紫外吸光光度法测定水源水中硝酸盐

氮的化合物 (主要包括氨态氮、亚硝酸盐氮、硝酸盐氮 )是饮用水水质评价的重要卫生指标之一。准确测定水源水中各种氮化合物含量 ,对于水质正确评价和是否适用于饮用有着重要意义。NO- 3 的测定 ,常用二酚磺酸法和镉柱还原法。前法因 Cl-严重干扰需脱氯处理 ,后法也繁琐费时。利用 NO- 3 在紫外区的吸收光谱特性对其进行定量测定 ,已有不少文献报道。本文对水源水中 NO- 3 的测定做了有关条件试验 ,结果较为理想。1　试验部分1 .1　仪器与试剂751 G紫外分光光度计NO- 3 标准溶液 :0 .1 0 0 0 mg.ml- 1 ,称取在 1 0 5～1 1 0°C干燥的硝…     

海藻酸钠水凝胶固定化血红蛋白的电化学性质

用海藻酸钠(SA)将血红蛋白(Hb)固定在热裂解石墨电极表面，制备了Hb—SA膜修饰电极。包埋在SA膜中的血红蛋白与电极直接传递电子。在pH7．0的磷酸盐缓冲溶液和磷酸盐／乙醇混合溶液中均可得到一对可逆氧化还原峰，这是血红蛋白辅基血红素Fe^3 ／Fe^2 电对的氧化还原。电势随溶液pH值增加而负移且呈线性关系，直线斜率为-41．0mV／pH，说明血红蛋白的电子传递过程伴随有质子的转移。并研究了Hb—SA膜修饰电极对O2、H2O2、NO和六氯乙烷(HCE)的电催化性质。     

血红蛋白在海藻酸钠膜中的电化学和电催化行为

用海藻酸钠(SodiumAlginate,SA)将血红蛋白(Hb)固定在热裂解石墨电极表面,制备了Hb SA膜修饰电极。包埋在海藻酸钠膜中的血红蛋白与电极直接传递电子。在pH7.0的磷酸盐缓冲溶液中可得到一对可逆的血红蛋白辅基血红素Fe(Ⅲ) Fe(Ⅱ)电对氧化还原峰,式电势为-0.364V(vs.SCE)。其式电势随溶液pH值增加而负移且成线性关系,直线斜率为-36.0mV pH,说明血红蛋白的电子传递过程伴随有质子的转移。并研究了Hb SA膜修饰电极对O2、H2O2和NO的电催化性质。     

血红蛋白在溶胶-凝胶纳米银修饰电极上的直接电化学

运用溶胶-凝胶技术将血红蛋白(Hb)固定于纳米银(AgNPs)修饰的玻碳电极(GCE)表面,制得溶胶-凝胶血红蛋白纳米银修饰电极(Sol-Gel/Hb/AgNPs/GCE)。优化了修饰电极的制备条件,研究了该修饰电极在B-R缓冲溶液(pH=4.10)中的电化学行为,探讨了Hb在AgNPs修饰电极表面的直接电子转移机理。结果表明:AgNPs不仅保持了Hb的生物活性,而且通过它的催化效应,实现了Hb与电极之间的直接电子转移。进一步的实验结果显示,固定在纳米银修饰电极表面的Hb能保持其对H2O2的生物电催化活性。     

分光光度法测定纯净水中亚硝酸盐含量

硝酸盐广泛存在于自然界中,在食物及饮用水中都含有一定量的硝酸盐。硝酸盐在硝酸盐还原酶的作用下产生亚硝酸盐;当摄入大量亚硝酸盐时,可使血红蛋白氧化成高铁血红蛋白,失去输氧能力,引起肠原性青紫症,而且亚硝酸盐与食品中固有的胺类化合物均能产生致癌的亚硝胺[1]。国家标准GB17324-2003[2]明确规定了饮用水中亚硝酸盐的限     

基于聚亚甲基蓝-碳纳米管复合膜固载血红蛋白的H2O2生物传感器

采用吸附和电化学聚合修饰方法,制得了聚亚甲基蓝-碳纳米管聚合膜玻碳电极(PMB-MWNTs/GCE),再将血红蛋白(Hb)固定在PMB-MwNTs/GCE表面,制备了稳定的Hb/PMB-MwNTs//GCE的H2O2生物传感器,并用循环伏安法对修饰电极的生物电催化行为进行了表征.研究结果表明,固定在PMB-MWNTs/...     

混合表面活性剂增效快速测定食品中亚硝酸盐含量

亚硝酸盐作为一种食品添加剂,具有一定的防腐性,同时还是一种发色剂,能够保持腌肉制品等色香风味,但同时也具有较强的致癌作用,过量食用会对人体产生危害。因此,我国规定肉制品和肉罐头的亚硝酸盐添加量不得超过150mg·kg-1,容许含量不得超过50mg·kg-1[1]。常见测定NO-2的方法为偶氮染料显色光度法[2]。经试验发现在稀盐酸介质中,NO-2与对氨基苯磺酸发生重氮反应,加NaOAc中和至pH5.2时,重氮化合物又与甲萘胺偶联成红色偶氮染料,其结构式如下:反应灵敏度较高[3],但染料分子中含有双键(-N=N-),易被日光、空气和氧化剂所分解,在水溶液…     

痕量亚硝酸盐的新分光光度测定方法

当亚硝酸盐与水中其他形态的氮相联系时,可以做为有机污染指标,在水质监测中它常列为重点分析项目之一。亚硝酸盐的测定,应用最广的是重氮偶合比色法,这些方法虽然有较高的灵敏度和选择性,但也受到某些离子如铜(Ⅱ),铁(Ⅲ),硫和碘离子的干扰,并且试剂都是毒性大的致癌物,有害于人体健康。近来,Gabby等人用间苯二酚在酸性条件下与亚硝酸盐反应,生成亚硝基产物再与氧锆基离子形成苍黄色螯合物,用于水中亚硝酸盐的测定,但灵敏度较低。我们在研究酚类的Ehrlish偶氮反应中,发现亚硝酸盐在酸性条件下与对氨基苯乙酮起     

基于血红蛋白、血红素的亚硝酸盐传感器的构建

构建了包埋于壳聚糖(CS)膜中的血红蛋白(Hb)和血红素(Hemin)的亚硝酸盐传感器并研究了它们在电极表面的电化学活性。在pH 4.0磷酸盐缓冲溶液中,利用示差脉冲伏安法分别研究CS/Hb-GCE、CS/Hemin-GCE对亚硝酸盐的电化学响应,基于此建立了对亚硝酸盐的电化学测定方法;结果表明,两种电极对亚硝酸盐响应的线性范围分别为0.069~25.86和0.50~16.67mmol/L,检测限分别为0.012和0.25mmol/L,而且对前者具有更低的检测限和更宽的线性范围的可能原因进行了探讨。     

琼脂糖水凝胶固定化血红素蛋白质的电化学研究

利用琼脂糖（agarose）水凝胶将肌红蛋白（Mb）、血红蛋白（Hb）、辣根过氧化物酶（HRP）和过氧化氢酶（Cat）4种血红素蛋白质固定在裂解石墨电极表面，形成稳定的血红素蛋白质-agarose膜修饰电极。在agarose膜中，Mb、Hb、HRP和Cat直接与电极传递电子。4种血红素蛋白质的式电势都随溶液pH的增加而负移且呈线性关系，表明电子传递过程伴随着质子转移。     

Density functional theory modeling of the proposed nitrite anhydrase function of hemoglobin in hypoxia sensing

The role of NO and nitrite-bound methemoglobin (Hb(III)NO(2)(-)) in hypoxic signaling is highly controversial. One provoking possibility is that hemoglobin (Hb) functions as a nitrite anhydrase, producing N(2)O(3) (from nitrite) as an NO carrier. The ability of Hb to generate N(2)O(3) would provide an intriguing means of NO release from red blood cells. We have investigated this proposed new reactivity of Hb using density functional theory (DFT) calculations. For this purpose, models of the Hb/myoglobin (Mb) active site have been constructed. Our results show that the O-bound (nitrito) form of Hb/Mb(III)NO(2)(-) is essential for the formation of N(2)O(3). The formation and release of N(2)O(3) is shown to be energetically favorable by 1-3 kcal/mol, indicating that the anhydrase function of Hb/Mb is biologically feasible.     

A new paramagnetic intermediate formed during the reaction of nitrite with deoxyhemoglobin

The reduction of nitrite by deoxygenated hemoglobin chains has been implicated in red cell-induced vasodilation, although the mechanism for this process has not been established. We have previously demonstrated that the reaction of nitrite with deoxyhemoglobin produces a hybrid intermediate with properties of Hb(II)NO(+) and Hb(III)NO that builds up during the reaction retaining potential NO bioactivity. To explain the unexpected stability of this intermediate, which prevents NO release from the Hb(III)NO component, we had implicated the transfer of an electron from the β-93 thiol to NO(+) producing ·SHb(II)NO. To determine if this species is formed and to characterize its properties, we have investigated the electron paramagnetic resonance (EPR) changes taking place during the nitrite reaction. The EPR effects of blocking the thiol group with N-ethyl-maleimide and using carboxypeptidase-A to stabilize the R-quaternary conformation have demonstrated that ·SHb(II)NO is formed and that it has the EPR spectrum expected for NO bound to the heme in the β-chain plus that of a thiyl radical. This new NO-related paramagnetic species is in equilibrium with the hybrid intermediate "Hb(II)NO(+) ? Hb(III)NO", thereby further inhibiting the release of NO from Hb(III)NO. The formation of an NO-related paramagnetic species other than the tightly bound NO in Hb(II)NO was also confirmed by a decrease in the EPR signal by -20 °C incubation, which shifts the equilibrium back to the "Hb(II)NO(+) ? Hb(III)NO" intermediate. This previously unrecognized NO hemoglobin species explains the stability of the intermediates and the buildup of a pool of potentially bioactive NO during nitrite reduction. It also provides a pathway for the formation of β-93 cysteine S-nitrosylated hemoglobin [SNOHb:S-nitrosohemoglobin], which has been shown to induce vasodilation, by a rapid radical-radical reaction of any free NO with the thiyl radical of this new paramagnetic intermediate.     

Detection of trace nitrite in waters using a QDs-based chemiluminescence analysis system

In the present work, a novel flow-injection chemiluminescence method based on CdTe quantum dots (QDs) was developed for the determination of nitrite. Weak chemiluminescence (CL) signals were observed from a CdTe QDs–H₂O₂ system under basic conditions. The addition of a trace amount of hemoglobin (Hb) caused the CL from the CdTe QDs–H₂O₂ system to increase substantially. In the presence of nitrite, the ferrous Hb reacted with the nitrate to form ferric Hb and NO. The NO then bound to ferrous Hb to generate iron nitrosyl Hb. As a result, the CL signal from the CdTe QDs–H₂O₂–Hb system was quenched. Thus, a flow-injection CL analytical system for the determination of trace nitrite was established. Under optimum conditions, there was a good linear relationship between CL intensity and the concentration of nitrite in the range 1.0?×?10^?9 to 8.0?×?10^?7 mol L^?1 (R ²?=?0.9957). The limit of detection for nitrite using this system was 3.0?×?10^?10 mol L^?1 (S/N?=?3). This method was successfully applied to detect nitrite in water samples.

A Nitrite Biosensor Based on the Direct Electron Transfer of Hemoglobin Immobilized on Carboxyl‐Functionalized Multiwalled Carbon Nanotubes/Polyimide Composite

The high electrically conductive carboxyl‐functionalized multiwalled carbon nanotubes (COOH‐MWCNTs) were used to combine with nonconducting polyimide (PI) to generate a PI/COOH‐MWCNTs membrane. PI served as a matrix to entrap COOH‐MWCNTs and hemoglobin (Hb). COOH‐MWCNTs can improve the conductivity of the composite. The direct electrochemistry measurement indicated that the PI/COOH‐MWCNTs composite enhanced the immobilization of Hb significantly. Besides, the Hb/PI/COOH‐MWCNTs/GCE biosensor possessed excellent electrocatalytic activity for the detection of nitrite. Therefore, PI is a good matrix for Hb immobilization and it has application in sensor construction. This work is promising in the development of sensitive biosensors based on PI/COOH‐MWCNTs composite film.     

Polyvinyl alcohol–ionic liquid composition for promoting the direct electron transfer and electrocatalysis of hemoglobin

The direct electron transfer and electrocatalysis of hemoglobin (Hb) entrapped in polyvinyl alcohol (PVA)–room temperature ionic liquid (i.e., 1-octyl-3-methylimidazolium hexafluorophosphate [OMIM]PF₆) composition has been investigated by using cyclic voltammetry and chronocoulometry. It is found that the composition can promote the direct electron transfer of Hb and the heterogeneous electron transfer rate constant (k_s) of immobilized Hb is enhanced to 19.9 s⁻¹. The immobilized Hb also shows high electro-catalytic activity towards the redox of oxygen, hydrogen peroxide and nitrite. The Michaelis constants (K_m) decrease to 1.2 × 10⁻⁴ M (for hydrogen peroxide) and 9.4 × 10⁻³ M (for nitrite). The surface concentration of electroactive Hb is estimated and it is ca. 1.4 × 10⁻¹⁰ mol cm⁻², meaning that several layers of immobilized Hb take part in the electrochemical reaction. When gold nanoparticles (GNP) is introduced into the composition, the resulting PVA–GNP–[OMIM]PF₆ composition presents better performance. The electrochemical characteristic of immobilized Hb is improved further. Thus PVA–GNP–[OMIM]PF₆ composition is more suitable for the immobilization of Hb. Therefore, it is a good strategy to prepare novel composition for protein immobilization by using several materials with different function.     

Hemoglobin as a nitrite anhydrase: modeling methemoglobin-mediated N2O3 formation

Nitrite has recently been recognized as a storage form of NO in blood and as playing a key role in hypoxic vasodilation. The nitrite ion is readily reduced to NO by hemoglobin in red blood cells, which, as it happens, also presents a conundrum. Given NO’s enormous affinity for ferrous heme, a key question concerns how it escapes capture by hemoglobin as it diffuses out of the red cells and to the endothelium, where vasodilation takes place. Dinitrogen trioxide (N₂O₃) has been proposed as a vehicle that transports NO to the endothelium, where it dissociates to NO and NO₂. Although N₂O₃ formation might be readily explained by the reaction Hb‐Fe³⁺+NO₂^?+NO?Hb‐Fe²⁺+N₂O₃, the exact manner in which methemoglobin (Hb‐Fe³⁺), nitrite and NO interact with one another is unclear. Both an “Hb‐Fe³⁺‐NO₂^?+NO” pathway and an “Hb‐Fe³⁺‐NO+NO₂^?” pathway have been proposed. Neither pathway has been established experimentally. Nor has there been any attempt until now to theoretically model N₂O₃ formation, the so‐called nitrite anhydrase reaction. Both pathways have been examined here in a detailed density functional theory (DFT, B3LYP/TZP) study and both have been found to be feasible based on energetics criteria. Modeling the “Hb‐Fe³⁺‐NO₂^?+NO” pathway proved complex. Not only are multiple linkage‐isomeric (N‐ and O‐coordinated) structures conceivable for methemoglobin–nitrite, multiple isomeric forms are also possible for N₂O₃ (the lowest‐energy state has an N? N‐bonded nitronitrosyl structure, O₂N? NO). We considered multiple spin states of methemoglobin–nitrite as well as ferromagnetic and antiferromagnetic coupling of the Fe³⁺ and NO spins. Together, the isomerism and spin variables result in a diabolically complex combinatorial space of reaction pathways. Fortunately, transition states could be successfully calculated for the vast majority of these reaction channels, both M_S=0 and M_S=1. For a six‐coordinate Fe³⁺‐O‐nitrito starting geometry, which is plausible for methemoglobin–nitrite, we found that N₂O₃ formation entails barriers of about 17–20 kcal mol^?1, which is reasonable for a physiologically relevant reaction. For the “Hb‐Fe³⁺‐NO+NO₂^?” pathway, which was also found to be energetically reasonable, our calculations indicate a two‐step mechanism. The first step involves transfer of an electron from NO₂^? to the Fe³⁺–heme–NO center ({FeNO}⁶) , resulting in formation of nitrogen dioxide and an Fe²⁺–heme–NO center ({FeNO}⁷). Subsequent formation of N₂O₃ entails a barrier of only 8.1 kcal mol^?1. From an energetics point of view, the nitrite anhydrase reaction thus is a reasonable proposition. Although it is tempting to interpret our results as favoring the “{FeNO}⁶+NO₂^?” pathway over the “Fe³⁺‐nitrite+NO” pathway, both pathways should be considered energetically reasonable for a biological reaction and it seems inadvisable to favor a unique reaction channel based solely on quantum chemical modeling.     

Nitrite reduction and detection at a carbon paste electrode containing hemoglobin and colloidal gold

A novel renewable reagentless nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) and a new sensing mechanism was proposed by combining the advantageous features of colloidal gold nanoparticle and carbon paste technology. The direct electrochemistry of immobilized Hb displayed a pair of redox peaks with a formal potential of -42 mV (vs. NHE) in 0.2 mol dm(-3) NaAc-HAc buffer (pH 5.5). The immobilized Hb displayed an excellent response to the reduction of NO2(-) with one interfacial charge transfer followed by a chemical reaction (EC) mechanism. Under optimal conditions, the interfacial EC process could be used for the sensitive determination of NO2(-) with a linear range from 0.1 to 9.7 micromol dm(-3) and a detection limit of 0.06 [micro sign]mol dm(-3) at 3sigma. The amperometric determination of high concentrations of NO2(-) based on the irreversible reduction of NO could be performed at pH 4.0 with a linear range from 0.1 to 1.2 mmol dm(-3). The surface of biosensor could be renewed quickly and reproducibly by a simple polish step. The biosensor has been used satisfactorily for nitrite determination in native water samples.     

Pencil Lead Electrode Modified with Hemoglobin Film as a Novel Biosensor for Nitrite Determination

In the present paper, the electrochemical reduction of nitrite at a hemoglobin modified pencil lead electrode (Hb/PLE) is described. The electrochemical properties of nitrite were studied by cyclic voltammetry and chronoamperometry. Results showed that the hemoglobin film has an excellent electrochemical activity towards the reduction of nitrite. By using voltammetric and chronoamperometric methods, α, n_α and n were calculated. Then the ability of the electrode for nitrite determination was investigated using differential pulse voltammetry. The electrocatalytic reduction peak currents were found to be linear with the nitrite concentration in the range from 10 to 220 µM with a detection limit of 5 µM. The relative standard deviation is 2 % for 3 successive determinations of a 100 µM nitrite solution. This modified electrode was successfully used for the detection of low amounts of NO₂^? in spinach sample and a spiked sample of tap water.     

Carbon-nanotube-enhanced direct electron-transfer reactivity of hemoglobin immobilized on polyurethane elastomer film

In this study, we investigate the direct electron-transfer reactivity of immobilized hemoglobin (Hb) on a polyurethane elastomer (PUE) film for biosensor designs. The PUE film synthesized by an additional polymerization possesses good biocompatibility, uniformity, and conformability and is ready for protein immobilization. Electrochemical and spectroscopic measurements show that the presence of multiwalled carbon nanotubes (MWNTs) increased the protein-PUE interaction, varied polymer morphology, improved the permeability and the conductivity of the PUE film, and thus facilitated the direct electron transfer between the immobilized Hb and the conductivity surface through the conducting tunnels of MWNTs. The immobilized Hb maintains its bioactivities and displays an excellent electrochemical behavior with a formal potential of -(334 +/- 7) mV. The addition of NaNO2 leads to an increase of the electrocatalytic reduction current of nitrite at -0.7 V. This allows us to develop a nitrite sensor with a linear response range from 0.08 to 3.6 mM. The proposed method opens a way to develop biosensors by using nanostructured materials mixed with low electrical conductivity matrixes.     

Direct electrochemistry of hemoglobin in layer-by-layer films with poly(vinyl sulfonate) grown on pyrolytic graphite electrodes

Stable layer-by-layer electroactive films were grown on pyrolytic graphite (PG) electrodes by alternate adsorption of layers of polyanionic poly(vinyl sulfonate) (PVS) and positively charged hemoglobin (Hb) from their aqueous solutions. Cyclic voltammetry (CV) of [PVS/Hb]n films showed a pair of well-defined and nearly reversible peaks at about - 0.28 V vs. SCE at pH 5.5, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The process of (PVS/Hb) bilayer growth was monitored and confirmed by CV, X-ray photoelectron spectroscopy (XPS) and UV-Vis spectroscopy. While the amount of Hb adsorbed in each bilayer was the same, the amount of electroactive Hb in each bilayer decreased dramatically with increase of the number of bilayer, and electroactivity was just extended to 8 [PVS/Hb] bilayers. CVs of [PVS/Hb]8 films maintained stable in buffers containing no Hb. Positions of Soret band of Hb in [PVS/Hb]n films grown on transparent glass slides suggest that Hb in the films keeps its secondary structure similar to its native state in a wide pH range. Trichloroacetic acid and nitrite were catalytically reduced by [PVS/Hb]8 films with significant lowering of the electrode potential required.