Phenothiazine derivatives as enhancers of peroxidase-dependent chemiluminescence

Some N-alkyl phenothiazines with different ionic groups were studied as enhancers of chemiluminescence catalyzed by soybean peroxidase. It was shown that under experimental conditions, the compounds with positively charged groups do not exhibit enhancing ability, while the addition of phenothiazines with negatively charged groups to a substrate mixture significantly increased the chemiluminescence intensity. The relationship between the enhancing activity of phenothiazines and their capacity for enzymatic oxidation by hydrogen peroxide was found. The enhancers discovered new opportunities for increasing the sensitivity of determination of analytes by chemiluminescent enzyme immunoassay.     

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.     

The comparative evaluation of spectrophotometric and chemiluminescent detection in an immunoenzyme test of antibodies to the antigens of the bovine leukosis virus]

Comparative evaluation of the sensitivity limit in the detection of antibodies to bovine leukemia virus in the enzyme immunoassay with the use of chemiluminescent and spectrophotometric detection techniques was carried out. In this assay 3-amino-1,4-phthalazinedion was used as chemiluminescent substrate and ortho-phenylenediamine, as chromogenic substrate. The chemiluminescent signal was registered by means of a special luminometer designed at the Institute of Biochemistry (Lithuanian Acad. Sci.). The use of the chemiluminescent substrate permitted the detection of proteins in amounts 2-3 times lower than those detected by the spectrophotometric technique.     

Sensitive fluorescent detection of protein on nylon membranes

Detection of antigen immobilized on membranes, as in Western transfers and dot enzyme linked immunosorbent assays (ELISAs), often employ antibody-enzyme conjugates and chemiluminescent or precipitated colored reaction products. Although chemiluminescent markers are sensitive, they are time-consuming because of their required exposure to X-ray film and the presence of background artifacts sometimes limits their use. This report demonstrates that direct fluorescent detection technique using nylon membranes that has higher sensitivity than chemiluminescent methods is easier to perform and has a uniform, low background. An alkaline phosphatase conjugated antibody was compared with antibody conjugated to a fluorescent phycobiliprotein (allophycocyanin) for sensitivity in both Western transfers and dot ELISA assays using mouse IgG as the membrane-bound antigen. Direct fluorescent detection of antigen-antibody complexes on positively charged nylon membrane provided better sensitivity and lower background than similar conditions using enzyme amplification and chemiluminescent detection on either nylon or PVDF membranes. Processing time was reduced by the elimination of steps associated with substrate incubation, washing and X-ray film exposures required for chemiluminescence detection. These data support the view that direct fluorescent detection can represent a significant improvement in assay sensitivity and reduction in time compared with more traditional chemiluminescent detection techniques employed in the conduct of Western transfers and dot ELISA studies.     

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. Copyright © 2013 John Wiley & Sons, Ltd.     

Measurement of urinary somatomedin C by a chemiluminescent method

We present a method for the measurement of the total Somatomedin C (SmC) content in human early morning urine samples after dialysis, extraction, and concentration. We modified a chemiluminescence immunoassay, previously developed for SmC determination in serum, for analysis of SmC in urine. Appropriate sensitivity was obtained by the preparation of a new chemiluminescent tracer (AEEI-COOH-SmC) and the optimization of a competitive non-equilibrium immunoassay system which had a detection limit of 0.24 fmol SmC per tube.     

High-sensitivity hybridization assay for quantitation of residual E. coli DNA

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.     

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.     

A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.     

Chemiluminescent detection of DNA on nylon membranes.

Two types of nonradioactive DNA detection systems were optimized for use with nylon membranes in Southern transfers. A luminol substrate system (consisting of an enhanced chemiluminescent reaction utilizing luminol enzyme substrate) was used with peroxidase-labeled probe DNA, and a dioxetane-based substrate was used with alkaline phosphatase/antibody and digoxigenin-labeled probe DNA. Chemiluminescence was detected by autoradiography. Methods for reprobing the membranes were also optimized for both systems; blots could be reprobed at least ten times. Results showed that excellent sensitivity and low background can be achieved on both amphoteric and positively charged nylon membranes, using either detection system.     

Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.

A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h.     

羧基化石墨烯/半胱胺修饰金电极对多巴胺的电化学行为研究

为了基于羧基化石墨烯/半胱胺修饰金电极建立更为先进的多巴胺生物传感器，以定量检测儿茶酚胺类神经递质多巴胺，利用自组装技术将半胱胺修饰于金电极上，再利用1-乙基-\[3-二甲基氨基丙基\]碳酰二亚胺盐酸化物/N-羟基琥珀酰亚胺（EDC/NHS）交联剂将羧基化石墨烯固定在修饰后的金电极上制成多巴胺电化学传感器。先对修饰电极进行表征以检验其灵敏度，再利用循环伏安法研究该电极在多巴胺溶液中的电化学行为，包括检测条件的优化和传感器性能的测定。修饰电极表征结果表明，羧基化石墨烯/半胱胺修饰金电极提高了电极传递电子的能力，具有较高的灵敏度。经单因素实验得出，最佳检测条件为利用pH 6.00的0.30 mol·L-1磷酸盐缓冲溶液（PBS）配制多巴胺溶液，扫描速率设定为200 mV·s-1。在最佳检测条件下，制备的多巴胺电化学传感器电流的大小随着多巴胺浓度的增大而增大，在1.0×10-3~3.5×10-3 mol·L-1范围内呈现良好的线性关系，线性回归方程为I=8.120 6C+7.017，相关系数R2为0.999 5。且该传感器精密度好，稳定性强，具有一定的抗干扰能力。研究结果为药物盐酸多巴胺注射液中多巴胺含量测定提供了支撑。     

Chemiluminescent measurement of oxalate in serum by detection of hydrogen peroxide generated through oxalate oxidase

Today, chemiluminescence detection reactions have become popular in analytical biochemistry essentially due to their high sensitivity. A chemiluminescent synthetic system (luminol/porphyrin) was successfully used to measure serum oxalate by determination of hydrogen peroxide generated through oxalate oxidase (EC 1.2.3.4.). This new method is efficient and simple, highly sensitive and the results obtained in normal adult subjects are in good agreement with those of approved methods. This original application of such a chemiluminescent system allowed us to achieve a sensitive serum oxalate assay (detection limit of 0.2 μmol/L) characterized by a low serum volume (200 μL) required for analysis. © 1997 John Wiley & Sons, Ltd.     

CCD camera imaging for the chemiluminescent detection of enzymes using new ultrasensitive reagents

We have designed and constructed an inexpensive imaging system based on charge coupled device (CCD) technology and utilized it to demonstrate the sensitivity and rapid detection possible with Lumigen® chemiluminescent reagents. We also report the development of two new chemiluminescent reagents, Lumi-Phos® Plus and Lumigen PS. Lumi-Phos Plus is an ehanced formulation for the rapid detection of alkaline phosphatase on membranes and in solution. It provides excellent images in blotting applications with exposures of under a minute. Lumigen PS represents a new generation of peroxidase detection reagents. The wide dynamic range with excellent linearity, higher signal and lower background than other chemiluminescent reagents make Lumigen PS of unsurpassed value in enzyme-linked immunoassays and nuclic acid probe assays using HRP conjugates.     

Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD‐methoxy‐pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets. Copyright © 2015 John Wiley & Sons, Ltd.     

Sensitive determination method of estradiol in plasma using high-performance liquid chromatography with electrochemical detection

The improvement in the sensitive determination method of estradiol using HPLC with electrochemical detection is described. The improvement was due to the optimization of the potential applied to the electrode of the analytical cell and employment of a guard cell. The detection conditions were optimized from the electrochemical properties of estradiol in acidic and alkaline eluents. The employment of the guard cell drastically decreased the background noise without any reduction in the response of estradiol, and contributed to improvement in the sensitivity. The optimized method combined with pretreatment by liquid-liquid extraction was applied to the determination of estradiol in rat plasma. The detection limit of 8 pg for the standard solution and 24 pg for the plasma sample, which was about 6-8-fold more sensitive compared to the previous reports, was attained.     

An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.     

化学发光法用于芳香酯酶抑制剂体外筛选

在前期的研究中,我们将9-(4-氯苯氧羰基)-10-甲基吖啶酯三氟甲基磺酸盐(CPOCMA)用于测定血清芳香酯酶活性,取得满意结果.在此基础上,本文以CPOCMA为底物,建立化学发光法评估药物对芳香酯活性影响的新方法.以硝酸甘油为模型药物,比较了化学发光法与UV方法的一致性.并将此法应用于评价三种抗炎药吲哚美辛、阿司匹林和乙酰氨基酚对芳香酯酶活性的影响.药物的加入使血清催化CPOCMA水解的动力学减缓,这表明这些药物均为抑制剂.吲哚美辛、阿司匹林和乙酰氨基酚表现出的IC50值分别为0.254、0.564和0.656 mmol/L,抑制常数ki分别为0.154、1.38和2.98 mmol/L.加入药物后的Lineweaver-Burk曲线表明这三种药物对PON的抑制均为竞争性抑制.根据加入药物后的动力学曲线,其IC50值、抑制常数和米氏常数的变化均表明这三种药物的抑制能力大小顺序:吲哚美辛阿司匹林乙酰氨基酚.CPOCMA可以作为PON底物体外评价药物对PON的抑制能力和抑制机理.UV法不适合评价紫外吸收峰与UV法的检测波长重叠的药物,而新建立的化学发光法对这类药物的筛选有独特优势.