Enhanced Chemiluminescence Reaction Research Articles

Chemiluminescent plate assay of microRNA-155 coupled with catalytic hairpin assembly with oligonucleotide release (CHAOR)

A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity. The detection limit of microRNA-155 assay was 0.4 pM. The coefficient of variation of the chemiluminescent signal, formed upon the heterogeneous determination of miRNA-155, was less than 12 % within the working range. The efficiency of CHAOR as an amplification method was similar to that of traditional CHA, as miRNA-155 assays based on CHAOR and CHA had similar analytical parameters. In addition, the proposed assay was highly specific. Contrary to traditional CHA, CHAOR, one of whose products is a single-stranded oligonucleotide, can be used in analytical methods based on cascade amplification.

A new enhanced chemiluminescence reaction based on polymer dots for the determination of metronidazole

Polymer dots (PDs) with non-conjugated functional groups are attracting nanomaterials due to their ease of synthesis, the biocompatibility of precursors, and low toxicity. In this work, PDs with non-conjugated groups were synthesized with a simple and straightforward method by Schiff base reaction. Then their possible application in the chemiluminescence (CL) reactions was explored. Results were shown that PDs increased the CL intensity of the NaIO4-fluorescein system about 15 times. Regarding the CL mechanism, we proved that the emitting species is fluorescein, which can be excited by the energy transfer from the excited-state PDs. It was observed that CL emission is promoted by the interaction of metronidazole (MND) with the PDs. Therefore, we designed a novel and sensitive assay for MND based on its enhancing effect on NaIO4-fluorescein-PDs CL system. The introduced assay showed a linear response in the range of 5.0–300 nM with a detection limit of 1.5 nM. The method was used for the determination of MND in spiked plasma samples.

Improving the Sensitivity of the miRNA Assay Coupled with the Mismatched Catalytic Hairpin Assembly Reaction by Optimization of Hairpin Annealing Conditions.

The mismatched catalytic hairpin assembly (mCHA), a programmable oligonucleotide circuit, is one of the promising isothermal amplification methods used in nucleic acid detection. Its limitations are related to a high background noise observed due to the target-independent hybridization of the reacting hairpins (HPs). In this work, it was shown that the introduction of salts such as NaCl and MgCl2 to HP1/HP2 annealing solutions sharply reduces the background in mCHA and simultaneously increases the signal-to-background (S/B) ratio. A comparison of the salts demonstrated the higher activity of MgCl2 as compared to NaCl. A similar effect of reducing the background was observed with a decrease in the concentration of H1/H2 probes in annealing solutions. Using the favorable annealing conditions allowed the development of an ultrasensitive chemiluminescence assay coupled with mCHA for miRNA quantitation. Except mCHA, the use of a streptavidin-polyHRP conjugate and an enhanced chemiluminescence reaction additionally increased the assay sensitivity. Notably, the optimization of the HP annealing diminished the detection limit of the assay by 2 orders of magnitude and increased the sensitivity and precision of miRNA-141 determination. The discovered fact of reducing the background by the variation of HP annealing conditions may be valuable not only for the mCHA performance but also likely for other HP-based biochemical methods.

Microplate Chemiluminescent Assay for DNA Detection Using Apoperoxidase-Oligonucleotide as Capture Conjugate and HRP-Streptavidin Signaling System.

A covalent conjugate of horseradish apoperoxidase and amino-containing oligonucleotide was synthesized for the first time. Using the obtained conjugate as a capture reagent chemiluminescent microtiter plate-based assay for detection of 35-mer fragment of hepatitis B virus (HBV) DNA (proof-of-concept analyte) was developed. To detect the target DNA, a signaling system consisted of biotinylated reporter oligonucleotide and HRP-streptavidin conjugate was used. The high sensitivity of the assay was due to the enhanced chemiluminescence reaction, where 3-(10′-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair was used as an enhancer. Under the optimized conditions the limit of detection and a working range of the assay were 3 pM and 6–100 pM, respectively. The assay sensitivity was 1.6 × 105 RLU/pM of target. The coefficient of variation (CV) for determination of HBV DNA within the working range was lower than 6%.

Enhancement effect of p-iodophenol on gold nanoparticle-catalyzed chemiluminescence and its applications in detection of thiols and guanidine

In the present study, the effects of nine compounds on the luminol-O2 chemiluminescence (CL) reaction catalyzed by gold nanoparticles (Au NPs) were investigated. p-Iodophenol (PIP), known as a traditional enhancer in horseradish peroxidase (HRP)-catalyzed luminol-H2O2 CL reaction, exhibited the highest enhancement effect on the luminol-O2-Au NPs CL reaction. The addition of PIP at a final concentration of 1.25 mM to luminol-O2-Au NPs CL system enhanced the CL signal almost 30-fold. Interestingly, the enhanced CL reaction exhibited a slow and intense CL signal which is different from the CL profile of luminol-H2O2-Au NPs CL system reported in previously. The experimental conditions of the PIP enhanced luminol-O2-Au NPs CL system were investigated systematically in the present study. And the mechanism studies showed that superoxide anion (O2•-) and singlet oxygen (1O2) generated from dissolved oxygen played an important role during the CL reaction. Furthermore, the effects of some organic compounds on the enhanced CL system were investigated. The results showed that compounds containing -SH and guanidine group can inhibit the signal of the enhanced CL reaction indicating the applicability of the proposed CL reaction for the detection of such compounds.

Microplate chemiluminescent assay for HBV DNA detection using 3-(10′-phenothiazinyl)propionic acid/N-morpholinopyridine pair as enhancer of HRP-catalyzed chemiluminescence

A sensitive sandwich assay for hepatitis B virus (HBV) DNA detection based on use of commercial CL-ELISA microplates was developed. To reveal the target the covalent conjugate of reporter oligonucleotide and horseradish peroxidase (HRP) was synthesized. An employment of enhanced chemiluminescence reaction, where 3-(10′-phenothiazinyl)propionic acid/N-morpholinopyridine pair was used as enhancer of HRP-catalyzed chemiluminescence, permitted to measure the enzyme activity of the conjugate with high sensitivity. Under the favorable conditions the limit of detection and a linear range of the assay were 3 pM and 0.07–2.0 nM, respectively. The coefficient of variation (CV) for determination of HBV DNA concentrations within the working range was lower than 4%. The obtained results demonstrated that the developed assay had high sensitivity and precision.

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented.

3-(10′-Phenothiazinyl)propionic acid is a potent primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low density lipoprotein

Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP) in the presence of 3-(10′-phenothiazinyl)propionic acid (PPA) as a primary enhancer was performed. The effect of concentrations of PPA, hydrogen peroxide, MORPH, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The detection limit value of HRP in ECR with PPA was 0.09pM. Using PPA the ultra-sensitive chemiluminescent ELISA for determination of methylglyoxal-modified low density lipoprotein was developed. The detection limit value for the developed method was 0.5ngmL−1. The obtained results open up very promising perspectives for using PPA to improve the sensitivity of enzyme immunoassay kits.

Mechanism of action of 4-dialkylaminopyridines as secondary enhancers in enhanced chemiluminescence reaction

Kinetic study of peroxidase-catalyzed oxidation of 3-(10′-phenothiazinyl)propane-1-sulfonate (SPTZ) by hydrogen peroxide demonstrated that the addition of 4-dialkylaminopyridines to a substrate solution significantly increased a rate of production of SPTZ cation radical and did not affect the rate of decomposition of this radical. These results explain the mechanism of action of 4-dialkylaminopyridines as secondary enhancers in enhanced chemiluminescence reaction used widely in analytical practice.

Enhanced chemiluminescence as an indicator of air pollution

AbstractInvestigations were conducted to determine the pollution levels of air condensate and rain samples using the enhanced chemiluminescence (ECL) reaction. Air condensate samples were initially collected from roadside sites, non‐roadside sites, smoking‐exposed sites and non‐smoking‐exposed sites using an automatic dew collector. Rainwater was also collected throughout the sampling period. Samples were subsequently analysed using the ECL reaction, and pollution levels (Eclox Units) were calculated. Air condensate samples collected from roadside‐ and smoking‐exposed sites generally had higher pollution levels than those from non‐roadside‐ and non‐smoking‐exposed sites. Samples from both smoking‐exposed and traffic‐exposed sites generally had higher pollution levels than rainwater. Finally, in samples collected from roadside‐exposed sites and rainwater, the main polluting constituents were determined to be cationic/anionic components. In samples collected from smoking‐exposed sites, the main polluting constituents were determined to be organic components.

Optimization of horseradish peroxidase-catalyzed enhanced chemiluminescence reaction by full factorial design

Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10′-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers was performed. The effect of concentrations of SPTZ, hydrogen peroxide, MORP, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The use of the full 25 factorial design instead of “one-variable-a time” method allowed to increase the sensitivity of HRP-C determination 2355 fold without a change of detection limit. The obtained results open up very promising perspectives for using HRP-C-catalyzed ECR to improve the sensitivity of chemiluminescent enzyme immunoassay.

Advantages of Soybean Peroxidase over Horseradish Peroxidase as the Enzyme Label in Chemiluminescent Enzyme-Linked Immunosorbent Assay of Sulfamethoxypyridazine

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.

Determination of Cytochrome c in Human Serum and Pharmaceutical Injections Using Flow Injection Chemiluminescence

It was found that the complex of cytochrome c (Cyt c) and hydrogen peroxide could significantly catalyze the chemiluminescence (CL) reaction from luminol-hydrogen peroxide, and a sensitive, rapid, and simple CL procedure was proposed for the determination of Cyt c in a flow injection system for the first time. The increment of CL intensity was linear over the concentration of Cyt c ranging from 5 to 700 ng ml(-1), with a detection limit of 2 ng ml(-1) (3sigma). At a flow rate of 2.0 ml min(-1), a complete analytical process could be performed in 30 s with a relative standard deviation of less than 4.0%. The proposed method was applied successfully for the assay of Cyt c in pharmaceutical injections and human serum, and the recoveries were from 98.0% to 108.8% and 92.5% to 109.0%. The possible mechanism of Cyt c enhanced CL reaction was also discussed.

Investigation on enhanced chemiluminescence reaction systems with bis(hydrogenperiodato) argentate(III) complex anion for fluoroquinolones synthetic antibiotics

A novel chemiluminescence (CL) reaction system with bis(hydrogenperiodato) argentate(III) complex anion (Ag(III) complex, [Ag(HIO 6) 2] 5−), for the first time, is developed for the determination of lomefloxacin (LMFX), enrofloxacin (ENLX) and pefloxacin (PFLX). The possible CL emission mechanism was discussed by comparing the fluorescence emission with CL spectra. The CL conditions of [Ag(HIO 6) 2] 5−–H 2SO 4–LMFX/ENLX/PFLX systems were investigated and optimized. Under the optimized experimental conditions, the CL intensity is proportional to the concentration of the drugs in the range 0.2994–36.80 × 10 −7 g mL −1 for LMFX, 4.00–30.0 × 10 −7 g mL −1 for ENLX and 1.54–27.64 × 10 −7 g mL −1 for PFLX. The limit of detection ( s/ n = 3) was 9.1 × 10 −9 g mL −1 for LMFX, 3.1 × 10 −9 g mL −1 for ENLX and 4.4 × 10 −9 g mL −1 for PFLX. The recovery of LMFX, ENLX and PELX from the spiked pharmaceutical preparations was in the range of 92.3–105% with the RSDs of 0.5–2.7%. For urine, serum and milk samples the recoveries of the three drugs were in the range of 85.1–107% for LMFX with the RSDs of 2.3–3.4%. 80.2–112% for ENLX with the RSDs of 1.4–2.8%, and 87.8–114% for PFLX with the RSDs of 1.6–2.7%. The proposed method was applied successfully to the determination of these compounds in real samples.

Toward high performance western blotting using vacuum-driven system and laser scanner; comparison between two signal detection methods based on chemiluminescent and immunofluorescent imaging

A vacuum-driven antibody-reaction system enables high-throughput western blotting, where the antibodies pass through protein-transferred membrane and antibody-antigen reaction is completed within 30 min. With the vacuum-based device, two protein detection methods compatible with by laser scanning were compared; one with enhanced chemiluminescence reaction and the other using fluorescence-labeled antibody. Considering sensitivity, quantitative capability, reproducibility and labor intensiveness, we concluded that fluorescence-based detection is more suitable in combination with the vacuum-driven antibody-reaction system and a laser scanner.

An ultrasensitive chemiluminescence biosensor for cholera toxin based on ganglioside-functionalized supported lipid membrane and liposome

A novel chemiluminescence biosensor based on a supported lipid layer incorporated with ganglioside GM1 was developed for the detection of cholera toxin. The planar supported lipid membrane was prepared as biosensing interface via spontaneous spread of ganglioside-incorporated phospholipid vesicles on the octadecanethiol-coated gold surface. The specific interaction of multivalent CT by ganglioside GM1 molecules enables the biosensor to be implemented via a sandwiched format using a liposome probe functionalized with GM1 and horseradish peroxidase (HRP). Then, the presence of the target CT could be determined via the HRP-catalyzed enhanced chemiluminescence reaction. The developed strategy offers several unique advantages over conventional biosensors in that it allows for an easy construction and renewal of the sensing interface, a small background signal due to low non-specific adsorption of serum constituents on the lipid membrane, and effective immobilization of multiple biocatalytic amplifiers and recognition components via common phospholipid reagents. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1 pg mL −1 to 1 ng mL −1 with readily achievable detection limit of 0.8 pg mL −1.

TWIK-related Acid-sensitive K+ Channel 1 (TASK1) and TASK3 Critically Influence T Lymphocyte Effector Functions

Two major K(+) channels are expressed in T cells, (i) the voltage-dependent K(V)1.3 channel and (ii) the Ca(2+)-activated K(+) channel KCa 3.1 (IKCa channel). Both critically influence T cell effector functions in vitro and animal models in vivo. Here we identify and characterize TWIK-related acid-sensitive potassium channel 1 (TASK1) and TASK3 as an important third K(+) conductance on T lymphocytes. T lymphocytes constitutively express TASK1 and -3 protein. Application of semi-selective TASK blockers resulted in a significant reduction of cytokine production and cell proliferation. Interference with TASK channels on CD3(+) T cells revealed a dose-dependent reduction ( approximately 40%) of an outward current in patch clamp recordings indicative of TASK channels, a finding confirmed by computational modeling. In vivo relevance of our findings was addressed in an experimental model of multiple sclerosis, adoptive transfer experimental autoimmune encephalomyelitis. Pretreatment of myelin basic protein-specific encephalitogenic T lymphocytes with TASK modulators was associated with significant amelioration of the disease course in Lewis rats. These data introduce K(2)P channels as novel potassium conductance on T lymphocytes critically influencing T cell effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.

Direct chemiluminescence determination of fenbufen by the enhancement of the % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX!% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeOuaiaabw% hadaqadaqaaiaabchacaqGObGaaeyzaiaab6gaaiaawIcacaGLPaaa% daqhaaWcbaGaaG4maaqaaiaaikdacqGHRaWkaaaaaa!3F82! $${\text{Ru}}\left( {{\text{phen}}} \right)_3^{2 + } $$ –cerium(IV) system

A method is described for determination of fenbufen that is based on the chemiluminescence (CL) reaction of the $${\text{Ru}}\left( {{\text{phen}}} \right)_3^{2 + } $$ –cerium(IV)–fenbufen system. An enhanced CL reaction was developed, and optimum conditions for CL were investigated. The CL was linearly dependent on fenbufen concentration in the range 4.0 × 10−8–9.0 × 10−6 mol L−1. The detection limit was 2.0 × 10−8 mol L−1. The relative standard deviation (RSD) was 2.8% for eleven measurements of 6.0 × 10−7 mol L−1 fenbufen standard solution. The new method enables simple, sensitive, and rapid determination of fenbufen and has been used for determination of fenbufen in pharmaceutical preparations in capsule, spiked serum and urine samples.

Interleukin-31 and Oncostatin-M Mediate Distinct Signaling Reactions and Response Patterns in Lung Epithelial Cells

Lung epithelial cells are primary targets of oncostatin M (OSM) and, to a lower degree, of interleukin (IL)-6 and IL-31, all members of the IL-6 cytokine family. The OSM receptor (OSMR) signals through activation of STAT and mitogen-activated protein kinase pathways to induce genes encoding differentiated cell functions, reduce cell-cell interaction, and suppress cell proliferation. IL-31 functions through the heteromeric IL-31 receptor, which shares with OSMR the OSMRbeta subunit, but does not engage gp130, the common subunit of all other IL-6 cytokine receptors. Because the response of epithelial cells to IL-31 is unknown, the action of IL-31 was characterized in the human alveolar epithelial cell line A549 in which the expression of the ligand-binding IL-31Ralpha subunit was increased. IL-31 initiated signaling that differed from other IL-6 cytokines by the particularly strong recruitment of the STAT3, ERK, JNK, and Akt pathways. IL-31 was highly effective in suppressing proliferation by altering expression of cell cycle proteins, including up-regulation of p27(Kip1) and down-regulation of cyclin B1, CDC2, CDK6, MCM4, and retinoblastoma. A single STAT3 recruitment site (Tyr-721) in the cytoplasmic domain of IL-31Ralpha exerts a dominant function in the entire receptor complex and is critical for gene induction, morphological changes, and growth inhibition. The data suggest that inflammatory and immune reactions involving activated T-cells regulate functions of epithelial cells by IL-6 cytokines through receptor-defined signaling reactions.

Proline-rich Motifs in the Parathyroid Hormone (PTH)/PTH-related Protein Receptor C Terminus Mediate Scaffolding of c-Src with β-Arrestin2 for ERK1/2 Activation

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.