两种萤火虫荧光素酶活性检测方法的一致性比较

目的：比较2种萤火虫荧光素酶活性检测方法的一致性。方法：分别采用化学发光技术（Che）及活体光学成像技术（Bio）,从细胞和动物水平检测在转染以萤火虫荧光素酶为报告基因的载体pCI-AAA-Fluc-neo后不同时间点,萤火虫荧光素酶的表达强度。结果：在细胞和动物水平,萤火虫荧光素酶的表达强度均随时间推移逐步递减。在HepG2细胞,萤火虫荧光素酶表达持续96h,活性从24h的2781±220mV（1．6×10^6±2．3×10^5光子）降至96h的49±3．5mV（6．4×10^4±2．5×10^4光子）。在动物水平得到相似的结果,BALB／c小鼠萤火虫荧光素酶表达持续20d,其活性从1d的16592±409mV（1．9×10^8±3．6×10^6光子）降至20d的798±139mV（3．37×10^5±3．8×10^4光子）。通过一致性检验,2种检测方法在细胞和动物水平的直线回归方程分别为lgChe=1．186·lgBio-3．764（r=-0．937,P〈0．001）和lgChe=0．451·lgBio＋0．64（r=0．915,P〈0．001）;进一步将理论数据与实验数据进行配对t检验,二者无统计学差异（P〉0．05）。结论：2种检测方法是一致的;从整个实验过程来看,活体光学成像技术较化学发光法更为简便、直观,可量化地对同一个体连续检测,减少了个体间差异和实验动物用量。     

Design of luminescence photobiosensors

The potential of immobilized enzyme membranes in biosensors has been explored in our group for several years. Although part of our work has been mainly devoted to electrochemical transducers and oxidases for the design of enzyme electrodes, the demand for ultrasensitive and highly selective sensors led us to consider the use of luminescent enzyme systems associated to optical transduction. When considering the need for operational and reliable biosensors in biotechnology, immobilization and stability of the sensing element still remain, in most cases, an unavoidable problem. We recently proposed a very fast and reliable procedure for preparing enzymatic membranes from Pall (Biodyne Immunoaffinity membranes) supplied in a pre-activated form. Both the firefly and bacterial systems as well as peroxidase for the chemiluminescent determination of various analytes, could be bound to such a support. Based on this approach, a fibre-optic sensor with immobilized enzymes has been designed which permits bio- or chemiluminescent analysis of ATP, NADH or H₂O₂ respectively. With the NADH-based system, other analytes could be detected using coupled dehydrogenases. This device appears very promising and includes the convenience of both the luminescence sensitivity as well as the handling of the biosensor design.     

Chemiluminescent and bioluminescent assays as innovative prospects for mycotoxin determination in food and feed.

Mycotoxin contamination of food and feedstuffs is among the top priorities for human and animal safety. The currently used techniques for mycotoxin determination, either chromatography or ELISA, are unsuitable for routine in-field assessment. There is an urgent need for other accurate, simple and cost-effective techniques that can be used as a screening tool for a rapid estimation of mycotoxin contamination in commodity lots. This paper reviews the literature on the use of chemiluminescence (CL) and bioluminescence (BL) assays for direct or indirect mycotoxin assessment. The chemiluminescence immunoassays, adenosine triphosphate (ATP) bioluminescence and bioassays are reviewed and their advantages and limitations discussed. These techniques used in food testing and the pharmaceutical industry offer promise as rapid techniques for mycotoxin determination. Chemiluminescence and bioluminescence bioassays are the most innovative alternatives to the conventional techniques used for mycotoxin determination in food and feed.     

Determination of cardamonin using a chemiluminescent flow-injection method

A sensitive and selective flow injection chemiluminescence method for the determination of cardamonin over the range 1.0 x 10(-8) to 8.0 x 10(-6) g/mL is described. The method is based on the enhancement by cardamonin of the chemiluminescence of the reaction between cerium (IV) and rhodamine 6G in sulphuric acid medium. The optimised flow injection procedure yielded a detection limit for cardamonin of 8.8 x 10(-9) g/mL, whilst the relative standard deviations of intraday and inter-day precision were below 2.5%. The method has the advantages of high sensitivity and a wide linear range. It was successfully applied to the determination of cardamonin in Alpinia katsumadai Hayata. The mechanism of the chemiluminescence reaction is proposed.     

Chemiluminescence inhibition assay for folic acid using flow injection analysis

A new flow injection method for the determination of folic acid is described. A fast oxidation reaction occurred when folic acid was mixed with potassium ferricyanide generating ferrocyanide which then inhibited the chemiluminescent reaction of ferricyanide and luminol in alkaline medium. The decrease of chemiluminescence intensity was correlated with the folic acid concentration in the range 0.1-21 microg/mL; the detection limit for the assay was 0.03 microg/mL (3sigma). A complete analysis of folic acid, including sampling and washing, could be performed within 2 min with a relative standard deviation of less than 4.0%. The proposed method has been applied successfully to the determination of folic acid in pharmaceutical preparations.     

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H₂O₂ (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.     

Determination of ampicillin and amoxycillin by flow injection chemiluminescence method based on their enhancing effects on the luminol–periodate reaction

In this work, a new flow injection chemiluminescence method is described for the determination of ampicillin and amoxycillin. The method is based on the strong enhancing effects of these antibiotics on the luminol–periodate reaction. The present method allows the measurements of ampicillin in the range 0.02–1.0 mg/L range and amoxycillin in the range 0.1–10.0 mg/L range with the relative standard deviations within 0.8–2.0%. The sampling frequency was calculated about 90/h. The method was successfully applied to the determination of ampicillin and amoxycillin in pharmaceutical preparations. A brief discussion on the possible chemiluminescence reaction mechanism is presented. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of procaine hydrochloride using flow injection inhibitory chemiluminescence

A new flow injection chemiluminescent method has been developed for the determination of procaine hydrochloride, based on the inhibition of the chemiluminescence reaction of luminol–hydrogen peroxide by procaine hydrochloride. The influence of several surfactants and β‐cyclodextrin on the chemiluminescence intensity were studied. It was found that β‐cyclodextrin enhanced the decrease in chemiluminescence intensity. The method is simple, convenient and sensitive, with a detection limit (3 σ) of 0.08 µg/mL. The decreased chemiluminescence intensity is linear, with the concentration of procaine hydrochloride in the range 0.2–100.0 µg/mL and 100.0–400.0 µg/mL. The relative standard deviation for 10 repeated measurements were 4.5% and 3.4% for 1.0 and 20.0 µg/mL procaine hydrochloride, respectively. The method has been successfully applied to the determination of procaine hydrochloride in injection solutions of this drug. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of bisphenol A using a flow injection inhibitory chemiluminescence method.

A new flow injection chemiluminescence (CL) method has been developed for the determination of bisphenol A (BPA), based on the inhibitory effect of BPA on the chemiluminescence reaction between luminol and potassium hexacyanoferrate. Under optimum conditions, the decrease in CL emission intensity was linear with BPA concentration in the range 8.0 x 10(-7)-1.2 x 10(-5) mol/L, and the detection limit was 3.1 x 10(-7) mol/L. The relative standard deviation (RSD) of 11 replicate measurements was 2.6% for 2.0 x 10(-6) mol/L BPA (n = 11). The sampling frequency was calculated to be ca. 120/h. This method has been successfully used to determine the content of BPA in aqueous solution of polycarbonate materials. A brief discussion on the possible chemiluminescence reaction mechanism is presented.     

Flow-injection chemiluminescence determination of catecholamines based on their enhancing effects on the luminol-potassium periodate system.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis is described for the determination of four catecholamines, dopamine, adrenaline, isoprenaline and noradrenaline, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. The optimized chemical conditions for the chemiluminescence reaction were 1.0 x 10(-4) mol/L luminol and 1.0 x 10(-5) mol/L potassium periodate in 0.2 mol/L sodium hydroxide (NaOH). Under the optimized conditions, the calibration graphs relating the CL signal intensity (peak height) to the concentration of the analytes were curvilinear and they were suitable for determining dopamine, adrenaline, isoprenaline, and noradrenaline in the range 0.1-10 ng/mL, 0.1-100 ng/mL, 1-100 ng/mL and 5-50 ng/mL, respectively, with the relative standard deviations of 0.8-1.7%. The detection limits of the method are 0.02 ng/mL for dopamine, 0.01 ng/mL for adrenaline, 0.1 ng/mL for isoprenaline and 2.0 ng/mL for noradrenaline. The sampling frequency was calculated to be about 60/h. The selectivity of the method was good, because a series of common ions or excipients, such as K(+), Ba(2+), CO(3)(2-), NO(3)(-), SO(4)(2-), PO(4)(3-), sodium citrate, sodium bisulphite, oxidate dopamine, starch, lactose, carbamide and gelatin, could not produce interference when their concentrations were 1000-fold than those of dopamine. The present method was successfully applied to the determination of the four catecholamines in pharmaceutical injections.     

Flow injection chemiluminescence determination of captopril based on its enhancing effect on the luminol–ferricyanide/ferrocyanide reaction

A new flow injection chemiluminescence method is described for the determination of captopril. It is based on the enhancing effect of captopril on the chemiluminescence reaction of luminol with potassium ferricyanide in alkaline solution in the presence of potassium ferrocyanide. The method allows the determination of captopril over 0.1–40 µg/mL range, with a relative standard deviation (SD) of 1.0% for the determination of 0.5 µg/mL captopril solution in 11 repeated measurements. The method was satisfactorily applied to the determination of captopril in commercial captopril tablets. The possible reaction mechanism is also discussed briefly. Copyright © 2002 John Wiley & Sons, Ltd.     

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: insights on the origin of protoluciferase activity and bioluminescence colours.

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of d-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site.     

Hydrazine-induced post-chemiluminescence phenomenon of permanganate-luminol reaction and its applications.

The post-chemiluminescence phenomenon arising from the permanganate-luminol reaction induced by hydrazine and isoniazid was investigated. When hydrazine or isoniazid was injected into the mixture after the end of the reaction of permanganate with alkaline luminol, a new chemiluminescence (CL) reaction was initiated and strong CL signal was detected. A possible CL mechanism is suggested, based upon the studies of the kinetic characteristics of the CL reaction, the UV-visible spectra, the CL spectra and some other experiments. The present reactions allow the determination of 0.1-10.0 mg/L hydrazine and 0.02-1.0 mg/L isoniazid, with detection limits of 0.03 mg/L and 0.006 mg/L, respectively. The method was applied to the determination of isoniazid in pharmaceutical preparations.