New nucleophilic catalysts for bright and fast peroxyoxalate chemiluminescence

Miniaturized detection applications based on chemiluminescence require fast reaction kinetics for optimum performance. In this work, high-intensity light from the analytically useful peroxyoxalate chemiluminescence reaction has been generated at high rates by employing both single-component and dual-component nucleophilic catalysis. 4-(Dimethylamine)pyridine and its derivatives were superior to all other bases in terms of reaction speed and intensity of the generated light and outshone imidazole, which hitherto has been considered as the best catalyst. The light intensity was related to the difference in pKa between the 4-aminopyridine catalyst and the leaving group of the reagent, and the optimum delta pKa was found to be close to 0. Similarly, high light intensities were obtained when mixtures of the imidazole analogue 1,2,4-triazole and the strong, nonnucleophilic base 1,2,2,6,6-pentamethylpiperidine acted as catalysts. The mechanism behind this was concluded to be a "base-induced nucleophilic catalysis", where the ancillary strong base assisted the production of the highly nucleophilic 1,2,4-triazolate anion, which as the actual catalyst then participated in the formation of a more reactive transient reagent. All the investigated catalysts reduced the light yield of the reaction due to base-catalyzed breakdown reactions of the reagents and/or intermediates. The intensity peak maximums of these bright and fast reactions typically appeared after less than 10 ms, whereafter the light decayed to darkness within a few seconds. These reaction characteristics are especially advantageous for sensitive detection applications where the observation volumes and times are limited, e.g., peaks emerging from a capillary-based separation process.     

化学发光免疫分析技术测定血清TSH及临床评价

探讨化学发光免疫技术检测血清TSH的临床应用。采用美国贝克曼库尔特ACCESS化学发光系统进行检测.健康人(n=100)血清TSH水平略呈偏态分布;甲减组水平升高;初诊甲亢组及甲亢治疗组水平降低。甲亢治愈组、甲状腺瘤组、甲状腺肿组、心脑血管组其水平与正常组无显著差异。与免疫放射法(IRMA)比较,两法结果符合率达90.8％(P<0.001);方法可靠性(CV％):0.3％～2.7％。用化学发光免疫技术测定TSH灵敏、特异、有效,在许多方面优于免疫放射法.     

A chemiluminescence reaction between hydrogen peroxide and acetonitrile and its applications.

Here we report a new chemiluminescence reaction between basic aqueous H2O2 and acetonitrile. Its ultraweak chemiluminescence could be greatly enhanced by luminol, isoluminol-labeled streptavidin, and an Edman-type fluorescent reagent. Light emission was intense and long-lived, and this facilitated the initiation of the reaction and the measurement of the light emission. The present results permit us to propose a series of convenient, highly sensitive, and enzyme-free techniques for the detection and quantification of luminol, related conjugates, acetonitrile, and amino acids. Overall, this new chemiluminescence reaction will be quite promising for numerous applications in immunoassay, DNA hybridization, environmental monitoring. and postcolumn chromatographic detection.     

流动注射化学发光体系测定金霉素

本文利用金霉素对KM_nO_4—Na_2SO_3化学发光体系的发光有增敏的作用,结合流动注射技术,建立了流动注射化学发光法测定金霉素的新方法。在最佳条件下,金霉素溶液的浓度在2.0×10~(-7)～5.0×10~(-5)mol/L范围内与化学发光强度呈良好的线性关系。该方法的检测限(S/N=3)为1.0×10~(-8)mol/L。对实际样品进行回收实验,回收率在98%-103%间,结果表明该方法适合对金霉素的测定。并对可能的发光机理进行了初步的探讨。     

A sensitive chemiluminescence procedure for the determination of carbon monoxide with myoglobin-luminol chemiluminescence system

A novel chemiluminescence method combined with the flow injection technique for the determination of carbon monoxide is presented in this paper. The chemiluminescence signal based on the reaction between myoglobin and luminol in an alkaline medium was remarkably enhanced by carbon monoxide. The enhanced chemiluminescence intensity was linear with carbon monoxide concentration in the range from 0.01 to 10.0 pmol.L(-1), and the detection limit was 3x10(-3) pmol.L(-1) (3sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a relative standard deviation of less than 4.0%. The proposed method was applied successfully in the assay of carbon monoxide in human serum and artificial water samples without any pretreatment procedure.     

Detection of enzymatically generated hydrogen peroxide by metal-based fluorescent probe

We developed a metal-based fluorescent probe for H(2)O(2) called MBFh1, which has an iron complex as a reaction site for H(2)O(2) and a 3,7-dihydroxyphenoxazine derivative as the fluorescent reporter unit. The iron complex reacts quickly with H(2)O(2) to form oxidants, and then the oxidants convert the closely appended nonfluorescent 3,7-dihydroxyphenoxazine moiety to resorufin in an intramolecular fashion. The quick response to H(2)O(2) allows us to plot the enzymatic evolution of H(2)O(2). A combination of N-acetyl-3,7-dihydroxyphenoxazine and horseradish peroxidase has been frequently used to detect enzymatically generated H(2)O(2), but this method has interference with phenol derivatives. The use of MBFh1 overcomes this drawback.     

Reagentless enzyme electrode for the determination of manganese through biocatalytic enhancement.

An amperometric enzyme electrode is described for the detection and determination of manganese(II). The biosensor is based on the stimulation by manganese of the aerobic oxidation of substrates by horseradish peroxidase. A mediator, 1,2-naphthoquinone, is used as the substrate and is incorporated with the enzyme into a carbon-paste electrode. The resulting electrode acts as an enzyme-based oxygen sensor, which is sensitive to manganese. Electrochemical control of enzyme activity is achieved through substrate promotion of catalysis. Enzyme modulation by manganese can be switched on and off or adjusted through the appropriate selection of the applied potential. Currents are generated due to the bioelectrocatalytic reduction of oxygen in response to the introduction of manganese sulfate. A sustained current is achieved which is dependent on manganese concentration. Concentrations of 0.5 microM manganese or greater can be measured, and the sensor is reversible, as demonstrated by manganese removal. Biological selectivity for manganese provides a sensor which does not respond to other divalent cations tested, with the possible exception of cobalt. Reagentless, continuous sensing is achieved through substrate cycling.     

Immobilized enzyme electrode for creatinine determination in serum

An immobilized enzyme electrode for continuous creatinine determination in blood serum is described. The enzymes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are coimmobilized to the surface of the polypropylene membrane of a Clark-type electrode responsive to oxygen. The immobilized enzymes catalyze the decomposition of creatinine with the consumption of oxygen and thus permit the creatinine measurement. The whole assay takes less than 1 min. Effects of pH and temperature on electrode response are also described. The proposed technique offers a rapid, simple, and inexpensive means to determine creatinine in blood serum within the normal and abnormal ranges. The repeatability of the creatine determination in serum is 2.5% (relative standard deviation), and the detection limit is 3 x 10(-6) mol L-1. The results obtained by this method were compared to those obtained with the Technicon AutoAnalyzer SMAC system based on the Jaffé reaction; the correlation factor between the two methods was found to be r = 0.9997.     

Sol-gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium.

An acid-stable soybean-peroxidase biosensor was developed by immobilizing the enzyme in a sol-gel thin film. Methylene blue was used as a mediator because of its high electron-transfer efficiency. The sol-gel thin film and enzyme membrane were characterized by FT-IR, and the effects of pH, operating potential, and temperature were explored for optimum analytical performance by using the amperometric method. The H2O2 sensor exhibited a fast response (5 s), high sensitivity (27.5 microA/mM), as well as good thermostability and long-term stability. In addition, the performance of the biosensor was investigated using flow-injection analysis (FIA).     

Simultaneous potentiometric determination of peracetic acid and hydrogen peroxide

A rapid and highly selective potentiometric method for the simultaneous analysis of peracetic acid (PAA) and hydrogen peroxide (H2O2) has been proposed, for the first time, using glassy carbon (GC) as an indicator electrode and I2/I- potential buffer. On the basis of the large difference in the reaction rates of PAA and H2O2 with I-, which was confirmed using stopped-flow spectrophotometry, a transient potential response corresponding to the reactions of the two species with I- was observed. The response times were typically a few seconds and several minutes for PAA and H2O2, respectively. The effects of the concentrations of molybdate catalyst, H+, I2, and I- in the potential buffer on the selectivity as well as the sensitivity were examined. The potential response obtained using the GC indicator electrode was found to be Nernstian over a wide range of their concentrations (typically from micromolar to millimolar) with slopes of 30.5 and 29.5 mV for PAA and H2O2, respectively (in close agreement with the theoretical value, that is, 29.6 mV). O2 was found to have no substantial effect on the potential change at the GC electrode in the present potential buffer.     

Quantitative determination of aqueous-phase ozone by chemiluminescence using indigo-5,5'-disulfonate

Indigo-5,5'-disulfonate (IDS) was found to be an efficient reagent for the determination of ozone by chemiluminescence (CL); hence it was applied to the continuous measurements of dissolved ozone (O3(aq]. The optimum reagent composition was determined as 10 mg L-1 IDS plus 2 mM phosphate (pH 7.2). The CL intensity was proportional to the O3(aq) concentration in the range of 0.025-410 ng mL-1. The detection limit was 0.006 ng mL-1, which is 3 orders of magnitude lower than that obtained by spectrophotometry using IDS as reported previously. The reduction of interferences from aldehydes and hydrogen peroxide was attempted. Furthermore, the mechanism of the CL was discussed from CL and fluorescence spectra measured.     

Simultaneous HPLC determination of peroxyacetic Acid and hydrogen peroxide

The first instrumental method for simultaneous determination of peroxyacetic acid (PAA) and hydrogen peroxide has been developed. The successive quantitative reaction of PAA with methyl p-tolyl sulfide (MTS) and hydrogen peroxide with triphenylphosphine (TPP) yields the corresponding sulfoxide MTSO and phosphine oxide TPPO. The reagents and their oxides are separated by HPLC on reversed-phase columns with acetonitrile/water gradient elution within 5 min. External calibration with the solid standards of MTSO and TPPO leads to a very accurate and reliable method. Samples are stable and can be stored after derivatization for several days prior to analysis. Real samples from brewery disinfection were analyzed in comparison to titration with excellent correlation.     

Immobilized enzyme electron spin resonance: a method for detecting enzymatically generated transient radicals

The study of enzymatically generated, transient radicals provides valuable information about radical reactivity as well as enzyme function. ESR methods to detect transient radicals are generally based on continuous flow and have the potential to consume large quantities of enzyme, substrate, and buffer. Experimental approaches have been pursued to minimize sample volumes, although none have made the continuous-flow ESR approach generally applicable for enzymes and substrates available in limited quantities. We have developed an alternative approach to the traditional continuous-flow ESR method that provides the same high-resolution ESR spectra, but does not consume large quantities of enzyme, substrate, or buffer. The method utilizes enzyme immobilized onto an inert substrate packed directly into an ESR flat cell. Flowing substrate solution over the immobilized enzyme generates in situ, transient radicals, which can then be observed on the submillisecond time scale. We have termed this method "immobilized enzyme ESR," abbreviated IE-ESR. In this paper, we have described the details of the IE-ESR technique and have presented data collected using the IE-ESR technique for transient radicals from limited quantity enzymes, limited quantity substrates, and D2O buffers. An extension of this technique to ESR spin trapping has also been discussed.     

Phthalhydrazide chemiluminescence method for determination of hydroxyl radical production: modifications and adaptations for use in natural systems

The hydroxyl radical is formed through a variety of processes pertinent to natural and anthropogenic systems. Here we report development of a simple and sensitive trap-and-trigger chemiluminescence method based upon the hydroxylation of phthalhydrazide to 5-hydroxy-2,3-dihydro-1,4-phthalazinedione, which emits chemiluminescence when oxidized under alkaline conditions. Cu(III) is employed as an oxidant and is shown to be relatively insensitive to a range of interferences likely to be encountered. The method has been standardized by use of γ-radiolysis of water as a primary source of hydroxyl radical, with a convenient secondary calibration procedure developed that uses Fenton chemistry. Detection limits varied from 7.4 nM (at pH 3) and 6.2 nM (at pH 8.1) of accumulated hydroxyl radical production in a simple 10 mM NaCl matrix to around 30 nM in an artificial seawater medium, due to competition for hydroxyl between the phthalhydrazide probe and bromide. The method has been used to characterize the kinetics of the Fenton system employed for calibration and is shown to be consistent with published models of this process over time scales of several hours. The application of this method to a range of matrices and for photochemical studies is also described.     

Catalysis-electrochemical determination of zeptomole enzyme and its application for single-cell analysis

A novel electrochemical method for determination of zeptomole amounts of enzyme was developed by a combination of on-capillary enzyme-catalyzed reaction and electrochemical detection. A limit of detection (LOD) of zeptomole (zmol, 10(-)(21) mol) was achieved by monitoring the product of the enzyme-catalyzed reaction. In this method, after enzyme molecules were electrokinetically injected into the capillary, they were electromigrated to the section of the capillary immersed in a warm water bath, where the enzyme molecules reacted with the enzyme substrates in the running buffer in the presence of the activator of the enzyme-catalyzed reaction. Then the electroactive product zone of the enzyme-catalyzed reaction was electromigrated to the horn-shaped outlet of the capillary and electrochemically detected by a carbon fiber disk bundle electrode at a constant potential. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as the model enzyme. A LOD of 1.3 zmol was achieved. This method was applied to determine zeptomoles of G6PDH in individual human erythrocytes.