碳纳米管／壳聚糖修饰电极的制备及其对NADH的电催化氧化

本文用浓硝酸活化多层碳纳米管,将壳聚糖与活化后的碳纳米管制备成复合材料,并将其滴涂于玻碳电极表面,制备出烟酰胺腺嘌呤二核苷酸(NADH)的电化学传感器。采用循环伏安法研究了该传感器的电化学性质以及对NADH的电催化氧化行为。实验结果表明,NADH在该电极上于 0.37V(vs.SCE)左右出现一氧化峰,与未修饰的玻碳电极相比,该修饰电极明显降低了NADH的氧化峰电位,消除了反应中间产物对电极表面的污染问题。对实验条件进行了优化,建立了碳纳米管/壳聚糖修饰电极微分脉冲伏安法测定NADH的方法。本文方法具有较好的重现性和选择性,对NADH检测的线性范围为1.0×10-4~9.0×10-3mol/L,检测限为9.2×10-5mol/L。     

Novel applications of fluorescence sensors

Typically, NAD(P)H-sensitive culture probes have been used to estimate biomass concentrations in suspended-cell cultivations, but these sensors have other uses as well. A number of applications, ranging from biosensors to immobilized-cell metabolic studies, are presented.     

Induced circular dichroism and uv spectra of inclusion complexes ofN-alkyl-dihydronicotinamides with cyclomaltoheptose and heptakis (2,6-di-O-methyl)cyclomaltoheptose

The mode of inclusion ofN-alkyldihydropyridines into native or modified cyclomaltooligosaccharides (cyclodextrins), as inferred from induced CD and UV spectra, was found to depend on the nature of theN-alkyl residue of the dihydronicotinamide, on the medium, and on the host-guest molar ratio.     

铝离子对辅酶NADH分子荧光增强和构象变化的影响

烟酰胺腺嘌呤二核苷酸(NADH)是生物体内重要的辅酶分子,在细胞能量代谢中发挥着关键作用。金属离子可以影响NADH所参与的酶促反应,其中铝离子（Al3+）对神经系统具有毒性,可以引发神经退行性疾病。因此,Al3+和NADH分子间相互作用的研究有助于了解Al3+对生物体内TCA循环和酶促反应的影响,具有重要的生物学意义。本文采用紫外-可见吸收和稳态荧光光谱,结合时间相关单光子计数技术(TCSPC),研究了Al3+对水溶液中NADH的本征荧光光谱和分子构象变化的影响。紫外-可见吸收光谱显示,NADH与Al3+的结合不会改变NADH分子腺嘌呤和烟酰胺两个本征发色团的吸收特性。为避免NADH分子内两个本征发色团之间的荧光共振能量转移效应的影响,采用340 nm作为激发波长,比较了NADH与Al3+作用前后的荧光特性。实验结果证实,Al3+可以与NADH焦磷酸盐桥上的两个氧原子相结合,使NADH分子的结构变得相对更加刚性,从而抑制NADH分子在溶液中的转动等非辐射过程,导致NADH分子平均荧光寿命增加,最终引起NADH分子荧光强度随Al3+浓度的增加而线性增强。进一步,采用NADH本征荧光寿命振幅比的研究方法表征了NADH分子在溶液中的两种主要构象形式：腺嘌呤和烟酰胺相互堆积的折叠构象以及腺嘌呤和烟酰胺相互分离的展开构象。研究发现,Al3+会打破溶液中NADH分子展开构象和折叠构象的平衡状态,促使辅酶NADH分子的展开构象转变为折叠构象,最终达到新的动态平衡,并且当NADH和Al3+以不大于1∶2的浓度比结合时,NADH分子两种构象的振幅比与铝离子浓度的对数间存在线性关系,在Al3+浓度检测等领域具有良好的应用前景。     

A Simple and Effective Way to Integrate Nile Blue Covalently onto Functionalized SWCNTs Modified GC Electrodes for Sensitive and Selective Electroanalysis of NADH

A simple and new way to assemble Nile blue (NB) covalently onto the surface of functionalized single‐walled carbon nanotubes (f‐SWCNTs) modified glassy carbon (GC) electrode (NB/f‐SWCNTs/GC electrode) was described. The NB/f‐SWCNTs/GC electrode catalyzes effectively the oxidation of NADH with a remarkably decreased overpotential (ca. 700 mV) compared with that at the bare GC. The reaction was found to obey a so‐called Michaelis–Menten kinetics and the related kinetic parameters were determined. This modified electrode possesses promising characteristics as NADH sensor; a wide linear dynamic range of 0.2 to 200 µM, low detection limit of 0.18 µM, fast response time (1–2 s), high sensitivity (24 µA cm⁻² mM⁻¹), anti‐interference ability and anti‐fouling.     

Consistency of the Mediated Electrochemical Method and the Fluorescence Method in Monitoring the Catabolic Activities of Yeasts

Abstract

The mediated electrochemical method and the intrinsic NADH fluorescence method were employed in evaluating the catabolic activities of three yeasts. The responses from the menadione/ferricyanide system were 70.00 ± 2.25 nA, 61.39 ± 1.76 nA, and 57.18 ± 1.51 nA, respectively, for Saccharomyces cerevisiae, Pachysolen tannophilus, and Pichia stipitis. The NADH fluorescence intensities were 1638 ± 25.46 FI, 1039 ± 18.67 FI, and 963.4 ± 15.78 FI, respectively,for S. cerevisiae, P. tannophilus, and P. stipitis. It was evident that there is a positive relationship between the mediated electrochemical method and the intrinsic NADH fluorescence method in cellular metabolic activity assays.     

Ultrasensitive photoelectrochemical sensing of nicotinamide adenine dinucleotide based on graphene-TiO2 nanohybrids under visible irradiation

In this paper, the photoelectrochemical behavior of graphene-TiO₂ (G-TiO₂) nanohybrids was investigated in the visible region and a new photoelectrochemical sensor for sensitive determination of nicotinamide adenine dinucleotide (NADH) was proposed. Under visible light, the G-TiO₂ nanohybrids possessed enhanced photocurrent, which was nearly 5 times than that of pure TiO₂ nanocrystals (NCs). Based on the enhanced photocurrent of G-TiO₂ nanohybrids toward NADH, a new photoelectrochemical methodology for ultrasensitive determination of NADH was developed. The proposed sensor showed linearly enhanced photocurrent by increasing the NADH concentration from 1.0 × 10⁻⁸ to 2.0 × 10⁻³ M with a low detection limit of 3.0 × 10⁻⁹ M. Furthermore, this sensor exhibited good selectivity and stability towards NADH determination. This strategy opens up a new avenue for the application of graphene-based hybrids in the field of photoelectrochemical sensing and monitoring.     

Homogeneous Amperometric Immunoassay for Digoxin

Abstract

A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.     

On-line Characterization of Physiological State in Poly(β-Hydroxybutyrate) Production by <Emphasis Type="Italic">Wautersia eutropha</Emphasis>

Culture fluorescence measurement technique has the potential for on-line characterization of metabolic status of fermentation processes. Many fluorophores present inside the living cells such as NADH + H⁺, tryptophan, pyridoxine, and riboflavin fluoresce at specific excitation and emission wavelength combinations. Since these key intracellular metabolites are involved in cell growth and metabolism, their concentration change at any time inside the cell could reflect the changes in cell metabolic activity. NADH + H⁺ spectrofluorometry was used for on-line characterization of physiological state during batch cultivation of poly-β-hydroxybutyric acid (PHB) production by Wautersia eutropha. The culture fluorescence increased with an increase in the biomass concentration with time. A linear correlation between cell mass concentration and net NADH + H⁺ fluorescence was established during active growth phase (13 to 38 h) of batch cultivation. The rate of change of culture fluorescence (dF/dt) exhibited a gradual increase during the predominantly growth phase of batch cultivation (till 20 h). Thereafter, a sudden drop in the dF/dt rate and its leveling was recorded indicating major changes in culture metabolism status which synchronized with the start-up of accumulation of PHB. After 48 h, yet another decrease in the rate of change of fluorescence (dF/dt) was observed primarily due to severe substrate limitation in the reactor. On-line NADH + H⁺ fluorescence signal and its rate (dF/dt) could therefore be used to distinguish the growth, product formation, and nutrient depletion stage (the metabolic state marker) during the batch cultivation of W. eutropha.     

Laser-induced autofluorescence for medical diagnosis

The naturally occurring autofluorescence of cells and tissues is based on biomolecules containing intrinsic fluorophores, such as porphyrins, the amino acids tryptophan and tyrosine, and the coenzymes NADH, NADPH, and flavins. Coenzymes fluoresce in the blue/green spectral region (fluorecence lifetimes: 0.5–6 ns) and are highly sensitive indicators of metabolic function. Steadystate and time-resolved blue-green autofluorescence is, therefore, an appropriate measure of the function of the respiratory chain as well as of cellular and tissue damage. Autofluorescence in the yellow/red spectral region is based mainly on endogenous porphyrins and metalloporphyrins, such as coproporphyrin, protoporphyrin (fluorescence lifetime of porphyrin monomers: >10 ns), and Zn-protoporphyrin (2 ns). Various pathological microorganisms such asPropionibacterium acnes, Pseudomonas aeruginosa, Actinomyces odontolyticus, Bacteroides intermedius, andSaccharomyces cerevisiae are able to synthesize large amounts of these fluorophores and can therefore be located. This permits fluorescence-based detection of a variety of diseases, including early-stage dental caries, dental plaque, acne vulgaris, otitis externa, and squamous cell carcinoma. The sensitivity of noninvasive autofluorescence diagnostics can be enhanced by time-gated fluorescence measurements using an appropriate time delay between ultrashort laser excitation and detection. For example, videocameras with ultrafast shutters, in the nanosecond region, can be used to create caries images of the teeth. Alternatively, autofluorescence can be enhanced by stimulating protoporphyrin biosynthesis with the exogenously administered porphyrin precursor 5-aminolevulinic acid (ALA). The fluorophore protoporphyrin IX (PP IX) is photolabile and photodynamically active. Irradiation of PP IX-containing tissue results in cytotoxic reactions which correlate with modifications in fluorescence due to photobleaching and singlet oxygen-dependent photoproduct formation. Therefore, on-line autofluorescence measurements during the phototreatment can yield information on the efficiency of ALA-based photodynamic therapy.