褪色光度法测定Fenton反应产生的羟自由基及其应用

建立检测Fenton反应产生羟自由基的新方法。Fenton反应产生的羟自由基与苋菜红反应，颜色发生变化，用分光光度计测定其△A510值的变化，可间接测定羟自由基的生成量。通过对测定条件的研究，确定了体系最佳实验条件。抗氧化药物甘露醇、硫脲与羟自由基清除率具有明显的量效关系。测定了阿魏酸、芦丁等几种中药活性成分清除羟自由基的功能，此法可用于羟自由基清除剂的筛选及抗羟自由基机理研究。     

罗丹明B显色检测Fenton反应产生的羟自由基

提出Fe2 H2O2 罗丹明B分析新体系并用于羟自由基的检测。方法用Fenton反应产生羟自由基,加入显色剂罗丹明B,羟自由基使罗丹明B的颜色发生变化,通过紫外 可见分光光度计测其ΔA值的变化,可间接测定羟自由基产生的量。通过测定条件的研究,探讨了最佳实验条件。该方法稳定,可作为一种简便的筛选抗氧化剂的方法。     

亚甲蓝光度法测定羟自由基

利用亚甲蓝被氧化后褪色的机理,使用Fenton试剂与亚甲蓝作用,验证了体系中起氧化作用的是羟自由基,从而建立了亚甲蓝光度法检验羟自由基的方法。试验结果表明,在过氧化氢与亚甲蓝的摩尔比为1~20范围内,亚甲蓝吸光度的降低值与Fenton试剂的加入量呈直线关系,因而可在660 nm处测定亚甲蓝的褪色程度(ΔA)对羟自由基作间接光度测定。试验同时对超声法产生的羟自由基进行了测定,表明羟自由基的生成量与超声时间成正比。     

HPLC-ECD检测羟自由基方法的研究

采用高效液相色谱结合库仑阵列多电极检测器体系(HPLC-ECD)建立了一套操作简便、高效、快速测定羟自由基的方法,并探究了检测的灵敏性,浓度和响应因子之间的线性关系,系统的稳定性等.2,3-二羟基苯甲酸(2,3-DHBA)、 2,5-二羟基苯甲酸(2,5-DHBA)以及水杨酸(SA)的相关系数分别为0.9999、 0.9996、 0.9968.2,3-DHBA和2,5-DHBA的检出限都低于0.1 ng/mL,SA的检出限均低于10 ng/mL,各种物质的方法回收率为100.5%～102.9%.     

Determination of the hydroxyl radical by its adduct formation with phenol and liquid chromatography/electrochemical detection

An HPLC-ECD method is described for the indirect determination of the hydroxyl (OH) radical. Fenton's reaction is used to produce OH, which simultaneously attacks phenols (phenol or pyrocatechol) to form the adducts, pyrocatechol or pyrogallic acid. Thus, [OH] quantification is based on the separation and detection of pyrogallic acid and/or pyrocatechol by an isocratic HPLC-ECD method. The quantification of OH is also performed alternatively by a chronoamperometric detection in an electrochemical cell, where simultaneously formed Fe^III (Fenton's reaction) combines [Fe^II(CN)₆]⁴⁻ to produce the Prussian blue (PB) molecules (Fe₄^III[Fe^II(CN)₆]₃). Newly formed PB molecules are then immediately converted to colorless Everitts salt (K₄Fe₄^II[Fe^II(CN)₆]₃) with the reduction of the high-spin Fe^III to Fe^II at the surface of a glassy carbon electrode at +0.150 V (versus Ag/AgCl). The calculated concentration of OH during incubation (0.626 ppm) can be detected with negative errors by the HPLC-ECD (0.595 and 0.615 ppm with the errors −5.2 and −1.8%, respectively) and by the chronoamperometric method (0.552 and 0.607 ppm with the errors −11.8 and −3.0%, respectively). For the comparison of the two sets of data, HPLC-ECD method is much more promising.     

Distinguishing the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical quenching effect from the hydroxyl radical scavenging effect in the ESR spin-trapping method

The spin-trapping method has been widely accepted to measure the hydroxyl radical scavenging activity in electron spin resonance (ESR) spectroscopy, however, the disappearance of the trapped signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical may lead to an erroneous conclusion, as the decreased signal must result from the rapid quenching of the DMPO-OH formed. The DMPO-OH quenching activity of a compound can be measured by tracing the signal decay resulting from its addition after the cessation of DMPO-OH formation. It was found that Fe(II) ions could quench the DMPO-OH radical quite rapidly, and phosphate ions could enhance the DMPO-OH radical quenching activity of the Fe(II) ions.     

Sensitive label-free electrochemical immunoassay by electrocatalytic amplification

A label-free electrochemical immunosensor for the detection of protein analytes without redox-active centers is for the first time developed based on the combination of gold nanoparticles and the mediated charge transport through the multilayer films that is related to an electrocatalytic process. Given the low detection limit, high sensitivity and selectivity, the success achieved here seems plausibly to serve as a significant step toward the development of versatile label-free immunoassay.     

DNA binding hydroxyl radical probes

The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.     

Assessing the non-specific hydroxyl radical scavenging properties of melanoidins in a Fenton-type reaction system

Soluble high molecular weight fraction (>10 kDa, named melanoidins) was isolated from Maillard reaction model systems, coffee, beer and sweet wine by ultrafiltration. Deoxyribose method was adjusted for measuring the hydroxyl radical scavenging properties of melanoidins. The presence of competitive melanoidins with deoxyribose for OH decrease the rate of deoxyribose degradation. Possible interferences to the deoxyribose method have been evaluated. Most of isolated melanoidins exhibited a variable and measurable non-site-specific hydroxyl scavenging activity in a Fenton-type reaction system. The iron reducing properties of melanoidins at the reaction conditions were evaluated with ferrozine. It has established a kinetic approach to assess the second rate constants of hydroxyl radical scavenging reactions of melanoidins. This approach may be a valuable tool for addressing the structure-activity relationships of melanoidins in a future. There is no correlation between browning (absorbance at 420 nm) and efficiency for scavenging hydroxyl radicals in solution.     

Sensitive electrochemical detection of the hydroxyl radical using enzyme-catalyzed redox cycling

Enzyme-catalyzed signal amplification was introduced to the electrochemical detection of the OH radical. In the presence of phenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-containing glucose dehydrogenase (PQQ-GDH) as a catalyst, the current signal for the trapping adducts (catechol and hydroquinone) produced by the hydroxylation of phenol could be amplified and detected sensitively. The limit of detection (S/N = 3) for catechol was 8 nM. The trapping efficiency of phenol was also estimated.     

Theoretical and experimental interpretations of phenol oxidation by the hydroxyl radical

Phenol oxidation by OH radicals produced by the Fenton reaction was studied and the oxidation process was monitored by the UV–visible, ¹³C NMR and LC techniques. The results show that benzoquinone is formed. In the NMR and LC experiments, since the peaks corresponding to isomers ortho and para- benzoquinones are unresolved, DFT was used to determine the branching ratios of the isomers formation that coincides with their ΔG values (ortho > para > meta): 72% for ortho, 23% for para and 5.0% for meta. Furthermore, the energy profile of the OH attack at ortho is quite similar to that at the para position while the meta position attack is less favored by 2.0 kcal/mol.     

Determination of hydroxyl radical by capillary electrophoresis and studies on hydroxyl radical scavenging activities of Chinese herbs

High-performance capillary electrophoresis (CE) with electrochemical detection (ED) was employed to determine hydroxyl radicals in the Fenton reaction. Hydroxyl radicals can react with salicylic acid to produce 2,3-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid, which can be analyzed by CE-ED. Based on this principle, hydroxyl radicals were determined indirectly. In a 20 mmol/L phosphate running buffer (pH 7.4), 2,3-dihydroxy benzoic acid and 2,5-dihydroxy benzoic acid would elute simultaneously from the capillary within 6 min. As the working electrode, a 300 m diameter carbon-disk electrode exhibits good responses at +0.60 V (vs. SCE) for the two analytes. Peak currents of the two analytes are additive. Excellent linearity was obtained in the concentration range from 1.0×10^-3 mol/L to 5.0×10^-6 mol/L for 2,3-dihydroxy benzoic acid. The detection limit (S/N=3) was 2.0×10^-6 mol/L. This method was successfully applied for studying hydroxyl radical scavenging activities of Chinese herbs. It is testified that Apocynum Venetum L., Jinkgo bibola L., Morus alba L. and Rhododendron dauricum L. have strong hydroxyl radical scavenging activities.     

Hydroxylation of 3-nitrotyrosine by hydroxyl radical

Hydroxylation of 3-nitrotyrosine (3-NT) and 3-NT containing peptide Gly-nitroTyr-Gly in aqueous solution by hydroxyl radical were investigated with gamma irradiation. The structures of the hydroxylated products were confirmed by electrospray ionization mass spectrometry and 1H NMR spectrometry. The reactivity of 3-nitrotyrosine has been investigated using density functional theory (DFT) calculation.     

Nanosilver resonance scattering spectral method for determination of hydroxyl radical and its application

Silver nanoparticles were prepared under a microwave high-pressure condition using citric acid sodium as a reducer while the excess citrate was removed under high speed centrifugation. There is a resonance scattering (RS) peak at 470 nm for silver nanoparticles. In a pH 4.0 HAc-NaAc buffer solution, hydroxyl radicals from the Fenton reaction can oxidize silver nanoparticles to Ag , resulting in the RS intensity decreasing. The decreased RS intensity at 470 nm (△I 470 nm) is linear with respect to the concentration of H2O2 (C) in the range of 0.27-7.56 μmol/L with a detection limit of 0.23 μmol/L. Its regression equation is △I 470 nm = 24.3 C 13.8 with a correlation coefficient of 0.9959. This method was applied to screening the antioxidants with satisfactory results.     

Covalent organic frameworks functionalized carbon fiber paper for the capture and detection of hydroxyl radical in the atmosphere

Developing a fast, sensitive and convenient method for the detection of hydroxyl radicals (OH) in the atmosphere could help us know the precursor levels of atmospheric species and control air pollution. In this work, the carbon fiber paper (CFP) functionalizing with a kind of covalent organic frameworks (COFs), formed from 1,3,5-triformylphloroglucinol (Tp) and benzidine (BD) (COF(TpBD)), was firstly used a new platform for OH trapping and detection. The COF(TpBD) modified CFP was acted as a filter to impregnate salicylic acid (SA) and a detector to detect 2,5-dihydroxybenzoic acid (2,5-DHBA) which was produced from the reaction between the impregnated SA and OH in the atmosphere. This method provided a linearity for 2,5-DHBA from 5.0 × 10⁻¹⁴ mol/L 1.0 × 10⁻⁹ mol/L with a detection limit of 6.9 × 10⁻¹⁵ mol/L, which is corresponding to the amount of OH from 3.0 × 10⁷ to 6.0 × 10¹¹ molecules/cm³ with the detection limit of 4.1 × 10⁶ molecules/cm³. This COF(TpBD)-CFP platform has been successfully applied for the detection of OH concentration under different conditions of Yangzhou when the sampling time was shortened to 30 min. This work has provided a new method for atmospheric OH detection with excellent sensitivity, simplicity, and high speed.     

Rapid screening,separation, and detection of hydroxyl radical scavengers from total flavonoids of Ginkgo biloba leaves by chromatography combined with molecular devices

Hydroxyl radicals are the most reactive free radical of human body, a strong contributor to tissue damage. In this study, liquid chromatography coupled to electrospray ionization mass spectrometry was applied to screen and identify hydroxyl radical scavengers from the total flavonoids of Ginkgo biloba leaves, and high‐performance counter current chromatography was used to separate and isolate the active compounds. Furthermore, molecular devices were used to determine hydroxyl radical scavenging activities of the obtained hydroxyl radical scavengers and other flavonoids from G. biloba leaves. As a result, six compounds were screened as hydroxyl radical scavengers, but only three flavonoids, namely, rutin, cosmos glycosides and apigenin‐7‐O‐Glu‐4’‐O‐Rha, were isolated successfully from total flavonoids by high‐performance counter current chromatography. The purities of the three obtained compounds were over 90%, respectively, as determined by liquid chromatography. Molecular devices with 96‐well microplates evaluation indicated that the 50% scavenging concentration values of screened compounds were lower than that of other flavonoids, they performed greater hydroxyl radical scavenging activity, and the evaluation effects were consistent with the liquid chromatography with mass spectrometry screening results. Therefore, chromatography combined with molecular devices is a feasible and an efficient method for systematic screening, identification, isolation, and evaluation of bioactive components in mixture of botanical medicines.     

次甲基蓝-Mn2+-H2O2体系光度法测定中药的抗氧化性

提出了Mn^2 -H2O2一次甲基蓝分析新体系并用于羟自由基的测定：醋酸介质中,用Mn^2 与RQ反应,类似Fenton试剂产生羟自由基,羟自由基与次甲基蓝反应,使次甲基蓝褪色,用分光光度汁测定其吸光度的变化(△A664),可间接测定羟自由基的产生量．实验结果表明．△A664与次甲基蓝、MnSO4及H2O2呈量效关系．中药提取物可以清除溶液中的羟自由基,使次甲基蓝溶液的褪色程度降低,据此建立了一种测定中药对羟自由基清除率的新方法．测定了五倍子、诃子、金银花等11种中药的抗氧化性,结果满意。     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

Development of a novel terbium(III) chelate-based luminescent probe for highly sensitive time-resolved luminescence detection of hydroxyl radical

Time-resolved (or time-gated) luminescence detection technique using lanthanide chelates as luminescent probes is a widely used and highly sensitive method for the biological applications. The developments of various functional lanthanide probes that can selectively recognize the biological targets are the essential objective of the technique. In this work, a unique Tb³⁺ chelate-based luminescent probe, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-(p-aminophenoxy)methylene-pyridine] tetrakis(acetate)-Tb³⁺(BMPTA-Tb³⁺), has been designed and synthesized for highly selective and sensitive time-resolved luminescence detection of one highly reactive oxygen species (ROS), hydroxyl radical (OH). The probe is almost non-luminescent, and can selectively react with hydroxyl radical to afford a highly luminescent Tb³⁺ chelate, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-hydroxymethyl-pyridine] tetrakis(acetate)-Tb³⁺ (BHTA-Tb³⁺), accompanied by a 49-fold increase in luminescence quantum yield with a long luminescence lifetime (2.76 ms). The luminescence response of the probe to hydroxyl radical is highly selective and insensitive to pH in the physiological pH range. For loading the probe into the living cells, the acetoxymethyl ester of BMPTA-Tb³⁺ was synthesized and used for the HeLa cell loading. Based on this probe, a background-free time-resolved luminescence imaging method for detecting hydroxyl radical in living cells was successfully established.     

Simple and sensitive determination of hydroxyl radical in atmosphere based on an electrochemically activated glassy carbon electrode

The hydroxyl radical (?OH) plays important roles in environment and health problems. However, the short life time and low concentrations of ?OH limited its detection. In this work, a simple method has been successfully performed for the sensitive detection of hydroxyl radical based on an activated glassy carbon electrode (AGCE).4-hydroxybenzoic acid (4-HBA) was used as a trapping agent for ?OH radicals, leading to the production of electroactive 3,4-dihydroxybenzoic acid (3,4-DHBA). Different procedures including polarisation and cyclic voltammetry in acid or base solutions have been used to activate the glassy carbon electrodes. The electrochemical behaviours of 3,4-DHBA on these activated electrodes were studied and compared. Experimental results showed that the glassy carbon electrode polarised in H₂SO₄ (AGCE-P/H₂SO₄) has the greatest sensitivity and reproducibility to 3,4-DHBA. 3,4-DHBA performed a linear relationship from 1.0 × 10^?7 to 1.0 × 10^?4 M on the AGCE-P/H₂SO₄. The detection limit was down to 6.2 × 10^?8 M. This method has been successfully applied for the detection of hydroxyl radical levels in atmosphere without separation and purification process.