RESUMO
The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.
Assuntos
Grafite , Metais Pesados , Grafite/química , Hidrogênio , Oxigênio , Sais , Água do MarRESUMO
Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.
RESUMO
Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.
Assuntos
Biofilmes , Fenômenos Eletromagnéticos , Geobacter/fisiologia , Grupo dos Citocromos c/metabolismo , Eletrodos , Elétrons , Heme/metabolismo , Oxirredução , Ressonância de Plasmônio de SuperfícieRESUMO
Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.
Assuntos
Adesivos/química , Amidas/química , Proteínas/química , Thoracica/química , Adesividade , Animais , Vidro/química , Ouro/química , Muda/fisiologia , Imagem Óptica , Oxirredução , Proteínas/metabolismo , Refratometria , Água do Mar , Thoracica/fisiologiaRESUMO
Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.
Assuntos
Biofilmes , Condutividade Elétrica , Transporte de Elétrons , Geobacter/metabolismo , TemperaturaRESUMO
An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10(2)-10(6) cells/mL of target bacteria. Reversal of magnets' rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.
Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo/instrumentação , Separação Imunomagnética/instrumentação , Análise em Microsséries/instrumentação , Integração de Sistemas , Bactérias/citologia , HumanosRESUMO
Bacterial infection and intoxication can present with common symptoms. The ability to identify a bacteria or toxin rapidly in clinical samples is critical for administering the appropriate treatment. The microflow cytometer has previously demonstrated the ability to test for six bacteria and toxins simultaneously in buffer. In this study, the number of bacteria and toxins analyzed was increased to ten, positive and negative controls were incorporated in all assays, and most importantly, multiplexed immunoassays were demonstrated in clinical matrices. The multiplexed assays using the microflow cytometer demonstrated detection limits similar to or better than other reported antibody-based methods for pathogen detection (ELISA, lateral flow, array biosensors). In most cases, detection from complex clinical matrices (serum and nasal wash) achieved limits of detection equivalent to those for spiked buffer samples. Clinical samples spiked with bacteria and/or toxins were also analyzed successfully in blind trials.
Assuntos
Bactérias/isolamento & purificação , Toxinas Bacterianas/análise , Citometria de Fluxo/instrumentação , Toxinas Bacterianas/sangue , Estudos de Casos e Controles , Citometria de Fluxo/métodos , Humanos , Imunoensaio , Limite de Detecção , Distribuição Aleatória , Ricina/análiseRESUMO
The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application.
Assuntos
Técnicas Biossensoriais/instrumentação , Hidrodinâmica , Técnicas Analíticas Microfluídicas/instrumentação , Microtecnologia/instrumentação , Desenho de Equipamento , Citometria de Fluxo/instrumentaçãoRESUMO
The phenomenon of "unmixing" has been demonstrated in microfluidic mixers, but here we manipulate laminar flow streams back to their original positions in order to extend the operational utility of an analytical device where no mixing is desired. Using grooves in the channel wall, we passively focus a sample stream with two sheath streams to center it in a microchannel for optical analysis. Even though the sample stream is completely surrounded by sheath fluid, reversing the orientation of the grooves in the channel walls returns the sample stream to its original position with respect to the sheath streams. We demonstrate the separation of the sample stream from the contiguous sheath streams and the recycling of the sheath fluid using the reversibility of laminar flow. Polystyrene microspheres and fluorescent dye were used to quantify the performance of the unsheathing process. We found that the maximum numbers of microspheres and all of the fluorescent dye were recaptured at sheath recycling levels <92%. The use of this sheathing technique has previously been demonstrated in a sensitive microflow cytometer; the unsheathing capability now provides the opportunity to recover particles from the sensor with minimal dilution or to recycle the sheath fluid for long-term unattended operation.
Assuntos
Citometria de Fluxo/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Modelos Teóricos , Citometria de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Microesferas , Poliestirenos/químicaRESUMO
With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ~35 µm × 40 µm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 µm × 125 µm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization.
Assuntos
Bioensaio/instrumentação , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Bioensaio/métodos , Linhagem Celular Tumoral , Humanos , Imunoensaio , Microesferas , Polimetil Metacrilato/químicaRESUMO
A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.
Assuntos
Escherichia coli/isolamento & purificação , Citometria de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Ficoeritrina/análise , Animais , Contagem de Colônia Microbiana , Desenho de Equipamento , Escherichia coli/imunologia , Citometria de Fluxo/métodos , Corantes Fluorescentes , Imunoglobulina G/imunologia , Técnicas Analíticas Microfluídicas/métodos , Microesferas , Ficoeritrina/imunologia , Sensibilidade e EspecificidadeRESUMO
A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.
Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo/métodos , Microfluídica/métodos , Toxinas Biológicas/análise , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Bactérias/química , Enterotoxinas/análise , Escherichia coli/química , Citometria de Fluxo/instrumentação , Corantes Fluorescentes/química , Listeria/química , Microfluídica/instrumentação , Ricina/análise , Salmonella/química , Processamento de Sinais Assistido por ComputadorRESUMO
Array-based biosensor technology offers the user the ability to detect and quantify multiple targets in multiple samples simultaneously (Analytical Sciences 23:5-10, 2007). The NRL Array Biosensor has been developed with the aim of creating a system for sensitive, rapid, on-site screening for multiple targets of interest. This system is fluorescence-based, using evanescent illumination of a waveguide, and has demonstrated the use of both sandwich and competitive immunoassays for the detection of both high and low molecular weight targets, respectively. The current portable, automated system has demonstrated detection of a wide variety of analytes ranging from simple chemical compounds to entire bacterial cells, with applications in food safety, disease diagnosis, homeland security and environmental monitoring.
Assuntos
Técnicas Biossensoriais/instrumentação , Imunoensaio/instrumentação , Análise em Microsséries/instrumentação , Espectrometria de Fluorescência/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Imunoensaio/métodos , Análise em Microsséries/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
A simple design capable of 2-dimensional hydrodynamic focusing is proposed and successfully demonstrated. In the past, most microfluidic sheath flow systems have often only confined the sample solution on the sides, leaving the top and bottom of the sample stream in contact with the floor and ceiling of the channel. While relatively simple to build, these designs increase the risk of adsorption of sample components to the top and bottom of the channel. A few designs have been successful in completely sheathing the sample stream, but these typically require multiple sheath inputs and several alignment steps. In the designs presented here, full sheathing is accomplished using as few as one sheath input, which eliminates the need to carefully balance the flow of two or more sheath inlets. The design is easily manufactured using current microfabrication techniques. Furthermore, the sample and sheath fluid can be subsequently separated for recapture of the sample fluid or re-use of the sheath fluid. Designs were demonstrated in poly(dimethylsiloxane) (PDMS) using soft lithography and poly(methyl methacrylate) (PMMA) using micromilling and laser ablation.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Simulação por Computador , Desenho de EquipamentoRESUMO
Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities-such as automated sample staining-are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.
Assuntos
Biotecnologia/métodos , Desenho de Equipamento , Citometria de Fluxo/métodos , Fenômenos Biomecânicos , Biotecnologia/instrumentação , Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Citometria de Fluxo/instrumentação , Microfluídica/instrumentação , Microfluídica/métodos , Microfluídica/tendências , Sistemas On-Line/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980's to investigate thin film conductive polymers.
Assuntos
Biofilmes/crescimento & desenvolvimento , Eletroquímica/métodos , Transporte de Elétrons/fisiologiaRESUMO
A study is presented that examines the effect of microfluidic mixing elements on direct and sandwich assays performed in microchannels. Patterned grooves were embossed in the top of microchannels made in PDMS using soft lithography. The grooves redirected the fluid flowing in the channel, enhancing delivery of the target from the bulk fluid to the surface and preventing the formation of a depletion layer at the surface. Comparing assays in grooved and plain channels demonstrated that the mixers improved assay results by 26-46%. A computational flow analysis showed that the grooves caused virtual particles in the bulk flow to come close to the surface ( approximately 11 microm) which is consistent with the signal increase seen experimentally. Direct assays for several concentrations of CY5-labeled biotin were performed in the microchannels. The mixers also improved signal intensity in sandwich assays for botulinum toxin which required mixing of the reagents as well as the direction of the target to the surface.
Assuntos
Técnicas Biossensoriais/instrumentação , Toxinas Botulínicas/análise , Sistemas de Liberação de Medicamentos/instrumentação , Fluorimunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Biossensoriais/métodos , Toxinas Botulínicas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Fluorimunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.
Assuntos
Técnicas Biossensoriais , Imunoensaio/instrumentação , Automação , Contaminação de Alimentos , Microbiologia de Alimentos , Indicadores e Reagentes , Peso Molecular , Proteínas/análiseRESUMO
A computational "toolbox" for the a priori design of optimized microfluidic components is presented. These components consist of a microchannel under low-Reynolds number, pressure-driven flow, with an arrangement of grooves cut into the top and bottom to generate a tailored cross-channel flow. An advection map for each feature (i.e., groove of a particular shape and orientation) predicts the lateral transport of fluid within the channel due to that feature. We show that applying these maps in sequence generates an excellent representation of the outflow distribution for complex designs that combine these basic features. The effect of the complex three-dimensional flow field can therefore be predicted without solving the governing flow equations through the composite geometry, and the resulting distribution of fluids in the channel is used to evaluate how well a component performs a specified task. The generation and use of advection maps is described, and the toolbox is applied to determine optimal combinations of features for specified mixer sizes and mixing metrics.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica , Modelos Teóricos , ViscosidadeRESUMO
A new microfluidic mixer is presented consisting of a rectangular channel with grooves placed in the top and bottom. This not only increases the driving force behind the lateral flow, but allows for the formation of advection patterns that cannot be created with structures on the bottom alone. Chevrons, pointing in opposite directions on the top and bottom, are used to create a pair of vortices positioned side by side. Stripes running the width of the channel generate a pair of vertically stacked vortices. Computational fluid dynamics (CFD) simulations are used to model the behavior of the systems and provide velocity maps at cross-sections within the mixer. Experiments demonstrate the mixing that results when two segregated species enter the mixer side-by-side and pass through two cycles of the mixer (i.e., two alternating sets of four stripes and four chevrons).