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1.
Langmuir ; 37(38): 11316-11329, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34529445

RESUMO

The biomimetic core-shell nanoparticles coated with membranes of various biological cells have attracted significant research interest, because of their extensive applications in targeted drug delivery systems. The cell membrane consists of a lipid bilayer, which can be regarded as a two-dimensional oriented viscous liquid with low dielectric permittivity, compared to a bulk aqueous medium. Such a liquid layer comprised of cell membrane may bear additional mobile charges, because of the presence of free lipid molecules or charged surfactant molecules, which further results in nonzero charge along the surface of the peripheral layer. In this article, we present an analytical theory for electrophoresis of such cell membrane coated functionalized nanoparticles in the extent of electrolyte solution, considering the combined effects of finite ion size and of ion partitioning. Going beyond the Debye-Huckel approximations, we propose an analytical theory for Donnan potential and electrophoretic mobility. The derived expressions are applicable for moderate to highly charged undertaken core-shell particles when the thickness of the peripheral liquid layer greatly exceeds the electric double layer thickness. The impact of pertinent parameters on the electrophoretic response of such a particle is further discussed.


Assuntos
Eletrólitos , Eletroforese , Membranas , Propriedades de Superfície
2.
Nanoscale ; 13(29): 12687-12696, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34477619

RESUMO

Measuring the electrophoretic mobility of molecules is a powerful experimental approach for investigating biomolecular processes. A frequent challenge in the context of single-particle measurements is throughput, limiting the obtainable statistics. Here, we present a molecular force sensor and charge detector based on parallelised imaging and tracking of tethered double-stranded DNA functionalised with charged nanoparticles interacting with an externally applied electric field. Tracking the position of the tethered particle with simultaneous nanometre precision and microsecond temporal resolution allows us to detect and quantify the electrophoretic force down to the sub-piconewton scale. Furthermore, we demonstrate that this approach is suitable for detecting changes to the particle charge state, as induced by the addition of charged biomolecules or changes to pH. Our approach provides an alternative route to studying structural and charge dynamics at the single molecule level.


Assuntos
Nanopartículas , Nanotecnologia , DNA , Eletroforese
3.
Nano Lett ; 21(16): 6835-6842, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34355908

RESUMO

Sorting of extracellular vesicles has important applications in early stage diagnostics. Current exosome isolation techniques, however, suffer from being costly, having long processing times, and producing low purities. Recent work has shown that active sorting via acoustic and electric fields are useful techniques for microscale separation activities, where combining these has the potential to take advantage of multiple force mechanisms simultaneously. In this work, we demonstrate an approach using both electrical and acoustic forces to manipulate bioparticles and submicrometer particles for deterministic sorting, where we find that the concurrent application of dielectrophoretic (DEP) and acoustophoretic forces decreases the critical diameter at which particles can be separated. We subsequently utilize this approach to sort subpopulations of extracellular vesicles, specifically exosomes (<200 nm) and microvesicles (>300 nm). Using our combined acoustic/electric approach, we demonstrate exosome purification with more than 95% purity and 81% recovery, well above comparable approaches.


Assuntos
Exossomos , Técnicas Analíticas Microfluídicas , Acústica , Eletricidade , Eletroforese
4.
Sensors (Basel) ; 21(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34372193

RESUMO

We present the integration of a flow focusing microfluidic device in a dielectrophoretic application that based on a tapered aluminum microelectrode array (TAMA). The characterization and optimization method of microfluidic geometry performs the hydrodynamic flow focusing on the channel. The sample fluids are hydrodynamically focused into the region of interest (ROI) where the dielectrophoresis force (FDEP) is dominant. The device geometry is designed using 3D CAD software and fabricated using the micro-milling process combined with soft lithography using PDMS. The flow simulation is achieved using COMSOL Multiphysics 5.5 to study the effect of the flow rate ratio between the sample fluids (Q1) and the sheath fluids (Q2) toward the width of flow focusing. Five different flow rate ratios (Q1/Q2) are recorded in this experiment, which are 0.2, 0.4, 0.6, 0.8 and 1.0. The width of flow focusing is increased linearly with the flow rate ratio (Q1/Q2) for both the simulation and the experiment. At the highest flow rate ratio (Q1/Q2 = 1), the width of flow focusing is obtained at 638.66 µm and at the lowest flow rate ratio (Q1/Q2 = 0.2), the width of flow focusing is obtained at 226.03 µm. As a result, the flow focusing effect is able to reduce the dispersion of the particles in the microelectrode from 2000 µm to 226.03 µm toward the ROI. The significance of flow focusing on the separation of particles is studied using 10 and 1 µm polystyrene beads by applying a non-uniform electrical field to the TAMA at 10 VPP, 150 kHz. Ultimately, we are able to manipulate the trajectories of two different types of particles in the channel. For further validation, the focusing of 3.2 µm polystyrene beads within the dominant FDEP results in an enhanced manipulation efficiency from 20% to 80% in the ROI.


Assuntos
Técnicas Analíticas Microfluídicas , Alumínio , Eletroforese , Microeletrodos , Microfluídica
5.
Nat Commun ; 12(1): 4994, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404799

RESUMO

We present a simple and effective scheme of a dynamic switch for DNA nanostructures. Under such a framework of toehold-free strand displacement, blocking strands at an excess amount are applied to displace the complementation of specific segments of paired duplexes. The functional mechanism of the scheme is illustrated by modelling the base pairing kinetics of competing strands on a target strand. Simulation reveals the unique properties of toehold-free strand displacement in equilibrium control, which can be leveraged for information processing. Based on the controllable dynamics in the binding of preformed DNA nanostructures, a multi-input-multi-output (MIMO) Boolean function is controlled by the presence of the blockers. In conclusion, we implement two MIMO Boolean functions (one with 4-bit input and 2-bit output, and the other with 16-bit input and 8-bit output) to showcase the controllable dynamics.


Assuntos
DNA/química , Nanoestruturas , Eletroforese , Simulação de Dinâmica Molecular , Recombinação Genética
6.
J Chromatogr A ; 1652: 462127, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34214833

RESUMO

In this work, the molecular mechanism of Lactobacillus paracasei bio-colloid clumping under divalent metal ions treatment such as zinc, copper and magnesium at constant concentrations was studied. The work involved experimental (electrophoretic - capillary electrophoresis in pseudo-isotachophoresis mode, spectroscopic and spectrometric - FT-IR and MALDI-TOF-MS, microscopic - fluorescent microscopy, and flow cytometry) and theoretical (DFT calculations of model complex systems) characterization. Electrophoretic results have pointed out the formation of aggregates under the Zn2+ and Cu2+ modification, whereas the use of the Mg2+ allowed focusing the zone of L. paracasei biocolloid. According to the FT-IR analysis, the major functional groups involved in the aggregation are deprotonated carboxyl and amide groups derived from the bacterial surface structure. Nature of the divalent metal ions was shown to be one of the key factors influencing the bacterial aggregation process. Proteomic analysis showed that surface modification had a considerable impact on bacteria molecular profiles and protein expression, mainly linked to the activation of carbohydrate and nucleotides metabolism as well with the transcription regulation and membrane transport. Density-functional theory (DFT) calculations of modeled Cu2+, Mg2+ and Zn2+ coordination complexes support the interaction between the divalent metal ions and bacterial proteins. Consequently, the possible mechanism of the aggregation phenomenon was proposed. Therefore, this comprehensive study could be further applied in evaluation of biocolloid aggregation under different types of metal ions.


Assuntos
Cátions Bivalentes , Eletroforese , Íons , Lactobacillus paracasei , Metais , Cátions Bivalentes/química , Íons/química , Lactobacillus paracasei/metabolismo , Metais/química , Proteômica , Espectroscopia de Infravermelho com Transformada de Fourier
7.
J Coll Physicians Surg Pak ; 30(7): 864-867, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34271795

RESUMO

The objective of this study was to investigate the diagnostic significance of serum protein electrophoresis and immunofixation electrophoresis detection in diagnosis of multiple myeloma (MM). One hundred and five patients were investigated. The detection rate of M protein by immunofixation electrophoresis detection was better (105 cases, 100%) than that of serum protein electrophoresis (101 cases, 96.19%, p<0.001). The M band was not detected by serum protein electrophoresis in four cases (3.81%), among which one case (0.95%) was identified as IgA type and 3 cases (2.86%) as light chain type after immunoglobulin analysis. Immunofixation electrophoresis detection technique can be used for screening M protein in patients with atypical MM; and immunofixation electrophoresis detection technique can increase the diagnosis accuracy in patients with atypical MM. Key Words: Multiple myeloma (MM), Serum protein electrophoresis, Immunofixation electrophoresis, Monoclonal immunoglobulin.


Assuntos
Mieloma Múltiplo , Eletroforese das Proteínas Sanguíneas , Eletroforese , Humanos , Imunoeletroforese , Cadeias Leves de Imunoglobulina , Mieloma Múltiplo/diagnóstico
8.
Lab Chip ; 21(16): 3076-3085, 2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34195745

RESUMO

Capture-to-translocation dynamics control is an important issue for single-particle and -molecule analyses by resistive pulse waveforms. Here, we report on regulated motions for accurate zeta-potential assessments of single nanoscale objects passing through an octet-nanochannel. We observed ionic spike signals consisting of eight consecutive sub-pulses signifying the ion blockage at the eight sensing zones in series upon electrophoretic translocation of individual nanoparticles. We find an exponential decrease to saturation of the channel-to-channel translocation duration as a nanobead moves forward, reflecting the more restricted radial motion degrees of freedom via inertial effects at the downstream side of the octet channel. This finding enabled a protocol for single-nanoparticle zeta potential estimation impervious to the uncertainty stemming from the stochastic nature of the translocation dynamics. The multi-channel approach presented in this study may be used as a useful tool for analyzing particles and molecules of variable sizes.


Assuntos
Nanopartículas , Eletroforese , Íons
9.
J Pharm Biomed Anal ; 204: 114284, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34332308

RESUMO

Protein concentration is an important attribute in the production of subunit or component-based vaccine antigens. Rigorous monitoring of protein concentration is required to identify potential areas for yield improvement. The current GMP method for quantitation is the plate-based ELISA which requires numerous hands-on steps and has low sensitivity in comparison to new microfluidic systems. To address this issue, a sensitive automated microCapillary Electrophoresis ImmunoAssay (mCE IA) method was developed to accurately separate and quantitate pertactin (PRN), an important antigen of the modern acellular Pertussis (aP) vaccine. PRN is reported to be a low-yielding antigen; thus, it is critical to observe its concentration throughout its manufacturing process. First, a primary antibody for PRN was identified to establish suitable immunoprobing conditions for detection of PRN over a wide linear dynamic range that spans 3 orders of magnitude. Next, the pre-adsorbed PRN Drug Substance (DS) was used as a reference standard to quantitate PRN samples against a calibration curve with adequate accuracy and precision. Four representative samples including three in-process steps and final adjuvanted drug product: Quadracel®, were examined to demonstrate the capability of mCE IA to quantitate PRN with high sensitivity and specificity. The matrices of the selected samples contain additional components (e.g. other proteins, growth factors, cell culture media, residual ammonium sulfate, and aluminum adjuvant) often making the quantitation of PRN challenging. The specificity and method linearity were demonstrated by spiking pre-adsorbed PRN DS into the four representative samples. In addition, it was shown that reportable concentrations of PRN for nine downstream process steps as analyzed by our method is comparable to concentrations obtained with ELISA. Most importantly, this study demonstrated that our method's quantitative accuracy is independent of matrix components, as each sample undergoes extensive dilution. This allows for seamless end-to-end analysis of PRN from fermenter harvest, through to complex downstream process samples to adjuvanted drug products. Finally, for the first time the developed and qualified mCE IA method was shown to quantify PRN throughout the entire manufacturing process to provide rapid feedback for process optimizations allowing for accurate yield and step-loss calculations.


Assuntos
Bordetella pertussis , Fatores de Virulência de Bordetella , Proteínas da Membrana Bacteriana Externa , Eletroforese , Vacina contra Coqueluche
10.
J Phys Chem Lett ; 12(28): 6469-6477, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34240883

RESUMO

Many biological assays require effectively and sensitively sorting DNA fragments. Here, we demonstrate a solid-state nanopore platform for label-free detection and separation of short single-stranded DNA (ssDNA) fragments (<100 nt), based on their length-dependent translocation behaviors. Our experimental data show that each sized pore has a passable length threshold. The negative charged ssDNA fragments with length smaller than the threshold can be electrically facilitated driven through the correspondingly sized nanopore along the direction of electric field. In addition, the passable length threshold increases with the pore size enlarging. As a result, this phenomenon is able to be applicable for the controllable selectivity of ssDNA by tuning nanopore size, and the selectivity limitation is up to 30nt. Numerical simulation results indicate the translocation direction of ssDNA is governed by the competition of electroosmosis and electrophoresis effects on the ssDNA and offer the relationship between passable length threshold and pore size.


Assuntos
DNA de Cadeia Simples/análise , DNA de Cadeia Simples/isolamento & purificação , Nanoporos , Nanotecnologia/métodos , Eletroforese , Limite de Detecção , Osmose
12.
Clin Chim Acta ; 520: 172-178, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34118239

RESUMO

BACKGROUND: The causal relationship between low-density lipoprotein (LDL) and atherosclerotic cardiovascular disease (CVD) has been firmly substantiated. LDL consists of a heterogeneous group of particles with different physicochemical and metabolic properties. Among them, small dense LDL (sdLDL) particles are considered an emerging CVD risk factor and a promising CVD risk biomarker. This paper reviews published analytical and calculation-based methods for sdLDL determination in plasma, present their principles, strengths, and weaknesses, and examine the challenges arising from method comparison. METHODS: A literature survey was conducted using the PubMed database. Subject headings and keywords facilitated the search strategy. Titles and abstracts were initially assessed, and the full-text article of the pre-selected ones was reviewed. RESULTS: A range of methods is currently available for the analysis of LDL subfractions and the measurement of sdLDL particle size, number, and cholesterol concentration. Ultracentrifugation (UC), vertical auto profile, gradient gel electrophoresis (GGE), nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography, ion mobility analysis, and a homogeneous assay are the most prevalent. To date, there is no "gold standard". UC and GGE are the most established techniques, albeit significantly sophisticated. NMR and the homogeneous assay are options with potential clinical use as they yield results rapidly and can be high-throughput. None of the proposed equations for the calculated sdLDL determination has been sufficiently validated to serve as a clinical tool. CONCLUSIONS: Many analytical procedures have been developed for the study of sdLDL particles. Their use remains largely restricted to research laboratories since their analytical and clinical performance, along with the clinical- and cost-effectiveness of sdLDL determination have not been fully established.


Assuntos
Aterosclerose , Lipoproteínas LDL , Biomarcadores , Eletroforese , Humanos , Ultracentrifugação
13.
ACS Sens ; 6(6): 2330-2338, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34138539

RESUMO

Understanding the dynamic behavior of charged particles driven by flow and electric field in nanochannels/pores is highly important for both fundamental study and practical applications. While a great breakthrough has been made in understanding the translocation dynamics of charged particles within the nanochannels/pores, studies on the dynamics of particles at the orifice of nanochannels/pores are scarcely reported. Here, we study particle motion at a smaller-sized orifice of a nanopipette by combining experimentally observed current transients with simulated force conditions. The theoretical force analysis reveals that dielectrophoretic force plays an equally important role as electrophoretic force and electroosmotic force, although it has often been neglected in understanding the particle translocation dynamics within the nanopipette. Under the combined action of these forces, it thus becomes difficult for particles to physically collide with the orifice of the nanopipette, resulting in a relatively low decrease in the current transients, which coincides with experimental results. We then regulate the dynamic behavior by altering experimental conditions (i.e., bias potential, nanopipette surface charge, and particle size), and the results further validate the presence and influence of forces being considered. This study improves the understanding of the relationship between particle properties and observed current transients, providing more possibilities for accurate single-particle analysis and single-entity regulation.


Assuntos
Eletricidade , Fenômenos Mecânicos , Eletroforese , Íons , Tamanho da Partícula
14.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065142

RESUMO

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes' protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.


Assuntos
Membrana Celular/metabolismo , Eletroforese , Mesembryanthemum/fisiologia , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica , Transporte Biológico , Biologia Computacional/métodos , Eletroforese/métodos , Espaço Intracelular/metabolismo , Espectrometria de Massas/métodos , Proteômica/métodos , Frações Subcelulares/metabolismo
15.
Anal Chem ; 93(19): 7204-7209, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33939916

RESUMO

We present a novel multi-emitter electrospray ionization (ESI) interface for the coupling of microfluidic free-flow electrophoresis (µFFE) with mass spectrometry (MS). The effluents of the µFFE outlets are analyzed in near real-time, allowing a direct optimization of the electrophoretic separation and an online monitoring of qualitative sample compositions. The short measurement time of just a few seconds for all outlets even enables a reasonable time-dependent monitoring. As a proof of concept, we employ the multi-emitter ESI interface for the continuous identification of analytes at 15 µFFE outlets via MS to optimize the µFFE separation of important players of cellular respiration in operando. The results indicate great potential of the presented system in downstream processing control, for example, for the monitoring and purification of products in continuous-flow microreactors.


Assuntos
Espectrometria de Massas por Ionização por Electrospray , Eletroforese
16.
Anal Chem ; 93(21): 7606-7615, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34003009

RESUMO

Prefocusing of cell mixtures through sheath flow is a common technique used for continuous and high-efficiency dielectrophoretic (DEP) cell separation. However, it usually limits the separation flow velocity and requires a complex multichannel fluid control system that hinders the integration of a DEP separator with other microfluidic functionalities for comprehensive biomedical applications. Here, we propose and develop a high-efficiency, sheathless particle/cell separation method without prefocusing based on flow-field-assisted DEP by combining the effects of AC electric field (E-field) and flow field (F-field). A hollow lemon-shaped electrode array is designed to generate a long-range E-field gradient in the microchannel, which can effectively induce lateral displacements of particles/cells in a continuous flow. A series of arc-shaped protrusion structures is designed along the microchannel to form a F-field, which can effectively guide the particles/cells toward the targeted E-field region without prefocusing. By tuning the E-field, two distinct modes can be realized and switched in one single device, including the sheathless separation (ShLS) and the adjustable particle mixing ratio (AMR) modes. In the ShLS mode, we have achieved the continuous separation of breast cancer cells from erythrocytes with a recovery rate of 95.5% and the separation of polystyrene particles from yeast cells with a purity of 97.1% at flow velocities over 2.59 mm/s in a 2 cm channel under optimized conditions. The AMR mode provides a strategy for controlling the mixing ratio of different particles/cells as a well-defined pretreatment method for biomedical research studies. The proposed microchip is easy to use and displays high versatility for biological and medical applications.


Assuntos
Técnicas Analíticas Microfluídicas , Separação Celular , Eletrodos , Eletroforese , Microfluídica , Fenômenos Físicos
17.
Anal Chem ; 93(21): 7635-7646, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34014074

RESUMO

Multiplex separation of mixed biological samples is essential in a considerable portion of biomedical research and clinical applications. An automated and operator-independent process for the separation of samples is highly sought after. There is a significant unmet need for methods that can perform fractionation of small volumes of multicomponent mixtures. Herein, we design an integrated chip that combines acoustic and electric fields to enable efficient and label-free separation of multiple different cells and particles under flow. To facilitate the connection of multiple sorting mechanisms in tandem, we investigate the electroosmosis (EO)-induced deterministic lateral displacement (DLD) separation in a combined pressure- and DC field-driven flow and exploit the combination of the bipolar electrode (BPE) focusing and surface acoustic wave (SAW) sorting modules. We successfully integrate four sequential microfluidic modules for multitarget separation within a single platform: (i) sorting particles and cells relying on the size and surface charge by adjusting the flow rate and electric field using a DLD array; (ii) alignment of cells or particles within a microfluidic channel by a bipolar electrode; (iii) separation of particles based on compressibility and density by the acoustic force; and (iv) separation of viable and nonviable cells using dielectric properties via the dielectrophoresis (DEP) force. As a proof of principle, we demonstrate the sorting of multiple cell and particle types (polystyrene (PS) particles, oil droplets, and viable and nonviable yeast cells) with high efficiency. This integrated microfluidic platform combines multiple functional components and, with its ability to noninvasively sort multiple targeted cells in a label-free manner relying on different properties, is compatible with high-definition imaging, showing great potential in diverse diagnostic and analysis applications.


Assuntos
Hidrodinâmica , Técnicas Analíticas Microfluídicas , Acústica , Separação Celular , Eletroforese , Microfluídica , Som
18.
Biomater Sci ; 9(13): 4671-4678, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34018505

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive form of gliomas. The development of supplementary approaches for glioblastoma diagnosis, limited to imaging techniques and tissue biopsies so far, is a necessity of clinical relevance. In this context, nanotechnology might afford tools to enable early diagnosis. Upon exposure to biological media, nanoparticles are coated with a layer of proteins, the protein corona (PC), whose composition is individual and personalized. Here we show that the PC of graphene oxide nanosheets has a capacity to detect GBM using a simple one-dimensional gel electrophoresis technique. In a range of molecular weights between 100 and 120 kDa, the personalized PC from GBM patients is completely discernible from that of healthy donors and that of cancer patients affected by pancreatic adenocarcinoma and colorectal cancer. Using tandem mass spectrometry, we found that inter-alpha-trypsin inhibitor (ITI) heavy chain H4 is enriched in the PC of all tested individuals but not in the GBM patients. Overall, if confirmed on a larger cohort series, this approach could be advantageous at the first level of investigation to decide whether to carry out more invasive analyses and/or to follow up patients after surgery and/or pharmacological treatment.


Assuntos
Adenocarcinoma , Glioblastoma , Neoplasias Pancreáticas , Coroa de Proteína , Eletroforese , Glioblastoma/diagnóstico , Grafite , Humanos
19.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946951

RESUMO

Flavonoid compounds are known for their antibacterial, anti-inflammatory, and anticancer properties. Therefore, they can influence membrane properties that interest us, modifying both their structure and functions. We used kaempferol (K) and myricetin (M) as representatives of this group. We investigated the influence of the abovementioned compounds on model cell membranes' properties (i.e., Langmuir monolayers and liposomes). The basic research methods used in these studies were the Langmuir method with Brewster angle microscopy and microelectrophoresis. The π-A isotherms were registered for the pure components and mixtures of these compounds with phosphatidylcholine (PC) in appropriate volume ratios. Using mathematical equations, we established that kaempferol, myricetin, and the lipids formed complexes at 1:1 ratios. We derived the parameters characterizing the formed complexes, i.e., the surfaces occupied by the complexes and the stability constants of the formed complexes. Using the microelectrophoretic method, we determined the dependence of the lipid membranes' surface charge density as a function of the pH (in the range of 2 to 10) of the electrolyte solution. The presented results indicate that the PC membrane's modification with kaempferol or myricetin affected changes in the surface charge density and isoelectric point values.


Assuntos
Derivados de Alilbenzenos/farmacologia , Dioxolanos/farmacologia , Eletroforese/métodos , Quempferóis/farmacologia , Membranas Artificiais , Microquímica/métodos , Fosfatidilcolinas/química , Lipossomas Unilamelares/química , Derivados de Alilbenzenos/química , Difusão , Dioxolanos/química , Concentração de Íons de Hidrogênio , Quempferóis/química , Microscopia de Polarização/métodos , Pressão , Refratometria , Eletricidade Estática , Propriedades de Superfície , Tensoativos , Viscosidade
20.
Methods Mol Biol ; 2290: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009591

RESUMO

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.


Assuntos
Biocombustíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Bacillus/genética , Bactérias/classificação , Bactérias/genética , Cellulomonas/genética , Clostridium/genética , DNA Bacteriano/genética , Eletroforese/métodos , Pseudomonas/genética , Rhodothermus/genética
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