辣根过氧化物酶的电化学固定化及其对H2O2的生物电催化…

利用电化学固定化方法制备了聚吡咯／辣根过氧化物酶（ＰＰ／ＨＲＰ）膜电极，并研究了其电化学行为，在除氧的磷酸盐缓冲液介质中，ＰＰ／ＨＲＰ电极加速Ｈ２Ｏ２的还原，归因子酶加成物的直接电子传递，探索ＨＲＰ与电子传递体Ｋ４Ｆｅ（ＣＮ）６在聚吡咯（ＰＰ）膜中的同时固定化条件及其膜电极的电化学行为，实验证实，Ｋ４Ｆｅ（ＣＮ）６在酶膜中的存在使得Ｈ２Ｏ２的还原电位强烈正移，在－０．０５Ｖ的工作电位下能对Ｈ２Ｏ２     

聚邻苯二胺膜电极上氧还原为过氧化氢

聚邻苯二胺膜电极上氧还原为过氧化氢陆兆锷，锺天耕，张关永（华东理工大学化学系，上海２００２３７）参考了ＯｈｓａｋａＴ等￣［１－４］的工作，制成聚邻苯二胺（ＰＰＤ）膜覆盖的石墨电极。研究了电极在酸性溶液中对氧还原的电催化作用，氧还原为过氧化氢的电流效率...     

功能聚间苯二胺膜用于免疫传感器分析

以电化学聚合法在石墨电极上获得聚间苯二胺膜（ＰＭＰＤ），并建立了快速、灵敏的膜质量检验方法：辣根过氧化物酶（ＨＲＰ）标记ＰＭＰＤ膜－邻苯二胺（ＯＰＤ）反应法，对聚合、活化等条件作了研究；进一步制成了检测乙肝表面抗原（ＨＢｓＡｇ）和检测鼠免疫蛋白（ＭＩｇＧ）的免疫电极，对血清样品作了检测．结果表明：２．５Ｖ电池电压下，于含０．１２ｍｏｌ／ＬＭＰＤ的１．２ｍｏｌ／ＬＨ２ＳＯ４中聚合２０ｍｉｎ，再以３０ｇ／Ｌ浓度的戊二醛活化４ｈ，所得ＨＲＰ标记电极及免疫电极重现性好；检测ＨＢ－ｓＡｇ和ＭＩｇＧ的线性范围分别为０．１～３．２ｍｇ／Ｌ及０．１～１０ｍｇ／Ｌ     

分子氢和二硫化铁对1，3─二苯丙烷分解的促进作用

以１，３─二苯丙烷（ＤＰＰ）作为煤相关模型化合物，考察了在４００℃下分子氢和催化剂对ＤＰＰ分解反应的作用。ＤＰＰ的分解反应主要经Ｃ＿烷─Ｃ＿烷键的断裂进行，由ＤＰＰ的分解产生的苄基自由基（ＰｈＣＨ＿２）等活性自由基显著促进ＤＰＰ中Ｃ＿烷─Ｃ＿烷键的断裂。分子氢与ＰｈＣＨ＿反应，产生可夺取ＤＰＰ中ａ－亚甲基上的氢或附加在ＤＰＰ中苯环的ｉｐｓｏ位上游离氢原子；分子氢还可以还原作为ＤＰＰ分解产物的苯乙烯，从而防止其与ＰｈＣＨ＿２反应。由于这些作用，分子氢促进ＤＰＰ的分解反应。在加压氢气存在下添加二硫化铁进一步加速ＤＰＰ分解反应的进行，而添加超细铁粉抑制ＤＰＰ的分解反应。     

优化固氮活性模型信息研究—双齿配体DPPE和DPPM对固氮酶促反应的影响

报道了某些双齿配体对固氮酶促反应影响的研究结果．发现１，２－双（二苯基磷）－乙烷（ＤＰＰＥ）对固氮酶酶促乙炔还原反应有促进作用，但同时又抑制了固氮酶的放氢反应。而比ＤＰＰＥ少一个－ＣＨ２－链的双（二苯基磷）－甲烷（ＤＰＰＭ）却不能表现出对固氮酶促乙炔还原活力的促进作用．对照固氮酶ＭｏＦｅ－蛋白Ｘ－光衍射结构分析结果和量子化学近似计算所导出的固氮酶活性中心结构模型提出了ＤＰＰＥ促进酶促乙炔还原反应的一种可能的解释．     

分子氢和二硫化铁对1,3—二苯丙烷分解的促进作用

以１，３－二苯丙烷（ＤＰＰ）作为煤相关模型化合物，考察了在４００℃下分子氢和催化剂对ＤＰＰ分解反应的作用。ＤＤＰ的分解反应主要经Ｃ烷＿Ｃ烷键的断裂进行，由ＤＰＰ的分解产生的苄基自由基（ＰｈＣＨ２）等活性自由基显著促进ＤＰＰ中Ｃ烷＿Ｃ烷键的断裂。分子氢与ＰｈＣＨ２反应，产生可夺取ＤＰＰ中ａ－亚甲基上的氢或附加在ＤＰＰ中苯环的ｉｐｓｏ位上游离氢原子；分子氢还可以还原作为ＤＰＰ分解产物的苯乙烯，从而防止     

微过氧化物酶-11修饰电极对O2和H2O2的电催化还原

运用电化学循环伏安法和旋转圆盘电极技术研究了Ｏ２和Ｈ２Ｏ２在Ｎａｆｉｏｎ膜固定的微过氧化物酶－１１修饰的玻磷（ＭＰ－１１＋Ｍａｆｉｏｎ／Ｇｃ）电极上的电化学还原,结果表明,饰电极 对Ｏ２和Ｈ２Ｏ２的还原均具电催化作用。测定和比较了Ｏ２和Ｈ２Ｏ２在ＭＰ－１１＋Ｎａｆｉｏｎ／ＧＣ的电极上电催化还原反应的一些动力学参数。发现Ｏ２在修饰电极上经历了四电子还原,且还原过程与溶液的ＰＨ值有关。     

偶合反应伏安酶联免疫分析测定人血清乙型肝炎E抗体的研究

本文提出用竞争性抑制偶合反应伏安酶联免疫分析法测定人血清乙型肝炎Ｅ抗体（ＨＢｅＡｂ）方法基于酶标ＨＢｅＡｂ辣根过氧化物酶（ＨＲＰ）催化Ｈ２Ｏ２氧化邻联甲苯胺（ＯＴ）的反应与邻联甲苯胺氧化产物在电极上的还原反应相偶合，测定标记在ＨＢｅＡｂ上ＨＲＰ量，以求得抑制免疫反应的乙型肝炎Ｅ抗体含量本法测定酶标ＨＢｅＡｂＨＲＰ及ＨＢｅＡｂ的灵敏度均高于经典的ＥＬＩＳＡ光度法方法用于病人血清样品分析，与ＥＬＩＳＡ光度法对照，其相关性很好     

邻苯二胺对1，4－二氧六环介质中HRP酶电极催化性能的影响

邻苯二胺对１,４－二氧六环介质中ＨＲＰ酶电极催化性能的影响陈红,吴辉煌（厦门大学固体表面物理化学国家重点实验室,厦门大学化学系,厦门３６１００５）１实验部分辣根过氧化物酶（ＨＲＰ,Ｅ．Ｃ．１．１１．１．７,２５０ｕ／ｍｇ,Ｒ．Ｚ．～３．０）及牛血清蛋...     

邻苯二胺-过氧化氢-辣根过氧化物酶酶联免疫示差脉冲伏安法测定人血清类风湿因子抗体

建立了邻苯二胺（ＯＰＤ）－Ｈ２Ｏ２－辣根过氧化物酶（ＨＲＰ）酶联示差脉冲伏安分析体系并用于测定人血清中类风湿因子（ＲＦ），ＨＲＰ催化Ｈ２Ｏ２氧化ＯＰＤ所形成酶催化产物在ｐＨ２．０磷酸盐－枸橼酸缓冲溶液中于－０．１８ Ｖ左右产生一灵敏示差脉冲伏安峰，ＲＦ浓度在１．２５～２０．０ Ｕ／ｍＬ之间与峰电流呈线性关系，应用此峰检测人血清ＲＦ的检测限低至 ０．２８Ｕ／ｍＬ。该法较相同条件下ＥＬＩＳＡ显色光度测定法的灵敏度增加５倍，且受干扰较少。     

Physical immobilization of laccase on an electrode by means of poly(ethyleneimine) microcapsules

Microcapsules of poly(ethyleneimine) were used to immobilize laccase on the surface of an electrode and its mediated electron transfer was studied with the redox mediator p-phenylenediamine (PPD). The microcapsules consisted of a cross-linked PEI wall generated from an emulsion of an aqueous phase containing the enzyme. The reaction of encapsulated laccase with PPD was studied by spectrophotometry and oxygen consumption. We found that the encapsulation resulted in a small shift for the optimum pH and a lower K_m value when compared to free laccase. These differences are attributed to the charged micro-environment offered by the microcapsules. The microcapsules were then deposited on a glassy carbon electrode and chronoamperometry was used to evaluate the mediated electron transfer between the enzyme and the electrode. No significant differences in term of optimum pH and K_m occurred upon capsules deposition on the electrode. The response time of the electrode for PPD oxidation was higher than those found in the literature, which suggests that the PEI capsule wall offers some resistance to mediator permeation, an hypothesis that was verified by RDE measurements. The charged nature of the PEI membrane appeared to affect several parameters of the laccase-mediator reaction and the effect of pH and mediator charge on this reaction are reported. The immobilization platform under study can be applied to different enzyme-mediator systems than the laccase-PPD used here and is relevant to the development of bioelectrocatalytic systems.     

Synthesis, structure, and photoelectrochemical properties of new tetrathiafulvalene-diphenyl-1,3,4-oxadiazole dyads

New donor-acceptor dyads containing tetrathiafulvalene (TTF) and 2,5-diphenyl-1,3,4-oxadiazole (PPD) moieties were synthesized to develop new photoconducting materials. Crystal structure analysis indicated the highly planar molecular skeleton of the dyad. Fluorescence from the PPD part was almost quenched by the intramolecular electron transfer from the TTF part to the PPD part. Photoelectrochemical measurements indicate that cathodic photocurrents can be generated from a thin film of the dyad spin-coated on ITO electrode.     

Design variations of a polymer–enzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependence

The glucose sensitivity and oxygen dependence of a variety of implantable biosensors based on glucose oxidase (GOx), incorporating an electrosynthesized poly-o-phenylenediamine (PPD) permselective barrier on 125-μm diameter Pt disks (Pt_D) and cylinders (Pt_C, 1-mm length), were measured and compared. Full glucose calibrations and experimental monitoring of solution oxygen concentration allowed us to determine apparent Michaelis–Menten parameters for glucose and oxygen. In the linear region of glucose response, the most sensitive biosensor design studied was Pt_D/PPD/GOx (enzyme deposited over polymer) that was 20 times more sensitive than the more widely used Pt_C/GOx/PPD (enzyme immobilized before polymer deposition) configuration. The oxygen dependence, quantified as K_M(O₂), of both active and less active designs was surprisingly similar, a finding that could be rationalized in terms of an increase in K_M(G) with increased enzyme loading. The Pt_D/PPD/GOx design will now enable us to explore glucose concentration dynamics in smaller and layered brain regions with good sensitivity and minimal interference from fluctuations in tissue pO₂.     

Formation of p-phenylenediamine-crown ether-[PMo12O40]4- salts

Electron transfer from the electron donor of p-phenylenediamine (PPD) to the electron acceptor of (H+)3[PMo12O40]3- forms a one-electron-reduced Keggin cluster of [PMo12O40]4-, bearing a S = 1/2 spin, while proton transfer from the proton donor of (H+)3[PMo12O40]3- to the proton acceptor of PPD yielded mono- and diprotonated cations of 4-aminoanilinium (HPPD+) and p-phenylenediammonium (H2PPD2+). By introduction of crown ether receptors during the crystallization process, supramolecular cations of (HPPD+)(crown ethers) and/or (H2PPD2+)(crown ethers) were successfully introduced into three new alpha-[PMo12O40]4- salts of (H2PPD2+)2([12]crown-4)4[PMo12O40]4- (1), (HPPD+)4([15]crown-5)4[PMo12O40]4- (2), and (HPPD+)2(H2PPD2+)([18]crown-6)4[PMo12O40]4- (3) as the countercation. The protonated states of PPD and molecular-assembly structures of the supramolecular cations depended on the size of the crown ethers. In salt 3, a novel mixed-protonated state of HPPD+ and H2PPD2+ was confirmed to be complexed in the cation structure. According to the changes in the cation structures, the anion arrangements were modulated from those of the two-dimensional layer for salt 1 to the isolated cluster for salts 2 and 3. The temperature-dependent magnetic susceptibilities of salts 1-3 were consistent with the isolated spin arrangements of [PMo12O40]4-. The electronic spectra of salts 1-3 indicated the intervalence optical transition from pentavalent Mo(V) to hexavalent Mo(VI) ions within the [PMo12O40]4- cluster. Temperature-dependent electron spin resonance spectra of salt 2 revealed the delocalization-localization transition of the S = 1/2 spin at 60 K. The spin on the [PMo12O40]4- cluster was localized on a specific Mo(V) site below 60 K, which was thermally activated with an activation energy of 0.015 eV.     

Purification and characterization of new special ginsenosidase hydrolyzing multi-glycisides of protopanaxadiol ginsenosides, ginsenosidase type I

In this paper, the new type ginsenosidase which hydrolyzing multi-glycosides of ginsenoside, named ginsenoside type I from Aspergillus sp.g48p strain was isolated, characterized and generally described. The enzyme molecular weight was about 80 kDa. Ginsenosidase type I can hydrolyze different glycoside of protopanaxadiol type ginsenosides (PPD); i.e., can hydrolyze the 3(carbon)-O-beta-glucoside of Rb1, Rb2, Rb3, Rc, Rd; can hydrolyze 20(carbon)-O-beta-glucoside of Rb1, 20-O-beta-xyloside of Rb3, 20-O-alpha-arabinoside(p) of Rb2 and 20-O-alpha-arabinoside(f) of Rc to produce mainly F2, compound-K (C-K) and small Rh2, but can not hydrolyze the glycosides of protopanaxatriol type ginsenoside (PPT) such as Re, Rf, Rg1. So, when the ginsenosidase type I hydrolyzed ginsenosides, the enzyme selected ginsenoside-aglycone type, can hydrolyze different glycosides of PPD type ginsenoside; however no selected glycoside type, can hydrolyze multi-glycosides of PPD type ginsenosides. These properties were novel properties, and differentiated with the other previously described glycosidases.     

Photovoltammetric behavior and photoelectrochemical determination of p-phenylenediamine on CdS quantum dots and graphene hybrid film

A photoelectroactive film composed of CdS quantum dots and graphene sheets (GS) was coated on F-doped SnO₂ (FTO) conducting glass for studying the electrochemical response of p-phenylenediamine (PPD) under photoirradiation. The result indicated that the cyclic voltammogram of PPD on CdS–GS hybrid film became sigmoidal in shape after exposed under visible light, due to the photoelectrocatalytic reaction. Such a photovoltammetric response was used to rapidly optimize the photoelectrocatalytic activity of hybrid films composed of different ratios of CdS to GS toward PPD. The influences of scan rate and pH on the photovoltammetric behavior of PPD on CdS–GS film revealed that although the controlled step for electrochemical process was not changed under photoirradiation, more electrons than protons might participate the photoelectrocatalytic process. Furthermore, the photoelectroactive CdS–GS hybrid film was explored for PPD determination based on the photocurrent response of film toward PPD. Under optimal conditions, the photocurrent signal on CdS–GS film was linearly proportional to the concentration of PPD ranging from 1.0 × 10⁻⁷ to 3.0 × 10⁻⁶ mol L⁻¹, with a detection limit (3S/N) of 4.3 × 10⁻⁸ mol L⁻¹. Our work based on CdS–GS hybrid film not only demonstrated a new facile photovoltammetric way to study the photoinduced electron transfer process of PPD, but also developed a sensitive photoelectrochemical strategy for PPD determination.     

人参二醇类皂苷的生物转化动态及人参稀有皂苷C-K或F2的制备

为了低成本有效制备人参稀有皂苷C-K或F₂, 将A. niger g.848菌酶用于转化含有人参皂苷(质量分数)分别为49.6% Rb₁, 25.9% Rd, 19.3% Rc和5.23% Rb₂的西洋参二醇混合皂苷. 霉菌发酵时, 采用人参二醇皂苷诱导物比人参提取液诱导物的产酶总活力提高10%~15%. 所产的2种诱导酶均能水解人参二醇皂苷的3-O-和20-O-多种糖基, 均为人参皂苷酶Ⅰ型; 但是人参二醇皂苷诱导物所产酶几乎全部转化人参二醇皂苷为C-K, 而人参提取液诱导物所产酶则残留中间产物. 使用黑曲霉人参二醇皂苷诱导所产酶, 在转化西洋参二醇皂苷的动态研究中发现, 酶反应1.5~2.5 h, 主要为产物F₂; 酶反应12 h后, 主要产物为C-K皂苷. 基于此, 40 g人参二醇类皂苷在45 ℃粗酶反应24 h, 经处理得到含C-K质量分数为87%的23 g酶反应产物, C-K转化率达85%(摩尔分数). 用40 g西洋参二醇皂苷在45 ℃粗酶反应2.5 h, 经处理得到含有质量分数为58%的F₂和27%的C-K的26 g酶反应产物, F₂转化率为50.4%, C-K转化率为29.5%. 通过人参二醇皂苷诱导的黑曲霉粗酶转化人参二醇类皂苷动态研究, 建立了C-K转化率为85%, F₂转化率为50%的制备方法, 为大批量制备提供了基础依据.     

对苯二胺在血红蛋白/聚L-谷氨酸/玻碳修饰电极上的可逆氧化

采用电聚合法在玻碳电极(GCE)表面得到导电性能良好的聚L-谷氨酸(PGA)薄膜,通过共价键合法将血红蛋白(Hb)固定于电极表面得到稳定且具有催化活性的Hb/PGA/GCE修饰电极,将其用于对苯二胺(PPD)的可逆氧化。 修饰电极交流阻抗及血红蛋白直接电化学实验表明,血红蛋白成功地固定于电极表面,保持良好的电催化活性,能有效催化H₂O₂的还原。 PPD在电极上表现为受吸附控制的准可逆氧化还原反应,I_p,a/I_p,c约为1.02,电极没有明显的钝化现象。 氧化还原峰电流与PPD的浓度均呈良好的线性关系,I_p,a(μA)=3.124+0.705c_PPD(mmol/L)(r=0.9973)。 H₂O₂的存在使PPD氧化还原峰型更对称,可逆性更好,表明体系中PPD氧化与过氧化酶催化途径一致。     

Bicomponent Microneedle Array Biosensor for Minimally‐Invasive Glutamate Monitoring

This article describes the design of a new and attractive minimally‐invasive bicomponent microneedle sensing device for the electrochemical monitoring of the excitatory neurotransmitter glutamate and glucose. The new device architecture relies on the close integration of solid and hollow microneedles into a single biosensor array device containing multiple microcavities. Such microcavities facilitate the electropolymeric entrapment of the recognition enzyme within each microrecess. The resulting microneedle biosensor array can be employed as a minimally‐invasive on‐body transdermal patch, obviating the extraction/sampling of the biological fluid, thereby simplifying device requirements. The new concept is demonstrated for the electropolymeric entrapment of glutamate oxidase and glucose oxidase within a poly(o‐phenylenediamine) (PPD) thin film. The PPD‐based enzyme entrapment methodology enables the effective rejection of coexisting electroactive interferents without compromising the sensitivity or response time of the device. The resulting microneedle‐based glutamate and glucose biosensors thus exhibit high selectivity, sensitivity, speed, and stability in both buffer and undiluted human serum. High‐fidelity glutamate measurements down to the 10 µM level are obtained in serum. The attractive recess design also serves to protect the enzyme layer upon insertion into the skin. This simple, yet robust microneedle design is well‐suited for diverse biosensing applications in which real‐time metabolite monitoring is a core requirement.     

Talaromyces thermophilus β-d-Xylosidase: Purification, Characterization and Xylobiose Synthesis

When grown on wheat bran as the only carbon source, the filamentous fungus Talaromyces thermophilus produces large amounts of beta-xylosidase activity. The presence of glucose drastically decreases the beta-xylosidase production level. The beta-xylosidase of T. thermophilus was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration (high-performance liquid chromatography). The molecular mass of the enzyme was estimated to be 97 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The enzyme activity was optimum at 50 degrees C and pH 7. The apparent Michaelis constant K(m) of the beta-xylosidase was 2.37 mM for p-nitrophenyl-beta-D-xylopyranoside, with a V(max) of 0.049 micromol min(-1) per milligram protein. Enzyme activity was inhibited by Cu(2+), Hg(2+), and Zn(2+) and activated by Ca(2+), Mn(2+), and Co(+) at a concentration of 5 mM. At high xylose concentration, this enzyme catalyses the condensation reaction leading to xylobiose production.