Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and -lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or -lactic acid in the range 0.05–2 × 10^-9 mol.     

Electrochemical enzymatic determinations of ethanol and l-lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and l-lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or l-lactic acid in the range 0.05–2 × 10^-9 mol.     

An enzymatic-fluorimetric method for monitoring of ethanol in ambient air

A method is described for the continuous monitoring of ethanol in ambient air. The system consists of a scrubber coil for enrichment of the analyte from air in an aqueous solution and a directly connected fluorescence detector. Because of using a reagent solution containing alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) for absorption, ethanol can react directly with ADH and NAD⁺ during air sampling, producing NADH, which can be measured by fluorescence detection. The influence of reagent concentrations, gas flow rate and scrubber solution flow rate on the performance of the instrument was tested. Possible ozone interferences can be avoided by placing a KI coated filter in front of the scrubber inlet. The response time of the system was found to be 2.3 min and the detection limit about 1 ppb_V. The applicability of the developed method was demonstrated during a field campaign in Brazil.     

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD⁺), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD⁺, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD⁺, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD⁺ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD⁺, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

Equilibrium (NAD+/NADH) potential on poly(Neutral Red) modified electrode

The electrochemical regeneration of nicotinamide adenine dinucleotide (NAD⁺/NADH) has been one of the central subjects of bioelectrochemistry during past three decades. We report on the unique chemical electrocatalyst for NAD⁺/NADH regeneration based on electropolymerized Neutral Red. Using poly(Neutral Red) modified electrodes, the reversible polarographic waves of nicotinamide adenine dinucleotide reduction–oxidation and the equilibrium (NAD⁺/NADH) potential were observed. This was impossible using all known catalytic and mediator systems. The unique poly(Neutral Red) based electrocatalyst allowed us to determine the standard (NAD⁺/NADH) potential more precisely (E^′≅0.59 V SCE, pH 6.0).     

脯氨酸保护的铜纳米团簇的制备及其在三硝基苯酚检测中的应用

以脯氨酸(Pro)为保护剂,盐酸羟胺为还原剂,通过一步化学还原法制备脯氨酸稳定的铜纳米团簇(Cu NCs).采用分子荧光仪和紫外可见吸收仪对Cu NCs的光学性质进行分析,通过透射电子显微镜(TEM)、X射线光电子能谱(XPS)和傅里叶变换红外波谱仪(FTIR)对Cu NCs的结构进行表征.TEM图像显示Cu NCs的...     

Sequential Injection Kinetic Flow Assay for Monitoring Glycerol in a Sugar Fermentation Process by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD⁺ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD⁺ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in agreement with those obtained with a high-performance liquid chromatography method.     

Fluorescein derivative-based, selective and sensitive chemosensor for NADH

Fluorescein mercury acetate (FMA) derivative can be used as a chemosensor for NADH that plays key roles in cellular energy metabolism and dehydrogenase enzymatic reactions. The sensor recognizes NADH by two mercury metal ions in the compound using the metal-anion interaction and its subsequent binding-induced fluorescence changes of FMA. The FMA can detect an aqueous NADH concentration of approximately 1.0 μM and has a high selectivity over various anions including NAD⁺, the oxidized form of NADH.     

A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition

In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5′-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1–2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.     

pH Resistant Ratiometric Measurement of Nicotinamide Adenine Dinucleotide Levels by Time-resolved Fluorescence Spectroscopy

Circular permutation fluorescent protein is a novel method to construct biosensors. The ratio of two excitation channels is employed to quantitatively calibrate the level of analysts. SoNar is one of them, which can be used to monitor cellular NADH/NAD⁺ levels. However, the 490 nm excitation channel of these biosensors is sensitive to pH environments, which is negative in real applications. In this work, we demonstrated that the fractional intensity ratio extracted from time-resolved fluorescence spectroscopy could be used to quantify NADH levels with one excitation (420 nm) and one emission channels. The 420-nm excitation channel was pH resistant. Comparing to average lifetime, the fractional intensity ratio had a 3.2-fold dynamic range, which was much wider than average lifetimes.     

Electrocatalytic oxidation of the reduced nicotinamide adenine dinucleotide at carbon ionic liquid electrode modified with polythionine/multi-walled carbon nanotubes composite

A carbon ionic liquid electrode (CILE) was modified with a polythionine (PTh)/multi-walled carbon nanotubes (MWCNTs) composite and used for the detection of reduced nicotinamide adenine dinucleotide (NADH). The electrode was prepared by electrochemical polymerization of thionine on the MWCNTs in neutral medium. Cyclic voltammetry indicated that the electrode was capable of mediating the oxidation of NADH at an overpotential as low as 0.03 V. Amperometric experiments showed that a sensitive and stable response towards NADH is obtained within 5 s. The linear range for the determination of NADH is from 0.8 μmol L^?1 to 422 μmol L^?1, with a detection limit of 0.26 μmol L^?1 (S/N = 3). The wide linear range, lower detection limit and faster response towards NADH suggests that the new method potentially is useful for developing NAD⁺-dependent enzyme-based biosensors.     

Cyclic regeneration of nicotinamide adenine dinucleotide with immobilized enzymes: Flow-injection spectrofluorimetric determination of ethanol

Ethanol (0.05–0.5%) in water is determined by injection of a 20-μl sample into a solution of 1.5 × 10^?3 M NAD⁺ in pH 8.0 phosphate buffer flowing from a reservoir. The solution passes through a minicolumn of yeast alcohol dehydrogenase immobilized on controlled-pore glass (CPG). The NADH formed is monitored spectrofluorimetrically in the flow system, before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on CPG in the presence of glutarate and ammonium ions, also in the flowing solution. The solution then returns to the reservoir. The regeneration of NAD⁺ allows the same coenzyme solution to be used for 50 ethanol determinations daily for 4 days.     

Cytidine-stabilized gold nanocluster as a fluorescence turn-on and turn-off probe for dual functional detection of Ag and Hg

In this study, we have developed a label-free, dual functional detection strategy for highly selective and sensitive determination of aqueous Ag⁺ and Hg²⁺ by using cytidine stabilized Au NCs and AuAg NCs as fluorescent turn-on and turn off probes, respectively. The Au NCs and AuAg NCs showed a remarkably rapid response and high selectivity for Ag⁺ and Hg²⁺ over other metal ions, and relevant detection limit of Ag⁺ and Hg²⁺ is ca. 10 nM and 30 nM, respectively. Importantly, the fluorescence enhanced Au NCs by doping Ag⁺ can be conveniently reusable for the detection of Hg²⁺ based on the corresponding fluorescence quenching. The sensing mechanism was based on the high-affinity metallophilic Hg²⁺–Ag⁺ interaction, which effectively quenched the fluorescence of AuAg NCs. Furthermore, these fluorescent nanoprobes could be readily applied to Ag⁺ and Hg²⁺ detection in environmental water samples, indicating their possibility to be utilized as a convenient, dual functional, rapid response, and label-free fluorescence sensor for related environmental and health monitoring.     

Albumin coated CuInS2 quantum dots as a near-infrared fluorescent probe for NADH, and their application to an assay for pyruvate

Water-soluble CuInS₂ quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD⁺). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD⁺ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.

Adsorption phenomena in the NAD+/NADH system at glassy carbon electrodes

A variety of electrochemical approaches has been used to investigate the adsorption of NAD⁺, NADH and the NAD-NAD dimer from aqueous solution at glassy carbon electrodes (GCE) with supplementary studies of adsorption at pyrolytic graphite and platinum electrodes from aqueous media and at GCE from DMSO solution. The following hypotheses are advanced concerning the adsorption orientation: at carbon electrodes, on which NADH is not adsorbed, NAD⁺ produced by anodic oxidation of the NADH is first rapidly adsorbed in a planar configuration relative to the electrode surface, which is probably bound to the surface through the adenine moiety; there is then a relatively slow reorientation of the adsorbed NADH molecules to a perpendicular orientation relative to the electrode surface, which adsorbate is more tightly bound to the surface than the planar oriented adsorbate and which likely involves interaction between parallel adenine and pyridinium rings. Reduction (one-electron process) of NAD⁺ at the GCE produces the NAD-NAD dimer, which, at a clean electrode surface, involves a diffusion-controlled process and an adsorption-controlled process; the latter is due to formation of adsorbed dimer, which is more strongly adsorbed than NAD⁺. The dimer is oxidized at the GCE only if it is adsorbed. The factors controlling and involved in the adsorption processes have been examined with particular reference to the use of anodic voltammetry for the analytical determination of NADH.     

Fluorescent sensing layer for the determination of<Emphasis Type="SmallCaps"> L</Emphasis>-malic acid in wine

An enzymatic method for determining L-malic acid in wine based on an L-malate sensing layer with nicotinamide adenine dinucleotide (NAD⁺), L-malate dehydrogenase (L-MDH) and diaphorase (DI), immobilized by sol-gel technology, was constructed and evaluated. The sol-gel glass was prepared with tetramethoxysilane (TMOS), water and HCl. L-MDH catalyzes the reaction between L-malate and NAD⁺, producing NADH, whose fluorescence (λ _exc = 340 nm, λ _em = 430 nm) could be directly related to the amount of L-malate. NADH is converted to NAD⁺ by applying hexacyanoferrate(III) as oxidant in the presence of DI. Some parameters affecting sol-gel encapsulation and the pH of the enzymatic reaction were studied. The sensing layer has a dynamic range of 0.1–1.0 g/L of L-malate and a long-term storage stability of 25 days. It exhibits acceptable reproducibility [s _r(%)≈10] and allows six regenerations. The content of L-malic acid was determined for different types of wine, and polyvinylpolypyrrolidone (PVPP) was used as a bleaching agent with red wine. The results obtained for the wine samples using the sensing layer are comparable to those obtained from a reference method based on UV-vis molecular absorption spectrometry, if the matrix effect is corrected for.     

Half‐sandwich Ruthenium (II) complexes with triphenylamine modified dipyridine skeleton and application in biology/luminescence imaging

Four half‐sandwich ruthenium^II (Ru^II) complexes with triphenylamine‐modifed dipyridine frameworks were synthesized and characterized. The cytotoxicity of target complexes toward A549 (lung cancer cells), HeLa (cervical cancer cells) and HepG2 (hepatoma cells) were obtained by the MTT assay, which were superior to cisplatin with the IC₅₀ values changed from 2.4 ± 0.1 μM to 9.2 ± 2.7 μM. Meanwhile, complexes possess the ability of antimetastasis to cancer cells. Ru^II complexes could be transported by serum albumin, catalyze the conversion of NADH (the reduced state of nicotinamide‐adenine dinucleotide) to NAD⁺ and induce the accumulation of reactive oxygen species, which confirmed the antineoplastic mechanism of oxidation. Ru^II complexes could enter A549 cells followed by a non‐energy dependent cellular uptake mechanism, target lysosomes with the Pearson's colocalization coefficient of 0.75, lead to lysosomal damage, disturb the cell cycle (S phase), and eventually induce apoptosis. The results demonstrate that these Ru^II complexes are potential anticancer agents with dual functions, including metastasis inhibition and lysosomal damage.     

2-巯基苯并咪唑保护的铜纳米团簇的制备及银离子检测

以2-巯基苯并咪唑为保护剂,聚乙烯吡咯烷酮为稳定剂,水合肼为还原剂,“一锅法”合成2-巯基苯并咪唑保护的,高稳定性、强荧光、大斯托克斯位移的铜纳米团簇(Cu NCs),并用于检测水样中的银离子含量。采用透射电子显微镜(TEM)和X射线光电子能谱(XPS)对铜纳米团簇的结构进行表征,通过荧光光谱和紫外可见光谱对其光学性能进行研究。研究表明:该铜纳米团簇的最大激发和发射波长分别为340和558 nm,在日光灯和紫外灯下分别为无色和橙色。Cu NCs具有较高的分散性,尺寸大小为2~3 nm。在最佳反应条件下,铜纳米团簇可以选择性地被银离子猝灭,且灵敏度高,线性范围为1~40μmol/L,检测限为0.5μmol/L。该方法用于检测实际水样(自来水、湖水)中银离子的浓度,结果令人满意,表明在环境监测中有非常高的应用价值。     

Preparation of an Electrode Modified with an Electropolymerized Film as a Mediator of NADH Oxidation and Its Application in an Ethanol/O2 Biofuel Cell

This paper reports a novel mediator for the oxidation of β‐nicotinamide adenine dinucleotide (NAD⁺/NADH), an electropolymeric film (pAPRu) of [Ru(NH₂‐phen)₃]²⁺. A pAPRu‐modified electrode was prepared via electropolymerization and exhibited catalytic activity toward the electrochemical oxidation of NADH due to the imine moieties of pAPRu. The electrochemical oxidation of ethanol was observed using an alcohol dehydrogenase (ADH)‐immobilized electrode. A compartmentless ethanol/O₂ biofuel cell composed of an ADH anode and a bilirubin oxidase cathode was constructed. The maximum current density and the maximum power density of the biofuel cell were 190 µA cm^?2 and 31 µW cm^?2 (at 0.29 V), respectively.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.