HEPES Buffer Research Articles

Modulation of aluminum sensing properties of a sulphone group containing chemosensor and its biological applications

A chemosensor with two binding pockets facilitates binding of one metal ion in either of the pockets providing a better chance for the interaction and hence recognition of the cation. We report here a chemosensor, namely 2,2′-(1E)-(5,5′-sulfonylbis(2-hydroxy-5,1-phenylene))bis(azan-1-yl-1-ylidene)bis(methan-1-yl-1-ylidene)dinaphthalen-1-ol (H4L-naph), for selective sensing of Al3+ in DMF- HEPES buffer (1:4, v/v, pH 7.4). It shows almost 100-fold fluorescence enhancement at 532 nm (λex = 482 nm) in the presence of Al3+. Its quantum yield and excited state lifetime enhances significantly with the cations. H4L-naph forms a 1:2 complex with Al3+ with an association constant value of 2.18 × 104 M−2. Fluorescence enhancement may be attributed to CHEFF mechanism and restriction of >CN isomerization. Effect of the presence of naphthyl rings instead phenyl ring of a previously reported probe has resulted shifting of excitation/emission peak towards longer wavelength. The probe has been applied to image Al3+ in L6 cells with no significant cytotoxicity.

A cyanobiphenyl-based fluorescent ''lighting-up'' sensor for highly selective and sensitive recognition of Al3+: Theoretical, practical and bioimaging studies.

The recognition of toxic Al3+ in foods and biosystems has of great interest to researchers. Herein, a novel cyanobiphenyl-based chemosensor CATH (E)-N'-((4'-cyano-4-hydroxy-[1,1'-biphenyl]-3-yl)methylene)thiophene-2-carbohydrazide was fabricated and shown to recognize Al3+ in HEPES buffer:EtOH (90:10, v:v, pH=7.4) by ''lighting-up'' fluorescence sensing. The CATH evidenced high sensitivity (LOD=13.1nM) and excellent selectivity to Al3+ over competing cations. The Job's plot, TOF-MS and theoretical computation studies were performed to probe the binding mechanism of Al3+ to CATH. Additionally; CATH was successfully utilized to practical applications and employed to recover of Al3+ from different food samples. More importantly, it was employed to intracellular Al3+ detection in living cells including THLE2 and HepG2.

Zwitterionic betaines over HEPES as the new generation biocompatible pH buffers for cell culture.

Good's buffers have been widely applied in cell/organ culture over the past half a century as biocompatible pH stabilizers. However, the emergence of severe adverse effects, such as cellular uptake, lysosomal autophagic activation, and visible light-induced cytotoxicity, raises serious questions over its biocompatibility while underlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) generates 1O2, ·OH, and O2 •- under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6μM of H2O2. Orders of magnitude elevated half-life of ROS in the medium caused severe cytotoxicity and systematic disorder of normal cell functions. We have further designed and validated zwitterionic betaines as the new generation biocompatible organic pH buffers, which is able to completely avoid the adverse effects that found on HEPES and derivate Good's buffers. These findings may also open a new avenue for zwitterionic betaine based materials for biomedical applications.

Iodide-Mediated Etching of Gold Nanostar for the Multicolor Visual Detection of Hydrogen Peroxide.

A multicolor visual method for the detection of hydrogen peroxide (H2O2) was reported based on the iodide-mediated surface etching of gold nanostar (AuNS). First, AuNS was prepared by a seed-mediated method in a HEPES buffer. AuNS shows two different LSPR absorbance bands at 736 nm and 550 nm, respectively. Multicolor was generated by iodide-mediated surface etching of AuNS in the presence of H2O2. Under the optimized conditions, the absorption peak Δλ had a good linear relationship with the concentration of H2O2 with a linear range from 0.67~66.67 μmol L-1, and the detection limit is 0.44 μmol L-1. It can be used to detect residual H2O2 in tap water samples. This method offered a promising visual method for point-of-care testing of H2O2-related biomarkers.

Adapted method for rapid detection and quantification of pathogen Campylobacter jejuni from environmental water samples.

Building on a previously developed workflow for rapid and sensitive pathogen detection by qPCR, this work has established a sample treatment strategy that produces consistent quantification efficiencies (QEs) for Campylobacter jejuni against a complex and highly variable sample matrix from a suburban river. The individual treatments most effective at minimizing the inhibitory effects of the sample matrix were pH buffering with HEPES (50mM, pH 5.7) and addition of the surfactant Tween 20 (2% v/v). Unexpectedly, sample acidification (pH 4-5) resulting from the use of aged Tween 20 that had undergone partial hydrolysis, appeared to play a key role in enhancing QE. This effect could be replicated by direct pH adjustment with dilute hydrochloric acid and may be linked to the solubilization and removal of inhibitory particles at an acidic pH. While the effectiveness of each individual treatment method varied, a combined treatment of either HEPES buffer + Tween 20, or direct pH adjustment + Tween 20, consistently produced QEs of 60%-70% and up to 100%, respectively, over a sampling period of one year. The consistency and scalability of this workflow make it a suitable alternative to culture-based ISO methods for detecting Campylobacter spp.

MOF-Fabric Composites Based on a Multi-Functional MOF as Luminescent Sensors for a Neurotransmitter and an Anti-Cancer Drug.

A biocompatible, reliable, fast, and nanomolar-level dual-functional sensor for a neurotransmitter (e.g., adrenaline) and an anti-cancer drug (e.g., 6-mercaptopurine (6-MP)) is still far away from the hand of modern-day researchers. To address this issue, we synthesized an aqua-stable, bio-friendly, thiourea-functionalized Zr(IV) metal-organic framework (MOF) for selective, rapid sensing of adrenaline and 6-MP with ultra-low limit of detection (LOD for adrenaline = 1.9 nM and LOD for 6-MP = 28 pM). This is the first MOF-based fluorescent sensor of both the targeted analytes. The sensor not only can detect adrenaline in HEPES buffer medium but also in different bio-fluids (e.g., human urine and blood serum) and pH media. It also exhibited 6-MP sensing ability in aqueous medium and in various wastewater specimens and pH solutions. For the quick and on-site detection of this neuro-messenger (adrenaline) and the drug (6-MP), cost-effective sensor-coated cotton fabric composites were fabricated. The MOF@cotton fabric composite is capable of detecting both the analytes up to the nanomolar level by the naked eye under UV light. The sensor can be recycled up to five times without significantly losing its efficiency. The Förster resonance energy transfer in the presence of adrenaline and inner-filter effect in the presence of 6-MP are the most likely reasons behind the quenching of the MOF's fluorescence intensity, which were proved with the help of appropriate instrumental techniques.

Terpyridine-Zn(II) based water-soluble fluorescent probes for selective detection of pyrophosphate ions in aqueous medium and its bioimaging application in living cells

Pyrophosphate (PPi) is a key biomarker for various cellular processes and human diseases and thus the development of receptors to bind selectively with PPi ions and facilitate the monitoring of intracellular PPi concentration in living cells is highly imperative. In the present study, novel water-soluble terpyridine-Zn(II) complexes-based fluorescent probes [TPD-Zn2]Cl4 and [TPTBr-Zn2]Cl4 have been designed and synthesized for the selective and sensitive detection of pyrophosphate (PPi) ions in aqueous environment as well as in living cells. The selectivity investigations of both the fluorescent probes towards different anions in HEPES buffer (pH = 7.4) showed highly selective binding towards PPi ions even in the presence of other concomitant nucleotides. Both the probes [TPD-Zn2]Cl4 and [TPTBr-Zn2]Cl4 have exhibited linear fluorescence enhancement upon the addition of different concentrations of PPi and this enhancement in the intensity could be attributed to the photoinduced electron transfer (PET) inhibition process. This proposed binding mechanism was demonstrated using fluorescence and NMR spectroscopic titrations and also the interaction of [TPD-Zn2]Cl4 and [TPTBr-Zn2]Cl4 probes with PPi ions was examined in detail using density functional theory (DFT) analysis. The Job’s plot has shown the binding of complexes between [TPD-Zn2]Cl4 and [TPTBr-Zn2]Cl4 and PPi in 1:2 stoichiometry and the probes have shown excellent binding affinity towards PPi ions with binding constants of 7.72 × 105 M−2 and 6.74 × 104 M−2, respectively. Noticeably, both the probes exhibited promising sensitivity towards PPi with detection limits as low as 0.47 nM and 0.17 nM, respectively. Cytotoxicity studies of [TPD-Zn2]Cl4 and [TPTBr-Zn2]Cl4 revealed that both the probes are biocompatible and subsequently their diagnostic efficacy as bioimaging probes was demonstrated through monitoring the intracellular PPi concentrations in breast cancer cells (MCF-7).

An optimization of blocking agents for designing reliable electrochemical biosensors for ovarian cancer

Here, we report development of an electrochemical biosensor for ovarian cancer diagnosis using an optimized blocking agent for reliable performance of the device. The sensitivity, specificity and reproducibility of the device are negatively impacted by non-specific binding of molecules present in sample - proteins, DNA, RNA, small drug molecules, hormones etc, other than the analyte of interest bind to biorecognition molecule on the biosensor surface. Electrochemical biosensor was fabricated by functionalizing carbon screen printed electrodes with citrate-reduced gold nanoparticles followed by immobilization of 5′-amine modified ssDNA probe against miRNA 204 (ovarian can biomarker). To avoid or minimize non-specific binding, blocking reagent was optimized. Blocking reagents and methods are typically chosen in an empirical manner since a single standardized procedure has not been determined suitable for all applications. This paper presents the commonly used blocking buffers with varying molecular weights to minimize the non-specific binding process on the surface of the electrode. Here, three different blocking buffers are used - Bovine serum albumin (BSA; MW: 66430.3 kDa), Gelatin (MW: 40 kDa), and Polyethylene Glycol (PEG MW 4 kDa and 6 kDa). Additionally, different surfactants like tween20, Tritonx100, and HEPES buffer are used for preparation of blocking agents. Further, to evaluate the performance of different blocking agents, the sample of miRNA-204 is spiked in 0.01 M Phosphate buffer saline (PBS) and fetal bovine serum (FBS) were tested using chroamperometry. The difference in the saturation current was recorded for both curves obtained for 0.01 M PBS and FBS. Optimum blocking buffer 1% Gelatin in Tween20 was found to be giving negligible nonspecific binding for DNA based ovarian cancer biosensor.

Clofazimine pKa Determination by Potentiometry and Spectrophotometry: Reverse Cosolvent Dependence as an Indicator of the Presence of Dimers in Aqueous Solutions.

The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. However, the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosolvent to approximate the true aqueous pKa as 9.43 ± 0.12 (25 °C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric UV/vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosolvent UV/vis data in our study showed reverse cosolvent dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 μM CFZ yielded the apparent pKa 9.51 ± 0.17 (I ≈ 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed. Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.

High-Efficiency Aluminum-Metal Organic Framework/HEPES Electrochemiluminescence System for Ultrasensitive Detection of HBV DNA.

In this work, a novel aluminum metal-organic framework (Al-MOF)/N-2-hydroxyethylpiperazine-N'-ethane-sulfonic acid (HEPES) system with an excellent electrochemiluminescence (ECL) property was developed. First, Al-MOF was successfully synthesized through a one-pot solvothermal method by using 9,10-di(p-carboxyphenyl)anthracene (DPA) as the organic luminescence ligand and Al3+ as the metal node. Compared with DPA, Al-MOF showed high ECL intensity and excellent stability without an additional coreactant in the HEPES buffer. The corresponding ECL mechanism was studied in detail, verifying HEPES was not only the buffer in the system but also the coreactant of Al-MOF. In particular, the system of Al-MOF/HEPES showed a high ECL efficiency of 30.0%, taking the Ru(bpy)32+ system as the standard. In addition, the ECL signal of Al-MOF was effectively quenched by dopamine (DA). The biosensor for HBV DNA detection was constructed through the ECL signal on-off-on mode of DNA specific recognition integrated with the DNA walker signal amplification strategy. The ultrasensitive detection for HBV DNA was achieved with a linear range of 100 aM to 10 pM and a limit of detection (LOD) of 62.1 aM. This work proposed a high-efficiency Al-MOF/HEPES system, providing a new viewpoint for a coreactant-free system in the field of ECL.

Nanomolar pyrophosphate detection in water using a zinc-terpyridine receptor and its applications in antiproliferative and antioxidant activity

A new fluorescent 4′-(3,4-diethoxyphenyl)-2,2′:6′,2′'-terpyridine ligand L and its 1:1 zinc complex ZnCl2L have been synthesized, characterized with the help of NMR, Mass spectroscopy, CHN analysis, and DFT study. In-situ prepared non-fluorescent ZnCl2L receptor was utilized for selective detection of pyrophosphate anion (P2O74-, PPi) in water (10 mM HEPES buffer, pH 7.4) in the vicinity of other competing analytes (10 equivalents of HPO42-, H2PO4-, CO32–, HCO3–, Cl−, OH–, Br−, CH3CO2–, F−, I-, NO3–, SO42-, SO32-, S2O32-, N3–) by employing UV–Visible spectroscopy and fluorescence “ON–OFF–ON” mechanism. ZnCl2L receptor formed a fluorescent 2:1 adduct with PPi in water during its detection at a nanomolar concentration (LOD ∼27.89 nM). The receptor is also successfully employed in low-cost PPi detection through real-time easily accessible paper strip application. Further, ZnCl2L exhibited significant cytotoxic activity against MDAMB-231 cells with an IC50 value of 53.85 µM and excellent antioxidant activity having an antioxidant percentage of 73.75%.

An AIE mechanism-based fluorescent probe for relay recognition of HSO3−/H2O2 and its application in food detection and bioimaging

In view of the important physiological role of HSO3− and H2O2, it is of great significance to develop fluorescent probes to detect HSO3− and H2O2 in aqueous medium. We herein report a new fluorescent probe (E)-3-(2-(4-(1,2,2-triphenylvinyl)styryl)benzo [d]thiazol-3-ium-3-yl)propane-1-sulfonate (TPE-y) possessing benzothiazolium salt based on tetraphenylethene (TPE) moiety with aggregation-induced emission (AIE) characteristics. TPE-y can sequentially recognize HSO3− and H2O2 through colorimetric and fluorescence dual-channel response in HEPES (pH = 7.4, 1% DMSO) buffer solution, and exhibits high sensitivity and selectivity, a large Stokes shift (189 nm), as well as a wide applicable pH range. The detection limits of TPE-y and TPE-y-HSO3 for HSO3− and H2O2 are 3.52 μM and 0.15 μM, respectively. The recognition mechanism is verified by 1H NMR and HRMS methods. Furthermore, TPE-y can detect HSO3− in sugar samples, and can image exogenous HSO3− and H2O2 in living MCF-7 cells. TPE-y can relay detect HSO3− and H2O2, which is of great significance to maintain the redox balance in organisms.

Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

Chemotaxis and Motility of Spongospora subterranea Zoospores in Response to Potato Root Exudate Constituents and pH.

Spongospora subterranea f. sp. subterranea is an important pathogen of potato responsible for major losses in most potato growing regions of the world. Infection is initiated by biflagellated motile zoospores released from long-lived resting spores. Zoospore chemotaxis to the host plant root is widely believed to be stimulated by host root exudate compounds, although direct evidence is lacking. This study refined the traditional chemotaxis capillary assay, with which we provided the first empirical evidence of S. subterranea zoospore chemotaxis. Individual potato root exudate metabolites were either taxis neutral, inhibitory, or attractant to the zoospores. L-Glutamine was the strongest chemoattractant, while spermine was the most inhibitory. Zoospore motility and chemotaxis were constrained by strongly acidic or alkaline solutions of pH < 5.3 and >8.5, respectively. Beyond pH, ionic constituents of the test solution affected zoospore motility as Sorensen's phosphate buffer stalled zoospore motility, but HEPES buffer at the same concentration and pH had little or no negative motility effect. Zoospore motility, as characterized by several parameters, influenced chemotaxis. Among the parameters measured, total distance traveled was the best predictor of zoospore chemotaxis. The characterization of environmental and ecological effects on zoospore motility and chemotaxis highlights useful targets for S. subterranea disease control through manipulation of zoospore taxis or selection of host resistance traits.

Inactivation of Lactobacillus brevis cells and Bacillus cereus spores as influenced by pressure, shear, thermal, and valve geometry

ABSTRACT This study investigated the influence of pressure, shear, temperature, and their interactions on the inactivation of two bacteria – Lactobacillus brevis and Bacillus cereus spores. Cell suspension of L. brevis (1.6 × 1010 CFU/mL) and spore suspension of B. cereus (3.2 × 108 CFU/mL) were suspended in HEPES buffer (pH 7.3) and subjected to ultra-shear treatment (UST) at 400 MPa, process temperatures of 40 and 70°C, and flow rate of 0.0012 ± 0.0003 kg/s. In separate equipment, thermal (0.1 MPa-70°C-0/5 min) and high-pressure processing (HPP-400 MPa-40°C and 70°C-0/5 min) experiments were carried out to evaluate thermal only, pressure only, and combined pressure-thermal effects. Pressure-only treatment at 400 MPa for 0 min (come-up time) at 70°C resulted in 8.4 and 2.3 log reductions of L. brevis cells and B. cereus spores, respectively. After UST treatment at 70°C, L. brevis and B. cereus spores were reduced by 7.1 and 1.6 logs, respectively. Both strains showed different sensitivities to thermal, pressure, shear, and their combinations. B. cereus spores were relatively more resistant to HPP and UST treatments. Three valve geometries, namely, ultra-shear valve, needle valve, and tubular valve, were used to compare UST treatment effect on L. brevis and B. cereus spores at 40 and 70°C. UST treatment using the ultra-shear valve produced inactivation of 2.0 and 7.1 log cycles for L. brevis during 40 and 70°C process temperatures, respectively, which was relatively higher than needle valve and tubular valve. This higher inactivation could be attributed to the smaller valve gap and higher shear rate in the ultra-shear valve. Further, the comparatively higher inactivation at 70°C UST (7.1 log) emphasized the role of temperature on inactivation during UST. For both L. brevis and B. cereus, the inactivation was least in the tubular valve with relatively lesser shear rate, which indicated the desirable contribution of high shear rate for microbial inactivation. The information from this study contributes to the development of safe harbor UST process conditions for liquid foods.

Fluorescence Sensing of pH and p-Nitrophenol Using an AIEE Active Pyridoxal Derived Schiff Base.

An easy-to-prepare aggregation-induced emission enhancement (AIEE) active Schiff base NPY was synthesized by condensing vitamin B6 cofactor pyridoxal with 3-hydroxy-2-naphthoic hydrazide, and employed for the fluorescent sensing of pH and p-nitrophenol (p-NP). The AIEE phenomenon of NPY was investigated in mixed DMSO/H2O medium. The weakly yellow-fluorescent NPY (λem = 535nm) in pure DMSO turned to a bright cyan-fluorescent NPY (λem = 490nm) upon addition of poor solvent water. The DLS and SEM analyses supported the self-aggregation of NPY that restricted the intramolecular rotation and activated the excited state intramolecular proton transfer (ESIPT) process. The AIEE luminogen (AIEEgen) NPY containing 90% of water fraction (fwater) was employed for the fluorescent sensing of pH. AIEEgen NPY displays three distinct fluorescent pH windows: non-fluorescent below pH 3.0 and above pH 10.0, cyan fluorescent between pH 3.0 to 8.0, and yellow fluorescent between pH 8.0 to 10.0. AIEEgen NPY was also applied for the detection of nitroaromatics in HEPES buffer (10% DMSO, 10 mM, pH 7.0). The addition of p-NP selectively quenched the fluorescent intensity of AIEEgen NPY with an estimated detection limit of 1.73 µM. The analytical utility of AIEEgen NPY was examined by quantifying p-NP in different real water samples.

A simple paper-based nickel nanocluster-europium mixed ratio fluorescent probe for rapid visual sensing of tetracyclines

In this work, a ratiometric fluorometric sensor based on nickel nanoclusters (NiNCs)-europium complex (NiNCs-Eu3+) was constructed for the highly selectivity detection of tetracyclines (TCs) in water samples. In the presence of TCs, the blue fluorescence of the sensor NiNCs-Eu3+ was quenched at 430 nm and the characteristic red fluorescence of Eu3+-TCs appeared at 620 nm because of the combined help of inner filter effect (IFE) and antenna effect. Under the optimized conditions (100 mM Eu3+ (100 µL); temperature (25℃); reaction time (10 min), HEPES buffer solution (pH = 7.0)), the sensor offered a wide detection range of tetracycline (TC) and oxytetracycline (OTC) from 0.1 to 50 μM with the detection limit (LOD) of 25 nM and 21 nM, respectively. Moreover, the sensor was able to detect of TC and OTC in tap and lake water with high recovery rate (89.10%-97.60%). In addition, the portable paper-based sensor was constructed using filter paper embedded with NiNCs-Eu3+. The distinct fluorescent color of the paper-based sensor varied from bright blue to red against different concentrations of TC and OTC. These above findings demonstrated the potential for wide application of as-prepared ratio metric fluorescence sensor for visual detection of TCs in water samples.

A Signal Amplification Strategy Using ATP as a Co‐Reaction Accelerator for the Electrochemiluminescence of Ru(bpy)32+/HEPES System and Detection of Iodide Anions**

AbstractAdenosine triphosphate is an important biological molecule that provides energy and substrate for various cellular biochemical processes,it's ubiquitous in our lives. In this work, a novel electrochemiluminescence (ECL) sensor composed of Ru(bpy)32+, 2‐[4‐(2‐hydroxyethyl)piperazin‐1‐yl] ethanesulfonic acid (HEPES) and adenosine 5’‐triphosphate disodium salt (ATP) was developed for the first time. In this ECL system, Ru(bpy)32+ was used as the luminophore, HEPES buffer was served as the co‐reactant, and ATP was added to the system to amplify the luminous signal to investigate the ECL behavior. Through fluorescence spectroscopy, ECL spectroscopy, and cyclic voltammetry (CV), it can be hypothesized that the possible ECL mechanism is that ATP acts as an advanced co‐reaction promoter to accelerate the oxidation of HEPES to HEPES+⋅, more HEPES⋅ can be generated, which then reacts with oxidized Ru(bpy)33+ to emit stronger ECL signals. Subsequently, the influence of the ATP concentrations and the CV scanning speed was investaged. Moreover, it was observed that I− could quench the ECL intensity, the linear relationship range of I− concentration is from 10−3 to 10−6 m, the limit of detection is 3.7×10−8 m (signal/noise=3), therefore, a new, highly sensitive, and selective ECL approach for I− detection was created.

Assessment of the interactions between pea and salivary proteins in aqueous dispersions

To understand and limit the unpleasant oral sensation of astringency felt during the consumption of pea-based drinks, we investigated the interaction between mixtures of salivary and pea proteins, as compared to mixtures where HEPES buffer (at pH 6.8) was used as a negative control for saliva. Since astringent compounds have the ability to bind with salivary proteins, mixes of freshly collected whole unstimulated saliva and a pea protein isolate (PPI) (a dispersion at 3.5% w/v) were prepared in ratios 95:5 and 1:1 saliva:PPI, to allow different stoichiometries to occur in the mouth. Samples were incubated at 37 °C during 30 min, after centrifugation at 16000g during 20 min to separate pellet from supernatant. Using techniques such as SEC, Native-PAGE and LC-MS, 7 pea proteins were identified as being capable of forming aggregates with at least 7 saliva proteins, some of which have been previously connected to astringency.

Oxidative damage to βL-crystallin in vitro by iron compounds formed in physiological buffers

βL-crystallin aggregation due to oxidative damage in the presence of H2O2 and ferric chloride was studied in-vitro under conditions close to physiological. It was shown that the protein aggregation characterized by the nucleation time and the aggregation rate significantly depended on the composition of the isoosmotic buffers used, and decreased in the series HEPES buffer > Tris buffer > PBS. Ferric chloride at neutral pH was converted into water-insoluble iron hydroxide III (≡FeIIIOH). According to the data of scanning electron microscopy the ≡FeIIIOH particles formed in HEPES buffer, Tris buffer, and PBS practically did not differ in structure. However, the sizes of ≡FeIIIOH floating particles measured by dynamic light scattering differed significantly and were 44 ± 28 nm, 93 ± 66 nm, 433 ± 316 nm (Zaver ± SD) for HEPES buffer, Tris buffer, and PBS, respectively. It was found by the spin trap method that the ability of ≡FeIIIOH to decompose H2O2 with the formation of a •OH decreases in the series HEPES buffer, Tris buffer, and PBS. The authors suggest that the ability to generate •OH during the decomposition of H2O2 is determined by the total surface area of ≡FeIIIOH particles, which significantly depends on the composition of the buffer in which these particles are formed.