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1.
Anal Bioanal Chem ; 412(16): 3799-3800, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32405678
2.
Anal Bioanal Chem ; 412(16): 3801-3811, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32314000

RESUMO

The ability to selectively move and trap proteins is core to their effective use as building blocks and for their characterization. Analytical and preparative strategies for proteins have been pursued and modeled for nearly a hundred years, with great advances and success. Core to all of these studies is the separation, isolation, purification, and concentration of pure homogeneous fractions of a specific protein in solution. Processes to accomplish this useful solution include biphasic equilibrium (chromatographies, extractions), mechanical, bulk property, chemical equilibria, and molecular recognition. Ultimately, the goal of all of these is to physically remove all non-like protein molecules-to the finest detail: all atoms in the full three-dimensional structure being identical down the chemical bond and bulk structure chirality. One strategy which has not been effectively pursued is exploiting the higher order subtle electrical properties of the protein-solvent system. The advent of microfluidic systems has enabled the use of very high electric fields and well-defined gradients such that extremely high resolution separations of protein mixtures are possible. These advances and recognition of these capabilities have caused a re-evaluation of the underlying theoretical models and they were found to be inadequate. New theoretical descriptions are being considered which align more closely to the total forces present and the subtlety of differences between similar proteins. These are focused on the interfacial area between the protein and hydrating solvent molecules, as opposed to the macroscale assumptions of homogeneous solutions and particles. This critical review examines all data which has been published that place proteins in electric field gradients which induce collection of those proteins, demonstrating a force greater than dispersive effects or countering forces. Evolving theoretical constructs are presented and discussed, and a general estimate of future capabilities using the higher order effects and the high fields and precise gradients of microfluidic systems is discussed. Graphical abstract.

3.
Analyst ; 145(8): 2904-2914, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32072998

RESUMO

Differentiating bacteria strains using biophysical forces has been the focus of recent studies using dielectrophoresis (DEP). The refinement of these studies has created high-resolution separations such that very subtle properties of the cells are enough to induce significant differences in measurable biophysical properties. These high-resolution capabilities build upon the advantages of DEP which include small sample sizes and fast analysis times. Studies focusing on differentiating antimicrobial resistant and susceptible bacteria potentially have significant impact on human health and medical care. A prime example is Staphylococcus aureus, which commonly colonizes adults without ill effects. However, the pathogen is an important cause of infections, including surgical site infections. Treatment of S. aureus infections is generally possible with antimicrobials, but antimicrobial resistance has emerged. Of special importance is resistance to methicillin, an antimicrobial created in response to resistance to penicillin. Here, dielectrophoresis is used to study methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA) strains, both with and without the addition of a fluorescent label. The capture onset potential of fluorescently-labeled MRSA (865 ± 71 V) and thus the ratio of electrokinetic to dielectrophoretic mobility, was found to be higher than that of fluorescently-labeled MSSA (685 ± 61 V). This may be attributable to the PBP2a enzyme present in the MRSA strain and not in the MSSA bacteria. Further, unlabeled MRSA was found to have a capture onset potential of 732 ± 44 V, while unlabeled MSSA was found to have a capture onset potential of 562 ± 59 V. This shows that the fluorescently-labeled bacteria require a higher applied potential, and thus ratio of mobilities, to capture than the unlabeled bacteria.

4.
Analyst ; 144(24): 7478-7488, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31720589

RESUMO

A central challenge in measuring the biophysical properties of cells with electrokinetic approaches is the assignment of these biophysical properties to specific biological characteristics. Changes in the electrokinetic behavior of cells may come from mutations, altered gene expression levels, post-translation modifications, or environmental effects. Here we assess the electrokinetic behavior of chemically surface-modified bacterial cells in order to gain insight into the biophysical properties that are specifically affected by changes in surface chemistry. Using E. coli as a scaffold, an amine coupling reaction was used to covalently attach glycine, spermine, bovine serum albumin (protein), or 7-amino-4-methyl-3-coumarinylacetic acid (fluorescent dye) to the free carboxylic acid groups on the surface of the cells. These populations, along with unlabeled control cells, were subject to electrokinetic and dielectrophoretic measurements to quantify any changes in the biophysical properties upon alteration. The properties associated with each electrokinetic force are discussed relative to the specific reactant used. We conclude that relatively modest and superficial changes to cell surfaces can cause measurable changes in their biophysical properties.

5.
Biomicrofluidics ; 13(5): 054112, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31673302

RESUMO

Transmission electron microscopy (TEM) of biological samples has a long history and has provided many important insights into fundamental processes and diseases. While great strides have been made in EM data collection and data processing, sample preparation is still performed using decades-old techniques. Those sample preparation methods rely on extensive macroscale purification and concentration to achieve homogeneity suitable for high-resolution analyses. Noting that relatively few bioparticles are needed to generate high-quality protein structures, this work uses microfluidics that can accurately and precisely manipulate and deliver bioparticles to grids for imaging. The use of microfluidics enables isolation, purification, and concentration of specific target proteins at these small scales and does so in a relatively short period of time (minutes). These capabilities enable imaging of more dilute solutions and obtaining pure protein images from mixtures. In this system, spatially isolated, purified, and concentrated proteins are transferred directly onto electron microscopy grids for imaging. The processing enables imaging of more dilute solutions, as low as 5 × 10-6 g/ml, with small total amounts of protein (<400 pg, 900 amol). These levels may be achieved with mixtures and, as proof-of-principle, imaging of one protein from a mixture of two proteins is demonstrated.

6.
Anal Chem ; 91(21): 13994-14001, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31638373

RESUMO

Electrochemically modulated liquid chromatography (EMLC) uses electrical potentials, applied to a conductive chromatographic stationary phase (e.g., porous graphitic carbon [PGC]), to manipulate analyte retention. This paper reports the design of a capillary EMLC column with a smaller internal diameter (ID; 250 µm) than that of the standard bore predecessor (3.3 mm ID). The new capillary EMLC columns are configured so that the PGC stationary phase serves as the working electrode in a two-electrode electrochemical cell and simplifies electrode placement by obviating the need for a counter electrode. This configuration also eliminates the internal Nafion sleeve that is critical to operation for the standard bore columns, thereby avoiding Nafion deformation as a source of chromatographic band broadening and rupturing as a mode of column failure. Indeed, values for chromatographic efficiency obtained on the capillary columns meet or exceed those measured for the standard columns (20 000-40 000 vs 14 000 plates/m, respectively) with near symmetric elution bands (asymmetry factors of 1.1-1.4 for well-packed capillaries) that surpass band symmetries observed in all prior studies. A test suite of aromatic sulfonates was used to characterize the chromatographic performance of the capillary EMLC columns. Separations of this test mixture showed that retention factors for individual analytes could be manipulated by as much as 21× by changing the applied potential at the PGC stationary phase. Changes in retention behavior at different potential ranges, hypothesized to result from differences in adsorption orientation, were also observed and are consistent with past work. Collectively, the retention behavior unique to EMLC is operative in this new capillary configuration and promises to open new avenues in tuning LC separations.

7.
Analyst ; 144(13): 4066-4072, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31165125

RESUMO

Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.

8.
Anal Chim Acta ; 1068: 41-51, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072476

RESUMO

Foodborne pathogens pose one of the greatest challenges facing public health in the modern day. One important pathogen, Listeria monocytogenes, is known to be challenging to detect and identify. Three serovars cause most of the Listeria related food-borne illnesses, which the Centers for Disease Control currently utilizes a combination of pulsed-field gel electrophoresis and whole genome sequencing for identification and the determination of clusters and outbreaks. There is a potential method for rapid collection of epidemiological information by exploiting the electrokinetic and dielectrophoretic properties of the L. monocytogenes serovars. Using dielectrophoresis, the three most commonly identified serovars of L. monocytogenes can be distinguished from each other. The electrokinetic and dielectrophoretic mobilities of each serovar was determined through a combination of electrokinetic velocity and dielectrophoretic trapping assessments, in conjunction with finite element multi-physics modeling. A mathematical model of the data, which defines the various factors of dielectrophoretic trapping, is utilized and verified based on the behavior of L. monocytogenes in the microchannel. The trapping condition for the serovars were evaluated as 2.8±0.2×109, 2.2±0.2×109, and 2.2±0.3×109Vm-2 and the electrokinetic mobility was assessed to be 19±0.7, 17±0.7, and for the L. monocytogenes serovars 1/2a, 1/2b, and 4b, respectively.


Assuntos
Listeria monocytogenes/isolamento & purificação , Eletroforese , Listeria monocytogenes/citologia , Técnicas Analíticas Microfluídicas
9.
Ecol Appl ; 29(4): e01881, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30939226

RESUMO

The development and expansion of wind energy is considered a key global threat to bat populations. Bat carcasses are being found underneath wind turbines across North and South America, Eurasia, Africa, and the Austro-Pacific. However, relatively little is known about the comparative impacts of techniques designed to modify turbine operations in ways that reduce bat fatalities associated with wind energy facilities. This study tests a novel approach for reducing bat fatalities and curtailment time at a wind energy facility in the United States, then compares these results to operational mitigation techniques used at other study sites in North America and Europe. The study was conducted in Wisconsin during 2015 using a new system of tools for analyzing bat activity and wind speed data to make near real-time curtailment decisions when bats are detected in the area at control turbines (N = 10) vs. treatment turbines (N = 10). The results show that this smart curtailment approach (referred to as Turbine Integrated Mortality Reduction, TIMR) significantly reduced fatality estimates for treatment turbines relative to control turbines for pooled species data, and for each of five species observed at the study site: pooled data (-84.5%); eastern red bat (Lasiurus borealis, -82.5%); hoary bat (Lasiurus cinereus, -81.4%); silver-haired bat (Lasionycteris noctivagans, -90.9%); big brown bat (Eptesicus fuscus, -74.2%); and little brown bat (Myotis lucifugus, -91.4%). The approach reduced power generation and estimated annual revenue at the wind energy facility by ≤ 3.2% for treatment turbines relative to control turbines, and we estimate that the approach would have reduced curtailment time by 48% relative to turbines operated under a standard curtailment rule used in North America. This approach significantly reduced fatalities associated with all species evaluated, each of which has broad distributions in North America and different ecological affinities, several of which represent species most affected by wind development in North America. While we recognize that this approach needs to be validated in other areas experiencing rapid wind energy development, we anticipate that this approach has the potential to significantly reduce bat fatalities in other ecoregions and with other bat species assemblages in North America and beyond.


Assuntos
Quirópteros , África , Animais , Europa (Continente) , América do Norte , Wisconsin
10.
Anal Bioanal Chem ; 411(10): 2223-2237, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30879117

RESUMO

Dielectrophoresis (DEP) brings about the high-resolution separations of cells and other bioparticles arising from very subtle differences in their properties. However, an unanticipated limitation has arisen: difficulty in assignment of specific biological features which vary between two cell populations. This hampers the ability to interpret the significance of the variations. To realize the opportunities made possible by dielectrophoresis, the data and the diversity of structures found in cells and bioparticles must be linked. While the crossover frequency in DEP has been studied in-depth and exploited in applications using AC fields, less attention has been given when a DC field is present. Here, a new mathematical model of dielectrophoretic data is introduced which connects the physical properties of cells to specific elements of the data from potential- or time-varied DEP experiments. The slope of the data in either analysis is related to the electrokinetic mobility, while the potential at which capture initiates in potential-based analysis is related to both the electrokinetic and dielectrophoretic mobilities. These mobilities can be assigned to cellular properties for which values appear in the literature. Representative examples of high and low values of properties such as conductivity, zeta potential, and surface charge density for bacteria including Streptococcus mutans, Rhodococcus erythropolis, Pasteurella multocida, Escherichia coli, and Staphylococcus aureus are considered. While the many properties of a cell collapse into one or two features of data, for a well-vetted system the model can indicate the extent of dissimilarity. The influence of individual properties on the features of dielectrophoretic data is summarized, allowing for further interpretation of data. Graphical abstract.


Assuntos
Algoritmos , Bactérias/química , Eletroforese/métodos , Bactérias/citologia , Bactérias/isolamento & purificação , Condutividade Elétrica , Eletro-Osmose , Cinética , Modelos Biológicos , Modelos Químicos , Eletricidade Estática , Propriedades de Superfície
11.
Electrophoresis ; 40(2): 304-314, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30350873

RESUMO

Electrophoretic exclusion (EE) is a counterflow gradient technique that exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. Resolution for this technique has been theoretically examined and the smallest difference in electrophoretic mobilities that can be completely separated is estimated to be 10-13  cm2 /Vs. Traditional and mesoscale systems have been used, whereas microfluidics offers a greater range of geometries and configurations towards approaching this theoretical limit. To begin to understand the impact of seemingly subtle changes to the entrance flow and the electric field configurations, three closely related microfluidic interfaces were modeled, fabricated, and tested. These interfaces consisted of systematically varying placement of an asymmetric electrode relative to a channel entrance: leading electrode placed outside the channel entrance, leading electrode aligned with the channel, and leading electrode placed within the channel. A charged fluorescent dye is used as a sensitive and accurate probe for the model and to test the concentration variation at these interfaces. Models and experiments focused on visualizing the concentration profile in areas of high temporal dynamics, thus providing a severe test of the models. Experimental data and simulation results showed strong qualitative agreement. The complexity of the electric and flow fields about this interface and the agreement between models and testing suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces.


Assuntos
Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Eletrodos , Desenho de Equipamento , Modelos Químicos
12.
Sci Rep ; 8(1): 10193, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976961

RESUMO

Invasive reptilian predators can have substantial impacts on native species and ecosystems. Tegu lizards are widely distributed in South America east of the Andes, and are popular in the international live animal trade. Two species are established in Florida (U.S.A.) - Salvator merianae (Argentine black and white tegu) and Tupinambis teguixin sensu lato (gold tegu) - and a third has been recorded there- S. rufescens (red tegu). We built species distribution models (SDMs) using 5 approaches (logistic regression, multivariate adaptive regression splines, boosted regression trees, random forest, and maximum entropy) based on data from the native ranges. We then projected these models to North America to develop hypotheses for potential tegu distributions. Our results suggest that much of the southern United States and northern México probably contains suitable habitat for one or more of these tegu species. Salvator rufescens had higher habitat suitability in semi-arid areas, whereas S. merianae and T. teguixin had higher habitat suitability in more mesic areas. We propose that Florida is not the only state where these taxa could become established, and that early detection and rapid response programs targeting tegu lizards in potentially suitable habitat elsewhere in North America could help prevent establishment and abate negative impacts on native ecosystems.


Assuntos
Distribuição Animal , Monitorização de Parâmetros Ecológicos/métodos , Espécies Introduzidas , Lagartos/fisiologia , Modelos Biológicos , Animais , Florida , Florestas , México
13.
PLoS One ; 13(2): e0192887, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29466401

RESUMO

Common vampire bats (Desmodus rotundus) occur throughout much of South America to northern México. Vampire bats have not been documented in recent history in the United States, but have been documented within about 50 km of the U.S. state of Texas. Vampire bats feed regularly on the blood of mammals and can transmit rabies virus to native species and livestock, causing impacts on the health of prey. Thus cattle producers, wildlife management agencies, and other stakeholders have expressed concerns about whether vampire bats might spread into the southern United States. On the other hand, concerns about vampire-borne rabies can also result in wanton destruction at bat roosts in areas occupied by vampire bats, but also in areas not known to be occupied by this species. This can in turn negatively affect some bat roosts, populations, and species that are of conservation concern, including vampire bats. To better understand the current and possible future distribution of vampire bats in North America and help mitigate future cattle management problems, we used 7,094 vampire bat occurrence records from North America and species distribution modeling (SDM) to map the potential distribution of vampire bats in North America under current and future climate change scenarios. We analysed and mapped the potential distribution of this species using 5 approaches to species distribution modeling: logistic regression, multivariate adaptive regression splines, boosted regression trees, random forest, and maximum entropy. We then projected these models into 17 "worst-case" future climate scenarios for year 2070 to generate hypotheses about how the vampire bat distribution in North America might change in the future. Of the variables used in this analysis, minimum temperature of the coldest month had the highest variable importance using all 5 SDM approaches. These results suggest two potential near-future routes of vampire bat dispersal into the U.S., one via southern Texas, and a second into southern Florida. Some of our SDM models support the hypothesis that suitable habitat for vampire bats may currently exist in parts of the México-U.S. borderlands, including extreme southern portions of Texas, as well as in southern Florida. However, this analysis also suggests that extensive expansion into the south-eastern and south-western U.S. over the coming ~60 years appears unlikely.


Assuntos
Distribuição Animal , Quirópteros/virologia , Mudança Climática , Vetores de Doenças , Modelos Biológicos , Vírus da Raiva , Animais , Ecossistema , Fósseis , Modelos Logísticos , México , Análise Multivariada , Dinâmica Populacional , Raiva/transmissão , Temperatura , Estados Unidos
14.
Anal Bioanal Chem ; 409(27): 6405-6414, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28852806

RESUMO

Blood is one of the most important biofluids used for clinical diagnostics. Cells and proteins in the blood can provide a rich source of information for the evaluation of human health. Efficient separation of blood components is a necessary process in order to minimize the interference of unwanted components during sensing, separation, and detection. In this paper, an insulator-based gradient dielectrophoretic device has been applied to separate red blood cells from model protein biomarkers for myocardial infarction in buffer. Within one min, red blood cells are largely depleted regardless of the minimum adherence on the channel wall. Considering the adhered red blood cells will not be transported further, a purified protein solution can be delivered for potential downstream processing or detection. Graphical Abstract ᅟ.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Separação Celular/instrumentação , Eletroforese/instrumentação , Eritrócitos/citologia , Infarto do Miocárdio/diagnóstico , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Desenho de Equipamento , Humanos
15.
PLoS One ; 12(7): e0180693, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28686737

RESUMO

Recent research has demonstrated that temperature and precipitation conditions correlate with successful reproduction in some insectivorous bat species that live in arid and semiarid regions, and that hot and dry conditions correlate with reduced lactation and reproductive output by females of some species. However, the potential long-term impacts of climate-induced reproductive declines on bat populations in western North America are not well understood. We combined results from long-term field monitoring and experiments in our study area with information on vital rates to develop stochastic age-structured population dynamics models and analyzed how simulated fringed myotis (Myotis thysanodes) populations changed under projected future climate conditions in our study area near Boulder, Colorado (Boulder Models) and throughout western North America (General Models). Each simulation consisted of an initial population of 2,000 females and an approximately stable age distribution at the beginning of the simulation. We allowed each population to be influenced by the mean annual temperature and annual precipitation for our study area and a generalized range-wide model projected through year 2086, for each of four carbon emission scenarios (representative concentration pathways RCP2.6, RCP4.5, RCP6.0, RCP8.5). Each population simulation was repeated 10,000 times. Of the 8 Boulder Model simulations, 1 increased (+29.10%), 3 stayed approximately stable (+2.45%, +0.05%, -0.03%), and 4 simulations decreased substantially (-44.10%, -44.70%, -44.95%, -78.85%). All General Model simulations for western North America decreased by >90% (-93.75%, -96.70%, -96.70%, -98.75%). These results suggest that a changing climate in western North America has the potential to quickly erode some forest bat populations including species of conservation concern, such as fringed myotis.


Assuntos
Quirópteros/fisiologia , Mudança Climática , Conservação dos Recursos Naturais , Reprodução/fisiologia , Animais , Florestas , Modelos Teóricos , América do Norte , Dinâmica Populacional , Temperatura
16.
Analyst ; 142(9): 1608-1618, 2017 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-28394391

RESUMO

The ability to separate analytes with increasingly similar properties drives the field of separation science. One way to achieve such separations is using trapping and streaming dielectrophoresis (DEP), which directly exploits the subtle differences in the electrophysical properties of analytes. The non-uniform fields necessary for DEP can be formed using various insulator shapes in microchannels. Current insulator shapes include triangles, diamonds, circles, and rectangles. However, all of these insulators pose problems for trapping, streaming, and sorting (deflection) as the induced fields/gradients are not behaviorally consistent across the lateral dimension. This leads to analytes experiencing different forces depending on their pathline in the microchannel and result in low resolution separations. Based on an iterative process that explored approximately 40 different insulator shapes, a design was chosen that indicated improved particle streamlines, better trapping efficiency, and consistent electrical environments across the lateral dimension. The design was assessed by simulations where the electric field, gradient of the electric field squared, and the ratio of the two were plotted. The improved design includes a unique new multi-length scale element. The multi-length scale structure streamlines the analyte(s) and improves homogeneity in the lateral dimension, while still achieving high gradients necessary for analyte separation using DEP. The design is calculated to keep analytes on the centerline which should improve resolution, and eliminate extraneous trapping zones. Behaviors consistent with the features of the simulations were observed in proof of principle experiments using representative test probes.

17.
Analyst ; 141(6): 1997-2008, 2016 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-26878279

RESUMO

Biotechnology, separation science, and clinical research are impacted by microfluidic devices. Separation and manipulation of bioparticles such as DNA, protein and viruses are performed on these platforms. Microfluidic systems provide many attractive features, including small sample size, rapid detection, high sensitivity and short processing time. Dielectrophoresis (DEP) and electrophoresis are especially well suited to microscale bioparticle control and have been demonstrated in many formats. In this work, an optimized gradient insulator-based DEP device was utilized for concentration of Sindbis virus, an animal virus with a diameter of 68 nm. Within only a few seconds, the concentration of Sindbis virus can be increased by two to six times in the channel under easily accessible voltages as low as about 70 V. Compared with traditional diagnostic methods used in virology, DEP-based microfluidics can enable faster isolation, detection and concentration of viruses in a single step within a short time.


Assuntos
Eletroforese/instrumentação , Vírus Sindbis/isolamento & purificação , Análise de Elementos Finitos , Dispositivos Lab-On-A-Chip
18.
Analyst ; 141(1): 352, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26626370

RESUMO

Correction for 'Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance' by Paul V. Jones et al., Analyst, 2015, 140, 5152-5161.

19.
Anal Methods ; 7(20): 8632-8639, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26527562

RESUMO

To achieve improved sensitivity in cardiac biomarker detection, a batch incubation magnetic microbead immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I. A sandwich immunoassay was performed in a simple micro-centrifuge tube allowing full dispersal of the solid capture surface during incubations. Following magnetic bead capture and wash steps, samples were analyzed in the presence of a manipulated magnetic field utilizing a modified microscope slide and fluorescent inverted microscope to collect video data files. Analysis of the video data allowed for the quantitation of myoglobin, heart-type fatty acid binding protein and cardiac troponin I to levels of 360 aM, 67 fM, and 42 fM, respectively. Compared to the previous detection limit of 50 pM for myoglobin, this offers a five-fold improvement in sensitivity. This improvement in sensitivity and incorporation of additional markers, along with the small sample volumes required, suggest the potential of this platform for incorporation as a detection method in a total sample analysis device enabling multiplexed detection for the analysis of clinical samples.

20.
Anal Bioanal Chem ; 407(28): 8605-15, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26342315

RESUMO

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.


Assuntos
Anticorpos/química , Antígenos/química , Imunoensaio/estatística & dados numéricos , Limite de Detecção , Humanos , Análise Multivariada , Ligação Proteica , Razão Sinal-Ruído
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