pH Value Of Buffer Research Articles

A mitochondrial-targeted fluorescent probe to sense pH and HOCl in living cells

A series of rosamine dyes were designed and synthesized through one-step reaction. Among them, bifunctional fluorescent probe RosPO(OH)2 showed excellent water solubility and large molar extinction coefficient. RosPO(OH)2 underwent a two-step ionization process as the pH value of the HEPES buffer increased, accompanied a blue shift of UV absorption and an enhancement of fluorescence intensity. This means it can be used as a pH sensor. After the addition of HOCl, the fluorescence of RosPO(OH)2 was quenched and the color of the solution changed from pink to colorless which permitted "naked eye" detection. RosPO(OH)2 could be used as a sensor for detecting HOCl with high selectivity, rapid response, low detection limit (52 nM) and a wide range of pH application (pH 1–10). Colocalization results showed that RosPO(OH)2 could specifically stain in mitochondria. Due to the low toxicity and the excellent water solubility of RosPO(OH)2, it had been successfully applied to image pH and HOCl in living cells.

Measurement of anti-influenza neuraminidase antibody induced by H7N9 influenza vaccine

Objective To develop an enzyme-linked lectin assay (ELLA) for measuring neuraminidase inhibition (NI) antibody titers in subjects vaccinated with H7N9 influenza vaccine. Methods Neuraminidase substrate, the dilution and incubation time of enzyme-labeled antibody, the concentration of influenza antigen for coating and pH value of the dilution buffer were optimized. Based on that, ELLA was established and used to detect anti-influenza neuraminidase antibody titers in serum samples of 34 subjects before and after vaccination with H7N9 influenza vaccine. Results The optimal neuraminidase substrate was fetuin at a coating concentration of 7.5 μg/ml. The optimal dilution of enzyme-labeled antibody was 1∶500. The virus strain of influenza H7N9 vaccine was used as antigen at a concentrations of 4.5lgCCID50/ml in solution with a pH of 6.5. Influenza-specific NI titers detected after immunization with vaccine were significantly higher than those before vaccination (P<0.001). In the 34 subjects receiving H7N9 vaccine, the seroconversion rate of NI antibody was 47% (≥40 in NI titer ), which was lower than that of HI antibody (P<0.05). Conclusions An ELLA with natural substrate for measurement of anti-influenza NI antibody was developed. It is simple and practical and might be used in the establishment of immune evaluation system for influenza vaccines and NI antibody. Key words: H7N9 influenza vaccine; Neuraminidase inhibition antibody; ELLA

Optimization of conditions for single-step purification of recombinant hepatitis B surface antigen produced in Pichia pastoris using ion exchange chromatography

ABSTRACTThe adsorption and release of rHBsAg extracted from the final dosage form on various ion exchange resins and under different pH conditions were investigated after its peptide map and isoelectric point (PI) determination. Efficient antigen adsorption to the anion exchange resins occurred when the pH value of the protein buffer was adjusted to 5.0. In purification of rHBsAg derived from the yeast crude extract using Q Sepharose FF column, with adjusting the pH value of the crude extract to 5.0 (i.e., near to the target protein PI) and using 2M NaCl, rHBsAg with high purity (up to >95%) was obtained.Abbreviations: rHBsAg, recombinant hepatitis B surface antigen; Alhydrogel, aluminum hydroxide; IEF, isoelectric focusing; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC, reversed-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; PBS, phosphate-buffered saline

Open-tubular capillary electrochromatography with β-cyclodextrin-functionalized magnetic nanoparticles as stationary phase for enantioseparation of dansylated amino acids.

Magnetic nanoparticles (MNPs) modified with β-cyclodextrin and mono-6-deoxy-6-(1-methylimidazolium)-β-cyclodextrin tosylate (an ionic liquid), which called MNP-β-CD and MNP-β-CD-IL, were coated into the capillary inner wall. Compared to anuncoated capillary, thenew systems show good reproducibility and durability. The systems based on the use of MNP-β-CD or MNP-β-CD-IL as stationary phases were established for enantioseparation of Dns-modifiedamino acids. Improved resolutions were obtained for both CEC systems. Primary parameters such as running buffer pH value and applied voltage were systematically optimized in order to obtain optimal enantioseparations. Under the optimized conditions, the capillaries exhibited excellent chiral recognition ability for six Dns-amino acids (the DL-forms of alanine, leucine, lsoleucine, valine, methionine, glutamic acid)and provided a promising way for the preparation of chiral column. Graphical Abstract Schematic presentation of the open-tubular capillary electrochromatography systems with MNP-β-CD and MNP-β-CD-IL as stationary phases for enantioseparation of dansylatedamino acids.

A magnetic nanoparticle based immunoassay for alternariol monomethyl ether using hydrogen peroxide-mediated fluorescence quenching of CdTe quantum dots.

The authors describe a fluorometric immunoassay for alternariol monomethyl ether (AME). It is making use of magnetic nanoparticles and quenching of the fluorescence of mercaptopropionic acid-capped CdTe quantum dots (MPA-CdTe QDs) by H2O2. Catalase (CAT) was labeled with AME as a competitive antigen to competitively bind to magnetic nanoparticles carrying monoclonal antibodies (mAbs) with free AME in samples. The effects of the concentration and pH value of buffer, the concentrations of H2O2 and CAT-AME, and the incubation time of H2O2 and MPA-CdTe QDs were optimized. Under optimal conditions and in combination with magnetic separation, the quenching of the fluorescence of the MPA-CdTe QDs (excitation at 310nm, emission at 599nm) can be used to quantify AME with a detection limit of 0.25pg·mL-1 and the linear range from 0.25 to 7.5pg·mL-1. The immunoassay also has a lower cross-reactivity to AME analogues. It was evaluated by analyzing fruit samples spiked with AME. The recoveries from spiked fruits ranged from 87.2% to 92.0%. Graphical abstract Schematic presentation of a fluorometric immunoassay for alternariol monomethyl ether (AME) using magnetic nanoparticles (MNPs) for the rapid separation and purification. The method is based on quenching of the fluorescence of mercaptopropionic acid-capped CdTe quantum dots (MPA-CdTe QDs) by H2O2 for the fluorescence signal output, and on the use ofcatalase (CAT) with its high catalytic activity.

Enantioseparation of propranolol, amlodipine and metoprolol by electrochromatography using an open tubular capillary modified with β-cyclodextrin and poly(glycidylmethacrylate) nanoparticles.

The inner wall of a capillary was coated with glycidyl methacrylate (GMA) to form tentacle-type coating, and poly(glycidyl methacrylate) nanoparticles (PGMA NPs) were then immobilized on the film. Ethanediamine-β-cyclodextrin as chiral selector was covalently bonded into the PGMA NPs through the ring-open reaction. The materials were characterized by SEM, TEM and FT-IR. The modified column was applied to the enantioseparation of the racemates of propranolol, amlodipine and metoprolol. Compared to a capillary with a single layer of CD-PGMA (without GMA coating) and to a CD-GMA system (without PGMA nanoparticles), the performance of the capillary is strongly improved. The effects of buffer pH value and applied voltage were optimized. Best resolutions (propranolol: 1.27, metoprolol: 1.01 and amlodipine: 2.93) were obtained when using the PGMA-coated capillary system. The run-to-run, day-to-day and column-to-column reproducibility were tested and found to be highly attractive. The new stationary phase is likely to have a large potential and scope in that it may also be applied to chiral separations of other enantiomers, such as amino acids and biogenic amines. Graphical abstract Schematic presentation of the preparation of a capillary column with glycidyl methacrylate (GMA) coating which was then immobilized with poly(glycidyl methacrylate) nanoparticles and ethanediamine-β-cyclodextrin. This novel open tubular column was applied to construct capillary electrochromatography system for separation of basic racemic drugs.

Pollution Abatement of Heavy Metals in Different Conditions by Water Kefir Grains as a Protective Tool against Toxicity

This research paper addresses the hypothesis that Water Kefir grains can be used as absorbers of metal ions and reports the first application of the Water Kefir grains as a protective tool against toxicity by heavy metal ions. The aim of this study is to evaluate the concentration of heavy metal ions in several Water Kefir solutions during the fermentation process under various conditions. Two colonies of Water Kefir grain were used, and the concentrations of Cd, Co, Cr, Cu, Mn, Ni, Pb, Ba, and Ca were measured in Water Kefir grain solutions at different contact times (0, 24, 48, and 72 hours), different pH values in citric and acetic buffers, and different Water Kefir grains/metal solution ratios, with and without sucrose (5%). Optical emission spectroscopy was used to measure the concentrations of metal ions. Among the tested experimental conditions, the best combination for pollution abatement is sucrose (5%), contact time 24 hours, starting pH = 4.5, acetate buffer, and Kefir grains/metal solution ratio 1 : 1. In these conditions, the heavy metal abatement by Water Kefir grains is particularly effective for Cr and Pb (70%) and good for Cu, Ni, and Mn (50%).

Optimalization of deoxyribonucleic acid extraction using various types of magnetic particles

Isolation of deoxyribonucleic acid is an important step in the molecular diagnostics of microorganisms. A high quality of isolated deoxyribonucleic acid is necessary for deoxyribonucleic acid amplification by the polymerase chain reaction. The conventional deoxyribonucleic acid isolation using phenol–chloroform extraction and deoxyribonucleic acid precipitation in ethanol is time-consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase deoxyribonucleic acid extraction. The amounts of deoxyribonucleic acid in separation mixtures were measured using ultraviolet spectrophotometry. The first experimental conditions were tested on chicken erythrocytes deoxyribonucleic acid. Phosphate buffer (pH 7, 7.6 and 8) was used for the adsorption of deoxyribonucleic acid on magnetic particles. Tested values of pH have no effects on deoxyribonucleic acid adsorption. It was shown that approximately almost one half of deoxyribonucleic acid was adsorbed to the particles. A number of different elution conditions (temperature, time and pH value of elution buffer) were investigated to determine their effect on elution efficiency. It was shown that only a small fraction of the bound DNA was released at 22 °C, while the release was more effective as the temperature was increased also the amount of elution deoxyribonucleic acid was more effective when time was increased. The higher amounts of deoxyribonucleic acid were eluted with TE buffer pH 9.0. Second, bacterial deoxyribonucleic acid was tested. This deoxyribonucleic acid eluted from the particles was in polymerase chain reaction ready quality.

Laccase‐catalysed coloration of wool and nylon

The potential for laccase (EC 1.10.3.2) to be used within the area of textile coloration, specifically for the generation of decorative surface pattern design, remains relatively unexplored. The current study presents a novel process for the coloration of wool and nylon 6,6 fibres via laccase oxidation of aromatic compounds as an alternative to conventional dyeing methods. Emphasis was placed on producing a diverse colour palette, which was achieved through the investigation of three different aromatic compounds as laccase substrates: 1,4‐dihydroxybenzene, 2,7‐dihydroxynapthalene and 2,5‐diaminobenzenesulphonic acid. Reaction processing parameters such as buffer systems and pH values, laccase and aromatic compound concentrations, and reaction times were investigated, all in the absence of additional chemical auxiliaries. Enzymatically dyed fabrics were tested against commercial standards, resulting in reasonably good colour fastness to wash. To demonstrate the coloration and design potential by laccase catalysation of aromatic compounds, specially constructed fabrics using a combination of undyed wool, nylon and polyester yarns were dyed using the one‐step laccase‐catalysed coloration process. The use of different fibre types and weave structures enabled simple colour variations to be produced. Shadow, reserve and contrasting effects were achieved with the laccase‐catalysed dyeing process developed. Important advantages over conventional processing methods include the use of simpler and milder processing conditions that eliminate additional chemical use and reduce energy consumption.

Microbial biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates as potential route to platform chemicals.

Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8gL-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1gL-1 , and 48H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2M Tris-HCl buffer (pH 9, strength=0.2M, 30°C) at 0.21gL-1 H-1 and 98.6±1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.

Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols.

The usefulness of Spotchem® Analyser (Arkray) in determining the risk in oral diseases in adolescents – a pilot study

Introduction. Saliva Check Buffer® has been long used for assessing saliva physical and chemical properties, while CRT Bacteria® is used for estimating the count of cariogenic bacteria. Spotchem® Analyser is a new device for the assessment of saliva properties. Aim. To determine the consistency of results obtained using Spotchem®, Saliva Check Buffer® and CRT Bacteria® kits, as well as to evaluate the correlations between the obtained results and oral hygiene status, ICDAS II indices in the diagnosis of the risk of oral diseases in children. Material and methods. Patients aged 12-17 years were evaluated for oral hygiene (% API, OHI), gingival inflammation (GI), and carious lesions (ICDAS II). Salivary tests using Saliva Check Buffer® (GC), CRT Bacteria® (Ivoclar Vivadent) and Spotchem® (Arkray) Analyser were conducted. The consent of the bioethical committee of Warsaw Medical University, as well as written consent from all the parents of all the subjects or legal guardians of all the subjects were obtained. Results. The study included 25 patients (mean age 13.7 ± 2.2 years). The following mean index values were obtained: OHI-S – 0.93 ± 0.43; API% – 72 ± 0.26; GI – 0.83 ± 0.61; DMFt – 6.44 ± 4.12. Active white lesions were observed in 13 patients (mean number of lesions 2.2 ± 2.92). Spearman’s rank correlation coefficient showed significant correlations between pH values according to Saliva Check Buffer® and salivary buffer capacity (r = 0.608) and acidity (r = -0.713) according to Spotchem®; as well as a negative correlation between salivary buffer capacity and pH values in Spotchem® (r = -0.845). High count of S. mutans (&gt; 105 CFU/mL) assessed by CRT Bacteria® correlated significantly with high bacteria count estimated by the Spotchem® Analyser (r = 0.54). Significant correlations were found between OHI and high (r = 0.46) and average (r = -0.54) metabolic activity of S. mutans; GI and salivary protein levels (r = 0.42); carious lesions and salivary protein levels (r = 0.40); salivary blood levels (r = 0.47) (Spotchem®) and the levels of S. mutans and Lactobacillus spp. (CRT Bacteria®) (0.47 and 0.42, respectively). Conclusions. The parameters estimated by the Spotchem® Analyser were correlated with the results obtained with the commonly known salivary kits and oral health indices. However, its clinical relevance should be confirmed by further studies.

Simple and green synthesis of piperazine-grafted reduced graphene oxide and its application for the detection of Hg(II)

In this paper, piperazine-grafted reduced graphene oxide (NH-rGO) was synthesized via a simple and green two-step procedure: (i) opening of the resulting epoxides of graphene oxide (GO) with piperazine (NH) through nucleophilic substitution; (ii) reduction of GO with ascorbic acid. Its structure and morphology were characterized by scanning electron microscopy and x-ray photoelectron spectroscopy. The NH-rGO modified glassy carbon electrode was explored as an electrochemical sensor for the determination of Hg(II) using a differential pulse anodic stripping voltammetry technique. Hg(II) can be efficiently accumulated and deposited on the surface of a modified electrode by strong coordination chemical bonds formed between Hg(II) and NH. And then the anodic stripping current can be significantly enhanced by rGO with the merits of large specific surface area and high conductivity, which served as a signal amplifier, finally realizing the highly sensitive determination of Hg(II). The experimental parameters including the pH value of the acetate buffer, deposition potential and deposition time were optimized. Under optimal conditions, the developed sensor exhibited a wide linear range from 0.4–12 000 nM with a low limit of detection of 0.2 nM, which is well below the guideline value in drinking water set by the WHO. Moreover, the practical application of this method was confirmed by an assay of Hg(II) in tap water samples with acceptable results.

A Small-Scale Process for Predicting Donnan and Volume Exclusion Effects During Ultrafiltration/Diafiltration Process Development

Achieving the desired final protein formulation using ultrafiltration/diafiltration (UF/DF) operations is an essential component of many protein purification processes. It is well documented that differences in the excipient and buffer concentrations exist between the DF and retentate solutions when they have achieved equilibrium. Several publications have proposed ways to calculate these differences. However, the accuracy of these methods has been limited by the use of an estimated protein charge value. In this article, a small-scale system is described, which can accurately determine the protein charge by making buffer and excipient concentration measurements and applying the determined values to the Donnan and volume exclusion equations. This information can be utilized to generate a standard curve, which in turn can be applied to at-scale UF/DF operations. For 2 different antibodies, the standard curve generated by the small-scale system yielded buffer concentrations and pH values that agreed well with those generated after UF/DF operations, whereas using the theoretical protein charge caused a departure from the measured results. This model also provides good estimates as to how the final formulation pH and buffer concentration vary as a function of the pH and buffer concentration in the DF buffer. This information is of important utility for the accurate formulation of high-concentration protein solutions (>100 mg/mL) where the coconcentration of buffers and the volume exclusion of certain excipients are amplified. The low material requirements of the small-scale system are a major benefit for early phase formulation and process development when sufficient time and material may not be available, in particular to ensure successful UF/DF operations for the development of high protein concentration formulations.

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6–6.5, low salt concentrations and 3 mM Mg2+ or 0.3–3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

Preparation of pH-controllable nanofibrous membrane functionalized with lysine for selective adsorption of protein

A pH-controllable electrospun nanofibrous membrane (NFM) was successfully fabricated by functionalizing polyacrylonitrile (PAN) NFM with lysine (LYS) for selective adsorption of protein. Due to the introduction of similar α- amino acid structure to PAN NFM, the surface charges of the obtained PAN-LYS NFM can switch via changing the pH value of adsorbate solution. Benefiting from the integrated merits of electrospun PAN NFM and LYS, PAN-LYS NFM exhibited excellent selectivity and reusability in protein adsorption. Pepsin and lysozyme were used as the model proteins, and the maximum adsorption capacity of pepsin and lysozyme were 425.49mgg−1 at pH 3 and 54.98mgg−1 at pH 8, respectively. Meanwhile, there was almost no adsorption of pepsin and lysozyme on the PAN-LYS NFM at pH 8 and pH 3, respectively, primarily owing to the strong repulsion of like charges between the NFM and protein. In view of this, PAN-LYS NFM could be used to realize the selective adsorption of protein by regulating buffer pH value based on the isoelectric point (pI) of protein. Furthermore, the protein adsorption can be also achieved via filtration by packing five layers of PAN-LYS NFM together and fixing them in a filter. More importantly, the PAN-LYS NFM can be regenerated and reused. The adsorption capacities of proteins kept relatively stable values within the four cycles, indicating that PAN-LYS NFM can meet the requirement of long service life. Therefore, PAN-LYS NFM exhibits a great potential in the applications of protein adsorption and purification.

New chiral ligand exchange capillary electrophoresis system with chiral amino amide ionic liquids as ligands

Using chiral amino amide ionic liquids as the ligands, a new chiral ligand exchange capillary electrophoresis method with Cu(II) as the central ion was constructed for enantioseparation of labeled D,L-amino acids. The effects of key parameters, including pH value of the running buffer, the ratio of Cu(II) to chiral amino amide ionic liquids, the concentration of complexes based on Cu(II)-chiral amino amide ionic liquids were investigated. It has been observed that eight pairs of labeled D,L-amino acids could be baseline-separated with a running buffer of 15.0mM ammonium acetate, 10.0mM Cu(II) and 20.0mML-phenylalaninamide based ionic liquid at pH 5.0. The quantitation of D,L-amino acids was conducted and good linearity (r2 ≥ 0.964) was obtained. Furthermore, an assay for determining the enantiomeric purity of D,L-amino acids was developed and the possible enantiorecognition mechanism was discussed briefly. The results indicated that the chiral amino amide ionic liquids could play the role of ligands in chiral ligand exchange capillary electrophoresis system and exhibit great potential in chiral analysis.

Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via online pre-concentration of field-amplified sample stacking

In this paper, polydopamine/gold nanoparticles (PDA/Au NPs) were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis (MCE) for the separation of reduced glutathione (GSH) and oxidized glutathione (GSSH). The formation of the PDA/Au NPs was characterized by scanning electron microscopy, transmission electron microscope, UV–Vis spectra and ATR-FTIR. An online pre-concentration strategy involving field-amplified sample stacking was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria (Escherichia coli, Staphylococcus aureus and Salmonella enterica serovar Typhimurium) and HaCaT cells samples by MCE with laser-induced fluorescence detection. The influences of the separation voltage, the concentration of the running buffer and the pH value of running buffer, were carefully investigated. Using this studied method, GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s. The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM, respectively (S/N = 3), which corresponded to approximately 180–301-fold improvements in peak height. Moreover, this method was successfully applied to the analysis of bacteria (E. coli, S. aureus and S. typhimurium) and HaCaT cell samples with a satisfactory recovery rate.

The Optimization and Characterization of an RNA-Cleaving Fluorogenic DNAzyme Probe for MDA-MB-231 Cell Detection.

Breast cancer is one of the most frequently diagnosed cancers in females worldwide and lacks specific biomarkers for early detection. In a previous study, we obtained a selective RNA-cleaving Fluorogenic DNAzyme (RFD) probe against MDA-MB-231 cells, typical breast cancer cells, through the systematic evolution of ligands by exponential process (SELEX). To improve the performance of this probe for actual application, we carried out a series of optimization experiments on the pH value of a reaction buffer, the type and concentration of cofactor ions, and sequence minimization. The length of the active domain of the probe reduced to 25 nt from 40 nt after optimization, which was synthesized more easily and economically. The detection limit of the optimized assay system was 2000 MDA-MB-231 cells in 30 min, which is more sensitive than the previous one (almost 5000 cells). The DNAzyme probe was also capable of distinguishing MDA-MB-231 cell specifically from 3 normal cells and 10 other tumor cells. This probe with high sensitivity, selectivity, and economic efficiency enhances the feasibility for further clinical application in breast cancer diagnosis. Herein, we developed an optimization system to produce a general strategy to establish an easy-to-use DNAzyme-based assay for other targets.

Enantioseparation of Tedizolid phosphate by RP-HPLC, using ??-Cyclodextrin as a Chiral Mobile Phase Additive

Enantiomeric separation of Tedizolid phosphate was developed using a reversed phase HPLC (RP-HPLC) with &#120573;-cyclodextrin (&#120573;-CD) as a chiral mobile phase additive. Effect of the pH value of aqueous buffer, concentration of chiral additive, composition of mobile phase, and column temperature on the enantioseparation of Tedizolid phosphate was investigated on the Phenomenex Luna, Phenyl-Hexyl, 250 x 4.6mm, 5μm HPLC column. A satisfactory resolution was achieved at column temperature 20⁰C using a mobile phase consisting of a mixture of aqueous buffer of pH 7.0 of disodium hydrogen phosphate with additive &#120573;-cyclodextrin, triethylamine and acetonitrile. This analytical method was evaluated by performing method validation as per ICH guideline. The calibration curve was plotted within the concentration range between 0.30 and 2.25 �g mL−1 and the recoveries between 96.9 and 105.3% were obtained, with regression coefficient R2 is 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) of S- enantiomer of Tedizolid phosphate is 0.10 and 0.30 μg mL−1 The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of Tedizolid phosphate drug substance.