Zwitterionic Buffers Research Articles

Interrupting marine fouling with active buffered coatings

Biofouling on marine surfaces causes immense material and financial harm for maritime vessels and related marine industries. Previous reports have shown the effectiveness of amphiphilic coating systems based on poly(dimethylsiloxane) (PDMS) against such marine foulers. Recent studies on biofouling mechanisms have also demonstrated acidic microenvironments in biofilms and stronger adhesion at low-pH conditions. This report presents the design and utilization of amphiphilic polymer coatings with buffer functionalities as an active disruptor against four different marine foulers. Specifically, this study explores both neutral and zwitterionic buffer systems for marine coatings, offering insights into coating design. Overall, these buffer systems were found to improve foulant removal, and unexpectedly were the most effective against the diatom Navicula incerta.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing.Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

P-157 Improved embryo development and clinical outcome using bicarbonate buffer as the oocyte holding medium during Intracytoplasmic sperm injection (ICSI) compared to MOPS buffer

Abstract Study question Does bicarbonate buffer used as the oocyte holding medium during ICSI result in improved embryo development and clinical outcome compared to the zwitterionic buffer MOPS? Summary answer Pre-equilibrated bicarbonate buffer is acceptable for ICSI use in atmospheric conditions, and results in superior sibling-oocyte cohort study outcomes compared to ICSI with MOPS buffer. What is known already Maintaining proper pH is of the utmost importance during IVF micromanipulation procedures. However, currently used zwitterionic buffers, HEPES and MOPS, may significantly influence intrinsic biological mechanisms outside pH maintenance, impacting oocyte competence, embryo development, and clinical outcome. Zwitterionic buffer influence is of concern during ICSI because piercing the oolemma allows the influx of the surrounding buffer into the oocyte (Mendola unpublished data, 2022). Bicarbonate is the physiological buffer for pH maintenance in all mammals providing numerous benefits for cellular development. Study design, size, duration An iSTAT handheld blood analyzer was used to analyze the pH shift of pre-equilibrated bicarbonate buffer dishes to determine the acceptablity for use during ICSI in atmospheric conditions. A clinical sibling-oocyte cohort study consisting of 115 patients and 1,997 oocytes (1,016 bicarbonate/981 MOPS) was analyzed to compare the embryo development and clinical outcomes of bicarbonate buffer vs. MOPS as the oocyte-holding medium during ICSI. Morphokinetic time points for all PGT-A tested embryos were also compared. Participants/materials, setting, methods We analyzed the pH shift of pre-equilibrated bicarbonate buffer in atmospheric conditions using 50x9mm culture dishes (Falcon, cat#351006) comparing light and high viscosity paraffin oil overlay, OvOil and Heavy OvOil (Vitrolife), after 5-minute, 10-minute, and 15-minute intervals. We analyzed all data points in triplicate. We analyzed the sibling-oocyte cohort study comparing fertilization rate, abnormal fertilization rate, useable blastocyst rate, mosaicism rate, euploidy/aneuploidy rate, morphokinetic time points, pregnancy rate, and ongoing pregnancy rate. Main results and the role of chance Pre-equilibrated (6.5%CO2/5.0%O2/7.301pH) bicarbonate buffer in 50 × 9mm ICSI culture dishes, with 5.0mL of OvOil overlay, maintained proper pH (7.301/7.328/7.354pH) for the time required to complete the ICSI procedure in atmospheric conditions (0min/5min/10min). The use of the high viscosity paraffin oil overlay, Heavy OvOil, reduced pH drift of the bicarbonate buffer ICSI dishes (7.301/7.323/7.342pH). In this clinical sibling-oocyte cohort study, ICSI was performed in pre-equilibrated bicarbonate buffer culture drops (6µL) in the 50x9mm dishes with 5.0mL Heavy OvOil overlay. Utilization of bicarbonate buffer as the oocyte-holding medium during ICSI is superior to MOPS buffer. Bicarbonate buffer used with ICSI resulted in higher fertilization rates (85.4% vs. 78.8%, P<0.0001), higher useable blastocyst rate per ICSI oocyte (50.6% vs. 46.2%, P<0.05), lower abnormal fertilization rate (4.5% vs. 6.7%, P<0.05), and a lower whole chromosome mosaicism rate (4.2% vs. 8.2%, P<0.03) compared to the MOPS buffer. Pregnancy rates were higher in the bicarbonate group compared to MOPS but not statistically significant. Morphokinetic analysis revealed a slight delay in all MOPS developmental time points compared to bicarbonate, but only statistically significant in the time to compaction (P<0.03). Limitations, reasons for caution During this study, different genetic testing companies, with differing mosaicism thresholds and decisions on included ploidy results, were used for PGT-A analysis of patient samples. Wider implications of the findings We determined that pre-equilibrated bicarbonate buffer can be used for ICSI in atmospheric conditions, therefore avoiding the potential detrimental effect of zwitterionic buffer after influx into the oocyte from the ICSI procedure. Bicarbonate buffer used during ICSI was determined to be superior to MOPS buffer. Trial registration number Not applicable

Interactions of Ti3C2 MXene with Aqueous Zwitterionic Biological Buffers: Implications for Applications in Biological Systems

MXenes, a family of ultrathin layered two-dimensional (2D) transition-metal carbides, nitrides, and carbonitrides, are steadily advancing as novel inorganic nanosystems for a broad range of applications. While high conductivity, solution processability, hydrophilic nature, and the presence of various surface functional groups enable the use of 2D MXenes in aqueous systems, applications that involve the use of MXenes in aqueous systems and biological solutions, including but not limited to water treatment, water desalination, and biological assays, are only beginning to emerge. For the successful application of MXene in aqueous systems, their interactions with common aqueous solution constituents must be better understood. This paper describes the structural and functional properties of Ti3C2Tx (Tx = O−, OH−, F−) MXenes in N-substituted commonly used biological buffers like N-(2-hydroxyethyl)-piperazine-N′-ethanesulfonic acid (HEPES) and 3-(N-morpholino)-propane sulfonic acid (MOPS) and with a series of piperazine-based molecules dissolved in phosphate buffer solutions. Dissolving MXenes in these buffers significantly impacts their optical, structural, and electrical properties. The interactions of Ti3C2Tx MXenes with N-substituted zwitterionic buffer molecules are structure-dependent and mostly but not fully reversible. X-ray photoelectron spectroscopy measurements reveal small but measurable surface oxidation through the formation of Ti–O bonds. Our results suggest a complex interaction pattern where some interactions are driven by hydrogen bonding and electrostatic forces, while others involve chemisorption, which results in a permanent impact on the MXene nanosheets. This study is an important step toward understanding the stability of MXenes in complex aqueous media and the impact of interactions of MXenes with nitrogen-containing molecules on MXene properties, which are especially important in applications of MXenes in biological systems. The nature of interactions between MXenes and biological buffer molecules, revealed in our study, suggests that surface chemistry modifications of MXenes are required to preserve their chemical stability and enable their applications in complex biological solutions.

A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography.

Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.

Rapid antimicrobial susceptibility profiling using impedance spectroscopy

The present antibiotic susceptibility testing (AST) techniques based on bacterial culture, gene amplification and mass spectrometry are highly time consuming, labour intensive or expensive. Impedance spectroscopy is an emerging tool for rapid bacterial analysis as it is label-free, real-time, affordable and high-throughput. The over-reliance of this technique on complex chip designs and cell enrichment strategies has, however, slowed its foray into clinical AST. We demonstrate a label-free approach in which a low conductivity zwitterionic buffer is used for boosting impedance sensitivity in simple interdigitated electrodes (IDEs) allowing rapid AST in just 20 min without any liquid flow, biofunctionalization or cell enrichment steps. The detection principle relies on measuring changes in solution resistance due to antibiotic-induced bacterial cell death or growth. While the death-based approach is faster (20 min), it's restricted to surface-acting bactericidal antibiotics. The cell growth approach is longer (60–80 min) but more versatile as it applies to all drug types. Results for antibiotic sensitivity analysis and minimum inhibitory concentration (MIC) determination are illustrated for Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus against a wide class of antibiotics (penicillins, cephalosporins, polymyxins, carbapenems etc.).

Effect of pH variation and annealing on covalently assembled nanodiamond films

This work examines the effect of using low-ionic strength zwitterionic buffer and post-deposition thermal annealing on the microstructure and thermal conductivity of continuous nanocrystalline diamond films that are deposited via the directed covalent assembly of detonation nanodiamonds. Variation of the zwitterionic buffer pH was found to tune the surface coverage, film thickness, thermal conductivity, and film morphology as quantified by apparent porosity and pore size distribution. The sequential annealing of these films up to 400 °C, showed the anticipated severance of amide bonds holding the nanodiamonds together, followed by the detrimental aggregation of the nanodiamonds to segregated islands, losing film continuity and increasing apparent porosity. The change in thermal conductivity with annealing was subjective to buffer pH, suggesting that the morphology changes associated with annealing may have a complex relationship with thermal conductivity. The thermal conductivity data were further analyzed using a phonon hopping model to quantify the degree of phonon movement across boundaries. Overall, the results demonstrate a way to achieving porous, low-cost nanocrystalline diamond thin films with tunable film morphology and thermal conductivity.

Zwitterions for impedance spectroscopy: The new buffers in town

Studying the role of buffers in impedance spectroscopy is a relatively unexplored area. We demonstrate a special class of biologically relevant buffers known as Good's zwitterionic buffers that show improved performance over standard electrolyte buffers (e.g. PBS) currently widely used in impedance spectroscopy measurements of bacterial suspensions. Our theoretical and experimental comparisons of conductivity of classical and zwitterionic buffers at various different concentrations show that ion-ion interaction effects are significantly higher in zwitterionic buffers as compared to classical buffers at the concentrations at which they are used. This and the fact that zwitterions have larger sizes leads to the lowering of their conductivity which significantly improves their impedance sensing ability. We illustrate through an example of heat-induced ionic release in model S. typhi and S. aureus bacteria that having a low conductivity buffer is indeed beneficial for biological impedance measurements. In fact, the best buffer for impedance studies can be chosen solely based on their electrical properties as long as they are also biologically compatible. This gives Good's zwitterionic buffers an edge over conventional media as they satisfy both these criteria.

Using mesoporous silica alternate nanomaterials in adsorption of phenols from aqueous solutions

For efficient adsorbents of hazardous phenols from samples of waste, composite materials utilizing mesoporous silica and organic ligands were used. The 4-amino antipyrine (4-AAP) ligand has been effectively conjugated to the mesoporous nanoparticles and adsorbs phenols under appropriate conditions. The scanning electron microscope (SEM), X-ray diffraction, and N2 adsorption/desorption isotherms process have been used to research this modern adsorbent content. The data highlighted that the proposed sensor, as per its selectivity, response, financially savvy and eco-accommodating method is highly appropriate for phenols collecting from polluted water. A batch process was used: 50 ml of 50 mg/L of each phenolic solutions at pH=8.0 with (zwitterionic buffer HEPES), was introduced to 100 mg 4-AAP sensor, then the mixture was submitted to filtration and the remaining phenols specified by Gas Chromatography in the supernatant solution. The sorbent capacity was 151.05, 82.03 and 83.47mg of phenol, m-cresol, o-cresol and quinol per gram of 4-AAP sensor, respectively with a correlation coefficient range of 0.998-0.999.

Influence of Zwitterionic Buffer Effects with Thermal Modification Treatments of Wood on Symbiotic Protists in Reticulitermes grassei Clément.

Simple SummaryOver the past thirty years, the thermal modification of wood has become a universally recognised and commercialised wood modification process. Thermal modifications may affect wood properties, either positively (dimensional stability and decay resistance) or negatively (mechanical properties). The combination of the impregnation of specific reagents with thermal modification may help to overcome the negative effects on wood properties. In this study, we evaluate the effect of a combination of two zwitterionic buffers, bicine and tricine, and thermal modification of two wood species (beech and spruce) against subterranean termites and their symbiotic fauna. Bicine and tricine treatments alone had a clear influence on wood mass loss and termite survival. The flagellate protist symbiotic community was affected by the treatments and responded differently to them, as a highly adaptable community. However, the combination of bicine with the thermal modification showed a negative effect on termites and their symbionts on both wood species. The combination of these different factors should be further investigated, as these results seem to be promising with regard to the enhancement of the termite resistance of wood.The majority of thermal modification processes are at temperatures greater than 180 °C, resulting in a product with some properties enhanced and some diminished (e.g., mechanical properties). However, the durability of thermally modified wood to termite attack is recognised as low. Recent attempts at combining thermal modification with chemical modification, either prior to or directly after the thermal process, are promising. Buffers, although not influencing the reaction systems, may interact on exposure to certain conditions, potentially acting as promoters of biological changes. In this study, two zwitterionic buffers, bicine and tricine, chosen for their potential to form Maillard-type products with fragmented hemicelluloses/volatiles, were assessed with and without thermal modification for two wood species (spruce and beech), with subsequent evaluation of their effect against subterranean termites (Reticulitermes grassei Clément) and their symbiotic protists. The effect of the wood treatments on termites and their symbionts was visible after four weeks, especially for spruce treated with tricine and bicine and heat treatment (bicine HT), and for beech treated with bicine and bicine and heat treatment (bicine HT). The chemical behaviour of these substances should be further investigated when in contact with wood and also after heat treatment. This is the first study evaluating the effect of potential Maillard reactions with zwitterionic buffers on subterranean termite symbiotic fauna.

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat.

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al3+ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al+3 -tolerant, high TaALMT1 expression) and ES8 (Al+3 -sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.

Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons.

Good's buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.

Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons

Good’s buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.

The benefit of poor mixing: kinetics of coacervation.

Complex coacervation has become a prominent area of research in the fields of food science, personal care, drug stabilization, and more. However, little has been reported on the kinetics of assembly of coacervation itself. Here, we describe a simple, low-cost way of looking at the kinetics of coacervation by creating poorly mixed samples. In particular, we examine how polymer chain length, the patterning and symmetry of charges on the oppositely charged polyelectrolytes, and the presence of salt and a zwitterionic buffer affect the kinetics of complex coacervation. Our results suggest an interesting relationship between the time for equilibration and the order of addition of polymers with asymmetric patterns of charge. Furthermore, we demonstrated that increasing polymer chain length resulted in a non-monotonic trend in the sample equilibration times as a result of opposing factors such as excluded volume and diffusion. We also observed differences in the rate of sample equilibration based on the presence of a neutral, zwitterionic buffer, as well as the presence and identity of added salt, consistent with previous reports of salt-specific effects on the rheology of complex coacervates. While not a replacement for more advanced characterization strategies, this turbidity-based method could serve as a screening tool to identify interesting and unique phenomena for further study.

Potentiometric and Electrochemical studies for the interaction of Zinc (II), Lead (II), Cadmium (II) with HEPES and HEPPSO as Zwitterionic Buffer Ligands at Different Temperature

The complexation of the divalent transition metal ions Zn (II), Pb (II) or Cd (II) with two important biologically zwitterionic buffers (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES) and (N-[2-hydroxyethyl] piperazine-N'-[2-hydroxy-propanesulfonic acid]) (HEPPSO) in an aqueous medium, were studied using potentiometric pH titration in 1:1 ratio at four different temperatures 288 K, 298 K, 308 K and 318 K at ionic strength 0.1 molL-1 KNO3. The formation constants and thermodynamic parameters of different protonated, normal, monohydroxy and dihydroxy complexes for the binary systems M(II) + HEPES and M(II) + HEPPSO have been evaluated. The electrochemical behavior of the free Pb(II) and its binary complexes with HEPES and HEPPSO was studied by cyclic voltammetry on glassy carbon electrode. Upon temperature elevation the rate of the reduction process of the Pb2+-HEPPSO is higher than that of Pb2+-HEPES. The diffusion coefficient, the activation energies and the standard rate constant (Ks) for the diffusion process of Pb(II) complexes were calculated.

Synthesis of Photothermally Stable Triangular Silver Nanoplates for SERS Applications, Photokilling of Bacteria

AbstractWe developed a facile seeded‐growth strategy to synthesize triangular silver nanoplates with enhanced photothermal stability, in which zwitterionic buffers, hydroquinone (HQ) served as shape‐directing, reducing agents, respectively. Compared to the Ag nanoplates synthesized by the thermal methods reported before, the Ag nanoplates prepared in this study still retained the sharp tips of nanoplates, maintained excellent near infra‐red (NIR) surface plasmon resonance (SPR) properties after repeated NIR irradiation. The enhanced photothermal stability could enable the as‐prepared Ag nanoplates serve as stable substrates for bacterial surface enhanced Raman scattering (SERS) applications. Meanwhile, the Ag nanoplates synthesized in this study also yielded a steady‐state photothermal conversion efficiency up to 40%, giving effective photothermal therapy (PTT) towards antibiotic resistant bacteria.

Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer

BackgroundTumours rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. We hypothesise that acidity, independent from lactate, can augment the pro-tumour phenotype of macrophages.MethodsWe analysed publicly available data of human prostate cancer for linear correlation between macrophage markers and glycolysis genes. We used zwitterionic buffers to adjust the pH in series of in vitro experiments. We then utilised subcutaneous and transgenic tumour models developed in C57BL/6 mice as well as computer simulations to correlate tumour progression with macrophage infiltration and to delineate role of acidity.ResultsActivating macrophages at pH 6.8 in vitro enhanced an IL-4-driven phenotype as measured by gene expression, cytokine profiling, and functional assays. These results were recapitulated in vivo wherein neutralising intratumoural acidity reduced the pro-tumour phenotype of macrophages, while also decreasing tumour incidence and invasion in the TRAMP model of prostate cancer. These results were recapitulated using an in silico mathematical model that simulate macrophage responses to environmental signals. By turning off acid-induced cellular responses, our in silico mathematical modelling shows that acid-resistant macrophages can limit tumour progression.ConclusionsThis study suggests that tumour acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

PH: the silent variable significantly impacting meiotic spindle assembly in mouse oocytes.

Temperature fluctuation negatively impacts the assembly and function of the meiotic spindle, but does pH have a similar effect? Polarized light microscopy was used to study the spindle in living mouse oocytes under different pH conditions. Female mice (n = 53) were superovulated, and oocytes collected, denuded and allocated to treatment groups. All experiments were performed at 37°C, and standard bicarbonate-buffered medium was used either pre-equilibrated in 6% CO2 or unequilibrated (in ambient CO2). Mean oocyte spindle retardance was measured over time in response to changing pH. Spindles were also assessed to understand whether this effect was reversible, by using a fixed pH in a zwitterionic buffer. The data show the spindle is impacted by pH fluctuation, with mean retardance significantly higher at pH 7.4-7.5 than at the point of media equilibration (P<0.001). This effect was found to be reversible; retardance significantly decreased after transition of the oocytes from pH 7.43 or pH 7.53 back to the original pre-equilibration pH of 7.32 (P<0.05). This study has shown that the meiotic spindle in mouse oocytes is highly sensitive to changes in oocyte culture media pH. If comparable in humans, this has significance as to the pH level of culture media currently used in assisted reproductive technology clinics worldwide, and reinforces the requirement for stringent control over extrinsic variables in the IVF laboratory.

Factors impacting protein adsorption on layer-by-layer assembled stimuli-responsive thin films

Studying the stimuli-response of polyelectrolyte multilayers (PEMs) towards protein adsorption requires coping with the multiplicity of factors influencing interfacial processes. In this study, we report the design of a nanostructured stimuli-responsive coating based on the sequential layer-by-layer assembly of poly(N-isopropylacrylamide) block copolymers with poly (allylamine hydrochloride) and scrutinize its ability to modulate protein adsorption. The results reveal that the stimuli-response of the film towards protein adsorption under T, pH and I changes is strongly influenced by the type of buffer. This latter parameter has indeed a significant impact on the adsorbed amount of proteins, while it has only slight effect on the stimuli-response of free block copolymer, on the mechanism of PEM growth and the properties of the obtained film. In conditions favorable to protein adsorption, the use of anionic buffer leads to the adsorption of an apparent monolayer of proteins, while in zwitterionic buffer, proteins adsorb in a significantly higher amount. This trend may be explained by a more efficient charge screening of macromolecules by zwitterions which reduce the repulsion between proteins, leading to their accumulation at the interface. Finally, the length of the poly(N-isopropylacrylamide) block is shown to be another key factor impacting the amount of adsorbed proteins. The findings reported in this paper provide relevant information and allow the evaluation of stimuli-response of PEM to be carried out under optimized conditions.

Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer

HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R2=0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.