Peroxyoxalate Chemiluminescence Reaction Research Articles

Introduction of fluorescence and chemiluminescence detection to capillary chromatography based on tube radial distribution of water–hydrophilic–hydrophobic organic mixture carrier solvents

Fluorescence and chemiluminescence detection were introduced into a tube radial distribution chromatography (TRDC) system using an open fused-silica capillary tube and a water–acetonitrile–ethyl acetate mixture carrier solution. Model analyte mixture solutions, such as Eosin Y and perylene as well as dansyl methionine and perylene, were injected into the capillary tube by a gravity method. The analyte solution was subsequently delivered through the capillary tube with the carrier solution by a microsyringe pump; the system worked under laminar flow conditions. The analytes were separated through the tube and detected by on-capillary with fluorescence detection or by end-capillary with chemiluminescence detection taking advantage of peroxyoxalate chemiluminescence reaction. Eosin Y and perylene as well as dansyl methionine and perylene were detected in this order with a carrier solution of water–acetonitrile–ethyl acetate (15 : 3 : 2 volume ratio), while they were detected in the reverse order with a carrier solution of water–acetonitrile–ethyl acetate (3 : 8 : 4 or 2 : 7 : 4 volume ratio) with fluorescence or chemiluminescence detection. The elution times of the analytes were reversed by changing the component ratio of the solvents in the carrier solution. A fluorescein isothiocyanate-labeled bovine serum albumin was also analyzed and separated from the coexisting labeling reagent with the present system.

Role of Catecholamine Metabolism in Blood Pressure Regulation Using Chemiluminescence Reaction Detection

Catecholamines, namely, dopamine, norepinephrine and epinephrine, play important roles in higher animals as neurotransmitters or hormones, and are metabolized by catechol-O-methyltransferase (COMT). To elucidate the role of COMT in blood pressure regulation, we have developed simultaneous determination methods for catecholamines and their 3-O-methyl metabolites using high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection. Using the developed method, we have found that inactivation of catecholamines by COMT is attenuated in hypertensive rats compared to normotensive rats. Furthermore, both the activities and the amounts of membrane-bound (MB-)COMT in the liver were found to be lower in hypertensive rats than in normotensive rats, which indicated that liver MB-COMT may be a relevant factor in blood pressure regulation.

Micro-channel Chemiluminescence Analysis Using a Peroxyoxalate Reaction that Works through Liquid-Liquid Interface Collapse under Laminar-Flow Conditions

An oxalate reagent-hydrogen peroxide-fluorescent compound chemiluminescence reaction, i.e., peroxyoxalate chemiluminescence, was introduced into micro-channel chemiluminescence analysis (MCCLA) to establish the concept of MCCLA through the direct observation of fluorescence and chemiluminescence using a fluorescence microscope-CCD camera and a microscope-CCD camera. It was confirmed from visual data that chemiluminescence in the MCCLA generated through the liquid-liquid interface collapsed during the course of molecular diffusion in the micro-channel. In addition, the visual data of chemiluminescence were transformed to line drawings on a computer to obtain chemiluminescence profiles. The chemiluminescence profiles were examined in detail at various flow rates and detection points; the relationship between the residence times and the chemiluminescence peak heights, or areas in the profiles, was easily represented as one smoothly changing reaction curve. Furthermore, the fluorescent compounds were detected with high sensitivity and good reproducibility in MCCLA by turning the syringe pumps on and off to produce determinable chemiluminescence signals; a photomultiplier tube was used as a detector. The chemiluminescence intensities in the signals of erythrosine, rhodamine B, Rose Bengal, fluorescein isothiocyanate, and eosin Y were examined; their intensities increased in this order, and eosin Y responded over the range of 1 x 10(-9) - 1 x 10(-6) M with a detection limit of 1 x 10(-9) M (S/N = 3). Introducing of the peroxyoxalate chemiluminescence reaction into MCCLA can extend the analysis system to the analysis of various types of sample and applications incorporating fluorescence labeling techniques.

Non‐enzymatic aqueous peroxyoxalate chemiluminescence immune detection using a CCD camera and a CMOS device

A new method for non-enzymatic aqueous peroxyoxalate chemiluminescence (POCL) biomolecular detection using imaging chip-based devices has been developed. A water-soluble amide of oxalic acid was synthesized and used in the investigation and characterization of POCL immunodetection in an aqueous environment. Six fluorescent dyes commonly used in biological detection were tested, and the intensity of light generated from the aqueous POCL reactions was characterized in the liquid phase. Direct detection sensitivity comparisons between a standard fluorescent method and this POCL method were performed in both liquid and solid phases. Results showed that detection sensitivity using the POCL method is comparable to that of the fluorescent method. POCL biomolecular detection on a nitrocellulose membrane was also investigated using a charge-coupled device (CCD) camera. Again, POCL detection sensitivity proved to be comparable to that using the fluorescent detection method. In an application of aqueous POCL biomolecular detection, Staphylococcus aureus enterotoxin B (SEB) and its antibody were used to demonstrate immuno- and affinity detection. For further applications, such as DNA and protein arrays, simultaneous detection of biomolecules labelled with different fluorescent labels was investigated, using a complementary metal oxide semiconductor (CMOS) colour imaging chip.

Studies on the mechanism of the peroxyoxalate chemiluminescence reaction: Part 2. Further identification of intermediates using 2D EXSY 13C nuclear magnetic resonance spectroscopy

Further consideration has been given to the reaction pathway of a model peroxyoxalate chemiluminescence system. Again utilising doubly labelled oxalyl chloride and anhydrous hydrogen peroxide, 2D EXSY 13C nuclear magnetic resonance (NMR) spectroscopy experiments allowed for the characterisation of unknown products and key intermediate species on the dark side of the peroxyoxalate chemiluminescence reaction. Exchange spectroscopy afforded elucidation of a scheme comprised of two distinct mechanistic pathways, one of which contributes to chemiluminescence. 13C NMR experiments carried out at varied reagent molar ratios demonstrated that excess amounts of hydrogen peroxide favoured formation of 1,2-dioxetanedione: the intermediate that, upon thermolysis, has been long thought to interact with a fluorophore to produce light.

Chemiluminescence imaging assay dipyridamole based on molecular imprinted polymer as recognition material

A simple, sensitive and specific method has been developed for high throughput detection of dipyridamole. The proposed method is based on chemiluminescence (CL) imaging assay combined molecularly imprinted polymer (MIP) recognition. Molecularly imprinted microspheres were prepared using precipitation polymerization with methacrylic acid (MAA) as functional monomer, trimethylolpropane trimethacrylate (TRIM) as the crosslinker and dipyridamole as the template. Polymer as the artificial biomimetic recognition element for dipyridamole was coated in 96-microtiter well plates with poly(vinyl alcohol) (PVA) as glue. The amount of polymer-bound dipyridamole was determination based on dipyridamole peroxyoxalate chemiluminescence (PO-CL) reaction. The emitting light was measured with a high-resolution charge couple device (CCD) camera. Influencing factors were investigated and optimized in detail. The proposed method in this paper exhibits high selectivity and sensitivity to dipyridamole. Under the optimum conditions, the relative CL imaging intensity (AVG) is proportional to the concentration of dipyridamole ranging from 0.02 to 10 μg/ml. The detection limit is 0.006 μg/ml. The method can perform 96 independent measurements simultaneously in 30 min. These results show that the MIP-based CL imaging method can become a useful analytical technology for quick detection of dipyridamole in real sample.

Molecular imprinted polymer-based chemiluminescence imaging sensor for the detection of trans-resveratrol

Sensitive, rapid and inexpensive chemiluminescence (CL) imaging has been developed based on molecular imprinted polymer (MIP) sensing elements. Imprinted uniform microspheres were synthesized by precipitation polymerization. Microtiter plates (96 wells) were coated with polymer microspheres imprinted with trans-resveratrol, which were fixed in place using poly(vinyl alcohol) (PVA) as glue. The amount of polymer-bound trans-resveratrol was quantified using imidazole (IMZ)-catalyzed peroxyoxalate chemiluminescence (PO-CL) reaction. The light produced was then measured with a high-resolution CCD camera. Calibration curve corresponding to analyte concentration ranging from 0.3 to 25 μg mL −1 was obtained with a limit of detection 0.1 μg mL −1. These results showed that the MIP-based CL imaging sensor can become a useful analytical tool for quick simultaneous detection of trans-resveratrol in a large number of real samples.

Enhancement in sample preconcentration by the on-line incorporation of cloud point extraction to flow injection analysis inside the chemiluminescence cell and the determination of total serum bilirubin

The on-line incorporation of cloud point extraction (CPE) to flow injection analysis (FIA) was previously based on the use of a cotton-packed column to entrap the analyte-containing surfactant aggregates after salt-induced CPE, and then the preconcentrated analyte was eluted into a separate detection cell for subsequent chemiluminescence (CL) detection ( via the peroxyoxalate CL reaction). In the work, the on-line CPE/FIA technique was improved by the following: (1) sample preconcentration and CL detection were both carried out directly inside the collection column, thus avoiding the decrease in detection sensitivity due to sample dispersion and dilution, and (2) CL detection was performed through the reaction between nitrite and hydrogen peroxide, which is compatible with aqueous samples and should allow for chemical excitation to occur more efficiently inside the collection column. In addition to more effective sample preconcentration, the CL detection of the entrapped analytes directly inside the collection column, i.e., a unique heterogeneous microenvironment in which analyte-containing surfactant aggregates were embedded within the densely packed filtering material, may also contribute to the overall increase in CL intensity (e.g., a CL enhancement factor of ca. 1000) . Under optimum experimental conditions, the calibration curve was found to be linear for the CL detection of bilirubin (5 to 120 μg L −1), the limit of detection (S/N = 3) was 1.8 μg L −1, and the R.S.D. was ca. 2.6% ( n = 30) for 20 μg L −1 bilirubin. Good agreements were obtained for the determination of total bilirubin in certified reference human serum samples between the present approach and an established clinical method.

The study of a chemiluminescence immunoassay using the peroxyoxalate chemiluminescent reaction and its application

The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)–hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H 2O 2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO–H 2O 2 chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0–625.0 pg/ml, and β-HCG in the range of 12.5–400.0 mIU/ml. The detection limits were 0.5 pg/ml for rHu IL-6 and 3 mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0 pg/ml rHu IL-6, and 3.9 for 50.0 mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results.

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.

Quantification of Cu 2+ using 1,1′-oxalyldiimidazole chemiluminescence

Emission from two different peroxyoxalate chemiluminescence (PO-CL) reaction pathways (i.e., 1,1′-oxalyldiimidazole (ODI) and bis(2,4,6-trichlorophenyl)oxalate (TCPO) CL reactions) is significantly reduced in the presence of increasing Cu 2+ concentrations. The CL intensity decrease in the ODI-CL reaction results from deactivation of the high-energy intermediate(s) by Cu 2+, whereas the apparent quenching in the TCPO-CL reaction results from both this effect and formation of Cu 2+–imidazole (ImH) complexes [Cu(ImH) n=1–4 ] 2+. Because the reaction between Cu 2+ and ImH occurs competitively with the formation of a high-energy intermediate (X) in TCPO-CL reactions, accurately determining Cu 2+ concentrations with a TCPO-CL-based system is more difficult versus an ODI-CL-based system. Using ODI-CL, the detection limit of Cu 2+ dissolved in water was determined to be 5.0 × 10 −8 M (signal:noise = 3) in a 1 cm fluorescence flow micro-cell. ODI-CL-based systems are expected to be useful for the rapid quantification of many quenchers in environmental and biological samples because the interactions between the high-energy intermediate(s) and quencher are very rapid relative to other competitive reactions.

Poly(benzyl ether) dendrimers with strongly fluorescent distyrylbenzene cores as the fluorophores for peroxyoxalate chemiluminescence: insulating effect of dendritic structures on fluorescent sites

Poly(benzyl ether) dendrimers containing strongly fluorescent distyrylbenzene cores were synthesized, and their fluorescence and electrochemical properties as well as the action as fluorophores in the chemiluminescence reactions were investigated. While all the dendrimers exhibited almost the same properties except for their intensities, a characteristic feature due to the dendritic structure was observed in the electrochemical behaviors. In the peroxyoxalate chemiluminescence reactions using these dendrimers as fluorophores, a bimolecular interaction between the high-energy intermediates and fluorophores was established, and a decrease in the chemiluminescence intensity with an increasing generation was observed, which was connected with the insulating effect of the dendritic structures on the core.

Improving the Quantification of Highly Overlapping Chromatographic Peaks by Using Product Unit Neural Networks Modeled by an Evolutionary Algorithm

This work investigates the ability of multiplicative (on the basis of product units) and sigmoidal neural models built by an evolutionary algorithm to quantify highly overlapping chromatographic peaks. To test this approach, two N-methylcarbamate pesticides, carbofuran and propoxur, were quantified using a classic peroxyoxalate chemiluminescence reaction as a detection system for chromatographic analysis. The four-parameter Weibull curve associated with the profile of the chromatographic peak estimated by the Levenberg-Marquardt method was used as input data for both models. Straightforward network topologies (one output) allowed the analytes to be quantified with great accuracy and precision. Product unit neural networks provided better information ability, smaller network architectures, and more robust models (smaller standard deviation). The reduced dimensions of the selected models enabled the derivation of simple quantification equations to transform the input variables into the output variable. These equations can be more easily interpreted from a chemical point of view than those provided by sigmoidal neural networks, and the effect of both analytes on the characteristics of chromatographic bands, namely profile, dispersion, peak height, and residence time, can be readily established.

A new strategy for the chemiluminescent screening analysis of total N-methylcarbamate content in water

A new procedure for the screening of total N-methylcarbamate (NMCs) content in water at different concentration levels is here presented. It is based on a previous off-line alkaline hydrolysis of the NMCs, for the production of methylamine (MA), and its subsequent derivatisation with o-phthalaldehyde (OPA). After that, the obtained derivative is involved in the peroxyoxalate chemiluminescent (PO-CL) reaction using imidazole as a catalyst being the CL emission proportional to the total NMC content. The chemiluminescent reaction is carried out in a flow injection analysis (FIA) manifold, employing sodium dodecyl sulphate micellar medium as carrier, which avoids the rapid degradation of POs in water solution. The applicability of the screening method is shown for the monitoring of NMCs in natural waters. Analytical features, as specification limit signal, screening uncertainty, sensitivity and specificity, are calculated.

Flow and evaporation cells for the detection of proteins on membranes with the peroxyoxalate chemiluminescent reaction in organic media.

The high-energy intermediates generated in the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with H2O2 can excite electronically different fluorophores with a high quantum yield in organic solvents. We have previously applied this peroxyoxalate chemiluminescent reaction to the detection of proteins labeled with the fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. In this work, we have investigated the possibility to enhance the sensitivity of this detection method using specially designed cells in which the reagents TCPO and H2O2 in acetone are continuously renewed. In the flow cell, two syringes are used to renew the reagents in the reaction chamber containing the PVDF membrane with blotted proteins labeled with MDPF. In the evaporation cell, a fresh solution of reagents continuously replaces the volume of acetone evaporated in the reaction chamber. Both cells show a low emission background but the observed elution of proteins from the membrane produced by the flow of reagents in acetone limits the maximum sensitivity attainable with these cells. The best result (detection of 1 ng of MDPF-labeled protein) has been obtained with the evaporation cell.

Heuristic extraction of rules in pruned artificial neural networks models used for quantifying highly overlapping chromatographic peaks.

The suitability of an approach for extracting heuristic rules from trained artificial neural networks (ANNs) pruned by a regularization method and with architectures designed by evolutionary computation for quantifying highly overlapping chromatographic peaks is demonstrated. The ANN input data are estimated by the Levenberg-Marquardt method in the form of a four-parameter Weibull curve associated with the profile of the chromatographic band. To test this approach, two N-methylcarbamate pesticides, carbofuran and propoxur, were quantified using a classic peroxyoxalate chemiluminescence reaction as a detection system for chromatographic analysis. Straightforward network topologies (one and two outputs models) allow the analytes to be quantified in concentration ratios ranging from 1:7 to 5:1 with an average standard error of prediction for the generalization test of 2.7 and 2.3% for carbofuran and propoxur, respectively. The reduced dimensions of the selected ANN architectures, especially those obtained after using heuristic rules, allowed simple quantification equations to be developed that transform the input variables into output variables. These equations can be easily interpreted from a chemical point of view to attain quantitative analytical information regarding the effect of both analytes on the characteristics of chromatographic bands, namely profile, dispersion, peak height, and residence time.

Comparative study of different fluorescent dyes for the detection of proteins on membranes using the peroxyoxalate chemiluminescent reaction

We have previously shown that the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H 2O 2 chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2 H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO–H 2O 2 reaction.

The key intermediates that interact with the fluorophores in the peroxyoxalate chemiluminescence reaction of 2,4,6-trichlorophenyl N-aryl-N-tosyloxamates.

A kinetic study of peroxyoxalate chemiluminescence reactions employing 2,4,6-trichlorophenyl N-aryl-N-tosyloxamates supports the 1,2-dioxetanones still bearing the eliminating group as the key intermediates that interact with the fluorophores rather than 1,2-dioxetanedione.

Rat liver and kidney catechol-O-methyltransferase activity measured by high-performance liquid chromatography with fluorescence detection.

We have previously reported a highly sensitive method for the measurement of catechol-O-methyltransferase (COMT) activities in rat erythrocytes with norepinephrine (NE), an endogenous native substrate, using high-performance liquid chromatography (HPLC)-fluorescence or peroxyoxalate chemiluminescence reaction detection. Applying this method to COMT activities in rat liver and kidney, known to have the highest activities of all organs, the optimum reaction conditions were investigated. Under the optimum conditions, soluble (S)-COMT and membrane-bound (MB)-COMT activities in rat liver, with NE as a substrate, were 2.17 +/- 0.33 and 0.16 +/- 0.02 nmol/min/mg protein (n = 5), respectively. In rat kidney, S-COMT and MB-COMT activities were 1.81 +/- 0.20 and 0.079 +/- 0.009 nmol/min/mg protein (n = 5), respectively. Since liver and kidney play important roles in inactivating catecholamines, using the proposed method would yield critical information to delineate the role of metabolism of catecholamines in rat tissues.

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.