A sensitive electrochemiluminescent sensor chip based on the ssDNA-Ru(II) complex and aptamer for the determination of thrombin

In this work, an electrochemiluminescence (ECL) sensor chip for sensitive detection of thrombin (TB) was prepared using a screen-printed electrode (SPE) as a working electrode and an aptamer as a specific recognition moiety. To produce an ECL sensor chip, a layer of pL-Cys was immobilized on the surface of the SPE using the cyclic voltammetry scanning method. A layer of gold nanoparticles (AuNPs) was assembled through an Au–S bond and hairpin DNA was further immobilized on the electrode surface. Ru(bpy)₂(mcpbpy)²⁺, as a luminescent reagent, was covalently bound to single-stranded DNA (ssDNA) to prepare a luminescence probe ssDNA-Ru. The probe was hybridized with TB aptamer to form a capture probe. In the presence of TB, the TB aptamer in the capture probe bound to TB, causing the release of ssDNA-Ru that could bind to hairpin DNA on the electrode surface. The Ru(II) complex as a luminescent reagent was assembled onto the electrode, and pL-Cys was used as a co-reactant to enhance the ECL efficiency. The ECL signal of the sensor chip generated based on the above principles had a linear relationship with log TB concentration at the range 10 fM to1 nM, and the detection limit was 0.2 fM. Finally, TB detection using this method was verified using real blood samples. This work provides a new method using an aptamer as a foundation and SPE as a material for the detection of biological substances.     

TiO(2) nanocrystals electrochemiluminescence quenching by biological enlarged nanogold particles and its application for biosensing

Electrochemiluminescence (ECL) of TiO(2) nanocrystals with different crystal styles modified fluorine-doped tin oxide (FTO) electrode was investigated in H(2)O(2) solution. The amorphous TiO(2) nanospheres were facilely synthesized by the hydrothermal and condensation method. Crystal TiO(2), namely anatase and rutile, were prepared by calcination of the amorphous TiO(2) nanospheres at 450 and 800°C, respectively. The transmission electron microscope (TEM) and electron diffraction pattern were used to characterize the obtained TiO(2) nanoparticles morphology and the corresponding crystal styles. The electrochemical and ECL behaviors were investigated by cyclic voltammetry. The ECL quenching was observed by introduction of gold nanoparticles. Based on the quenching effect, a sensitive glucose ECL biosensor as a model was fabricated by in-situ growing-up gold seeds in AuCl(4)(-) solution induced by biologically generated H(2)O(2). The linear range to detect glucose is from 5.0×10(-7)M to 4.0×10(-3)M with the limit of detection of 2.5×10(-7)M.     

The oxidizing power of the glutathione thiyl radical as measured by its electrode potential at physiological pH

The oxidizing power of the thiyl radical (GS*) produced on oxidation of glutathione (GSH) was determined as the mid-point electrode potential (reduction potential) of the one-electron couple E(m)(GS*,H+/GSH) in water, as a function of pH over the physiological range. The method involved measuring the equilibrium constants for electron-transfer equilibria with aniline or phenothiazine redox indicators of known electrode potential. Thiyl and indicator radicals were generated in microseconds by pulse radiolysis, and the position of equilibrium measured by fast kinetic spectrophotometry. The electrode potential E(m)(GS*,H+/GSH) showed the expected decrease by approximately 0.06 V/pH as pH was increased from approximately 6 to 8, reflecting thiol/thiolate dissociation and yielding a value of the reduction potential of GS*=0.92+/-0.03 V at pH 7.4. An apparently almost invariant potential between pH approximately 3 and 6, with potentials significantly lower than expected, is ascribed at least in part to errors arising from radical decay during the approach to the redox equilibrium and slow electron transfer of thiol compared to thiolate.     

A novel pH sensitive water soluble fluorescent nanomicellar sensor for potential biomedical applications

Herein we report on the synthesis and sensor activity of a novel pH sensitive probe designed as highly water-soluble fluorescent micelles by grafting of 1,8-naphthalimide–rhodamine bichromophoric FRET system (RNI) to the PMMA block of a well-defined amphiphilic diblock copolymer—poly(methyl methacrylate)–b-poly(methacrylic acid) (PMMA₄₈–b-PMAA₂₇). The RNI-PMMA₄₈–b-PMAA₂₇ adduct is capable of self-assembling into micelles with a hydrophobic PMMA core, containing the anchored fluorescent probe, and a hydrophilic shell composed of PMAA block. Novel fluorescent micelles are able to serve as a highly sensitive pH probe in water and to internalize successfully HeLa and HEK cells. Furthermore, they showed cell specificity and significantly higher photostability than that of a pure organic dye label such as BODIPY. The valuable properties of the newly prepared fluorescent micelles indicate the high potential of the probe for future biological and biomedical applications.     

Effects of external pH,fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by 31P-nuclear magnetic resonance spectroscopy

³¹P-Nuclear magnetic resonance spectroscopy was used to measure the cytoplasmic pH (pH_c) in barley (Hordeum vulgare L.) root tips. As the external pH was raised from 4–10, pH_c was found to increase from 7.44 to 7.75. The sensitivity of pH_c to changes in external pH decreased with increasing external pH. Metabolic inhibition by sodium azide caused pH_c to fall by 0.3 units. Addition of 10 mM butyrate resulted in a gradual decline in pH_c, by approx. 0.3 units over 90 min. At a concentration of 1 mM, butyrate had no effect on pH_c even after 2 h. Fusicoccin caused pH_c to rise by 0.1–0.2 units. In maize (Zea mays L.) root tips, pH_c was shown to have a similar sensitivity to fusicoccin. The results are discussed in relation to the regulation of pH_c and the possible role of pH_c in determining transmembrane electrical potential differences.Abbreviations and symbols FC Fusicoccin - NMR nuclear magnetic resonance - p.d. membrane electrical potential difference - pH_c cytoplasmic pH - P1 inorganic phosphate - chemical shift     

Solution‐Processed TiO2 Nanoparticles as the Window Layer for CuIn(S,Se)2 Devices

As a wide‐bandgap semiconductor, titanium dioxide (TiO₂) with a porous structure has proven useful in dye‐sensitized solar cells, but its application in low‐cost, high‐efficiency inorganic photovoltaic devices based on materials such as Cu(InGa)Se₂ or Cu₂ZnSnS₄ is limited. Here, a thin film made from solution‐processed TiO₂ nanocrystals is demonstrated as an alternative to intrinsic zinc oxide (i‐ZnO) as the window layer of CuInS_xSe_1?x solar cells. The as‐synthesized, well‐dispersed, 6 nm TiO₂ nanocrystals are assembled into thin films with controllable thicknesses of 40, 80, and 160 nm. The TiO₂ nanocrystal films with thicknesses of 40 and 80 nm exhibit conversion efficiencies (6.2% and 6.33%, respectively) that are comparable to that of a layer of the typical sputtered i‐ZnO (6.42%). The conversion efficiency of the devices with a TiO₂ thickness of 160 nm decreases to 2.2%, owing to the large series resistance. A 9‐hour reaction time leads to aggregated nanoparticles with a much‐lower efficiency (2%) than that of the well‐dispersed TiO₂ nanoparticles prepared using a 15‐hour reaction time. Under optimized conditions, the champion TiO₂ nanocrystal‐film‐based device shows even higher efficiency (9.2%) than a control device employing a typical i‐ZnO film (8.6%).     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138-624 nmol l^-1 EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC-H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC-pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

Membrane potential,pH and the activation of surf clam oocytes

Unfertilized oocytes of the surf clam, Spisula solidissima, have resting membrane potentials of ?18 ± 7 mV (n = 20). Within five seconds of sperm addition, an electrophysiologically detectable response was apparent, which was characterized by a rapid and prolonged depolarrization depolarization followed four to five minutes post-insemination by the beginning of the beginning of a steady hyperpolarization to approximatelv ?70 mV. This final hyperpolarization was completed within ten minutes of sperm addition. The initial rapid depolarization following insemination may result from a transient increase in sodium conductance, and it may be crucial in preventing polyspermy, since the degree of polyspermy in Spisula oocytes was sensitive to external sodium ion concentrations. Evidence was obtained that changes in intracellular pH are essential for oocvte activation. Using germinal vesical breakdown (GVB) as a marker for activation, it was shown that agents that raise intracellular pH (ammonia and procaine) induced GVB, whereas agents that lower intracellular pH pH (Na-acetate or Na-propionate seawater) inhibited GVB.     

A Facile Method to Improve the Photocatalytic and Lithium‐Ion Rechargeable Battery Performance of TiO2 Nanocrystals

TiO₂ has been well studied as an ultraviolet (UV) photocatalyst and electrode material for lithium‐ion rechargeable batteries. Recent studies have shown that hydrogenated TiO₂ displayed better photocatalytic and lithium ion battery performances. Here it is demonstrated that the photocatalytic and battery performances of TiO₂ nanocrystals can be successfully improved with a facile low‐temperature vacuum process. These TiO₂ nanocrystals extend their optical absorption far into the visible‐light region, display nanometer‐scale surface atomic rearrangement, possess superoxide ion characteristics at room temperature without light irradiation, show a 4‐fold improvement in photocatalytic activity, and has 30% better performance in capacity and charge/discharge rates for lithium ion battery. This facile method could provide an alternative and effective approach to improve the performance of TiO₂ and other materials towards their practical applications.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Influence of the pH and redox potential on phosphate activity in the Parana Medio system

The effect of redox potential and pH on the phosphate mobility in two sediments were investigated using both consolidated and suspended sediments from the area where the Parana Medio long reservoir (Atgentina) is to be built (Smirnov, 1984). In addition to direct chemical sediment analysis, extraction techniques were carried out with a stepwise NH₄Cl-NaOH-HCl shaking method, the latter supposedly separating the weakly bound, the Fe- and Al- bound and the Ca- bound phosphates in the sediments.Phosphate released into water depends upon redox potential and pH, which both were modified in an experimental setup. The source of the phosphate was the fraction of Fe and/or Al bound phosphate present both in the sediment and in the suspended solids.Abbreviations cm centimeter - km kilometer - gg gram - l liter - ¬m micrometer - °C grade centigrades - km² square kilometer - m.s^–1 meter per second - m³.s^–1 cubic meter per second - mg.1¹ miligram per liter     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

温度、pH及CO2对白藜芦醇与过氧亚硝基相互作用的影响

白藜芦醇(Resveratrol,Res)是植物在遇到紫外线照射、真菌感染等不利条件下自然产生的抗毒素,存在于多种植物中,具有明显的消炎、抗癌、抗血栓等作用。本文利用紫外-可见光谱法研究了Res与过氧亚硝基阴离子(Peroxynitrite anion,ONOO-)的相互作用,提出了反应机理。研究了温度、pH及CO2对Res与ONOO-反应的影响,结果表明低温、偏碱性pH有利于反应的进行;CO2存在时Res仍能与ONOO-反应,但反应机理与前面不同。     

Time-resolved chemiluminescence study of the TiO2 photocatalytic reaction and its induced active oxygen species.

The time-resolved chemiluminescence (CL) method has been applied to study the TiO(2) photocatalytic reaction on a micros-ms timescale. The experimental set-up for time-resolved CL was improved for confirmation of the unique luminol CL induced by the photocatalytic reaction. The third harmonic light (355 nm) from an Nd:YAG laser was used for the light source of the TiO(2) photocatalytic reaction. Luminol CL induced by this reaction was detected by a photomultiplier tube (PMT) and a preamplifier was used for amplifying the CL signal. Experimental conditions affecting the photocatalytically induced CL were discussed in detail. The involvement of active oxygen species such as .OH, O(2) (.-) and H(2)O(2) in the CL was examined by adding their scavengers. It is concluded that .OH was greatly involved in the CL on a micros-ms timescale, especially in time periods <100 micros after illumination of the pulse laser. On the other hand, CL generated by O(2) (.-) began to increase after 100 micros and became dominant after 2.5 ms. A small part of the CL might be generated by H(2)O(2) on the whole micros-ms timescale. A CL reaction mechanism related with .OH and dissolved oxygen was proposed to explain the photocatalytically induced luminol CL on a micros-ms timescale, especially in periods <100 micros.     

温度、pH对非光合微生物菌群固碳效率的影响及其成因分析

CO2是主要的温室气体,也是地球上最丰富的碳源和可再生资源,本实验通过研究温度和pH对非光合固碳微生物固碳效率的影响,以期获得非光合固碳微生物适宜的固碳条件,并通过16SrDNA序列分析,研究了不同条件对固碳微生物群落结构的影响及其与固碳效率的相关性。结果表明,好氧／厌氧固碳微生物菌群生长的最适pH均在7左右。好氧固碳微生物菌群可在较大的温度范围（10℃-40℃）内良好生长,但其固碳速率在10℃左右时最高,之后微生物固碳速率随温度上升而下降,在40℃时,微生物固碳速率开始回升。厌氧时微生物的固碳效率随温度的变化情况与好氧时相似,但总体效果要比其在好氧时低20％～25％。由DGGE图谱及戴斯系数分析可得培养温度和pH对群落结构有显著影响,不同pH和温度条件下优势菌差异显著,这可能是固碳效率变化的重要原因之一。     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na⁺ and K⁺ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl₂ and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na⁺ and K⁺ without significantly altering the ratio of pumped $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between ?10 and +60 mV at constant internal pH did not affect the rates of active transport of Na⁺ or K⁺.     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)