Employment of 4‐(1,2,4‐triazol‐1‐yl)phenol as a signal enhancer of the chemiluminescent luminol–H2O2–horseradish peroxidase reaction for detection of hepatitis C virus in real samples

Highly sensitive detection of hepatitis C virus (HCV) in serum is a key method for diagnosing and classifying the extent of HCV infection. In this study, a p‐phenol derivative, 4‐(1,2,4‐triazol‐1‐yl)phenol (4‐TRP), was employed as an efficient enhancer of the luminol–hydrogen peroxide (H₂O₂)–horseradish peroxidase (HRP) chemiluminescence (CL) system for detection of HCV. Compared with a traditional enhancer, 4‐TRP strongly enhanced CL intensity with the effect of prolonging and stabilizing light emission. The developed CL system was applied to detecting HCV core antigen (HCV‐cAg) using a sandwich structure inside microwells. Our experimental results showed that there was good linear relationship between CL intensity and HCV‐cAg concentration in the 0.6–3.6 pg/mL range (R = 0.99). The intra‐ and inter‐assay coefficients of variation were 4.5–5.8% and 5.0–7.3%, respectively. In addition, sensitive determination of HCV‐cAg in serum samples using the luminol–H₂O₂–HRP–4‐TRP CL system was also feasible in clinical settings. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence of the luminol–AgNO3 system by Ag nanoparticles

The oxidation reaction of luminol with AgNO₃ can produce chemiluminescence (CL) in the presence of silver nanoparticles (NPs) in alkaline solution. Based on the studies of UV‐vis absorption spectra, photoluminescence (PL) spectra and CL spectra, a CL enhancement mechanism is proposed. The CL emission spectrum of the luminol–AgNO₃–Ag NPs system indicated that the luminophore was still 3‐aminophthalate. On injection of silver nanoparticles into the mixture of luminol and AgNO₃, they catalysed the reduction of AgNO₃ by luminol. The product luminol radicals reacted with the dissolved oxygen, to produce a strong CL emission. As a result, the CL intensity was substantially increased. Moreover, the influences of 18 amino acids, e.g. cystine, tyrosine and asparagine, and 25 organic compounds, including gallic acid, tannic acid and hydroquinone, on the luminol–AgNO₃–Ag NPs CL system were studied by a flow‐injection procedure, which led to an effective method for detecting these compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over–the‐counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol–HRP–H₂O₂ system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol–HRP–H₂O₂ system. A putative enhancement mechanism for the luminol–H₂O₂–HRP–acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O‐H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol–H₂O₂–HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%. Copyright © 2013 John Wiley & Sons, Ltd.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol–H₂O₂–horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO^•/PhOH) vs. luminol radicals (L^•/LH⁻) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (σ) for their substituents similar or greater than 0.20 are enhancers of the luminol–H₂O₂–horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (σ) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted. © 1998 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence of the luminol–potassium periodate system enhanced by TGA–capped CdTe quantum dots for the determination of theophylline

The chemiluminescence (CL) behaviour of the luminol–potassium periodate system enhanced by CdTe quantum dots capped with thioglycolic acid (TGA–CdTe QDs) was studied using kinetic experiments, CL spectra, UV–vis absorption spectra and fluorescence spectra. The production of oxygen‐containing reactant intermediates (O₂^?? and OH^?) in the present CL system was verified by CL. The possible CL mechanism was discussed in detail. Furthermore, theophylline (THP) was determined based on its enhancement of the CL intensity of the CdTe QDs–luminol–potassium periodate system coupled with a flow‐injection technique. Under these optimized conditions, the linear range was found to be from 1.0 × 10^?8 to 1.0 × 10^?5 g/mL with a detection limit of 2.8 × 10^?9 g/mL (3σ). The recoveries for the determination of THP in tablets were from 98.2 to 99.6%.     

A highly sensitive and temporal visualization system for gaseous ethanol with chemiluminescence enhancer

A two‐dimensional gaseous ethanol visualization system has been developed and demonstrated using a horseradish peroxidase–luminol–hydrogen peroxide system with high‐purity luminol solution and a chemiluminescence (CL) enhancer. This system measures ethanol concentrations as intensities of CL via the luminol reaction. CL was emitted when the gaseous ethanol was injected onto an enzyme‐immobilized membrane, which was employed as a screen for two‐dimensional gas visualization. The average intensity of CL on the substrate was linearly related to the concentration of standard ethanol gas. These results were compared with the CL intensity of the CCD camera recording image in the visualization system. This system is available for gas components not only for spatial but also for temporal analysis in real time. A high‐purity sodium salt HG solution (L‐HG) instead of standard luminol solution and an enhancer, eosin Y (EY) solution, were adapted for improvement of CL intensity of the system. The visualization of gaseous ethanol was achieved at a detection limit of 3 ppm at optimized concentrations of L‐HG solution and EY. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of phenol using an enhanced chemiluminescent assay

Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidation reaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para‐iodophenol. In this work, the effects of phenol on the para‐iodophenol‐enhanced HRP‐catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrations stimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in the sodium bis(2‐ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation with phenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only an inhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed. Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were 0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer‐containing system, the AOT‐applied system with phenol standard solutions in water and for the AOT‐applied system with phenol standard solutions in octane. Copyright © 2002 John Wiley & Sons, Ltd.     

A novel chemiluminescent flow‐injection analysis of transferrin by its reduction of the luminol—hydrogen peroxide reaction catalysed by meso‐tetra‐(3‐methoxyl‐4‐hydroxyl) phenyl manganese porphyrin

Based on the inhibition effect of transferrin (Tf) on the reaction of the luminol–hydrogen peroxide (H₂O₂) chemiluminescence (CL) system, catalysed by meso‐tetra‐(3‐methoxyl‐4‐hydroxyl) phenyl manganese porphyrin (MnP) as a mimetic enzyme of peroxides, a sensitive flow‐injection CL method has been developed for the determination of Tf in an alkaline medium. The CL reaction was carefully investigated by examining the variations of reaction conditions. Under optimum conditions, the linear range for the determination of transferrin was 0.04–20.0 μg/mL and the detection limit was 1.62 ng/mL. This proposed method was sensitive, convenient and simple, and has been successfully applied to the determination of transferrin in a serum sample. Copyright © 2011 John Wiley & Sons, Ltd.     

DPPH·–luminol chemiluminescence system and its application in the determination of scutellarin in pharmaceutical injections and rat plasma with flow injection analysis

In this article, a DPPH·–luminol chemiluminescence (CL) system was reported and the CL mechanism was discussed according to the CL kinetic properties after sequence injecting DPPH· into the DPPH·–luminol reaction mixture. It was observed that scutellarin could inhibit the CL response of the DPPH·–luminol system. Based on this observation, a simple and rapid flow injection CL method was developed for the determination of scutellarin using the inhibition effect in alkaline medium. The optimized chemical conditions for the CL reaction were 5 × 10^?6 mol/L DPPH · and 1.0 × 10^?4 mol/L luminol in 0.01 mol/L NaOH. Under optimized conditions, the CL intensity was inversely proportional to the concentration of scutellarin over the ranges 5–2000 and 40–3200 ng/ml in pharmaceutical injection and rat plasma, respectively. The limits of detection (S/N  = 3) were 5 and 40 ng/ml in preparations and rat plasma, respectively. Furthermore, the precision, recovery and stability of the validated method were acceptable for the determination of scutellarin in both pharmaceutical injections and rat plasma. The presented method was successfully applied in the determination of scutellarin in pharmaceutical injections and real rat plasma samples.     

Prospects for using a new sequential chemiluminescence strategy for monitoring the caffeine content in soft and energy drinks via the catalytic activities of different nano‐metal oxides

This article suggests a new sequential injection analysis chemiluminescence (SIA‐CL) strategy for monitoring the caffeine (CAF) content in soft and energy drinks using the catalytic activities of different nano‐metal oxides. The present study describes three different SIA‐CL systems (luminol–ferricyanide (III) coupled with Fe₂O₃ or ZnO nanoparticles (NPs), and luminol–H₂O₂ coupled with CuONPs. All experimental conditions were optimized and the linear concentration ranges of pure CAF were evaluated using the calibration graphs. The selectivity of the developed SIA‐CL systems was studied under the influence of various interfering species that may be present in soft or energy drinks such as sodium ions, sucrose, glucose, sodium benzoate, sodium citrate, riboflavin, niacin, citric, phosphoric and ascorbic acids. International Council for Harmonization (ICH) guidelines were obeyed for the validation of the suggested CL methods. The developed SIA‐CL systems displayed linear relationships over the concentration ranges 1.0–350, 5.0–400 and 10.0–400 μg ml^?1 with Fe₂O₃ NPs, ZnO NPs and CuO NPs, respectively. The recorded lower limits of detection and quantification were 0.7, 2.7 and 7.8 μg ml^?1, and 1.0, 5.0 and 10.0 μg ml^?1 for the previously mentioned SIA‐CL systems. The results revealed high selectivity for CAF determination and were in good agreement with those obtained by other reported methods.     

Post‐chemiluminescence determination of chloramphenicol based on luminol–potassium periodate system

A post‐chemiluminescence (PCL) phenomenon was observed when chloramphenicol was injected into a mixture of luminol and potassium periodate after the chemiluminescence (CL) reaction of luminol–potassium periodate had finished. The possible reaction mechanism was proposed based on studies of the CL kinetic characteristics, the CL spectra, the fluorescence spectra and the UV‐vis absorption spectra of the related substances. Based on the PCL reaction, a rapid and sensitive method for the determination of chloramphenicol was established. The linear response range was 6.0 × 10^?7–1.0 × 10^?5 mol/L, with a correlation coefficient of 0.9986. The relative standard deviation (RSD) for 5.0 × 10^?6 mol/L chloramphenicol was 2.3% (n = 11). The detection limit was 1.6 × 10^?7 mol/L. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of silver nanoparticles to the chemiluminescence determination of cefditoren pivoxil using the luminol–ferricyanide system

A new simple, accurate and sensitive sequential injection analysis chemiluminescence (CL) detection method for the determination of cefditoren pivoxil (CTP) has been developed. The developed method was based on the enhancement effect of silver nanoparticles on the CL signal arising from a luminol–potassium ferricyanide reaction in the presence of CTP. The optimum conditions relevant to the effect of luminol, potassium ferricyanide and silver nanoparticle concentrations were investigated. The proposed method showed linear relationships between relative CL intensity and the investigated drug concentration at the range 0.001–5000 ng/mL, (r = 0.9998, n = 12) with a detection limit of 0.5 pg/mL and quantification limit of 0.001 ng/mL. The relative standard deviation was 1.6%. The proposed method was employed for the determination of CTP in bulk drug, in its pharmaceutical dosage forms and biological fluids such as human serum and urine. The interference of some common additive compounds such as glucose, lactose, starch, talc and magnesium stearate was investigated. In addition, the interference of some related cephalosporins was tested. No interference was recorded. The obtained sequential injection analysis‐CL results were statistically compared with those from a reported method and did not show any significant differences. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of lophine derivatives as L‐012 (luminol analog)‐dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2

8‐Amino‐5‐chloro‐7‐phenylpyrido[3,4‐d]pyridazine‐1,4(2H,3H)dione (L‐012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L‐012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine‐based CL enhancers of the horseradish peroxidase (HRP)‐catalyzed CL oxidation of luminol, namely 2‐(4‐hydroxyphenyl)‐4,5‐diphenylimidazole (HDI), 2‐(4‐hydroxyphenyl)‐4,5‐di(2‐pyridyl)imidazole (HPI), 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenylboronic acid (DPA), and 4‐[4,5‐di(2‐pyridyl)‐1H‐imidazol‐2‐yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L‐012 and evaluated these as L‐012‐dependent CL enhancers. In addition, to detect HRP and/or H₂O₂ with higher sensitivity, each detection condition for the L‐012–HRP–H₂O₂ enhanced CL was optimized. All the derivatives enhanced the L‐012‐dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L‐012‐dependent CL using 4‐iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H₂O₂ detection, the detection limits for enhanced CL with HPI and 4‐iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L‐012‐dependent CL enhancer. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a sequential injection analysis system for monitoring of trehalose concentrations

A sequential injection analysis (SIA) system was developed to on-line monitor the concentrations of trehalose in biological processes. The system is based on the enzymatic reaction of trehalose to glucose in an immobilized trehalase reactor and the subsequent reaction of glucose in the presence of glucose oxidase. The peroxide produced is detected using horseradish peroxidase with a redox indicator (ABTS, 2,2′-azino-di(3-ethylbezthiazoli-sulfonic acid-6)) and a spectrophotometer set at 435 nm. The SIA was fully automated using software written in the LabVIEW^TM development environment. A number of system variablese.g. flow rate of the carrier buffer solution and volume ratio of sample to reagents, were evaluated to increase the sensitivity and performance of the SIA system. The performance of the trehalose-SIA system was linear under partially optimized operating conditions in the concentration range of 0.3 to 2.0 g/L (R²=0.999) with a sample frequency of 6 1/h. The SIA system was successfully employed to on-line monitor the concentrations of trehalose in a continuously stirred tank reactor. The on-line monitored data were in good agreement with the off-line data measured by a HPLC with a refractive index detector (n=14, R²=0.9865).     

Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a microflow injection–chemiluminescence detection system

A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H₂O₂). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H₂O₂) possible by micro?ow injection based on horseradish‐catalysed luminol chemiluminescence. For reproducible determination of H₂O₂ with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H₂O₂ must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro?ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within‐run reproducibility, CV = 4.0%) and the detection limit for H₂O₂ was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Non‐competitive immunoassay for luteinizing hormone in human serum using capillary electrophoresis with chemiluminescence detection

A non‐competitive immunoassay based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of luteinizing hormone (LH) in human serum. The work involved the development of separation and CL conditions, allowing for routine analysis of serum samples. In this study, horseradish peroxidase (HRP)‐labelled monoclonal anti‐LH can catalyse the luminol–hydrogen peroxide reaction. The determined LH can react with excessive amount of HRP‐labelled anti‐LH. Within 14 min, free enzyme conjugate and immune complex could be separated in alkaline borate buffer by means of a high voltage (15 kV). To improve sensitivity, a series of measures were adopted, including the choice of para‐iodophenol as a CL enhancer, unique design in detect window. Under the optimal conditions, the calibration curve for LH was established in the concentration range 1–200 mIU/mL and the detection limit was 0.08 mIU/mL. Compared with ELISA, this method decreased the detection limit by about 12 times, and it has been successfully employed in the determination of LH in human serum. Copyright © 2007 John Wiley & Sons, Ltd.     

Effect of oxygen abstraction on the peroxidase–luminol–perborate system: Relevance to the HRP enhanced chemiluminescence mechanism

Abstraction of oxygen from the HRP enhanced chemiluminescence system has no significant effect on the chemiluminescence generated. It is, therefore, proposed that in the peroxidase-luminol-perborate system at pH 7.3, chemiluminescence is generated by a direct reaction of diazaquinones with hydrogen peroxide and not, as generally assumed, from the reaction of luminol radicals with the molecular oxygen.     

Electrophoresis–chemiluminescence detection of phenols catalyzed by hemin

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis–chemiluminescence (CE‐CL) detection method for phenols using a hemin–luminol–hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br^? and F^? could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE‐CL detection system because of the self‐polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin–luminol afforded a stable CE‐CL baseline. The indirect CE‐CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10^?8 mol/L (o‐sec‐butylphenol), 4.9 × 10^?8 mol/L (o‐cresol), 5.4 × 10^?8 mol/L (m‐cresol), 5.3 × 10^?8 mol/L (2,4‐dichlorophenol) and 7.1 × 10^?8 mol/L (phenol). Copyright © 2013 John Wiley & Sons, Ltd.