Determination of peroxidase encapsulated in liposomes using homogentisic acid y-lactone chemiluminescence.

Homogentisic acid gamma-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in piodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

Droplet-based micro-flow chemiluminescence system for in vivo glucose determination by microdialysis sampling.

A micro-flow chemiluminescence (CL) system in vivo for glucose determination by the on-line microdialysis sampling is described in this paper. The micro-flow CL system uses discrete sample droplets, which formed at the tip of the capillary with the sampling volume of 4.5 microl. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and glucose oxidase (GOD) on the inside surface of the micro-flow cell which was fabricated in polymethyl methacrylate (PMMA). The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H2O2, then H2O2 oxidizing luminol to produce CL in presence of HRP. The microdialysis probe was utilized for sampling in the rabbit blood; the sample throughput was 20 h(-1). The glucose level in blood of the rabbit was on-line monitored with good results.     

Effect of the luminol signal enhancer selection on the curve parameters of an immunoassay and the chemiluminescence intensity and kinetics

In the present study, three luminol signal enhancers {4-methoxyphenol, 4-hydroxybiphenyl and 4-(1H-pyrrol-1-yl)phenol} were utilized in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP). The latter was applied in a heterogenous enzyme immunoassay that has been previously described. The employment of these molecules greatly affected important assay parameters, such as detection limit and the range of the calibration curve and the results were compared with those obtained from other two similar enhancers that have been described from our group. Practically, the use of a novel enhancer, even if this is a slightly changed 4-substituted phenol derivative, can affect assay properties so dramatically, one can assume that another substrate/enzyme system was applied. Furthermore, the use of different luminol signal enhancers in the luminol/HRP/H₂O₂ system affected not only the intensity of the obtained signal, which is well known, but also its kinetics. It was monitored that the stronger intensity was combined with a more rapid decrease of the CL signal.     

毛细管电泳免疫化学发光检测鼠血管平滑肌细胞中zmol骨形成蛋白-2

近年来,结合毛细管电泳的免疫分析研究在不断加强.特别是毛细管电泳免疫激光诱导荧光(CEIA-LIF)检测由于具有较高的灵敏度而十分引人注目.常文保等用CEIA-LIF检测雌三醇,检出限为31.6ng/L,可用于血清和尿样分析.Kennedy等用芯片CEIA-LIF检测了鼠胰腺细胞中的胰岛素,     

Highly sensitive trivalent copper chelate-H2O2 system for CE-chemiluminescent detection of luminol-type compounds

Luminol-type compounds can be used as chemiluminescent (CL) derivatization reagents for amines, carboxylic acids and protein. Copper chelate diperiodatocuprate(III) (K5[Cu(HIO6)2], DPC) was synthesized by complexation of copper at trivalent oxidation state and periodate in a strong basic medium. It was found that DPC can greatly enhance the reaction between luminol-type compounds and H2O2 to produce very strong CL emission. Based on this fact, a rapid CE method combined with high-sensitive end-column CL detection was established to simultaneously analyze luminol and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) with wide concentration range of 3.0-300 nmol/L in 5 min. The RSDs of the signal intensity and the migration time were less than 3.9 and 7.0% for a standard sample containing 100 nmol/L luminol and ABEI (n=5), respectively. The investigation implies that DPC is a promising sensitizer for CE-CL detection of a great variety of biomolecules and drugs in biological samples after derivatization using luminol derivatives.     

流动注射-化学发光法测定十六烷基三甲基溴化铵的临界胶束浓度

采用流动注射技术，研究了CTAB-Luminol-H2O2体系的化学发光行为。CTAB对Luminol-H2O2体系的化学发光强度有明显的增强作用，并且在临界胶束浓度出现最大值，从而建立了流动注射-化学发光法直接测定CTAB的CMC的一种新方法，并给出了可能的作用机理。     

Study on the chemiluminescence behavior of bovine serum albumin with luminol and its analytical application

In this paper, the luminescence behavior of bovine serum albumin (BSA) and luminol was first studied by flow injection chemiluminescence (CL). It was found that the hyperchromic effect of luminol in the presence of BSA led to the acceleration of the electrons transferring rate of excited 3-aminophthalate, which greatly enhanced the CL intensity of luminol/dissolved oxygen reaction. The increments of CL intensity were proportional to the concentrations of BSA with a linear range from 0.01 to 7 nmol L(-1). It was also found that azithromycin could inhibit the CL intensity of luminol/BSA reaction. The decrements of CL intensity were logarithm over the concentrations of azithromycin ranging from 0.1 to 700 ng mL(-1). At a flow rate of 2.0 mL min(-1), a complete analytical process, which included sampling and washing, could be performed within 30s with relative standard deviations of less than 3.1%. This proposed method was successfully applied in assaying azithromycin in pharmaceutical and human serum samples with recoveries from 91.0 to 104.3%. The possible luminescence mechanism of luminol/BSA/azithromycin reaction was discussed in detail by CL, UV and fluorescence methods.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

Inactivation of horseradish peroxidase by phenoxyl radical attack

To test the hypothesis that horseradish peroxidase (HRP) can be inactivated by phenoxyl radicals upon reaction with H(2)O(2)/phenol, we probed HRP-catalyzed phenol oxidation at various phenol/H(2)O(2) concentrations. To this end the total protein, phenolic product, active protein, and iron concentrations in the aqueous phase were determined by protein assay, phenol-(14)C isotopic labeling, resonance Raman and atomic absorption spectroscopy, respectively. Additionally, resonance Raman and FTIR measurements were carried out to probe possible structural changes of the enzyme during the reaction. The data obtained provide the first experimental support for the hypothesis that HRP can be inactivated by a phenoxyl radical attack. The heme macrocycle destruction involving deprivation of the heme iron occurs as a result of the reaction. An intermediate type of the active protein was observed by Raman difference spectra at low concentrations which features a stabilization of the quantum mixed state of the heme iron and a significant amount of phenoxylphenol-type oligomers in solution and probably also in the heme pocket. This work provides a basis for evaluating the relative contributions of different HRP inactivation mechanisms and is thus critical for optimizing engineering applications involving HRP reactions.     

The enhanced electrochemiluminescence of luminol on the nickel phthalocyanine modified electrode

A glassy carbon electrode (GCE) modified with nickel(II) tetrasulfophthalocyanine (NiTSPc) and Nafion was used for the investigation of the catalytic oxidation of luminol. The modified electrode was found to much more effectively improve the emission of electrochemiluminescence(ECL) of luminol in a solution containing hydrogen peroxide. The enhanced ECL signal corresponded to the catalytic oxidation of both luminol and H(2)O(2) by NiTSPc. Attached Ni(II) on GCE was oxidised to Ni(III) and then used as the catalyst for the chemiluminescence of luminol. The enhanced stability of the ECL signal with Nafion would mainly result from the prevention of the dissolution of NiTSPc and the adsorption of the oxidation product of luminol on the electrode surface. The proposed method enables a detection limit for luminal of 6.0 x 10(-8) mol L(-1) to be achieved in the presence of H(2)O(2) in the neutral solution. The enhanced ECL intensity had a linear relationship with the concentration of luminol in the range of 1.0 x 10(-7)-8.0 x 10(-6) mol L(-1).     

Existence of a new reactive intermediate oxygen species in hypoxanthine and xanthine oxidase reaction

We investigated a hypoxanthine (HPX) and xanthine oxidase (XOD) reaction by using a luminol analog 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-mediated chemiluminescence (CL) response. Addition of a high activity of superoxide dismutase (SOD), a potent O2* scavenger, and of a high concentration of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a potent spin trapping agent, diminished completely the CL response. Whereas a high concentration of dimethyl sulfoxide (DMSO), as a potent *OH scavenger could not attain to the complete diminishment of the CL response. It has been reported that luminol monoanion reacts with *OH to form luminol radical, and then resultant luminol radical reacts with O2* to elicit CL response. Complete scavenging for *OH is assumed to result in lack of luminol radical, which in turn induces lack of CL response. However, our results did not support the idea. Furthermore, we examined the effect of L-012 on the DMPO-OOH formation in the presence or absence of DMSO in the HPX-XOD system by applying an electron spin resonance (ESR)-spin trapping method. The DMPO-OOH formation was inhibited even in the presence of DMSO, and the rate constant (k2) between L-012 and O2* obtained in the presence of DMSO was 9.77 x 10(2) M(-1) s(-1) and the constant in the absence of DMSO was 2.97 x 10(3) M(-1) s(-1). The data suggests that L-012 is converted to a radical form that reacts with O2* even under the conditions of the absence of *OH. From these, we postulate that the existence of a reactive intermediate oxygen species in the HPX-XOD system.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

Chemiluminescence biosensor chip based on a microreactor using carrier air flow for determination of uric acid in human serum

A chemiluminescence biosensor on a chip coupled to a microfluidic system and a microreactor is described in this paper. The chemiluminescence biosensor measured 25 x 75 x 6.5 mm in dimension, and was readily produced in an analytical laboratory. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and luminol in the microreactor, and to immobilize uricase in the enzymatic reactor. The main characteristic of the biosensor was to introduce air as the carrier flow instead of the more common solution carrier for the first time. The uric acid was determined by a chemiluminescent (CL) reaction between the hydrogen peroxide produced from the enzymatic reactor and luminol under the catalysis of HRP in the microreactor. The linear range of the uric acid concentration was 1 to 100 mg L(-1) and the detection limit was 0.1 mg L(-1) (3sigma).     

增强辣根过氧化物酶催化鲁米诺化学发光反应的新型增强剂四苯硼钠

报道了新型增强剂四苯硼钠对过氧化物酶催化鲁米诺－过氧化氢发光反应的增强作用，建立了流动注射化学发光测定或辣过氧化物酶（ＨＲＰ）的新体系。用该体系测定ＨＲＰ线性范围为１．０×１０－１２×１．２×１０－１３ｍｏｌ／Ｌ；检测限为０．６×１０－１３ｍｏｌ／Ｌ。对０．６×１０－１３ｍｍｏｌ／Ｌ的 ＨＲＰ进行１１次平行测定，相对标准偏差为 １． ５％。     

Enzyme immobilization on poly(ethylene-co-acrylic acid) films studied by quartz crystal microbalance with dissipation monitoring

In this study, we use the quartz crystal microbalance with dissipation monitoring (QCM-D) to study the immobilization of the enzyme horseradish peroxidase (HRP) on poly(ethylene-co-acrylic acid) (PEAA) films. The surface polarity of spin-coated PEAA films was varied by heat treatments in air or in a 30% NaOH aqueous solution leading to COOH-depleted or COOH-enriched surfaces, respectively. Two reaction schemes, direct adsorption and amine coupling, were employed for HRP immobilization on the two surfaces. The shifts in frequency and dissipation, Deltaf and DeltaD, measured by QCM-D and the ratio DeltaD/Deltaf were used to evaluate the binding amount and the conformation of the adsorbed enzyme. It is found that HRP immobilized via covalent linkages forms rigid and little dissipative films. In contrast, directly adsorbed HRP films exhibit a highly dissipative structure. HRP-catalyzed oxidation of the 4-chloro-1-naphthol in the presence of H(2)O(2) was used to characterize the catalytic activity of the HRP films. The results show that the enzymatic activity of the covalently immobilized HRP tends to be higher.     

Amperometric measurement of ds-DNA content using a peroxidase-modified electrode

An enzyme electrode with a chemically amplified response for methylene blue (MB) was constructed from a glassy carbon electrode and a layer containing immobilized horseradish peroxidase (HRP). MB is reduced on the electrode but regenerated through the HRP-catalyzed reaction in the presence of H(2)O(2). The electroreduction/regeneration cycle for MB resulted in an amplified electrode response. The enzyme electrode was applied to the highly sensitive measurement of ds-DNA. The current for MB decreased in association with its complexation with DNA, and the current response caused by DNA was also amplified through the recycling processes. The detection limit of ds-DNA (from salmon testes) was as low as 5 ng ml(-1).     

Dual‐signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF–BB in capillary electrophoresis

Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE‐CL). This work describes a novel dual‐signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet‐derived growth factor–BB (PDGF–BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol–H₂O₂ CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof‐of‐concept, the proposed method was employed to quantify the concentration of PDGF–BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF–BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Sensitive determination of sub-nanogram amounts of rutin by its inhibition on chemiluminescence with immobilized reagents

An interesting inhibitory effect of rutin on the chemiluminescence (CL) reaction between luminol and periodate was reported, and this effect was used for the determination of rutin in medicine and human urine. The CL reagents, luminol and periodate, were both immobilized on an anion-exchange column. The CL signal produced by the reaction between luminol and periodate, which were eluted from the column through water injection, was decreased in the presence of rutin. Rutin was sensed by measuring the decrement of CL intensity, and which was observed to be linear over the logarithm of 0.1-30 ngml(-1) rutin concentration range, and the limit of detection was 0.03 ngml(-1) (3sigma). At a flow rate of 2.0 mlmin(-1), both sampling and washing could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The method proposed offered reagent-less procedures and remarkable stability in the determination of rutin, and could be easily reused over 80 h. The method proposed was applied successfully in the determination of rutin in pharmaceutical preparations and monitoring the excretion of rutin in human urine.     

Detection of hydrogen peroxide in rainwater based on Mg-Al-carbonate layered double hydroxides-catalyzed luminol chemiluminescence

Using a green catalyst of luminol chemiluminescence (CL), Mg-Al-carbonate layered double hydroxides (denoted as Mg-Al-CO(3) LDHs), a novel, sensitive and rapid CL method was developed for the determination of hydrogen peroxide (H(2)O(2)). The corresponding linear regression equation was established in the range of 0.05-10 μM for H(2)O(2). The detection limit (S/N = 3) is 0.02 μM and the relative standard deviation (RSD) for nine repeated measurements of 1.0 μM H(2)O(2) was 2.9%. This proposed method has been successfully applied to detect H(2)O(2) in rainwater samples with good accuracy and precision. The novel methodology is expected to provide a general protocol for the determination of H(2)O(2) as well as for numerous other oxidase-based reactions giving H(2)O(2) as a product (e.g., glucose).