior analyte extraction performance compared to the magnetite, and analyte uptake was observed across a broader pH range. We show that the uptake behavior of the various radiotracers on these two materials at different pH levels can generally be explained by the amphoteric nature of the nanoparticle surfaces. Finally, the rate of sorption of the radiotracers on the two materials in unacidified ground water was evaluated. The uptake curves generally indicate that equilibrium is obtained within a few minutes, which is attributed to the high surface areas of the nanomaterials and the high level of dispersion in the liquids. Overall, the results indicate that these nanomaterials may have the potential to be employed for a range of applications to extract radionuclides from aqueous solutions.

6.
J Microbiol Methods ; 118: 18-24, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26295278

RESUMO

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.


Assuntos
Bacillus anthracis/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Proteoma/análise , Proteoma/isolamento & purificação , Proteômica/métodos , Esporos Bacterianos/química , Bacillus anthracis/efeitos dos fármacos , Esporos Bacterianos/efeitos dos fármacos , Ácido Tricloroacético/metabolismo
7.
Bioconjug Chem ; 26(3): 593-601, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25730280

RESUMO

Methods to covalently conjugate Alexa Fluor dyes to cellulose nanocrystals, at limiting amounts that retain the overall structure of the nanocrystals as model cellulose materials, were developed using two approaches. In the first, aldehyde groups are created on the cellulose surfaces by reaction with limiting amounts of sodium periodate, a reaction well-known for oxidizing vicinal diols to create dialdehyde structures. Reductive amination reactions were then applied to bind Alexa Fluor dyes with terminal amino-groups on the linker section. In the absence of the reductive step, dye washes out of the nanocrystal suspension, whereas with the reductive step, a colored product is obtained with the characteristic spectral bands of the conjugated dye. In the second approach, Alexa Fluor dyes were modified to contain chloro-substituted triazine ring at the end of the linker section. These modified dyes then were reacted with cellulose nanocrystals in acetonitrile at elevated temperature, again isolating material with the characteristic spectral bands of the Alexa Fluor dye. Reactions with Alexa Fluor 546 are given as detailed examples, labeling on the order of 1% of the total glucopyranose rings of the cellulose nanocrystals at dye loadings of ca. 5 µg/mg cellulose. Fluorescent cellulose nanocrystals were deposited in pore network microfluidic structures (PDMS) and proof-of-principle bioimaging experiments showed that the spatial localization of the solid cellulose deposits could be determined, and their disappearance under the action of Celluclast enzymes or microbes could be observed over time. In addition, single molecule fluorescence microscopy was demonstrated as a method to follow the disappearance of solid cellulose deposits over time, following the decrease in the number of single blinking dye molecules with time instead of fluorescent intensity.


Assuntos
Microambiente Celular , Celulose/análise , Corantes Fluorescentes/química , Nanopartículas/química , Compostos de Quinolínio/química , Celulose/química , Microscopia de Fluorescência/métodos
8.
Analyst ; 139(10): 2440-8, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24695634

RESUMO

This paper presents results of designed experiments performed to study the effect of four factors on the detection of RDX vapors from desorption into an atmospheric flow tube mass spectrometer (AFT-MS). The experiments initially included four independent factors: gas flow rate, desorption current, solvent evaporation time and RDX mass. The values of three detection responses, peak height, peak width, and peak area were recorded but only the peak height response was analyzed. Results from the first block of experiments indicated that solvent evaporation time was not statistically significant at the 95% confidence level. A second round of experiments was designed and executed using flow rate, current, and RDX mass as factors and the results were used to create a model to predict conditions resulting in maximum peak height. Those conditions were confirmed experimentally and used to obtain data for a calibration model. The calibration model represented RDX amounts ranging from 1 to 25 pg desorbed into an air flow of 7 L min(-1). Air samples from a shipping container that held 2 closed explosive storage magazines were collected on metal filaments for varying amounts for time ranging from 5 to 90 minutes. RDX was detected from all of the filaments sampled by desorption into the AFT-MS. From the calibration model, RDX vapor concentrations within the shipping container were calculated to be in the range of 1 to 50 parts-per-quadrillion (ppqv) from data collected on 2 separate days.

9.
Langmuir ; 28(18): 7182-8, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22364481

RESUMO

The use of air-water, θ(wa), or air-liquid contact angles is customary in surface science, while oil-water contact angles, θ(ow), are of paramount importance in subsurface multiphase flow phenomena including petroleum recovery, nonaqueous phase liquid fate and transport, and geological carbon sequestration. In this paper we determine both the air-water and oil-water contact angles of silica surfaces modified with a diverse selection of silanes, using hexadecane as the oil. The silanes included alkylsilanes, alkylarylsilanes, and silanes with alkyl or aryl groups that are functionalized with heteroatoms such as N, O, and S. These silanes yielded surfaces with wettabilities from water wet to oil wet, including specific silanized surfaces functionalized with heteroatoms that yield intermediate wet surfaces. The oil-water contact angles for clean and silanized surfaces, excluding one partially fluorinated surface, correlate linearly with air-water contact angles with a slope of 1.41 (R = 0.981, n = 13). These data were used to examine a previously untested theoretical treatment relating air-water and oil-water contact angles in terms of fluid interfacial energies. Plotting the cosines of these contact angles against one another, we obtain the relationship cos θ(wa) = 0.667 cos θ(ow) + 0.384 (R = 0.981, n = 13), intercepting cos θ(ow) = -1 at -0.284, which is in excellent agreement with the linear assumption of the theory. The theoretical slope, based on the fluid interfacial tensions σ(wa), σ(ow), and σ(oa), is 0.67. We also demonstrate how silanes can be used to alter the wettability of the interior of a pore network micromodel device constructed in silicon/silica with a glass cover plate. Such micromodels are used to study multiphase flow phenomena. The contact angle of the resulting interior was determined in situ. An intermediate wet micromodel gave a contact angle in excellent agreement with that obtained on an open planar silica surface using the same silane.

10.
Langmuir ; 28(8): 3931-7, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22329500

RESUMO

A method for tuning the analyte affinity of magnetic, inorganic nanostructured sorbents for heavy metal contaminants is described. The manganese-doped iron oxide nanoparticle sorbents have a remarkably high affinity compared to the precursor material. Sorbent affinity can be tuned toward an analyte of interest simply by adjustment of the dopant quantity. The results show that following the Mn doping process there is a large increase in affinity and capacity for heavy metals (i.e., Co, Ni, Zn, As, Ag, Cd, Hg, and Tl). Capacity measurements were carried out for the removal of cadmium from river water and showed significantly higher loading than the relevant commercial sorbents tested for comparison. The reduction in Cd concentration from 100 ppb spiked river water to 1 ppb (less than the EPA drinking water limit of 5 ppb for Cd) was achieved following treatment with the Mn-doped iron oxide nanoparticles. The Mn-doped iron oxide nanoparticles were able to load ~1 ppm of Cd followed by complete stripping and recovery of the Cd with a mild acid wash. The Cd loading and stripping is shown to be consistent through multiple cycles with no loss of sorbent performance.


Assuntos
Compostos Férricos/química , Manganês/química , Nanopartículas Metálicas/química , Metais Pesados/química , Adsorção , Cádmio/química , Poluentes Químicos da Água/química , Purificação da Água/métodos
11.
Environ Sci Technol ; 45(17): 7581-8, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21774502

RESUMO

Permeability contrasts exist in multilayer geological formations under consideration for carbon sequestration. To improve our understanding of heterogeneous pore-scale displacements, liquid CO(2) (LCO(2))-water displacement was evaluated in a pore network micromodel with two distinct permeability zones. Due to the low viscosity ratio (logM = -1.1), unstable displacement occurred at all injection rates over 2 orders of magnitude. LCO(2) displaced water only in the high permeability zone at low injection rates with the mechanism shifting from capillary fingering to viscous fingering with increasing flow rate. At high injection rates, LCO(2) displaced water in the low permeability zone with capillary fingering as the dominant mechanism. LCO(2) saturation (S(LCO2)) as a function of injection rate was quantified using fluorescent microscopy. In all experiments, more than 50% of LCO(2) resided in the active flowpaths, and this fraction increased as displacement transitioned from capillary to viscous fingering. A continuum-scale two-phase flow model with independently determined fluid and hydraulic parameters was used to predict S(LCO2) in the dual-permeability field. Agreement with the micromodel experiments was obtained for low injection rates. However, the numerical model does not account for the unstable viscous fingering processes observed experimentally at higher rates and hence overestimated S(LCO2).


Assuntos
Dióxido de Carbono/química , Sedimentos Geológicos/química , Modelos Teóricos , Água/química , Permeabilidade , Porosidade , Viscosidade
12.
Health Phys ; 101(2): 196-208, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21709509

RESUMO

In the event of an accidental or intentional release of radionuclides into a populated area, massive numbers of people may require radiobioassay screening as triage for dose-reduction therapy or identification for longer-term follow-up. If the event released significant levels of beta- or alpha-emitting radionuclides, in vivo assays would be ineffective. Therefore, highly efficient and rapid analytical methods for radionuclide detection from submitted spot urine samples (≤50 mL) would be required. At present, the quantitative determination of alpha-emitting radionuclides from urine samples is highly labor intensive and requires significant time to prepare and analyze samples. Sorbent materials that provide effective collection and enable rapid assay could significantly streamline the radioanalytical process. The authors have demonstrated the use of magnetic nanoparticles as a novel method of extracting media for four alpha-emitting radionuclides of concern (polonium, radium, uranium and americium) from chemically-unmodified and pH-2 human urine. Herein, the initial experimental sorption results are presented along with a novel method that uses magnetic nanoparticles to extract radionuclides from unmodified human urine and then collect the magnetic field-induced particles for subsequent alpha-counting-source preparation. Additionally, a versatile human dose model is constructed that determines the detector count times required to estimate dose at specific protective-action thresholds. The model provides a means to assess a method's detection capabilities and uses fundamental health physics parameters and actual experimental data as core variables. The modeling shows that, with effective sorbent materials, rapid screening for alpha-emitters is possible with a 50-mL urine sample collected within 1 wk of exposure/intake.


Assuntos
Magnetismo , Nanopartículas Metálicas/química , Monitoramento de Radiação/métodos , Radioisótopos/urina , Urinálise/métodos , Partículas alfa , Humanos , Concentração de Íons de Hidrogênio , Monitoramento de Radiação/instrumentação , Sensibilidade e Especificidade , Fatores de Tempo , Urinálise/instrumentação
13.
Proteomics ; 11(2): 309-18, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21204257

RESUMO

Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, whereas the conventional immobilization of covalently attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. A single model protein, a five-protein mixture, and a whole mouse brain proteome were digested at atmospheric pressure and 37°C for 12 h or in combination with pressure cycling technology at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both the identified peptide/protein number and the digestion reproducibility. In addition, the concomitant use of EC-TR/NPs and pressure cycling technology resulted in very rapid (∼1 min) and efficient digestions with more reproducible digestion results.


Assuntos
Enzimas Imobilizadas/metabolismo , Magnetismo , Nanopartículas/química , Proteínas/metabolismo , Proteômica/métodos , Tripsina/metabolismo , Animais , Encéfalo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Pressão , Proteoma/metabolismo , Proteômica/economia
14.
ACS Appl Mater Interfaces ; 2(10): 2749-58, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20939537

RESUMO

Surface-functionalized nanoporous silica, often referred to as self-assembled monolayers on mesoporous supports (SAMMS), has previously demonstrated the ability to serve as very effective heavy metal sorbents in a range of aquatic and environmental systems, suggesting that they may be advantageously utilized for biomedical applications such as chelation therapy. Herein we evaluate surface chemistries for heavy metal capture from biological fluids, various facets of the materials' biocompatibility, and the suitability of these materials as potential therapeutics. Of the materials tested, thiol-functionalized SAMMS proved most capable of removing selected heavy metals from biological solutions (i.e., blood, urine, etc.) Consequentially, thiol-functionalized SAMMS was further analyzed to assess the material's performance under a number of different biologically relevant conditions (i.e., variable pH and ionic strength) to gauge any potentially negative effects resulting from interaction with the sorbent, such as cellular toxicity or the removal of essential minerals. Additionally, cellular uptake studies demonstrated no cell membrane permeation by the silica-based materials generally highlighting their ability to remain cellularly inert and thus nontoxic. The results show that organic ligand functionalized nanoporous silica could be a valuable material for a range of detoxification therapies and potentially other biomedical applications.


Assuntos
Materiais Biocompatíveis/química , Sangue , Teste de Materiais/métodos , Metais Pesados/química , Dióxido de Silício/química , Urina/química , Adsorção , Células CACO-2 , Humanos , Porosidade
15.
Langmuir ; 26(14): 12285-92, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20550201

RESUMO

Multifunctional organic molecules represent an interesting challenge for nanoparticle functionalization due to the potential for undesirable interactions between the substrate material and the variable functionalities, making it difficult to control the final orientation of the ligand. In the present study, UV-induced thiol-ene click chemistry has been utilized as a means of directed functionalization of bifunctional ligands on an iron oxide nanoparticle surface. Allyl diphosphonic acid ligand was covalently deposited on the surface of thiol-presenting iron oxide nanoparticles via the formation of a UV-induced thioether. This method of thiol-ene click chemistry offers a set of reaction conditions capable of controlling the ligand deposition and circumventing the natural affinity exhibited by the phosphonic acid moiety for the iron oxide surface. These claims are supported via a multimodal characterization platform which includes thermogravimetric analysis, X-ray photoelectron spectroscopy, and metal contact analysis and are consistent with a properly oriented, highly active ligand on the nanoparticle surface. These experiments suggest thiol-ene click chemistry as both a practical and generally applicable strategy for the directed deposition of multifunctional ligands on metal oxide nanoparticle surfaces.


Assuntos
Difosfonatos/química , Compostos Férricos/química , Magnetismo , Nanopartículas/química , Compostos de Sulfidrila/química , Ligantes , Modelos Moleculares , Conformação Molecular
16.
ChemSusChem ; 3(6): 749-57, 2010 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-20468024

RESUMO

We describe the synthesis and characterization of high-performance, superparamagnetic, iron oxide nanoparticle-based, heavy metal sorbents, which demonstrate excellent affinity for the separation of heavy metals in contaminated water systems (i.e., spiked Columbia River water). The magnetic nanoparticle sorbents were prepared from an easy-to-synthesize iron oxide precursor, followed by a simple, one-step ligand exchange reaction to introduce an affinity ligand to the nanoparticle surface that is specific to a heavy metal or class of heavy metal contaminants. The engineered magnetic nanoparticle sorbents have inherently high active surface areas, allowing for increased binding capacities. To demonstrate the performance of the nanoparticle sorbents, river water was spiked with specific metals and exposed to low concentrations of the functionalized nanoparticles. In almost all cases, the nanoparticles were found to be superior to commercially available sorbent materials as well as the unfunctionalized iron oxide nanoparticles.


Assuntos
Nanopartículas Metálicas/química , Metais Pesados/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água/métodos , Adsorção , Água Doce/química , Magnetismo , Rios/química
17.
Biosens Bioelectron ; 25(1): 179-84, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19643593

RESUMO

A fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H(C)-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 h was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 h. The beads were subsequently captured and concentrated in a rotating rod "renewable surface" flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-H(C)-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.


Assuntos
Anticorpos Antibacterianos/imunologia , Toxinas Botulínicas/análise , Imunoensaio/métodos , Pontos Quânticos , Toxinas Botulínicas/imunologia , Desenho de Equipamento , Fluorescência , Imunoensaio/instrumentação , Modelos Moleculares , Sensibilidade e Especificidade
18.
Anal Chem ; 81(14): 5783-93, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19530657

RESUMO

A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (approximately 50 pg/mL for BoNT/A-HC-fragment) for the 15 min fluidic assay in buffer.


Assuntos
Técnicas Biossensoriais/métodos , Toxinas Botulínicas Tipo A/análise , Citometria de Fluxo/métodos , Microesferas , Animais , Anticorpos/imunologia , Automação , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/imunologia , Soluções Tampão , Processamento Eletrônico de Dados , Epitopos/imunologia , Humanos , Proteínas Imobilizadas/análise , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Camundongos , Peso Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Fatores de Tempo
19.
Analyst ; 134(5): 987-96, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19381395

RESUMO

A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain ( approximately 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin ( approximately 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 microL column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.


Assuntos
Toxinas Botulínicas/química , Imunoensaio/instrumentação , Imunoensaio/métodos , Anticorpos , Técnicas Biossensoriais , Fluorescência , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Fc
20.
Langmuir ; 25(9): 4900-6, 2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19256464

RESUMO

The development of more efficient and environmentally benign methods for the synthesis and manipulation of nanomaterials has been a major focus of research among the scientific community. Supercritical (ScFs) and near-critical fluids (NcFs) offer numerous advantages over conventional solvents for these purposes. Among them, ScFs and NcFs offer dramatic reductions in the volume of organic waste typically generated during advanced material processes with the feasibility of changing a number of physicochemical properties by discrete variations in solvent pressure or temperature. In this work, we study the dispersibility of gold nanocrystals with a 3.7 nm core size stabilized by different ligand shells in NcF ethane and propane over a wide range of densities by fine-tuning the pressure of these fluids. Dispersibility vs density plots are obtained by following the variation in the surface plasmon resonance (SPR) absorption spectra of the nanoparticles. To understand the results obtained in this study, three models are briefly discussed: the total interaction theory, the sedimentation coefficient equation, and the Chrastil method. The dispersibility and behavior of the nanocrystals with variations in fluid density are strongly dependent on the surface chemistry of the nanocrystal and the solvent employed. A correlation between measured dispersibility values and calculated sedimentation coefficients was observed in both compressed solvents. In addition, we successfully applied the Chrastil equation to predict and describe the dispersibility of gold nanocrystals with different shells as a function of density, determining that the reason for the high stabilities of some of the nanocrystal dispersions is the strong solvent-nanocrystal interactions. While NcF propane showed higher nanocrystal dispersibilities, using NcF ethane led to improved tunability of nanoparticle dispersions formed in the pressure range studied. Therefore, with a judicious selection of the fluid, NcFs seem to offer a remarkable advantage over conventional solvents for manipulation of nanomaterials, which could be applied to transport, purification, and separation of nanocrystals.

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