BACKGROUND: The quality and yield of DNA specimens are essential for downstream analyses. Complex biological or environmental samples contain interfering compounds requiring advanced protocols for removing salts, small organic molecules, and/or proteins without compromising the DNA fragments distribution in the original sample. High-molecular-weight DNA fragments are often discriminated by standard extraction methods in attempts to remove the highly concentrated inorganic sample components. RESULTS: Here, we introduce a simple device for the simplification, purification, desalting, and buffer exchange of complex biological samples based on electrodialysis in discontinuous electrolyte systems. The device combines a 3D-printed body with two electrolyte reservoirs and commercially available cassettes with dialysis membranes as inserts. Upon applying an electric current via electrodes inside the electrolyte reservoirs, the sample ions, injected into the cassette or one of the electrolyte reservoirs, electromigrate across the dialysis membranes. During this process, small sample ions pass through the membrane and are substituted by the ions of the selected buffers loaded into the electrolyte reservoirs. The larger ions, such as DNA fragments or proteins, stay in the sample reservoir. The performance of the device was evaluated using DNA samples in blood plasma. Several milliliters of a plasma sample were purified in 30 min with 90 % DNA recovery. SIGNIFICANCE: The device enables fast and effective purification of highly complicated samples. It can be easily scaled for the available sample amount (ranging from microliters to milliliters) and the analyte size of interest by selecting appropriate membrane pore sizes. The device's applicability was optimized on DNA samples with high salt content and tested on plasma samples. But it also has the potential for purification/desalting of a wide range of biological compounds, including proteins, peptides, or charged oligosaccharides, as well as organelles, cells, viruses, or bacteria.
- Klíčová slova
- 3D printed device, Buffer replacement, Desalting, Electromigration, Sample purification,
- MeSH
- 3D tisk * MeSH
- dialýza přístrojové vybavení MeSH
- DNA * krev izolace a purifikace MeSH
- elektrochemické techniky * přístrojové vybavení MeSH
- elektrolyty * chemie MeSH
- lidé MeSH
- pufry MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA * MeSH
- elektrolyty * MeSH
- pufry MeSH
It has been demonstrated that freezing-induced acidity changes have an impact on the structural integrity, degree of aggregation, and chemical stability of frozen food and pharmaceutical products. The stability of the compounds in solutions is maintained by the presence of buffers. However, many buffers are unsuitable for applications involving freezing as this process substantially alters the acidity. In this study, we determine the effect of initial pH, concentration, and cooling rate on the freezing-induced change in acidity of phosphate buffered saline (PBS) in the frozen state via UV-VIS spectroscopy. Furthermore, we examine the impact of individual salts present in PBS and discuss the mechanisms affecting the resulting acidity that we approximate via Hammett acidity function (H2-).
- Klíčová slova
- Acidification, Buffering capacity, Freezing, PBS, Preferential precipitation,
- MeSH
- fosfáty * chemie MeSH
- koncentrace vodíkových iontů MeSH
- pufry MeSH
- solný roztok * chemie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfáty * MeSH
- pufry MeSH
- solný roztok * MeSH
The effect of buffer species on biomolecules and biomolecule-nanoparticle interactions is a phenomenon that has been either neglected, or not understood. Here, we study the formation of a BSA protein corona (PC) around amino-functionalized mesoporous silica nanoparticles (MSN-NH2) in the presence of different buffers (Tris, BES, cacodylate, phosphate, and citrate) at the same pH (7.15) and different concentrations (10, 50, and 100 mM). We find that BSA adsorption is buffer specific, with the adsorbed amount of BSA being 4.4 times higher in the presence of 100 mM Tris (184 ± 3 mg/g) than for 100 mM citrate (42 ± 2 mg/g). That is a considerable difference that cannot be explained by conventional theories. The results become clearer if the interaction energies between BSA and MSN-NH2, considering the electric double layer (EEDL) and the van der Waals (EvdW) terms, are evaluated. The buffer specific PC derives from buffer specific zeta potentials that, for MSN-NH2, are positive with Tris and negative with citrate buffers. A reversed sign of zeta potentials can be obtained by considering polarizability-dependent dispersion forces acting together with electrostatics to give the buffer specific outcome. These results are relevant not only to our understanding of the formation of the PC but may also apply to other bio- and nanosystems in biological media.
- MeSH
- adsorpce MeSH
- koncentrace vodíkových iontů MeSH
- nanočástice * chemie MeSH
- oxid křemičitý * chemie MeSH
- poréznost MeSH
- povrchové vlastnosti MeSH
- proteinová korona * chemie MeSH
- pufry MeSH
- sérový albumin hovězí * chemie MeSH
- skot MeSH
- velikost částic MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- oxid křemičitý * MeSH
- proteinová korona * MeSH
- pufry MeSH
- sérový albumin hovězí * MeSH
Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- Klíčová slova
- Diffuse reflectance spectroscopy, Lyophilization, Solid-state NMR spectroscopy, Solid-state acidity, UV/VIS,
- MeSH
- fosfáty * chemie MeSH
- histidin * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová chemie MeSH
- lyofilizace * metody MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- pufry MeSH
- spektrofotometrie ultrafialová metody MeSH
- trehalosa * chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfáty * MeSH
- histidin * MeSH
- kyselina citronová MeSH
- pufry MeSH
- trehalosa * MeSH
Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.
- Klíčová slova
- Cryomicroscopy, Differential Scanning Calorimetry, Freeze-concentrated solution, Glass Transition Temperature, Hammett acidity function, Stabilization, Sulfonephthalein indicators,
- MeSH
- citráty * MeSH
- diferenciální skenovací kalorimetrie MeSH
- koncentrace vodíkových iontů MeSH
- lyofilizace MeSH
- pufry MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- citráty * MeSH
- pufry MeSH
In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( μ L eff ( x ) $\mu _{\rm{L}}^{{\rm{eff}}}( x )$ / κ ( x ) $\kappa ( x )$ values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, 675-88) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on. In this paper, we demonstrate some of the operational features of the tbdITP model using the new ITP/IEF version of Simul 6.
- Klíčová slova
- Simul 6, capillary isoelectric focusing, isoelectric focusing, simulation, transient bidirectional isotachophoresis,
- MeSH
- amfolytové směsi * MeSH
- gely MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza * MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amfolytové směsi * MeSH
- gely MeSH
The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H3 O+ and OH- ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pKa values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.
- Klíčová slova
- isoelectric focusing, simulation, transient bidirectional isotachophoresis,
- MeSH
- amfolytové směsi MeSH
- elektrická vodivost MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza * MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amfolytové směsi MeSH
In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.
- Klíčová slova
- capillary isoelectric focusing, isoelectric focusing, polyacrylamide gel isoelectric focusing, steady-state pH gradient, two-stage model of isoelectric focusing,
- MeSH
- amfolytové směsi * chemie MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza * MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- amfolytové směsi * MeSH
We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds' pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.
- Klíčová slova
- Capillary, Colored, Focusing, Isoelectric point, Marker,
- MeSH
- isoelektrická fokusace metody MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- protonmotorická síla * MeSH
- pufry MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- pufry MeSH
The buffering capacity of the soil is a very important property of the soil, which determines the ability of the soil to resist external influences, especially changes in pH and thus create good living conditions for plants and microorganisms in the soil. The buffering capacity thus significantly contributes to maintaining the health and quality of the soil. Buffering capacity is an important indicator of soil quality, because it is related to the overall condition of the soil ecosystem and other soil properties. The goal of this paper is to determine the effect of applying different soil amendments on the soils, 10 years after application. We compared the effect of 6 different treatments in closed plots: Natural conditions (N = control); Bare soil (B); Straw mulching (S); Pine mulch (P); TerraCottem hydroabsorbent polymers (H); Prescribed burn (F); and Sewage sludge (M). Our results have shown that the application of different amedments leads to an effect on the plowing capacity of the soil. While in the case of the control variant (Natural conditions, N) the buffering capacity of the soil was measured at 144.93 ± 0.25, the addition of different amendments decreased the buffering capacity in the following order: Bare soil (B) 142.73±0.21 > TerraCotem hydroaborbent polymer (H) 142.23±.15 > Pine mulch (P) 140.40±0.30, Prescribed burn (F) 138.20±0.30, Sludge (S) 127.47±0.15. In the case of all variants, these are statistically significant differences (p ≤ 0.05). Thus, soil amendments have been shown to have a statistically significant effect on soil buffering capacity.
- MeSH
- biodegradace MeSH
- borovice chemie fyziologie MeSH
- ekosystém MeSH
- látky znečišťující půdu chemie MeSH
- lidé MeSH
- odpadní vody chemie MeSH
- půda chemie MeSH
- pufry MeSH
- regenerace a remediace životního prostředí metody MeSH
- skládková zařízení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- odvolaná publikace MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Španělsko MeSH
- Názvy látek
- látky znečišťující půdu MeSH
- odpadní vody MeSH
- půda MeSH
- pufry MeSH
