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1.
Biotechniques ; 74(1): 23-29, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36597257

RESUMO

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 µg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).


Assuntos
DNA , Trombose , Humanos , Genótipo , DNA/genética , Eletroforese , Congelamento
2.
Anal Chem ; 95(2): 1409-1418, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36599093

RESUMO

Presented here is the first continuous separation of microparticles and cells of similar characteristics employing linear and nonlinear electrokinetic phenomena in an insulator-based electrokinetic (iEK) system. By utilizing devices with insulating features, which distort the electric field distribution, it is possible to combine linear and nonlinear EK phenomena, resulting in highly effective separation schemes that leverage the new advancements in nonlinear electrophoresis. This work combines mathematical modeling and experimentation to separate four distinct binary mixtures of particles and cells. A computational model with COMSOL Multiphysics was used to predict the retention times (tR,p) of the particles and cells in iEK devices. Then, the experimental separations were carried out using the conditions identified with the model, where the experimental retention time (tR,e) of the particles and cells was measured. A total of four distinct separations of binary mixtures were performed by increasing the level of difficulty. For the first separation, two types of polystyrene microparticles, selected to mimic Escherichia coli and Saccharomyces cerevisiae cells, were separated. By leveraging the knowledge gathered from the first separation, a mixture of cells of distinct domains and significant size differences, E. coli and S. cerevisiae, was successfully separated. The third separation also featured cells of different domains but closer in size: Bacillus cereus versus S. cerevisiae. The last separation included cells in the same domain and genus, B. cereus versus Bacillus subtilis. Separation results were evaluated in terms of number of plates (N) and separation resolution (Rs), where Rs values for all separations were above 1.5, illustrating complete separations. Experimental results were in agreement with modeling results in terms of retention times, with deviations in the 6-27% range, while the variation between repetitions was between 2 and 18%, demonstrating good reproducibility. This report is the first prediction of the retention time of cells in iEK systems.


Assuntos
Escherichia coli , Saccharomyces cerevisiae , Reprodutibilidade dos Testes , Modelos Teóricos , Eletricidade , Eletroforese/métodos
3.
Curr Protoc ; 3(1): e651, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36688368

RESUMO

Digital imaging is the method of choice for documentation and analysis of electrophoretic separations of protein and DNA. Digital images of gel electropherograms can be obtained rapidly using sCMOS and CCD-based cameras, and the images can be easily archived and analyzed using image analysis software. This overview defines important key terms and calculations for imaging, explains the capture process, reviews the range of technologies used for image capture, and provides an introduction to the software and methods used for one- and two-dimensional digital image analysis. © 2023 Wiley Periodicals LLC.


Assuntos
Processamento de Imagem Assistida por Computador , Software , Eletroforese/métodos , Processamento de Imagem Assistida por Computador/métodos , DNA/análise , Proteínas/análise
4.
Nanotechnology ; 34(13)2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36571849

RESUMO

We model the dielectrophoretic response ofE. colibacterial cells and red blood cells, upon exposure to an electric field. We model the separation, capture, and release mechanisms under flow conditions in a microfluidic channel and show under which conditions efficient separation of different cell types occurs. The modelling work is aimed to guide the separation electrode architecture and design for experimental validation of the model. The dielectrophoretic force is affected both by the geometry of the electrodes (the gradient of the electric field), the Re{CM(ω)} factor, and the permittivity of the medium ϵm. Our modelling makes testable predictions and shows that designing the electrode structure to ensure structure periodicity with spacing between consecutive traps smaller than the length of the depletion zone ensures efficient capture and separation. Such electrode system has higher capture and separation efficiency than systems with the established circular electrode architecture. The simulated, modelled microfluidic design allows for the separated bacteria, concentrated by dedicated dielectrophoretic regions, to be subsequently detected using label-free functionalized nanowire sensors. The experimental validation of the modelling work presented here and the validation of the theoretical design constraints of the chip electrode architecture is presented in the companion paper in the same issue (Weber MUet al2022 Chip for dielectrophoretic Microbial Capture, Separation and Detection II: Experimental Study).


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Eletrodos , Eletricidade , Bactérias , Separação Celular , Eletroforese
5.
J Colloid Interface Sci ; 634: 921-929, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36571855

RESUMO

HYPOTHESIS: The synthesis of compositionally heterogeneous particles is central to the development of complex colloidal units for self-assembly and self-propulsion. Yet, as the complexity of particles grows, synthesis becomes more prone to "errors". We hypothesize that alternating-current dielectrophoretic forces can efficiently sort Janus particles, as a function of patch size and material, and colloidal dumbbells by size. EXPERIMENTS: We prepared Janus particles with different patch size and material by physical vapor deposition and colloidal dumbbells via capillarity-assisted particle assembly. We then performed sorting experiments in a microfluidic chip comprising electrodes with asymmetric orifices, specifically exploiting the dielectric contrast between different portions of the particles or their size difference to steer them towards different outlets. FINDINGS: We calculated that the DEP force for Janus particles may switch from positive to negative as a function of composition at a critical AC frequency, thus enabling sorting different particles crossing the electrodes' region. The predictions are confirmed by optical microscopy experiments. We also show that intact and "broken" dumbbells can be simply separated as they experience different DEP forces. The integration of multiple asymmetric orifices leads a larger zone with high field gradient to increase separation efficiency and makes it a promising tool to select precise particle populations, isolating fractions with narrowly distributed characteristics.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Eletroforese , Eletrodos , Coloides
6.
Langmuir ; 39(1): 101-110, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36541659

RESUMO

A dielectrophoretic device employing a planar array of microelectrodes is designed for controlled transport of individual microparticles. By exciting the electrodes in sequence, a moving dielectrophoretic force is created that can drag a particle across the electrodes in a straight line. The electrode shapes are designed to counter any lateral drift of the trapped particle during transport. This facilitates single particle transport by creating a narrow two-dimensional corridor for the moving dielectrophoretic force to operate on. The design and analysis processes are discussed in detail. Numerical simulations are performed to calculate the electromagnetic field distribution and the generated dielectrophoretic force near the electrodes. The Langevin equation is used for analyzing the trajectory of a microparticle under the influence of the external forces. The simulations show how the designed electrode geometry produces the necessary lateral confinement required for successful particle transport. Finally, experimental results are presented showing controlled bidirectional linear transport of single polystyrene beads of radius 10 and 5 µm for a distances 840 and 1100 µm, respectively. The capabilities of the proposed platform make it suitable for micro total analysis systems (µTAS) and lab-on-a-chip (LOC) applications.


Assuntos
Dispositivos Lab-On-A-Chip , Poliestirenos , Microeletrodos , Eletroforese/métodos
7.
An. R. Acad. Nac. Farm. (Internet) ; 88(número extraordinario): 237-256, diciembre 2022. ilus, tab, graf
Artigo em Espanhol | IBECS | ID: ibc-JHG-318

RESUMO

Las guías ICH más recientes en el apartado Q incluyen el concepto de Calidad por Diseño (QbD) que queda definida como un enfoque sistemático para el desarrollo que comienza con unos objetivos previamente definidos y que enfatiza la comprensión de los productos y de los procesos, y el control del proceso basándose en el rigor científico y en la gestión de riesgos para la calidad.Aunque en esta definición no se menciona expresamente a los métodos analíticos, desde el año 2007 ha habido autores que han defendido la posibilidad de aplicar los principios de la QbD al desarrollo y validación de métodos analíticos para asegurar su precisión intermedia y su robustez, especialmente en el ámbito de los métodos físicos y físico-químicos relacionados con el análisis cuantitativo de medicamentos.Sin embargo, en nuestra experiencia, el ámbito real de aplicación de la QbD para métodos analíticos puede abarcar no sólo el análisis cuantitativo de medicamentos, sino también otros métodos analíticos en los que se determinan otras propiedades, como es el caso de la citoferometría, modalidad específica de electroforesis de células y partículas en suspensión, con la que es posible determinar la movilidad electroforética de eritrocitos normales y patológicos.Los datos del desarrollo original de un método citoferométrico realizado en 1988 se han reprocesado de acuerdo con los principios QbD actuales, y se han comparado los resultados obtenidos con la metodología tradicional y con la metodología QbD.Con esta comparación se demuestra que los resultados procesados siguiendo la QbD aportan un mayor conocimiento y control del método citoferométrico, así como una mayor flexibilidad en el manejo de los parámetros físicos y físico-químicos necesarios y unas herramientas adecuadas para controlar las principales fuentes de variabilidad analítica. (AU)


Quality by Design (QbD) is defined as a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management, according to ICH Q section guidelines definition.Although this definition does not specifically mention analytical methods, since 2007 there have been authors who have defended the possibility of applying the principles of QbD to the development and validation of analytical methods in order to ensure their ruggedness and robustness, especially in the field of physical and physicochemical methods related to the quantitative drug analysis.However, in our experience, the real scope of application of QbD for analytical methods can include not only the quantitative analysis of drugs, but also other analytical methods in which other properties are determined, such as cytoferometry, a specific type of cell and particle suspension electrophoresis, with which it is possible to determine the electrophoretic mobility of normal and pathological erythrocytes.Data of the original development of a cytopherometric method carried out in 1988 have been reprocessed according to current QbD principles, and the results obtained with the traditional methodology and with the QbD methodology have been compared.This comparison demonstrates that the results processed following QbD show an enhancement in the knowledge and control of the cytopherometric method, giving more flexibility on physical and physico-chemical parameters management and appropriate tools to control the principal sources of analytical variability. (AU)


Assuntos
Humanos , Eletroforese , Material Particulado , Eritrócitos
8.
ACS Sens ; 7(12): 3906-3914, 2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36512685

RESUMO

Miniaturization and integration of chemical reactions into fluidic systems in combination with product purification or buffer exchange can reduce the amount of solvents and reactants required while increasing synthesis efficiency. A critical step is the regulation of flow rates to realize optimal synthesis conditions and high purification rates, so real-time, label-free monitoring is required in methods such as free-flow electrophoresis. Optical detection methods are widely used, but they often have complex excitation and detection setups that are disadvantageous for point-of-care applications. The method we have chosen is electrochemical impedance spectroscopy for detecting charged compounds in aqueous buffers with low ionic strength. Propranolol was selected for proof of concept and was separated from the organic solvent and the precursor oxirane by free-flow electrophoresis. For this purpose, electrode structures were fabricated in microfluidic channels by photolithographic lift-off technique and optimized in terms of positioning, electrode size and distance for sensitive detection, and quantification of propranolol in the nanomolar range. It is also noteworthy that the organic solvent dimethyl sulfoxide (DMSO) could be detected and quantified by an increased impedance magnitude. Subsequently, the optimized interdigital electrode structures were integrated into the outlet channels of the electrophoretic separation chamber to monitor the various outgoing fluidic streams and provide in-line control of the fluidic flows for the purification step. In conclusion, we can provide a microfluidic chip to monitor the separation efficiency of a substance mixture during free-flow electrophoresis without the need of complex analytical techniques using electrochemical impedance spectroscopy.


Assuntos
Técnicas Analíticas Microfluídicas , Técnicas Analíticas Microfluídicas/métodos , Espectroscopia Dielétrica , Propranolol , Eletroforese , Eletrodos
9.
Int J Mol Sci ; 23(24)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36555186

RESUMO

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation.


Assuntos
Endopeptidases , Serina Proteases , Abelhas , Animais , Larva/metabolismo , Endopeptidases/metabolismo , Eletroforese , Serina Proteases/metabolismo , Proteínas de Insetos/metabolismo , Concentração de Íons de Hidrogênio
10.
Langmuir ; 38(46): 14044-14052, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36343201

RESUMO

The colloidal stability of non-motile algal cells in water drives their distribution in space. An accurate description of the interfacial properties of microalgae is therefore critical to understand how microalgae concentrations can change in their biotope or during harvesting processes. Here, we probe the surface charges of three unicellular algae─Chlorella vulgaris, Nannochloropsis oculata, and Tetraselmis suecica─through their electrophoretic mobility. Ohshima's soft particle theory describes the electrokinetic properties of particles covered by a permeable polyelectrolyte layer, a usual case for biological particles. The results appear to fit the predictions of Ohshima's theory, proving that all three microalgae behave electrokinetically as soft particles. This allowed us to estimate two characteristic parameters of the polyelectrolyte external layer of microalgae: the volume charge density and the hydrodynamic penetration length. Results were compared with transmission electron microscopy observations of the algal cells' surfaces, and in particular of their extracellular polymeric layer, which was identified with the permeable shell evidenced by electrophoretic measurements. Noticeably, the algal surface potentials estimated from electrophoretic mobility using the soft particle theory are less negative than the apparent zeta potentials. This finding indicates that electrostatics are expected to play a minor role in phenomena of environmental and industrial importance, such as microalgae aggregation or adhesion.


Assuntos
Microalgas , Polieletrólitos , Eletroforese/métodos , Membrana Celular
11.
Anal Chem ; 94(48): 16560-16569, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36418026

RESUMO

In stochastic blocking electrochemistry, microparticles generate individual current steps when they adsorb on a microelectrode and decrease the current and flux of a redox mediator reacting at the surface. The amplitude of the current step informs on particle size and landing locus, while step frequency correlates with particle transport. Here, we report a new method to estimate the average arrival velocities of single rod-shaped bacteria (bacilli). The method relies on simulating the nearby threshold distance from the surface where the bacillus no longer perturbs mediator flux and the current step approaches zero. We estimated the average velocities of bacillus arrival by dividing the threshold distance over the current step duration, a parameter that here we detect for the first time and increases with bacillus length. By comparing diffusional fluctuations to bacillus average velocity, we estimated diffusion and migration contributions as a function of bacterium size. Average arrival velocities increase with bacillus length at the same time as migration intensifies and diffusion weakens. Our analysis is universal and more effective in determining transport mode contributions than the present approach of comparing theoretical and experimental step frequencies. Uncertainty in landing locus is inconsequential because the step duration used to calculate the average arrival speed already contains such information and knowing bacillus electrophoretic mobility or ζ-potential is not needed. Additionally, by simulating and assigning edge landings to the most repeated values of current steps in a recording, we obtain bacilli lengths and widths similar to scanning electron microscopy, from which we infer landing orientation.


Assuntos
Eletroquímica , Difusão , Tamanho da Partícula , Eletroforese , Microeletrodos
12.
PLoS One ; 17(11): e0277138, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36342943

RESUMO

DNA extraction, a vital pre-requisite for most biological studies, continues to be studied extensively. According to some studies, DNA shows a certain degree of absorbability on filter paper made of plant fiber-based adsorbent material. However, the principle underlying such specific adsorption as well as plant species associated with plant fiber-based adsorbents and optimized extraction conditions have not yet been studied. This study demonstrates the tight correlation between crystallinity and hygroscopicity in plant fiber-based adsorbents used for DNA extraction and proposes the concept of DNA adsorption on plant fiber-based adsorbents, for the first time. We also explored optimal extracting and eluting conditions and developed a novel plant fiber-based DNA extraction method that was quadruple times more powerful than current approaches. Starting with the screening of various types of earthed plant fiber-based adsorbents, we went on to mine new plant fiber-based adsorbents, bamboo paper and degreased cotton, and succeeded in increasing their efficiency of DNA extraction to 4.2 times than that of current approaches. We found a very strong correlation between the crystallinity and hygroscopicity of plant fiber-based adsorbents which showed efficiency for DNA extraction, and thus propose a principle that potentially governs such specific adsorption processes, in the hope that this information may guide related multidisciplinary research studies in the future. Nanodrop, electrophoresis and PCR were selected to demonstrate the quantity, quality, integrity and utility of the extracted DNA. Furthermore, crystallinity, hygroscopicity, pore size distribution and composition of plant fiber-based adsorbents were studied to explore their correlation in an attempt to understand the principle underlying this particular type of adsorption. The findings of this study may be further extended to the extraction of other types of nucleic acids with similar biochemical properties.


Assuntos
Estruturas Vegetais , Molhabilidade , Adsorção , DNA de Plantas/genética , Eletroforese
13.
Biosensors (Basel) ; 12(11)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36354455

RESUMO

Glioblastoma multiforme is one of the most aggressive malignant primary brain tumors. To design effective treatment strategies, we need to better understand the behavior of glioma cells while maintaining their genetic and phenotypic stability. Here, we investigated the deformation and migration profile of U87 Glioma cells under the influence of dielectrophoretic forces. We fabricated a gold microelectrode array within a microfluidic channel and applied sinusoidal wave AC potential at 3 Vpp, ranging from 30 kHz to 10 MHz frequencies, to generate DEP forces. We followed the dielectrophoretic movement and deformation changes of 100 glioma cells at each frequency. We observed that the mean dielectrophoretic displacements of glioma cells were significantly different at varying frequencies with the maximum and minimum traveling distances of 13.22 µm and 1.37 µm, respectively. The dielectrophoretic deformation indexes of U87 glioma cells altered between 0.027-0.040. It was 0.036 in the absence of dielectrophoretic forces. This approach presents a rapid, robust, and sensitive characterization method for quantifying membrane deformation of glioma cells to determine the state of the cells or efficacy of administrated drugs.


Assuntos
Glioma , Microfluídica , Humanos , Eletroforese/métodos , Microeletrodos
14.
Isr Med Assoc J ; 24(10): 629-633, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36309856

RESUMO

BACKGROUND: Multiple myeloma (MM) accounts for approximately 10% of hematological malignancies. The monoclonal immunoglobulin G kappa (IgG-κ) daratumumab can bind to CD38 on MM cells and be detected in serum immunofixation (IF), causing pitfalls in M-protein quantification. OBJECTIVES: To determine the efficacy of mitigating the interference of IgG MM treated with daratumumab. METHODS: Levels of Ig, free light chains (FLC) kappa (κ) and lambda (λ), serum protein electrophoresis (SPE)/IF, and Hydrashift 2/4 assays were assessed following manufacturer's instructions in three patients. RESULTS: Patient 1 was a 70-year-old male diagnosed with IgG-λ MM. The IF distinguished two monoclonal bands (IgG-κ and IgG-λ). With the Hydrashift assay, the daratumumab-anti-daratumumab immune complex shifted the IgG-κ to the α zone, suggesting that the monoclonal IgG-κ band corresponded to daratumumab. Patient 2 was a 63-year-old male with IgG-κ MM who was receiving daratumumab once every other week. SPE/IF assay revealed a faint monoclonal IgG-κ band in the  zone. A stronger monoclonal band was observed after administration. The IgG-κ band disappeared on the Hydrashift assay, while the daratumumab-anti-daratumumab complex appeared as a broad smear in the α-region. Patient 3, a 63-year-old male diagnosed with IgG-λMM, was receiving daratumumab once every other month. The IF assay showed two distinct bands (IgG-κ and IgG-λ) post-daratumumab administration. The shift to the α zone of the IgG-κ bands on the Hydrashift assay confirmed that the additional band observed post-infusion was due to the daratumumab. CONCLUSIONS: The Hydrashift assay can help distinguish daratumumab from endogenous M-spike.


Assuntos
Cadeias Leves de Imunoglobulina , Mieloma Múltiplo , Masculino , Humanos , Idoso , Pessoa de Meia-Idade , Imunoeletroforese/métodos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/tratamento farmacológico , Imunoglobulina G , Eletroforese
15.
Nanoscale ; 14(42): 15845-15858, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36259692

RESUMO

The electrical and biological interfacial properties of invasive electrodes have a significant impact on the performance and longevity of neural recordings in the brain. In this study, we demonstrated rapid electrophoretic deposition and electrochemical reduction of graphene oxide (GO) on metal-based neural electrodes. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and other characterizations confirmed the existence of a uniform and effectively reduced graphene oxide coating. Electrochemically reduced graphene oxide (ErGO) coated Pt/Ir neural electrodes exhibited 15.2-fold increase in charge storage capacity (CSC) and 90% decrease in impedance with only 3.8% increase in electrode diameter. Patch clamp electrophysiology and calcium imaging of primary rat hippocampus neurons cultured on ErGO demonstrated that there was no adverse impact on the functional development of neurons. Immunostaining showed a balanced growth of excitatory and inhibitory neurons, and astrocytes. Acute recordings from the auditory cortex and chronic recordings (19 days) from the somatosensory cortex found ErGO coating improved the performance of neural electrodes in signal-to-noise ratio (SNR) and amplitude of signals. The proposed approach not only provides an in-depth evaluation of the effect of ErGO coating on neural electrodes but also widens the coating methods of commercial neural electrodes.


Assuntos
Grafite , Animais , Ratos , Grafite/química , Eletrodos , Espectroscopia Fotoeletrônica , Eletroforese
16.
Analyst ; 147(23): 5395-5408, 2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36286388

RESUMO

A novel microfluidic device containing a bi-gap electrode pair is presented in this paper, and it is capable of continuously separating three different populations of particles using dielectrophoresis. A mixture of 5, 10, and 20 µm polystyrene particles is focused by a sheath flow and then separated based on size after flowing over a bi-gap electrode pair. A new solver is developed in OpenFOAM to investigate the effects of various parameters such as the flow rate, gaps, and electrode pair angles to achieve an appropriate configuration of the bi-gap electrode pair for efficient particle separation. Based on the numerical simulation results, three different configurations of bi-gap electrode pairs are fabricated. The paths of three populations of polystyrene particles under various operating conditions are experimentally examined and compared with numerical results. Then, by examining the purity of the separated particles with three different electrode configurations at different flow rates, the performance of the device is experimentally investigated. The results showed that by employing the proposed electrode configuration, at a maximum flow rate of 100 µL h-1 (25 µL h-1 for the sample), particles are separated precisely (with more than 99% purity for all particles at desired outlets) using a 20 Vpp sinusoidal electric potential with a frequency of 100 kHz. This novel microfluidic device is thus a practical device for continuously separating three different populations of particles/cells according to size in a heterogeneous admixture.


Assuntos
Técnicas Analíticas Microfluídicas , Eletroforese/métodos , Técnicas Analíticas Microfluídicas/métodos , Poliestirenos , Eletrodos , Dispositivos Lab-On-A-Chip
17.
Anal Chim Acta ; 1233: 340476, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36283775

RESUMO

In this article, we report significant improvements in the resolving power of pressure-driven charge based separations performed in sub-micrometer deep glass channels upon introducing an electrokinetic backflow in the system. Such improvements are realized as axial electrophoresis aids the pressure-driven separation process in negatively charged glass conduits under these conditions. In addition, the electroosmotic backflow slows down the bulk transport of the background electrolyte subjecting the sample to the separation field for prolonged periods and yields a higher fluid shear across the channel depth further assisting the separation process. Although this increased shear also contributes to additional hydrodynamic dispersion, such contributions are usually small due to fast diffusion across the flow streamlines in sub-micrometer deep channels. In the present work, the pressure-driven flow was generated on-chip by fabricating a polyacrylamide based gel membrane within a chosen access hole upstream of the separation channel. Upon application of an electric field across this structure, the electroosmotic flow generated in the open channel interfacing the membrane was partially blocked producing the needed pressure-gradient. Optimization of the electrical voltage applied to the downstream end of the separation channel then yielded a suitable electrokinetic backflow that significantly improved the resolving power of our separations. For a sample comprising of three 5-TAMRA, SE-labeled amino acids, the noted strategy improved the separation resolution by over an order of magnitude compared to the case when no electrokinetic backflow was present. The band broadening in these separations was also assessed to understand its dependence on the operating conditions.


Assuntos
Aminoácidos , Eletro-Osmose , Eletroforese , Hidrodinâmica , Difusão
18.
Electrophoresis ; 43(21-22): 2175-2183, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36209396

RESUMO

Due to its characteristics of noncontact, non-damage, high flux, and easy-to-achieve flexible manipulation, optically induced dielectrophoresis (ODEP) technology has been employed to manipulate microspherical biological particles, including separation, enrichment, capture, arrangement, and fusion. However, in nature, biomolecules are morphologically diverse, and some of them are rodlike. In order to illustrate the electrodynamics of rodlike particles under the action of ODEP, a transient multi-physical field coupling model of ODEP chip under the hypothesis of electrical double layer thin layer was established in this paper. The arbitrary Lagrangian-Eulerian method is used to track single-rod particle in the strong coupled flow field and electric field simultaneously. The influence of several key factors, including the applied alternating current (AC) electric voltage, the width of optical bright area, and the initial position of particle, on the trajectory of particle center was analyzed in positive dielectrophoresis (DEP) action and negative DEP action, respectively. Especially, the planar motion process of rodlike particles was discussed together. The research results reveal the electrodynamics behavior of rodlike particles based on the action of ODEP, which may provide theoretical support for the further design of rodlike biological cells manipulation chip based on AC ODEP technology in the future.


Assuntos
Eletroforese , Eletroforese/métodos
19.
Methods Enzymol ; 675: 323-350, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36220275

RESUMO

RNA 5' ends are remarkably heterogeneous. In addition to the eukaryotic 5' methyl-7-Guanosine (m7G) cap, a number of primarily metabolite-based cap structures have been identified both in prokaryotic and eukaryotic systems. These metabolite caps include Nicotinamide Adenine Dinucleotide (NAD+/NADH), dephosphoCoenzyme A (dpCoA), Flavin Adenine Dinucleotide (FAD), dinucleotide polyphosphates and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) (Chen et al., 2009; Kowtoniuk et al., 2009; Wang et al., 2019). The most highly studied of these new cap structures, 5' NAD, has significant effects on RNA stability (Bird et al., 2016; Jiao et al., 2017). Both prokaryotes and eukaryotes have decapping enzymes specific to these metabolite caps and decapping is an integral step in the control of RNA stability (Cahová et al., 2015; Jiao et al., 2017; Sharma et al., 2020; Zhang et al., 2020). To better study how these 5' metabolite RNAs are decapped, we present a method to (1) generate radiolabeled dinucleotide and "full length" 5' capped RNA substrates for use in decapping assays, (2) a simple decapping assay to test the activity of various enzymes on different 5' capped transcripts and (3) a gel electrophoresis-based method for the visualization and differentiation of 5' capped transcripts.


Assuntos
NAD , Capuzes de RNA , Eletroforese , Endorribonucleases/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Guanosina , NAD/metabolismo , Polifosfatos , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA , Uridina Difosfato N-Acetilglicosamina
20.
Methods Enzymol ; 675: 425-437, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36220280

RESUMO

Identification of target molecules of new bioactive compounds is still a challenge in drug development. Various proteomics-based methods have been developed to analyze the interaction between compounds and target proteins. Among these methods, cellular thermal shift assay (CETSA) has been frequently applied in recent years for validation studies of compound-protein interactions using antibodies. Combining CETSA with comprehensive proteomic analysis has been successful in narrowing down the target(s) of a new compound from the enormous number of proteins in cell. In this chapter, we introduce 2DE-CETSA, which combines CETSA with proteome analysis using two-dimensional electrophoresis as a method for identification of target proteins.


Assuntos
Proteoma , Proteômica , Descoberta de Drogas/métodos , Eletroforese , Eletroforese em Gel Bidimensional , Proteoma/metabolismo , Proteômica/métodos
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