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Disasters can create situations in which blood donations can save lives. However, in emergency situations and when resources are depleted, on-site blood donations require the rapid and accurate detection of blood-borne pathogens, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Techniques such as PCR and antibody capture by an enzyme-linked immunosorbent assay (ELISA) for HIV-1 and HIV-2 are precise but time-consuming and require sophisticated equipment that is not compatible with emergency point-of-care requirements. We describe here a prototype biosensor based on piezoelectric materials functionalized with specific antibodies against HIV-1 and HIV-2. We show the rapid and accurate detection of HIV-1 and HIV-2 in both simple and complex solutions, including human serum, and in the presence of a cross-confounding virus. We report detection limits of 12 50% tissue culture infective doses (TCID50s) for HIV-1 and 87 TCID50s for HIV-2. The accuracy, precision of measurements, and operation of the prototype biosensor compared favorably to those for nucleic acid amplification. We conclude that the biosensor has significant promise as a successful point-of-care diagnostic device for use in emergency field applications requiring rapid and reliable testing for blood-borne pathogens.
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Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , HIV-2/isolamento & purificação , Virologia/métodos , Técnicas Biossensoriais/instrumentação , Técnicas de Laboratório Clínico/instrumentação , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Virologia/instrumentaçãoRESUMO
A 3D finite element model was developed to optimize the kinetics and mass transfer characteristics of low concentration, 18 bp ssDNA targets in bulk media solution, to 18 bp complimentary oligonucleotide probes immobilized on electrochemical detection electrodes positioned along the length of a microfluidic channel. Conditions considered in the model were fluid flow rate, diffusion time, DNA melting temperature, number of matching base pairs, and temperature of the fluid in the channel. System optimization was based on maximizing the uniformity and surface concentration of the specifically bound hybridized DNA, minimizing waste volume generation and the hybridization time. With the coupled simulation method used, the total experiment time was reduced from 150 to 60 min and the simulated results were consistent with experimental results found in the literature. A stopped flow procedure was investigated as a means to improve hybridization. This procedure can not only improve uniformity and capture efficiency, and reduce waste, but can also decrease overall signal intensity relative to continuous flow operation. Finally, the use of temperature in reducing mismatched hybridization and improving duplex stability was also successfully modeled and simulated.
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Simulação por Computador , DNA/análise , Técnicas Analíticas Microfluídicas/métodos , Modelos Químicos , Cinética , Movimento (Física) , Hibridização de Ácido Nucleico/métodos , TemperaturaRESUMO
This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.
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Electrostatic interaction plays a leading role in nanoparticle interactions with membrane architectures and can lead to effects such as nanoparticle binding and membrane disruption. In this work, the effects of nanoparticles (NPs) interacting with mixed lipid systems were investigated, indicating an ability to tune both NP binding to membranes and membrane disruption. Lipid membrane assemblies (LBAs) were created using a combination of charged, neutral, and gel-phase lipids. Depending on the lipid composition, nanostructured networks could be observed using in situ atomic force microscopy representing an asymmetrical distribution of lipids that rendered varying effects on NP interaction and membrane disruption that were domain-specific. LBA charge could be localized to fluidic domains that were selectively disrupted when interacting with negatively charged Au nanoparticles or quantum dots. Disruption was observed to be related to the charge density of the membrane, with a maximum amount of disruption occurring at â¼40% positively charged lipid membrane concentration. Conversely, particle deposition was determined to begin at charged lipid concentrations greater than 40% and increased with charge density. The results demonstrate that the modulation of NP and membrane charge distribution can play a pivitol role in determining NP-induced membrane disruption and NP surface assembly.
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Ouro/química , Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Pontos Quânticos , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica , Tamanho da Partícula , Eletricidade EstáticaRESUMO
We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.
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Since the introduction of micro total analytical systems (µTASs), significant advances have been made toward development of lab-on-a-chip platforms capable of performing complex biological assays that can revolutionize public health, among other applications. However, use of these platforms in low-resource environments (e.g. developing countries) has yet to be realized as the majority of technologies used to control microfluidic flow rely on off-device hardware with non-negligible size, cost, power requirements and skill/training to operate. In this paper we describe a magnetic-adhesive based valve that is simple to construct and operate, and can be used to control fluid flow and store reagents within a microfluidic device. The design consists of a port connecting two chambers on different planes in the device that is closed by a neodymium disk magnet seated on a thin ring of adhesive. Bringing an external magnet into contact with the outer surface of the device unseats and displaces the valve magnet from the adhesive ring, exposing the port. Using this configuration, we demonstrate on-device reagent storage and on-demand transport and reaction of contents between chambers. This design requires no power or external instrumentation to operate, is extremely low cost ($0.20 materials cost per valve), can be used by individuals with no technical training, and requires only a hand-held magnet to actuate. Additionally, valve actuation does not compromise the integrity of the completely sealed microfluidic device, increasing safety for the operator when toxic or harmful substances are contained within. This valve concept has the potential to simplify design of µTASs, facilitating development of lab-on-a-chip systems that may be practical for use in point-of-care and low-resource settings.
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Adesivos , Recursos em Saúde/provisão & distribuição , Dispositivos Lab-On-A-Chip , Imãs , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
This paper describes an approach for fabricating multi-layer microfluidic systems from a combination of glass and plastic materials. Methods and characterization results for the microfabrication technologies underlying the process flow are presented. The approach is used to fabricate and characterize multi-layer plastic/glass microfluidic systems containing electrical and mechanical functionality. Hot embossing, heat staking of plastics, injection molding, microstenciling of electrodes, and stereolithography were combined with conventional MEMS fabrication techniques to realize the multi-layer systems. The approach enabled the integration of multiple plastic/glass materials into a single monolithic system, provided a solution for the integration of electrical functionality throughout the system, provided a mechanism for the inclusion of microactuators such as micropumps/valves, and provided an interconnect technology for interfacing fluids and electrical components between the micro system and the macro world.
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Vidro/química , Membranas Artificiais , Microfluídica/instrumentação , Plásticos/química , Materiais Biocompatíveis/química , Condutividade Elétrica , Desenho de Equipamento , Microeletrodos , Microscopia Eletrônica de Varredura , Fotografação/métodos , PressãoAssuntos
Ouro/química , Membranas Artificiais , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fotoquímica/métodos , Cristalização/métodos , Temperatura Alta , Íons , Substâncias Macromoleculares/química , Conformação Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin. Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements. This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH, which are important metabolic indicators of disease states (e.g., cancer proliferation) and exercise-induced acidosis.
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Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Microinjeções/instrumentação , Agulhas , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodosRESUMO
The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure(®)) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-h period.
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Acidose/metabolismo , Técnicas Biossensoriais/métodos , Glucose/metabolismo , Ácido Láctico/análise , Acidose/diagnóstico , Técnicas Biossensoriais/instrumentação , Carbono , Adesão Celular , Materiais Revestidos Biocompatíveis , Técnicas Eletroquímicas , Exercício Físico , Glucose Oxidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/biossíntese , Macrófagos/fisiologia , Microeletrodos , AgulhasRESUMO
Herein is described the fabrication and use of a plastic multilayer 3-channel microfluidic fixture. Multilayer devices were produced by laser machining of plastic polymethylmethacrylate and polyethyleneterapthalate laminates by ablation. The fixture consisted of an array of nine individually addressable gold or gold/ITO working electrodes, and a resistive platinum heating element. Laser machining of both the fluidic pathways in the plastic laminates, and the stencil masks used for thermal evaporation to form electrode regions on the plastic laminates, enabled rapid and inexpensive implementation of design changes. Electrochemiluminescence reactions in the fixture were achieved and monitored through ITO electrodes. Electroaddressable aryl diazonium chemistry was employed to selectively pattern gold electrodes for electrochemical multianalyte DNA detection from double stranded DNA (dsDNA) samples. Electrochemical detection of dsDNA was achieved by melting of dsDNA molecules in solution with the integrated heater, allowing detection of DNA sequences specific to breast and colorectal cancers with a non-specific binding control. Following detection, the array surface could be renewed via high temperature (95 °C) stripping using the integrated heating element. This versatile and simple method for prototyping devices shows potential for further development of highly integrated, multi-functional bioanalytical devices.
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Nonenzymatic glucose oxidation is demonstrated on highly faceted palladium nanowflower-modified porous carbon electrodes fabricated by interference lithography. Varying electrodeposition parameters were used to control the final shape and morphology of the deposited nanoparticles on the 3D porous carbon which showed a 12 times increase in the electrochemically active surface area over analogous planar electrodes. Extremely fast amperometric glucose responses (achieving 95% of the steady state limiting current in less than 5s) with a linear range from 1 to 10mM and a detection limit of 10 µM were demonstrated. The unusual surface properties of the pyrolyzed photoresist films produced strongly adhered palladium crystal structures that were stable for hundreds of cycles towards glucose oxidation without noticeable current decay.
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Técnicas Eletroquímicas/métodos , Eletrodos , Glucose/análise , Nanoporos , Ácido Ascórbico/química , Catálise , Oxirredução , Paládio/químicaRESUMO
In this study, carbon fiber electrodes were incorporated within a hollow microneedle array, which was fabricated using a digital micromirror device-based stereolithography instrument. Cell proliferation on the acrylate-based polymer used in microneedle fabrication was examined with human dermal fibroblasts and neonatal human epidermal keratinocytes. Studies involving full-thickness cadaveric porcine skin and trypan blue dye demonstrated that the hollow microneedles remained intact after puncturing the outermost layer of cadaveric porcine skin. The carbon fibers underwent chemical modification in order to enable detection of hydrogen peroxide and ascorbic acid; electrochemical measurements were demonstrated using integrated electrode-hollow microneedle devices.
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Increased mass transport due to hemispherical diffusion is observed to occur in 3D porous carbon electrodes defined by interferometric lithography. Enhanced catalytic methanol oxidation, after modifying the porous carbon with palladium nanoparticles, and uncharacteristically uniform conducting polymer deposition into the structures are demonstrated. Both examples result in two regions of hierarchical porosity that can be created to maximize surface area, via nanostructuring, within the extended porous network, while taking advantage of hemispherical diffusion through the open pores.
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A microscale thermal field-flow fractionation (micro-TFFF) system has been designed, fabricated, and characterized. Motivation for miniaturization of TFFF systems was established by examining the geometrical scaling of the fundamental TFFF theory. Miniaturization of conventional macroscale TFFF systems was made possible through utilization of micromachining technologies. Fabrication of the micro-TFFF system was discussed in detail. The micro-TFFF system was characterized for plate height versus flow rate, single-component polystyrene retention, and multicomponent polystyrene separations. Retention, thermal diffusion coefficients, and maximum diameter-based selectivity values were extracted from separation data and found comparable with macroscale TFFF system results. Retention values ranged from 0.33 to 0.46. Thermal diffusion coefficients were between 3.0 x 10(-8) and 5.4 x 10(-8) cm2/s x K. The maximum diameter-based selectivity was 1.40.