以3,3′,5,5′-四甲基联苯胺为底物的伏安酶联免疫分析体系研究

对 3 ,3′,5 ,5′ 四甲基联苯胺 (TMB) H2 O2 辣根过氧化物酶 (HRP)伏安酶联免疫分析体系进行了研究。HRP催化H2 O2 氧化TMB ,其氧化产物在汞电极上可以发生还原反应 ,在B R缓冲溶液中 ,-0 .76V(vs.SCE)处产生较为灵敏的伏安波。将此伏安波应用于测定HRP和HRP的标记物。测定HRP的线性范围为 6.0×1 0 - 1 1 ～ 5 .0× 1 0 - 9g mL ,检测限为 5 .0× 1 0 - 1 1 g mL。对该体系酶催化反应机理及产物的电极还原过程进行了探讨     

自来水中余氯测定之TMB配制探讨

对TMB的配制的介质浓度，配制方法及其助溶温度等诸因素进行了探讨，拟定了自来水中余氯测定的TMB溶液配制的具体方法，助溶温度和介质酸度等，解决了生活饮用水卫生规范中测定水中余氯用TMB显色无法与标准色阶进行比色的实际问题。完全能满足日常生产的现场检测的需要。     

镧离子对植物体内过氧化物酶活性的影响

本文首次比较了在植物体内La³⁺与过氧化物酶(POD)的相互作用和在植物体外La³⁺与辣根过氧化物酶(HRP)的相互作用。在植物体内的研究表明,La³⁺能够明显改变POD的活性,并呈现低促高抑的“Hormesis效应”。用圆二色谱(CD)、紫外-可见(UV-Vis)吸收光谱和傅立叶变换红外(FTIR)光谱研究在植物体外La³⁺与HRP相互作用的结果表明,在模拟生理条件的溶液中,La³⁺对HRP的活性同样呈现低促高抑的＂Hormesis 效应＂。其作用机理可能是La³⁺通过与HRP中酰胺基的O或N键合,导致HRP二级结构变化,进而改变活性中心血红素中铁卟啉的化学结构,最终改变HRP的催化活性。     

新型显色剂3,3,5,5''''-四甲基联苯胺分光光度法测定余氯的研究

本文报道了采用新型氧化还原剂3,3’,5,5’-四甲基联苯胺(TMB)测定水中总余氯的分光光度法,并对其灵敏度、稳定性、准确性作了较为详细的研究,探讨了分析条件,提出了分析方法。在pH≤2时,最大吸收峰为450 nm,表观摩尔吸光系数为6.89×10~4L·mol~(-1)·cm~(-1),可测范围为0.01～2.5 mg/L。与目前使用的传统显色剂邻联甲苯胺(DMB)相比较,具有灵敏度高,显色稳定,重现性好及使用安全等优点。     

TMB目视比色法检测水中余氯的改进

为解决GB/T 5750.11-2006生活饮用水标准检验方法中的3,3',5,5'-四甲基联苯胺(TMB)目视比色法检测余氯时出现的评价方法准确性欠佳、TMB显色剂发黄、显色体系偏绿等问题,对TMB目视比色法的评价方法、TMB显色剂的配制条件和水样的检测条件进行了研究。结果表明,次氯酸钠不宜作为余氯标准物质来评价TMB目视比色法的改进效果,而通过检测显色体系的A450和目视比色值相结合的方法来判断方法改进效果更为准确。采用优级纯浓盐酸和无氯纯水室温搅拌溶解,可使显色剂无色透明,从而解决TMB显色剂发黄的问题,符合国标要求。将显色剂配制用酸(盐酸)的浓度由国标中0.1mol/L变更为0.6 mol/L,可解决余氯显色体系偏绿的问题。显色体系pH变化后,显色时间、显色温度、显色剂用量可仍按照国标方法的要求进行操作。通过以上改进措施,进一步提高了余氯检测结果的准确度。     

辣根过氧化物酶直接电化学传感器检测过氧化物的研究

该文以高比表面积的泡沫镍电极(Ni foam)为基础,通过电沉积碳纳米管(CNTs)制备了CNTs/Ni foam。然后在十六烷基三甲基溴化铵(CTAB)的辅助下,通过一步法电沉积纳米金(AuNPs)将辣根过氧化物酶(HRP)固定到电极表面,制备了HRP-AuNPs/CNTs/Ni foam直接电化学酶传感器。并采用SEM、能谱(EDS)和电化学方法对该电极进行了表征,优化了测试电位和pH值,将该传感器对过氧化氢及2种有机过氧化物进行了检测。结果表明,该传感器性能良好,对过氧化氢、过氧化氢异丙苯、2-过氧化丁酮具有良好的催化检测性能,其检出限分别为1.2×10~(-7)、4.5×10~(-7)、2.5×10~(-7) mol/L。     

固定化辣根过氧化物酶和硫堇的过氧化物传感器

利用卡拉胶水凝胶将辣根过氧化物酶和硫堇同时固定在玻碳电极表面,制备以硫堇为媒介体的过氧化物电化学传感器.包埋在卡拉胶水凝胶中的硫堇在pH=7.0的磷酸缓冲溶液中出现了1对氧化还原峰,氧化峰电位和还原峰电位分别在-0.176和-0.264 V,电位差为88 mV,电流比近似为1,说明硫堇在电极表面发生准可逆的电化学反应.硫堇能作为辣根过氧化物酶催化还原过氧化物中的电子媒介体,加速催化还原过程中的电子传递,减少了催化还原过程中的其它氧化物的干扰.传感器检测过氧化物(过氧化氢、异丙苯基过氧化氢、过氧化丁酮、叔丁基过氧化氢)具有较快的响应时间和良好的灵敏度、重现性、稳定性及较长的使用寿命.     

反相胶束中辣根过氧化物酶的停流光谱研究

用停流光谱法研究了HRP在AOT、CTAB和SDS反相胶束中的吸收光谱和反应动力学，实验结果显示在AOT反相胶束中，HRP的吸收峰位置与水相中相同；而另外两种反相胶束对HRP的分子结构产生了较大影响，快速反应动力学研究显示在反相胶束中HRP形成化合物Ⅰ的速率常数远远高于化合物Ⅰ形成HRP—Ⅱ的反应速率常数，推测这是反相胶束的特殊性质造成的结果。     

循环连续流动分析法测定辣根过氧化物酶的活性

基于H2O2-辣根过氧化物酶(HRP)-邻苯二胺(OPDA)催化反应体系,建立了测定游离及固定化HRP酶活性的循环连续流动分析法(CCFA).CCFA实现了反应液、反应过程的循环连续检测.用CCFA法对酶催化反应条件进行研究,得到的最佳反应条件是:pH 5.0,反应温度27 ℃, 66.0 μmol/L H2O2 , 2 mg/L OPDA,缓冲液为0.1 mol/L柠檬酸-柠檬酸钠缓冲液; 测定游离HRP的线性范围为0～10 U/L,检出限为0.21 U/L,RSD＜1.03%.使用CCFA法测定了固定化HRP的活性,并对HRP酶促动力学进行了研究,得到的游离HRP的米氏常数为0.078 mmol/L,最大反应速度为0.26 mmol/(L min),催化常数为5.2 × 104 min-1.CCFA法操作简便、准确度高、节省试剂、易于实现自动化.     

Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers

Films constructed layer-by-layer on electrodes with architecture {protein/hyaluronic acid (HA)}n containing myoglobin (Mb) or horseradish peroxidase (HRP) were protected against protein damage by H2O2 by using outer catalase layers. Peroxidase activity for substrate oxidation requires activation by H2O2, but {protein/HA}n films without outer catalase layers are damaged slowly and irreversibly by H2O2. The rate and extent of damage were decreased dramatically by adding outer catalase layers to decompose H2O2. Comparative studies suggest that protection results from catalase decomposing a fraction of the H2O2 as it enters the film, rather than by an in-film diffusion barrier. The outer catalase layers controlled the rate of H2O2 entry into inner regions of the film, and they biased the system to favor electrocatalytic peroxide reduction over enzyme damage. Catalase-protected {protein/HA}n films had an increased linear concentration range for H2O2 detection. This approach offers an effective way to protect biosensors from damage by H2O2.     

Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis.

A fast, sensitive and direct method has been developed for the determination of glutathione peroxidase activity (both selenium- and non-selenium-dependent) in cell-free preparations. The assay is based on the separation and quantitation of reduced and oxidized glutathione by capillary electrophoresis. The electrophoretic separation buffer was 100 mM sodium tetraborate (pH 8.2) containing 100 mM sodium dodecylsulphate. A micellar electrokinetic mechanism took place under these conditions, and a total mass recovery was observed for both peptides. The reproducibility of migration times was excellent (less than 3% variability). A linear detector response range was observed in the range 5-50 U/ml, and both the reproducibility and accuracy were satisfied. Samples out of this linear range could be analysed by either increasing the reaction time or diluting the enzyme preparation. The results obtained with the new direct capillary electrophoresis assay were compared with those derived from a reversed phase high-performance liquid chromatographic and spectrophotometric coupled assay. A very good agreement was found between the two direct assay methods in all samples. Capillary electrophoresis is a versatile technique that allows the automation of the glutathione peroxidase assay in a reproducible manner and within a relatively short time with sufficient accuracy and precision.     

Studies in peroxidase action—XXV : Stereospecificity of peroxidase action

The oxidations of some 4-n-alkyl substituted aromatic amines by peroxidase have been studied. 4-n-Decylaniline inhibits the enzyme as do amines higher in the homologous series while amines lower in the series are oxidised normally although the rate of oxidation is slower as the length of the chain increases.     

Measurement of pork freshness using potentiometric sensor

This study evaluated pork freshness using potentiometric solid-state electrodes in order to detect chemical indices such as reduced compounds, organic compounds and sulfides, which are produced during the initial stage of putrefaction in meat. Pt, CuS and Ag₂S electrodes selected as solid-state electrodes have, respectively, been used to detect the organic compounds (regarded as chemical indices of deterioration in meat freshness). The outputs of these electrodes have been analyzed by principal component analysis (PCA) and multiple regression analysis (MRA) in order to find the correlation with the results of viable bacterial counts. By using the potentiometric sensor, the pork freshness was evaluated and the PCA and MRA corresponded to the degree of bacterial increases more simply and rapidly than other methods such as viable bacterial counts or a biosensor.     

A quantitative western blot technique using TMB: Comparison with the conventional technique

Numerous molecular biological experiments performed throughout the world require the detection or quantification of a protein of interest. Western blotting is one of the most popular techniques used for this purpose and offers quantitative information with the aid of specialized software. However, its dependence on the picture that is captured, and the background and the absence of a common protocol prevent the technique from being completely quantitative. To overcome these obstacles, we present a simple and reliable assay that is similar to the regular technique, with the exception of the last stage of band visualization and quantification. We propose that small pieces of the blot that include the protein of interest can be cut and dipped in a small volume of 3,3',5,5'-tetramethylbenzidine solution, giving a colorimetric signal with linear dependence on the quantity of the protein. The reaction is stopped with H₂SO₄, and the signal is measured in a plate reader. This modification shows high linearity without additional costs and can be applied for both purified proteins and proteins found in a lysate. The results obtained with our proposed technique were compared with those obtained by the conventional method and proved to be more reliable.     

Impregnation of tungstophosphoric acid on poly(methacrylamide-co-methyl methacrylate) and its acid catalytic activity in TMB alkylation

A poly(methacrylamide-co-methylmethacrylate) (abbreviated PMAA-MMA) polymer support was studied for supporting a heteropolyacid (tungstophosphoric acid, H₃PW₁₂O₄₀) with its surface positively charged in the polymerization step. PMAA-MMA supports could be obtained in a porous form by eliminating template reagent molecules (benzylmalonic acid) combined with properly selected monomer (methacrylamide). The amount of amine groups in PMAA-MMA directly determined the amount of H₃PW₁₂O₄₀ impregnated, because the amine groups induced a positive charge on the PMAA-MMA surface. Finally, H₃PW₁₂O₄₀/PMAA-MMA showed better acid catalytic activities than unsupported H₃PW₁₂O₄₀ in alkylation of 1,3,5-trimethylbenzene with cyclohexene, which confirmed that PMAA-MMA supported H₃PW₁₂O₄₀ effectively.     

Bioimprinted protein exhibits glutathione peroxidase activity

A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H₂O₂ by glutathione (GSH) were found to be 101-817 U μmol⁻¹, which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis-Menten kinetics for both H₂O₂ and GSH, e.g. the pesudo-first-order rate constant k_cat (H₂O₂) and the apparent Michaelis constant K_m (H₂O₂) at 1 mM GSH were calculated to be 784 min⁻¹ and 1.24×10⁻³ M, respectively, and the apparent second-order rate constant k_cat (H₂O₂)/K_m (H₂O₂) was determined to be 6.33×10⁵ (M min)⁻¹. The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.     

Semisynthetic tellurosubtilisin with glutathione peroxidase activity

Reaction of the hydroxyl group of serine-221 of subtilisin with phenylmethanesulfonylfluoride followed by nucleophilic substitution with sodium hydrogen telluride, a semisynthetic telluroprotein, tellurosubtilisin, was prepared. Tellurosubtilisin, which displays high substrate specificity for aromatic thiols, exhibits remarkable peroxidase activity and catalyzes the reduction of hydrogen peroxide by 3-carboxy-4-nitrobenzenethiol 20 000 times more efficiently than diphenyl diselenide.     

Backbone modification promotes peroxidase activity of G-quadruplex-based DNAzyme

In this work we studied how backbone chemical modifications, such as 2'-O-methyl, phosphorothioate, L-form nucleotides and locked nucleic acid, on G-quadruplex based DNAzymes would affect their peroxidase activity. Our results indicate that 2'-O-methyl modification facilitates the formation of a perfectly compacted parallel structure and significantly promotes peroxidase activity of G-quadruplex based DNAzymes.