Photographic detection of fluorescent-labelled oligodeoxynucleotide in the blotting format by peroxyoxalate chemiluminescence

The preparation of a fluorescent labelled oligonucleotide and its photographic detection by peroxyoxalate chemiluminescence (PO-CL) are described. Fluorescent labelling of an oligonucleotide (15-mer) was performed with naphthalene-2,3-dicarboxaldehyde to give an N-substituted 1-cyanobenz[f]isoindole (CBI) derivative (CBI-15-mer). For the photographic detection of CBI-15-mer, the bis(2,6-difluorophenyl) oxalate (DFPO)-dimethyl phthalate (DMP) system was selected to obtain a long-lived CL emission. After optimizing the conditions for the CL reaction, the system was applied to the photographic detection, and as little as 250 fmol per spot of CBI-15-mer on a membrane were detected as a visible spot with an instant photographic film. © 1998 John Wiley & Sons, Ltd.     

Kinetic studies on the peroxyoxalate chemiluminescence reaction: determination of the cyclization rate constant

Although more currently utilized as analytical tool because of its high sensitivity and good reproducibility, the mechanism of the peroxyoxalate system, a chemiluminescence reaction with quantum yields only comparable to bioluminescence systems, has been extensively studied. The light emission mechanism can be divided in the pathway before chemiexcitation, which contains the rate‐limiting steps, and the fast and kinetically non‐observable chemiexcitation step. In this work, we obtain information on the mechanism of the slow pathways, attribute values to several rate constants prior to chemiexcitation and suggest a mechanistic scheme that could help optimization of conditions when the peroxyoxalate reaction is used as analytical tool. Copyright © 2002 John Wiley & Sons, Ltd.     

Amines for detection of dopamine by generation of hydrogen peroxide and peroxyoxalate chemiluminescence

A range of nitrogen-containing compounds (alkyl amines, piperazines, cyclohexylamines and nitrogen heterocyclics) were investigated for generation of hydrogen peroxide from dopamine and detection by peroxyoxalate chemiluminescence. Imidazole, ethyleneurea and allantoin among the nitrogen heterocyclic compounds tested generated hydrogen peroxide from dopamine following incubation at 60°C, pH 9.5–10.5, for 0–30 min. Imidazole was the most effective for generation of hydrogen peroxide, but imidazole derivatives with a primary amine side chain (histamine) or thiol (ethylenethiourea) were not effective. The presence of a ketone group (ethyleneurea, allantoin) did not hinder the reaction. Under optimal conditions (30 min incubation, 50 mmol/L imidazole) 10.5 nmol of dopamine could be detected. The cyclohexylamines tested produced low amounts of hydrogen peroxide (0.09–2.74% of light intensity with imidazole), and the piperazines and the alkyl amines tested produced no detectable hydrogen peroxide. Imidazole reacts with the phenolic groups of dopamine in a different manner from monoamine oxidase, and a reagent containing imidazole, ethyleneurea or allantoin was useful for non-enzymatic detection of dopamine by peroxyoxalate chemiluminescence.© John Wiley & Sons, Ltd.     

Detection of substances with alcoholic or phenolic hydroxyl groups by generation of hydrogen peroxide with imidazole and peroxyoxalate chemiluminescence

On-line detection of substances with an alcoholic or phenolic hydroxyl group using imidazole and peroxyoxalate chemiluminescence was investigated qualitatively using a flow-injection method. The substances tested included six polyphenols, five monophenols and six sugars. After incubation at 80°C with an imidazole buffer (pH 9.5) the substances were detected by peroxyoxalate chemiluminescence. The polyphenols tested (e.g., pyrogallol, purpurogallin, and dopamine) showed the strongest light emission. The sugars with hydroxyl groups (e.g., fructose and lactose) and the monophenols (e.g., phenol, serotonin, and β-estradiol) produced only a weak light emission. Reaction of hydroxyl compounds and imidazole generated hydrogen peroxide. Imidazole served two roles, it catalysed the reaction with the hydroxyl compound and initiated peroxyoxalate chemiluminescence on-line. A novel reactor formed by packing glass beads into a flow cell (Teflon) of a chemiluminometer improved the sensitivity of light detection.     

Determination of aromatic compounds by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection.

This paper describes a novel high-performance liquid chromatographic (HPLC) method for the determination of aromatic compounds with peroxyoxalate chemiluminescence (PO-CL ) detection following on-line UV irradiation. Aromatic compounds were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide, which was determined via PO-CL detection using a mixture of bis(2,4,6-trichlorophenyl)oxalate (aryloxalate) and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine (fluorophore) as a post-column CL reagent. Generation of hydrogen peroxide from aromatic compounds was confirmed using a flow injection analysis (FIA) system incorporating an enzyme column reactor immobilized with catalase. The conditions for UV irradiation were optimized using benzene and monosubstituted benzenes (phenol, benzaldehyde, nitrobenzene and N,N-dimethylaniline) by an HPLC system to evaluate the analytical performance of the proposed system. The detection limits for benzene and monosubstituted benzenes were in the range 2.1-124 pmol/injection at signal:noise (S:N) ratio = 3. Monocyclic and polycyclic hydrocarbons were also employed to investigate their CL properties. The possibility of PO-CL detection for a wide variety of aromatic compounds was shown for the first time.     

Unprecedented chemiluminescence behaviour during peroxyoxalate chemiluminescence of oxalates with fluorescent or electron-donating aryloxy groups.

A series of diaryl and bis(4-styrylphenyl) oxalates with electron-donating substituents or fluorescent moieties were subjected to the peroxyoxalate chemiluminescence (PO-CL) reaction, some of which were found to behave in a unprecedented manner. The reaction of bis(p-methyoxyphenyl) oxalate, as a representative example, emits light due not only to the emission from the externally added excited fluorophore, but also from the presumable excimer of p-methoxyphenol. Also, during the reaction of the bis(4-styrylphenyl) oxalates, the emission based on the fluorescence as well as the excimer of the eliminating group were observed. These experimental results suggest that such emitting species would be formed by an intra- and intermolecular electronic interaction with a high-energy intermediate, such as a dioxetanone.     

Hybridization of horseradish peroxidase-labeled probes and detection by enhanced chemiluminescence

This article describes the use of probes directly labeled with horseradish peroxidase in conjunction with enhanced chemiluminescence, which allows a flexible approach to hybridizations and detections. This system may be used with the following applications: Southern blots, Northern blots, colony and plaque screening for positive clones, YAC clone screening, and PCR products detection. The major steps required for the use of directly labeled HRP probes are hybridization, stringent washes, and detection.     

Development of a novel capillary electrophoresis chemiluminescence system for amino acid analysis

In the present study, a novel peroxyoxalate CE–CL system was developed to achieve high signal stability and sensitivity based on a design of a new interface including a new mixing mode and a new grounding electrode mode. Amino acids fluorescently tagged with dansyl chloride and naphthalene‐2,3‐dicarboxaldehyde(NDA) were used for the study. Experiment results show this new system is quite effective to separate and detect amine acid with high stability and resolute. The detection limits were 1.1 nmol/L for dansyl‐leucine (Leu) and 2.0 nmol/L for dansyl‐aspartic acid (Asp). The relative standard deviations of peak height and migration time were in the ranges of 2.3–3.8% and 1.2–1.5%, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Estimation of tyrosine by the attenuation of 4-iodophenol enhanced chemiluminescence

Tyrosine markedly attenuates the chemiluminescence output intensity from the 4-iodophenol enhanced chemiluminescence assay system in a manner consistent with competition between the amino acid and luminol for the 4-iodophenoxy radical. This effect provides the basis for a sensitive assay of tyrosine. Interference by the other amino acids has been assessed; major interference by cysteine can be removed by incubation with iodoacetic acid.     

Determination of serum oxalate using peroxyoxalate chemiluminescence of free oxalic acid

We describe a new sensitive and specific method for determination of oxalate in human serum. By using the chemiluminescence decay of monoperoxyoxalic acid very low concentrations of oxalate (200 nmol/L) can be determined. The mean serum oxalate level in apparently healthy controls was 14.5 ± 8.5 m?mol/L. Supplementation of ascorbic acid leads to an increase in serum oxalate level. While serum oxalate concentrations of calcium oxalate stone formers (x = 16.4 ± 9.8 m?mol/L) are not significantly different from the control group, an extreme increase of serum oxalate is evident in haemodialysis patients. The serum oxalate concentration decreased during dialysis treatment from 141.4 ± 32.1 m?mol/L to 36.4 ± 12.7 m?mol/L.     

Microflow‐injection chemiluminescence of luminol and hypochlorite enhanced by phloxine B

We report for the first time that the sensitivity of the luminol–hypochlorite chemiluminescence (CL) reaction was enhanced approximately 10 times by the addition of phloxine B. The maximum wavelength of CL emission shifted from 431 to 595 nm in the absence and presence, respectively, of phloxine B, suggesting that an efficient chemiluminescence resonance energy transfer occurred between a luminol donor and a phloxine B acceptor in the luminol–hypochlorite–phloxine B system. Based on this observation, a simple, rapid and sensitive microflow injection CL method, using a microchip with spiral channel configurations, was developed for the determination of hypochlorite. Under optimized conditions, a linear calibration curve (R² = 0.9944) over the range 0.1–10.0 µmol/L was obtained, with a detection limit of 0.025 µmol/L (S:N = 3). The relative standard deviation (RSD) was found to be 4.2% (n = 10) for 2.5 µmol/L hypochlorite. The sample consumption was only 2 μL, with a sample throughput of 90/h. The method has been used for determining trace amounts of hypochlorite in water samples with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

A lab‐on‐a‐chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab‐on‐a‐chip device. Peroxyoxalate‐CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis‐(2,4,6‐trichlorophenyl)oxalate‐CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10–100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.     

Determination of vitamin B6 using an optimized novel TCPO–indolizine–H2O2 chemiluminescence system

Indolizine derivatives are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence. The reaction of peroxyoxalates such as bis‐(2,4,6‐trichlorophenyl) oxalate (TCPO) with H₂O₂ can transfer energy to fluorescer via the formation of dioxetanedione intermediate. Four indolizine derivatives were used as a novel fluorescer in the chemiluminescence (CL) systems in this study. The relationship between CL intensity and the concentration of fluorescer, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Optimum conditions were obtained for four fluorescers and it was found that the indolizine can be used as an efficient green fluorescence emitter. Vitamin B₆ induces a sharp decrease in the CL intensity of the TCPO–hydrogen peroxide–sodium salicylate system. A simple, rapid and sensitive CL method for the determination of vitamin B₆ has been developed. The results showed a linear relationship between vitamin B₆ concentration and peroxyoxalate CL intensity in the range 7.0 × 10⁻⁸–1.0 × 10⁻⁴. A detection limit of 2.3 × 10⁻⁸ M and relative standard deviation (RSD) of < 4.5% were obtained. Copyright © 2014 John Wiley & Sons, Ltd.     

On the mechanism of peroxyoxalate chemiluminescence. Quenched chemiluminescence as a detection method in HPLC

Several analytes such as the inorganic anions bromide, iodide, sulphite and nitrite and organic compounds as substituted anilines and sulphur compounds cause quenching of peroxyoxalate chemiluminescence. A detection method for liquid chromatography based on the quenching phenomenon has been developed. It makes use of an immobilized luminophore, i.e. 3-aminofluoranthene covalently bound via an alkyl-spacer on controlled pore glass, packed in the detector cell. The mechanism behind the quenching has been elucidated by investigating the roles of luminophores (both in the liquid and in solid state) and oxalates in peroxylate CL with respect to quenchers. Most probably the quencher destroys the radical ion pair produced after electron transfer in the last stage of the CIEEL reaction scheme, thus preventing the formation of electronically excited luminophore.     

Enhanced chemiluminescence of lucigenin with epinephrine in cationic surfactant micelles containing periodate

Epinephrine (EP) species involved in the lucigenin chemiluminescence (CL) were identified in alkaline solution by comparing the time course of the CL response and the formation of EP oxidation products. EP quinone and adrenolutine (AL) were found to be responsible for the lucigenin-CL reaction. The mechanism of the lucigenin-CL enhancement was investigated using cationic micellar hexadecyltrimethylammonium hydroxide (CTAOH), periodate, and a mixture of micellar CTAOH and periodate. The CL enhancement in the presence of micellar CTAOH and periodate could be explained in terms of increases in the oxidation rate of EP to EP quinone and the intramolecular oxidation rate of adrenochrome to AL.     

Response surface optimized peroxyoxalate chemiluminescence of octahydro‐Schiff base derivative as new luminophor and study of the quenching effect of some cations,amino acids and cholesterol

We report the first detailed study of the characteristics of an octahydro‐Schiff base derivative as a new luminophor in the peroxyoxalate chemiluminescence (POCL) system. The effect of reagents on this new POCL system was investigated. In addition, the response surface methodology was used for evaluating the relative significance of variables in this POCL system, establishing models and determining optimal conditions. The quenching effect of some cations and compounds such as Cu²⁺, Fe³⁺, Hg²⁺, imidazole, histidine and cholesterol on an optimized POCL reaction were studied. The dynamic ranges were up to approximaterly 100 and 175 × 10^?6 M for Cu²⁺ and cholesterol respectively. The detection limits were 3.3 × 10^?6 m and 2.58 × 10^?6 m for Cu²⁺ and histidine, respectively. In all cases the relative standard deviations were 4–5% (n = 4). Copyright © 2014 John Wiley & Sons, Ltd.     

Oxidation and chemiluminescence of catechol by hydrogen peroxide in the presence of Co(II) ions and CTAB micelles.

The oxidation of catechol in neutral and slightly alkaline aqueous solutions (pH 7-9.6) by excess hydrogen peroxide (0.002-0.09 mol/L) in the presence of Co(II) (2.10(-7)-2.10(-5) mol/L) is accompanied by abrupt formation of red purple colouration, which is subsequently decolourized within 1 h. The electron spectra of the reaction mixture are characterized by a broad band covering the whole visible range (400-700 nm), with maximum at 485 nm. The reaction is initiated by catechol oxidation to its semiquinone radical and further to 1,2-benzoquinone. By nucleophilic addition of hydrogen peroxide into the p-position of benzoquinone C=O groups, hydroperoxide intermediates are formed, which decompose to hydroxylated 1,4-benzoquinones. It was confirmed by MS spectroscopy that monohydroxy-, dihydroxy- and tetrahydroxy-1,4-benzoquinone are formed as intermediate products. As final products of catechol decomposition, muconic acid, its hydroxy- and dihydroxy-derivatives and crotonic acid were identified. In the micellar environment of hexadecyltrimethylammonium bromide the decomposition rate of catechol is three times faster, due to micellar catalysis, and is accompanied by chemiluminescence (CL) emission, with maxima at 500 and 640 nm and a quantum yield of 1 x 10(-4). The CL of catechol can be further sensitized by a factor of 8 (maximum) with the aid of intramicellar energy transfer to fluorescein.     

流动注射化学发光法检测剑麻皂苷元

采用Luminol-K3Fe(CN)6化学发光体系,建立流动注射化学发光法检测从剑麻残渣和麻膏中分离得到的皂苷元。当用0.1 mol·L-1NaOH作为溶剂配制鲁米诺浓度为1.0×10-5mol·L-1,用去离子水作为溶剂配制K3Fe(CN)6浓度为1.6×10-5mol·L-1,主副蠕动泵转数均在50~80 r·min-1时,用无水乙醇溶解的皂苷元流入体系具有最强的化学发光。在该条件下,剑麻皂苷元最低检出限为3.0×10-3mg·mL-1,标准曲线相关系数为0.999 6,平均回收率为98.5%,相对标准偏差在2.9%~4.2%之间。同时与HPLC检测方法对样品检测结果进行了比较。     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.