酶循环荧光法定量检测人血红细胞NADH浓度

用酶循环荧光测定法,多功能微孔板检测系统检测人血清还原型烟酰胺腺嘌呤二核苷酸(NADH)的浓度。通过底物和酶的循环反应将待测物质NADH的量扩大至荧光可检测范围。该方法的荧光强度与NADH的质量在0.00—0.80pmol范围内呈良好的线性关系,建立的线性方程式为:y=99.377x+13.481,r=0.9941,加标回收率为95.5%—100.0%,批内RSD为1.0%—2.7%,批间RSD为1.9%—12.6%。该方法准确度、灵敏度高、稳定性好,可用于NADH的定量检测。     

Synthesis of Chiral NADH Analog Based on Proline Template Including Thiourea and Nicotinic Acid Moieties

Chiral reductase-mimicking organic molecule built on proline template incorporating a covalently bound NADH mimic via thiourea, and related reducing agent Hantzsch dihydropyridine, was designed. A synthetic path was developed involving interlinking of chiral proline derivatives with thiourea and subsequent coupling reaction with nicotinoyl chloride. The structure of target compound was studied by x–ray, indicating a double H bond with thiourea hydrogens and oxygen O1 of benzylcarbamate fragment. The reduction of benzil and imines was performed.

含C^N-螯合配体的环戊二烯基铱抗癌配合物

合成、表征了8个半三明治结构环戊二烯基金属铱配合物[（η⁵-Cp^x）Ir（C^N）Cl],其中Cp^x分别为四甲基环戊二烯基（C₅Me₄H）,五甲基环戊二烯基（Cp^*）,四甲基（苯基）环戊二烯基（Cp^xph）,四甲基（联苯）环戊二烯基（Cp^xbiph）,C^N为苯亚甲基甲胺（BIMA）,N-（4-甲氧基苯亚甲基）苯胺（MBIA）。测定了其中3个配合物的单晶结构。所有配合物对Hela人宫颈癌细胞显示出很强的细胞毒性,IC₅₀值为1.7~32.9 μmol·L^-1。经检测Cp^x铱配合物的抗癌活性顺序为Cp^xbiph > Cp^xph > C₅Me₄H > Cp^*。配合物[（η⁵-Cp^xbiph）Ir（BIMA）Cl] （A4）和[（η⁵-Cp^xbiph）Ir（MBIA）Cl] （B4）表现出了最高的抗癌活性,比临床铂类药物顺铂活性高4倍以上。经检测,铱配合物A1~B4不与9-甲基腺嘌呤和9-乙基鸟嘌呤反应,与pBR322 DNA也没有作用,但这些配合物能够作为氢转移催化剂,将辅酶NADH转化为NAD⁺。机理研究表明IC₅₀浓度的配合物A4、B4处理Hela细胞时会引起明显的细胞凋亡和细胞周期的变化,并大幅增加细胞内活性氧（ROS）的水平。     

聚苯乙烯固载NADH辅酶模型化合物的合成及应用

设计、合成了一类新型的高分子还原试剂--聚苯乙烯固载烟酰胺辅酶模型化合物1-苄基-1,4-二氢烟酰胺(BNAH)4.该还原剂可以在温和条件下有效的还原活化烯烃,而且可以循环再生,但循环再生后有效BNAH含量下降.     

Charge at the migrating hydrogen in the transition state of hydride transfer reactions from CH groups to hydride acceptors. Dynamics of the redox‐couple NADH‐NAD+

We consider the controversial conclusions of the charge at the migrating hydrogen in the transition state of hydride‐transfer reactions from CH‐groups to hydride acceptors. Quantum chemical calculations were performed on elementary organic reactions involving carbenium ions, which can be considered as hydride acceptors. We also discuss the biochemical hydride‐transfer reactions in which the coenzyme NADH‐NAD⁺ plays an important role. With the calculations and the experimental model systems, an answer is given for the stereospecificity of the hydride transfer. Generally, the hydride transfer occurs via a trigonal pyramidal geometry in which the transferred hydride of the CH‐group is located in the axial position. In the case of the coenzyme NADH‐NAD⁺, the hydride transfer is coupled with an out‐of‐plane orientation of the carboxamide group of the pyridinium moiety, resulting in an increased stereospecificity. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Electrocatalytic oxidation of NADH at carbon paste electrodes modified with meldola blue adsorbed on zirconium phosphate: effect of Ca2+ and polyethyleneimine

The basic electrochemistry of carbon paste electrodes modified with Meldola Blue adsorbed on zirconium phosphate (MB-ZP-CPEs) and their ability to oxidize NADH have been investigated. Three types of carbon powder (graphite and glassy carbon-type Sigradur K and G) were used to obtain MB-ZP-CPEs. On comparing cyclic voltammograms recorded at MB-ZP-CPEs, similarly prepared from the three different carbon powders, those made with Sigradur K exhibited the lowest background current, and the best MB electrochemistry, seen as the highest peak intensities and smallest peak separation. Using MB-ZP-CPEs based on Sigradur K a study on NADH oxidation was done focusing on the effect of the Ca²⁺ concentration in the contacting solution and on the addition of polyethyleneimine (PEI) into the paste. It can be stated that MB-ZP-CPEs based on Sigradur K and containing 1.23% (w/w) PEI exhibited the best behavior for NADH oxidation, measured by the highest electrocatalytic rate constant (8.2×10³ M^–1 s^–1).     

Low-potential stable detection of β-NADH at sol–gel derived carbon composite electrodes

Sol–gel derived carbon composite electrodes, prepared from different non-silicate metal alkoxide precursors, offer a substantial decrease in the overvoltage of the NADH oxidation reaction (compared to ordinary carbon electrodes). Such promotion is attributed to acceleration of the proton-transfer step by the metal-oxide component of the composite. Passivation problems, accrued by accumulation of reaction products, are also greatly minimized. Both titania–, zirconia–sol–gel carbon composite electrodes thus offer a highly sensitive and stable anodic detection of NADH at +0.2 V. Greatly improved retention of the redox mediator Meldola Blue within the sol–gel network permits convenient measurements at NADH at −0.1 V. These improvements indicate great promise for the design of dehydrogenase-based amperometic biosensors. An intrinsic activation action by the metal-oxide component is also reported towards the oxidation of hydrazine, hydrogen peroxide, ascorbic acid and catechol. Low-potential detection of NADH is also illustrated at microfabricated titania/carbon screen-printed electrodes.     

A hyaluronic acid dispersed carbon nanotube electrode used for a mediatorless NADH sensing and biosensing

A biocompatible nanocomposite consisting of single-walled carbon nanotubes (CNTs) dispersed in a hyaluronic acid (HA) was investigated as a sensing platform for a mediatorless electrochemical detection of NADH. The device was characterised by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and extensively by electrochemistry. CNT-HA bionanocomposite showed more reversible electrochemistry, higher short-term stability of NADH sensing and higher selectivity of NADH detection compared to frequently used CNT-CHI (chitosan) modified GCE. Finally the performance of the sensor modified by CNT-HA was tested in a batch and flow injection analysis (FIA) mode of operation with basic characteristics revealed. The NADH sensor exhibits a good long-term operational stability (95% of the original sensitivity after 22 h of continuous operation). Subsequently a d-sorbitol biosensor based on such a nanoscale built interface was prepared and characterised with a d-sorbitol dehydrogenase used as a biocatalyst.     

Bio-electrocatalysis of NADH and ethanol based on graphene sheets modified electrodes

Characterization and application of graphene sheets modified glassy carbon electrodes (graphene/GC) have been presented for the electrochemical bio-sensing. A probe molecule, potassium ferricyanide is employed to study the electrochemical response at the graphene/GC electrode, which shows better electron transfer than graphite modified (graphite/GC) and bare glassy carbon (GC) electrodes. Based on the highly enhanced electrochemical activity of NADH, alcohol dehydrogenase (ADH) is immobilized on the graphene modified electrode and displays a more desirable analytical performance in the detection of ethanol, compared with graphite/GC or GC based bio-electrodes. It also exhibits good performance of ethanol detection in the real samples. From the results of electrochemical investigation, graphene sheets with a favorable electrochemical activity could be an advanced carbon electrode materials for the design of electrochemical sensors and biosensors.     

核黄素与NADH在紫光波段的激光诱导荧光光谱研究

基于细胞代谢荧光体的激光诱导荧光探测,在生物反应过程监测与生物活性物质探测中具有广泛的应用.本文利用波长可调谐激光光源,结合液体射流进样装置,研究了核黄素与NADH等生物荧光体在紫光波段(389 nm～404 nm)激发的荧光光谱,并考察了激光强度、样品浓度等参数对荧光光谱特性的影响.实验观察到核黄素的激发光谱在402.5 nm处出现"波谷",具有特征性,选择403.5 nm激光激发,核黄素的浓度灵敏度约为NADH的八百分之一.这些结果为发展生物荧光探测与识别技术提供了新的基础数据.