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1.
Vaccine ; 36(41): 6061-6069, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30219365

RESUMO

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.


Assuntos
Estomatite Vesicular/virologia , Vesiculovirus/isolamento & purificação , Vesiculovirus/patogenicidade , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Técnicas Citológicas , Células Vero , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vesiculovirus/imunologia
2.
Analyst ; 139(6): 1472-81, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24492491

RESUMO

The rapid and robust identification of viral infections has broad implications for a number of fields, including medicine, biotechnology and biodefense. Most detection systems rely on specific molecules, such as nucleic acids or proteins, to identify the target(s) of interest. These molecules afford great specificity, but are often expensive, labor-intensive, labile and limited in scope. Label free detection methods seek to overcome these limitations by instead using detection methods that rely on intrinsic properties as a basis for identifying and separating species of interest and thus do not rely on specific prior knowledge of the target. Optical chromatography, one such technique, uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we present the application of individual optical force measurements as a means of investigating pseudorabies virus infection in Vero cells. Optical force differences are seen between cells from uninfected and infected populations at a multiplicity of infection as low as 0.001 and as soon as 2 hours post infection, demonstrating the potential of this technique as a means of detecting viral infection. Through the application of a pattern recognition neural network, individual cell size data are combined with optical force as a means of classifying cell populations. Potential applications include the early detection of bloodborne pathogens for the prevention of sepsis and other diseases as well as the detection of biological threat agents.


Assuntos
Herpesvirus Suídeo 1/isolamento & purificação , Pseudorraiva/diagnóstico , Células Vero/virologia , Animais , Técnicas Biossensoriais , Chlorocebus aethiops , Lasers , Técnicas Analíticas Microfluídicas , Microscopia , Imagem Óptica
3.
Anal Chem ; 85(18): 8647-53, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-23964641

RESUMO

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.

4.
Electrophoresis ; 34(8): 1175-81, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23404174

RESUMO

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.


Assuntos
Eletro-Osmose/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Simulação por Computador , Microesferas , Tamanho da Partícula , Software
5.
Anal Chim Acta ; 718: 11-24, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22305893

RESUMO

On-line sample preconcentration is an essential tool in the development of microfluidic-based separation platforms. In order to become more competitive with traditional separation techniques, the community must continue to develop newer and more novel methods to improve detection limits, remove unwanted sample matrix components that disrupt separation performance, and enrich/purify analytes for other chip-based actions. Our goal in this review is to familiarize the reader with many of the options available for on-chip concentration enhancement with a focus on those manuscripts that, in our assessment, best describe the fundamental principles that govern those enhancements. Sections discussing both electrophoretic and nonelectrophoretic modes of preconcentration are included with a focus on device design and mechanisms of preconcentration. This review is not meant to be a comprehensive collection of every available example, but our hope is that by learning how on-line sample concentration techniques are being applied today, the reader will be inspired to apply these techniques to further enhance their own programs.

6.
Lab Chip ; 12(6): 1128-34, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22315144

RESUMO

A unique microfluidic system is developed which enables the interrogation of a single particle by using multiple force balances from a combination of optical force, hydrodynamic drag force, and electrophoretic force. Two types of polystyrene (PS) particles with almost identical size and refractive index (plain polystyrene (PS) particle - mean diameter: 2.06 µm, refractive index: 1.59; carboxylated polystyrene (PS-COOH) particles - mean diameter: 2.07 µm, refractive index: 1.60), which could not be distinguished by optical chromatography, reveal different electrokinetic behaviors resulting from the difference in their surface charge densities. The PS-COOH particles, despite their higher surface charge density when compared to the PS particles, experience a lower electrophoretic force, regardless of ionic strength. This phenomenon can be understood when the more prominent polarization of the counter ion cloud surrounding the PS-COOH particles is considered. The surface roughness of the carboxylated particles also plays an important role in the observed electrokinetic behavior.

7.
Anal Chem ; 83(14): 5666-72, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21634802

RESUMO

There is a compelling need to develop systems capable of processing blood and other particle streams for detection of pathogens that are sensitive, selective, automated, and cost/size effective. Our research seeks to develop laser-based separations that do not rely on prior knowledge, antibodies, or fluorescent molecules for pathogen detection. Rather, we aim to harness inherent differences in optical pressure, which arise from variations in particle size, shape, refractive index, or morphology, as a means of separating and characterizing particles. Our method for measuring optical pressure involves focusing a laser into a fluid flowing opposite to the direction of laser propagation. As microscopic particles in the flow path encounter the beam, they are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. On the basis of the flow rate at which this balance occurs, the optical pressure felt by the particle can be calculated. As a first step in the development of a label-free device for processing blood, a system has been developed to measure optical pressure differences between the components of human blood, including erythrocytes, monocytes, granulocytes, and lymphocytes. Force differentials have been measured between various components, indicating the potential for laser-based separation of blood components based upon differences in optical pressure. Potential future applications include the early detection of blood-borne pathogens for the prevention of sepsis and other diseases as well as the detection of biological threat agents.


Assuntos
Células Sanguíneas/citologia , Lasers , Óptica e Fotônica/instrumentação , Linhagem Celular Tumoral , Tamanho Celular , Desenho de Equipamento , Humanos
8.
Anal Chim Acta ; 670(1-2): 78-83, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20685420

RESUMO

In this work, microscopic particles in a fluid flow are manipulated using forces generated by a high power laser beam. The resulting manipulations on the particles are imaged using a microscope lens connected to a CCD camera. Differential forces on particles of varying physical and chemical composition have been measured. The goal is to measure the optical forces on a diverse range of particles and catalog the associated chemical and physical differences to understand which properties and mechanisms result in the largest force differentials. Using these measurements our aim is to better understand differences between similar microspheres in terms of size, morphology, or chemical composition. Particles of the same size, but different composition show large variations in optical pressure forces and are easily discernable in the present analytical system. In addition, we have demonstrated the ability to differentiate a 70 nm size difference between two NIST precision size standard polystyrene microspheres, corresponding to a 2.0 pN difference in optical force. Lastly, the instrument was used to measure differences between biological samples of similar size, demonstrating the ability to make precise analytical measurements on microorganism samples.


Assuntos
Bacillus anthracis/química , Bacillus thuringiensis/química , Geobacter/química , Óptica e Fotônica , Tamanho da Partícula , Espectrometria de Fluorescência
9.
Lab Chip ; 10(13): 1729-31, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20376381

RESUMO

We present the construction and operation of a microfluidic nozzle created using several standard fluidic parts available commercially. By elegantly combining several pieces from a standard assembly, a capillary and a few other standard parts, we were able to develop a novel device. Using this device, precise axisymmetric flow focusing of particles was achieved and observed at the exit of the nozzle and within a connected microfluidic device several centimetres away. Sheath and core flow rates were varied to show influence and control over the width of the focused particles.


Assuntos
Análise de Injeção de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento
10.
Appl Environ Microbiol ; 75(22): 7163-72, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19767469

RESUMO

The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms.


Assuntos
Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Bacillus cereus/genética , Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , Glicoproteínas de Membrana/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Variação Genética , Glicoproteínas de Membrana/química , Modelos Genéticos , Polimorfismo Genético/genética , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/genética
11.
Opt Express ; 17(3): 2024-32, 2009 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-19189033

RESUMO

Optical chromatography achieves microscale optical manipulation through the balance of optical and hydrodynamic forces on micron sized particles entrained in microfluidic flow traveling counter to the propagation of a mildly focused laser beam. The optical pressure force on a particle is specific to each particle's size, shape and refractive index. So far, these properties have been exploited in our lab to concentrate, purify and separate injected samples. But as this method advances into more complex optofluidic systems, a need to better predict behavior is necessary. Here, we present the development and experimental verification of a robust technique to simulate particle trajectories in our optical chromatographic device. We also show how this new tool can be used to gather better qualitative and quantitative understanding in a two component particle separation.

12.
Biomicrofluidics ; 3(4): 44106, 2009 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-20216968

RESUMO

Optical chromatography involves the elegant combination of opposing optical and fluid drag forces on colloidal samples within microfluidic environments to both measure analytical differences and fractionate injected samples. Particles that encounter the focused laser beam are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. In our recent devices particles are pushed into a region of lower microfluidic flow, where they can be retained and fractionated. Because optical and fluid forces on a particle are sensitive to differences in the physical and chemical properties of a sample, separations are possible. An optical chromatography beam focused to completely fill a fluid channel is operated as an optically tunable filter for the separation of inorganic, polymeric, and biological particle samples. We demonstrate this technique coupled with an advanced microfluidic platform and show how it can be used as an effective method to fractionate particles from an injected multicomponent sample. Our advanced three-stage microfluidic design accommodates three lasers simultaneously to effectively create a sequential cascade optical chromatographic separation system.

13.
Opt Express ; 16(23): 18782-9, 2008 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-19581966

RESUMO

Optical chromatography, used for particle separation, involves loosely focusing a laser into a fluid flowing opposite the direction of laser propagation. When microscopic particles in the flow path encounter this beam they are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. Because optical and fluid forces are sensitive to differences in the physical and chemical properties of a particle, separations are possible. An optical chromatography beam which completely fills a fluid channel can operate as an optically tunable filter for the preparative separation of polymeric/colloidal and biological samples. We show how the technique can be used to separate injected samples containing large numbers of colloids. The power of optical chromatographic separations is illustrated through combination with epi-fluorescence microscopy and sample purification for real-time polymerase chain reaction (RT-PCR) detection of Bacillus anthracis spores.


Assuntos
Lasers , Microscopia de Fluorescência/instrumentação , Dispositivos Ópticos , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Opt Express ; 15(5): 2724-31, 2007 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-19532509

RESUMO

Optical chromatography is a technique for the separation of particles that capitalizes on the balance between optic and fluidic forces. When microscopic particles in a fluid flow encounter a laser beam propagating in the opposite direction, they are trapped axially along the beam. They are then optically pushed upstream from the laser focal point to rest at a point where the optic and fluidic forces on the particle balance. Because optical and fluid forces are sensitive to differences in the physical and chemical properties of a particle, both coarse and fine separations are possible. We describe how an optical chromatography beam directed into a tailored flow environment, has been adapted to operate as an optical filter for the concentration / bioenrichment of colloidal and biological samples. In this work, the demonstrated ability to concentrate spores of the biowarfare agent, Bacillus anthracis, may have significant impact in the biodefense arena. Application of these techniques and further design of fluidic and optical environments will allow for more specific identification, concentration and separation of many more microscopic particle and biological suspensions.

15.
Anal Chem ; 78(9): 3221-5, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16643018

RESUMO

A significant difference between two closely related Bacillus spores has been discovered using optical chromatography. This difference can be harnessed for the separation of microscopic particles using opposing laser and fluid flow forces. Particles of different size, composition, and shape experience different optical and fluid forces and come to rest at unique equilibrium positions where the two forces balance. Separations in excess of 600 mum have been observed between Bacillus anthracis Sterne strain and its genetic relative, Bacillus thuringiensis. These findings open new possibilities for detection and characterization of the biological warfare agent, B. anthracis, the causative agent of anthrax, the deadly mammalian disease. The large optical separation between these species is surprising given their close genetic relationship but may be explained by differences in their shape and exosporium morphology, which may result in differences in fluid drag force. The observation of large differences due to less common variables indicates the complex nature of the force balance in optical chromatography, which may in the future be used to separate and characterize microbiological samples. In general, the discovery of such large differences between such closely related biological species suggests new possibilities for the separation and characterization of microorganisms using the full range of emerging techniques that employ radiation pressure (optical filtering, laser tweezers, optical chromatography, etc.).


Assuntos
Bacillus anthracis/química , Bacillus thuringiensis/química , Lasers , Óptica e Fotônica , Esporos Bacterianos/química , Microfluídica , Microscopia Eletrônica de Transmissão/métodos , Tamanho da Partícula , Sensibilidade e Especificidade , Esporos Bacterianos/isolamento & purificação , Fatores de Tempo
16.
Anal Bioanal Chem ; 374(3): 385-9, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12373382

RESUMO

A small volume flowcell for fluorescence detection in capillary flow injection (CFI) analysis has been created by using a low cost, commercially available fluidic device. Fluorescence detection is achieved using an optical fiber to deliver excitation light to the sample flowing through the device and another optical fiber to collect fluorescence emission. The flowcell is a standard fluidic cross with a swept volume of 721 nL. Optical fibers were oriented at right angles using standard sleeves and ferrules to set their position near the cross intersection. Multiple excitation sources were used including a low power UV laser and blue and UV light emitting diodes (LED). The full emission spectrum detection limits, using the laser, for fluorescein and bovine serum albumin (BSA) were 0.30 ppb and 2.1 x 10(-4)% (w/w), respectively. Two fluidic crosses were used in series for multi-wavelength fluorescence excitation using fiber-optically coupled LED.

17.
Anal Bioanal Chem ; 372(1): 205-15, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11939196

RESUMO

A multi-channel detection system utilizing fiber optics has been developed for the laser-induced fluorescence (LIF) analysis of chromatographic eluents. It has been applied to the detection of polycyclic aromatic hydrocarbons (PAH) in a chromatographically overlapped standard mixture and to a complex soil sample extract obtained during fieldwork. The instrument utilizes dual-fiber optic arrays, one to deliver multiple excitation wavelengths (258-342 nm) generated by a Raman shifter, and the other to collect fluorescence generated by the sample at each excitation wavelength; the collected fluorescence is dispersed and detected with a spectrograph/CCD combination. The resulting data were arranged into excitation emission matrices (EEM) for visualization and data analysis. Rapid characterization of PAH mixtures was achieved under isocratic chromatographic conditions (1.5 mL min(-1) and 80% acetonitrile in water), with mid microg L(-1) detection limits, in less than 4 minutes. The ability of the instrument to identify co-eluting compounds was demonstrated by identifying and quantifying analytes in the rapid analysis of a 17 component laboratory-prepared PAH mixture and a soil extracted sample. Identification and quantification were accomplished using rank annihilation factor analysis (RAFA) using pure component standards and the EEMs of mixtures measured during the rapid high-performance liquid chromatography (HPLC) method as the unknowns. The percentage errors of the retention times (RTs) determined using RAFA compared to the known RTs measured with a standard absorbance detector were between 0 and 11%. For the standard PAH mixture, all 17 components were identified correctly and for the soil extracted sample, all 8 analytes present were correctly identified with only one false positive. Overall, the system achieved excellent qualitative performance with semi-quantitative results in the concentration predictions of both the standard mixture and the real-world sample. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00216-001-1125-6.

18.
Analyst ; 127(12): 1693-9, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12537381

RESUMO

An excitation emission matrix (EEM) fluorescence instrument has been developed using a linear array of light emitting diodes (LED). The wavelengths covered extend from the upper UV through the visible spectrum: 370-640 nm. Using an LED array to excite fluorescence emission at multiple excitation wavelengths is a low-cost alternative to an expensive high power lamp and imaging spectrograph. The LED-EEM system is a departure from other EEM spectroscopy systems in that LEDs often have broad excitation ranges which may overlap with neighboring channels. The LED array can be considered a hybrid between a spectroscopic and sensor system, as the broad LED excitation range produces a partially selective optical measurement. The instrument has been tested and characterized using fluorescent dyes: limits of detection (LOD) for 9,10-bis(phenylethynyl)-anthracene and rhodamine B were in the mid parts-per-trillion range; detection limits for the other compounds were in the low parts-per-billion range (< 5 ppb). The LED-EEMs were analyzed using parallel factor analysis (PARAFAC), which allowed the mathematical resolution of the individual contributions of the mono- and dianion fluorescein tautomers a priori. Correct identification and quantitation of six fluorescent dyes in two to six component mixtures (concentrations between 12.5 and 500 ppb) has been achieved with root mean squared errors of prediction (RMSEP) of less than 4.0 ppb for all components.


Assuntos
Espectrometria de Fluorescência/instrumentação , Eletrônica , Luz , Sensibilidade e Especificidade , Raios Ultravioleta
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