Simple Buffer Solution Research Articles

Reverse Iontophoretic Extraction of Skin Cancer-Related Biomarkers.

Non-invasive methods for early diagnosis of skin cancer are highly valued. One possible approach is to monitor relevant biomarkers such as tryptophan (Trp) and kynurenine (Kyn), on the skin surface. The primary aim of this in vitro investigation was, therefore, to examine whether reverse iontophoresis (RI) can enhance the extraction of Trp and Kyn, and to demonstrate how the Trp/Kyn ratio acquired from the skin surface reflects that in the epidermal tissue. The study also explored whether the pH of the receiver medium impacted on extraction efficiency, and assessed the suitability of a bicontinuous cubic liquid crystal as an alternative to a simple buffer solution for this purpose. RI substantially enhanced the extraction of Trp and Kyn, in particular towards the cathode. The Trp/Kyn ratio obtained on the surface matched that in the viable skin. Increasing the receiver solution pH from 4 to 9 improved extraction of both analytes, but did not significantly change the Trp/Kyn ratio. RI extraction of Trp and Kyn into the cubic liquid crystal was comparable to that achieved with simple aqueous receiver solutions. We conclude that RI offers a potential for non-invasive sampling of low-molecular weight biomarkers and further investigations in vivo are therefore warranted.

Impact of Intestinal Concentration and Colloidal Structure on the Permeation-Enhancing Efficiency of Sodium Caprate in the Rat.

In this work, we set out to better understand how the permeation enhancer sodium caprate (C10) influences the intestinal absorption of macromolecules. FITC-dextran 4000 (FD4) was selected as a model compound and formulated with 50–300 mM C10. Absorption was studied after bolus instillation of liquid formulation to the duodenum of anesthetized rats and intravenously as a reference, whereafter plasma samples were taken and analyzed for FD4 content. It was found that the AUC and Cmax of FD4 increased with increasing C10 concentration. Higher C10 concentrations were associated with an increased and extended absorption but also increased epithelial damage. Depending on the C10 concentration, the intestinal epithelium showed significant recovery already at 60–120 min after administration. At the highest studied C10 concentrations (100 and 300 mM), the absorption of FD4 was not affected by the colloidal structures of C10, with similar absorption obtained when C10 was administered as micelles (pH 8.5) and as vesicles (pH 6.5). In contrast, the FD4 absorption was lower when C10 was administered at 50 mM formulated as micelles as compared to vesicles. Intestinal dilution of C10 and FD4 revealed a trend of decreasing FD4 absorption with increasing intestinal dilution. However, the effect was smaller than that of altering the total administered C10 dose. Absorption was similar when the formulations were prepared in simulated intestinal fluids containing mixed micelles of bile salts and phospholipids and in simple buffer solution. The findings in this study suggest that in order to optimally enhance the absorption of macromolecules, high (≥100 mM) initial intestinal C10 concentrations are likely needed and that both the concentration and total dose of C10 are important parameters.

Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice

Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice

Electrochemical Characterization of Nicotinamide Riboside

AbstractTo improve quality of life, nicotinamide riboside (NR) has become a go‐to supplement to serve as a precursor to nicotinamide adenine dinucleotide (NAD+/NADH). NAD+ is a cofactor used in various extra‐ and intra‐cellular metabolic pathways by enzymes that maintain physiological functions like vision, hearing, and cognitive behavior. Unfortunately, innate NAD+ levels decline over time giving rise to a need for NAD+ boosters like NR to fight age‐related diseases. To better understand redox pathways of NR in biological systems, we chose to study the electrochemistry of NR. Here, we electrochemically characterize NR in aqueous solutions using voltammetry, amperometry, bulk electrolysis, and mass spectrometry. We found the reduction of NR (Ep,c=−1.1 V vs. Ag/AgCl) is a diffusion‐controlled reaction and calculated a diffusion coefficient of 4.6 0.1×10−6 cm2 ⋅ s−1. Square wave voltammetry indicated NR is reduced by one electron, and cyclic voltammetry suggested a major kinetic product, oxidizable at Ep,a=+0.15 V vs. Ag/AgCl. Bulk electrolysis confirmed the species at +0.15 V vs. Ag/AgCl was a kinetic product, and liquid chromatography‐mass spectrometry of the bulk electrolysis products revealed the thermodynamic product was a dimer (NR2). Finally, we studied the reduction of NR in whole cell lysate, which is shown to differ markedly from simple buffer solutions.

Multiplexed surface plasmon imaging of serum biomolecules: Fe3O4@Au Core/shell nanoparticles with plasmonic simulation insights

Nano-biosensors that are not only sensitive and selective, but also enable multiplex detection of ultra-low levels of both large and small biomolecules in clinical sample matrices are essential for in vitro diagnostics. We present herein a multiplex surface plasmon microarray design that employs citrate-stabilized Fe3O4@Au core/shell nanoparticles (NPs) as the plasmon signal amplification label for combined detection of serum proteins and nucleotide markers. The multiplex sensing is demonstrated using two interleukins (IL-6 and IL-8) and two microRNAs (miRNA-21 and miRNA-155) in 10% serum, which is clinically relevant than simple buffer solution based biosensors. We observed that the surface plasmon signal change for larger proteins even at higher concentrations was less than the relatively smaller miRNA molecules. We draw two conclusions from this result: (i) the number of selectively bound analytes onto the sensor (i.e., antigen for an antibody or miRNA for a capture nucleotide) influences the signal change, and (ii) the extent of interaction of the detection probe carrying core/shell NP labels with the sensor surface plasmons influences the amount of signal change. Results indicate that both factors, (i) and (ii), are greater for small oligonucleotide hybridization assembly than a large sandwich protein immunoassembly. The core/shell NPs offered several fold enhanced sensitivity and wider dynamic range of detection over assays without using them. With recently growing attention on in vitro diagnostics for painless/minimally-invasive detection of diseases and abnormalities, findings presented herein are important for designing novel multiplex biosensors for real sample analysis in complex matrices.

Surface Plasmon Immunoarrays for Non-Glucose Diabetes Biomarkers

Diabetes is a very complex, chronic immune disorder that requires extensive medical care with multifactorial risk-reduction strategies beyond glycemic control. In recent years, the prevalence of diabetes, in particular, type 1 diabetes (T1D), has significantly increased from 5% to 10%, and this has in turn has impacted the incidence of associated lethal complications in children and adults. Personalized glucometers allow diabetes management by easy monitoring of the high millimolar blood-glucose concentration levels. However, the importance of non-glucose based diabetes biomarkers has recently gained considerable attention for early prediction and providing insights about the diabetes metabolic pathways and understanding diabetic disorders. Nevertheless, non-glucose markers of diabetes are present at much lower concentrations (picomolar to femtomolar) than the glucose levels in blood. High abundances of interfering components such as cell lysates and serum proteins (matrix effect) in clinical matrices affect reliable detection and sensitivity. Hence, detection of biomarker levels in complex clinical matrices compared to simple buffer solutions poses a considerable challenge in assay development and validation. Herein we present results from our surface plasmon resonance immunoarrays for measurements of ultra-low levels of non-glucose based diabetes markers present in serum and whole blood. Acknowledgements: This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health.

Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.

Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the ‘cytosol’ of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells.

Size-Dependent Diffusion of Dextrans in Excised Porcine Corneal Stroma.

Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. We evaluated the rate of diffusive transport in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Fluorescence correlation spectroscopy (FCS) was used to measure diffusion coefficients of dextran molecules in the excised porcine corneal stroma. The preferential sensitivity of FCS to diffusion along two dimensions was used to differentially probe diffusion along the directions parallel to and perpendicular to the collagen lamellae of the corneal stroma. In order to develop an understanding of how size affects diffusion in cornea, diffusion coefficients in cornea were compared to diffusion coefficients measured in a simple buffer solution. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. This indicates that, for dextrans in the 1.3 to 34 nm range, the diffusion landscape of corneal stroma can be represented as a simple liquid with a viscosity approximately 1.5 times that of water. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Our results support the notion that the corneal stroma is highly permeable and isotropic to transport of hydrophilic molecules and particles with hydrodynamic radii up to at least 34 nm.

A water-soluble pH fluorescence probe based on quaternary ammonium salt for bioanalytical applications

A novel fluorescence probe Rhodamine–Ethanediamine–Iodomethane (REI) was successfully prepared to serve as an efficient sensing platform for H+ with fully reversibility mainly between the pH 4.2 and 7.2 in simple buffer solution. The introduction of quaternary ammonium salt with positive charge can not only manage to increase the solubility and sensitivity of probe REI, but also avoid the “alkalizing effect” due to charge-induced effect compared to the reference probe Rhodamine–Ethanediamine (RE). In particular, probe REI was well used for monitoring the weak acid pH fluctuations in lysosome of the live HeLa cells due to its excellent biological properties, including low cytotoxicity, high selectivity, good sensitivity and membrane permeability.

Quantification of proteins using 13C7-labeled and unlabeled iodoacetanilide by nano liquid chromatography/nanoelectrospray ionization and by selected reaction monitoring mass spectrometry

The combination of cysteine-specific modifiers, iodoacetanilide (IAA) and 13C7-labeled iodoacetanilide (13C7-IAA), has been applied to absolute quantification of proteins. The selected reaction monitoring (SRM) with the use of nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS) analysis was applied to precise quantification of three commercial proteins. Good correlation was observed between the theoretical ratios and observed ratios for all these proteins both in a simple buffer solution and in a complex protein environment. Due to efficient tagging, this method does not require separate synthesis of isotope-labeled peptides for the SRM studies. Therefore, this method is expected to be a useful tool for proteomics research.

Performance of enzymatic fuel cell in cell culture

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25μWcm−2 at 0.5V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode.

Comparison of the Thermal Denaturing of Human Serum Albumin in the Presence of Guanidine Hydrochloride and 1-Butyl-3-methylimidazolium Ionic Liquids

The interaction of proteins with aqueous solutions of ionic liquids (ILs) has attracted considerable recent attention owing to the challenges of finding biocompatible water-free ILs. These systems remain of great interest because of the potential for using ILs as designer solvents for biocatalytic processes. Increasing evidence demonstrates that aqueous solutions of water-miscible ILs, such as the well-studied 1-alkyl-3-methylimidazolium ILs, disrupt the native fold of proteins and can drive the formation of non-native aggregates that could negatively impact catalytic function. Here, we present a study comparing the thermal unfolding of human serum albumin (HSA) in a 1 M solution of the protein denaturant guanidine hydrochloride with two 1 M aqueous solutions of 1-butyl-3-methylimidazolium ILs, namely the chloride and the acetate. Small-angle neutron scattering (SANS) measurements found qualitative agreement between the thermally driven unfolding process for the three denaturants, as well as with a Tris buffer solution. HSA irreversibly aggregates and unfolds in the three denaturant solutions upon heating to temperatures below that required to drive the same process in a simple Tris buffer solution. The results reveal subtle differences in the interaction of the ILs and guanidine hydrochloride with the protein, although the final states of the protein were similar in all cases. The results indicate that the ions of water-miscible ILs and guanidine hydrochloride have specific roles in disrupting protein structure and driving aggregation. The experimental approach employed has the potential to provide new insights into protein interactions with ionic liquids that may aid in the search for more biocompatible ionic liquids.

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody–antigen interactions: Part 2

Within this paper we describe the use of scanning electrochemical microscopy (SECM) to fabricate a dotted array of biotinylated polyethyleneimine which was then used to immobilise first neutravidin and then a biotinylated antibody towards a relevant antigen of interest (PSA, NTx, ciprofloxacin). These antigens were selected both for their clinical relevance but also since they display a broad range of molecular weights, to determine whether the size of the antigen used effects the sensitivity of this approach. The SECM was then used to image the binding of both complementary and non-complementary antigens in a label-free assay. Imaging of the arrays before and following exposure to various concentrations of antigen in buffer showed clear evidence for specific binding of the complementary antigens to the antibody functionalised dots. Non-specific binding was also quantified by control experiments with other antigens. This demonstrated non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen of interest at the surface of the dots. The binding of ciprofloxacin was investigated both in simple buffer solution and in a more complex media, bovine milk.

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4°C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4°C for 3years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.

Visualization of microscale particle focusing in diluted and whole blood using particle trajectory analysis.

Inertial microfluidics has demonstrated the potential to provide a rich range of capabilities to manipulate biological fluids and particles to address various challenges in biomedical science and clinical medicine. Various microchannel geometries have been used to study the inertial focusing behavior of particles suspended in simple buffer solutions or in highly diluted blood. One aspect of inertial focusing that has not been studied is how particles suspended in whole or minimally diluted blood respond to inertial forces in microchannels. The utility of imaging techniques (i.e., high-speed bright-field imaging and long exposure fluorescence (streak) imaging) primarily used to observe particle focusing in microchannels is limited in complex fluids such as whole blood due to interference from the large numbers of red blood cells (RBCs). In this study, we used particle trajectory analysis (PTA) to observe the inertial focusing behavior of polystyrene beads, white blood cells, and PC-3 prostate cancer cells in physiological saline and blood. Identification of in-focus (fluorescently labeled) particles was achieved at mean particle velocities of up to 1.85 m s(-1). Quantitative measurements of in-focus particles were used to construct intensity maps of particle frequency in the channel cross-section and scatter plots of particle centroid coordinates vs. particle diameter. PC-3 cells spiked into whole blood (HCT = 45%) demonstrated a novel focusing mode not observed in physiological saline or diluted blood. PTA can be used as an experimental frame of reference for understanding the physical basis of inertial lift forces in whole blood and discover inertial focusing modes that can be used to enable particle separation in whole blood.

Shift of pH-Value During Thermal Treatments in Buffer Solutions and Selected Foods

The pH value is one of the most important process parameters during thermal treatments, regardless if the medium is a simple buffer solution or a complex food matrix. When the temperature increases after an initial measurement of the pH at ambient temperature (25°C), a significant pH shift could occur, which could produce incomparable results in different buffer solutions or lead to side reactions during food preservation. Consequently, a measurement cell was constructed to record online the pH-value and temperature up to 130°C. By applying the Nernst equation, it was possible to exclude the temperature-dependent influence of the pH glass electrode on the total pH value. The pH shift was measured over a wide temperature range (ΔT 20–130°C) in the most commonly used buffer solutions and some selected food matrices. The ΔpH of certain buffer solutions, namely TRIS and ACES, showed a significant pH decrease of −2.01 ± 0.08 (ΔT 20–130°C) and −1.27 ± 0.1 (ΔT 20–130°C), respectively, whereas the pH of PBS buffer solution was nearly independent of temperature. The ΔpH decrease recorded in milk (−0.89 ± 0.6, ΔT 20–130°C) as well as commercial and self-made baby food (−0.56 ± 0.05, ΔT 20–130°C) is of special interest for the food industry to get a deeper insight in occurring reactions during thermal preservation processes.

Evaluation of the ability of aqueous black walnut extracts to induce the production of reactive oxygen species

To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum. 3 BWEs. Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity. BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG. The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.

Determination of yessotoxins and pectenotoxins in shellfish by capillary electrophoresis-electrospray ionization-mass spectrometry

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and pectenotoxins (PTX)-6, were investigated with capillary electrophoresis coupled to mass spectrometry (MS) with an electrospray ionization source. After optimization, a simple and MS compatible alkaline volatile buffer solution of ammonium acetate was selected as background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with ammonium acetate used at the electrospray ionization (ESI) source. Previously to capillary electrophoresis (CE) separations, the application of an on-line sample pre-concentration approach based on field-amplified sample stacking was accomplished to increase sensitivity. As a result, the limits of detection provided by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) were 0.02 µg ml−1 (0.01 µg g−1), which corresponded to 1.25 pg for yessotoxin and 0.25 µg ml−1 (0.13 µg g−1 and 13.25 pg on capillary) for PTX-6. Accuracy tests showed 97.7% recovery from spiked blank mussel samples that showed no significant matrix influence running under optimal conditions. Intermediate precision was close to 4% relative standard deviation (RSD) for the migration time, and an RSD of 7.5% for peak areas. The method was successfully applied to naturally contaminated seafood samples in which yessotoxins and pectenotoxins-6 were clearly determined. This work demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of lipophilic toxins from the marine environment as alternative to liquid chromatography-electrospray ionization-single quadrupole mass spectrometry (LC-ESI-MS) for this purpose.

Microbial mass-dependent fractionation of chromium isotopes

Mass-dependent fractionation of Cr isotopes occurs during dissimilatory Cr(VI) reduction by Shewanella oneidensis strain MR-1. Cells suspended in a simple buffer solution, with various concentrations of lactate or formate added as electron donor, reduced 5 or 10 μM Cr(VI) to Cr(III) over days to weeks. In all nine batch experiments, 53Cr/ 52Cr ratios of the unreacted Cr(VI) increased as reduction proceeded. In eight experiments covering a range of added donor concentrations up to 100 μM, isotopic fractionation factors were nearly invariant, ranging from 1.0040 to 1.0045, with a mean value somewhat larger than that previously reported for abiotic Cr(VI) reduction (1.0034). One experiment containing much greater donor concentration (10 mM lactate) reduced Cr(VI) much faster and exhibited a lesser fractionation factor (1.0018). These results indicate that 53Cr/ 52Cr measurements should be effective as indicators of Cr(VI) reduction, either bacterial or abiotic. However, variability in the fractionation factor is poorly constrained and should be studied for a variety of microbial and abiotic reduction pathways.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 microg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.