Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87^N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.     

Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents

Fungal contamination is a major cause of deterioration in libraries and archives. Curators and conservators increasingly need rapid microbiological analyses. This paper presents a rapid detection method for the fungal contaminants on documents. A previous study showed that the calculation of energy charge, using bioluminescence ATP assays, provides a useful indicator to determinate the viability of fungal strains. We argue that this sensitive and time‐saving method is better than traditional culture techniques. However, the procedure needs to be modified to make it usable for lay persons. An improved and simplified protocol is proposed here for the extraction of adenylate nucleotides (AN) from fungal spores and for their measurements. Our new procedure can detect the existence of viable fungal strains on documents, presenting suspect spots within minutes. The extraction is performed by filtration with DMSO–TE solution as extractant. The different step of the measurement of AN content is carried out successively in a single test tube instead of the three tubes necessary in the initial method. The new procedure was tested on 12 strains among those most frequently found in archives and libraries and validated on swab samples from real documents. Copyright © 2008 John Wiley & Sons, Ltd.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

Allosteric regulation of mouse brain serine racemase

Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg²⁺ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for l-serine decreased from 13 mM to 1.8 mM with little change in V _max, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on l-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. l-serine was also converted to pyruvate at about twice the racemization rate. l-serine O-sulfate dehydration was inhibited by ATP, while l-serine dehydration, like racemization, was activated by nucleotides, indicating that, for l-serine, dehydration and racemization take place at the same site.     

Bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in clinical blood cultures

A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10^?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10^?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture.     

三磷酸腺苷发光法快速检测结核分枝杆菌药敏技术的研究

目的 建立一种利用三磷酸腺苷( ATP) 与荧光素酶反应测定结核分枝杆菌( 简称结核杆菌) 释放的ATP 来判断结核杆菌药敏的技术。方法 ATP 生物发光法( 简称ATP 法) 通过裂解液体培养基中的结核杆菌, 释放活菌中的ATP, 加入荧光素酶使之发光以检测结核杆菌的活性。共采用H37Rv 标准株和10 株临床分离菌株, 用ATP 法与BACTEC 3D 法同步平行进行利福平药敏检测, 连续7 d 检测结核杆菌释放的ATP, 观察其生长曲线, 并以此判断对药物的敏感性。结果 在生长5 ～7 d的培养基中ATP法可以检测到敏感菌释放的ATP, 并且显著高于耐药菌所释放的ATP, 通过与BACTEC 3D 法相比确定其判断药敏的临界值, 检测结果与L-J 法及同步平行的BACTEC 3D 法对照组符合率达100% 。结论 ATP法可用于结核杆菌对抗结核药物敏感性的检测, 且因其价格较低, 无放射性元素的存在, 作为一种新型的结核杆菌药敏检测技术具有巨大的临床应用潜力。     

Detection of viable fungal spores contaminant on documents and rapid control of the effectiveness of an ethylene oxide disinfection using ATP assay

Filamentous fungi are able to damage and even destroy archival and library materials. Nowadays the conventional method for detecting such micro‐organisms is to put them in cultures but such methods are laborious and time‐consuming. ATP methodology has been widely applied in other domains and its success on bacteria and yeast has been demonstrated. Several commercial reagent kits are available but they did not give satisfactory results on spores mould. We have elaborated new extraction strategies specific to fungi. A comparison of 42 extraction protocols of ATP from fungal spores was carried out. Extraction at 100°C with DMSO 90% in a Tris–acetate–EDTA buffer proved to be the best method. The viability of cells is estimated by the determination of adenylate energy charge (EC). We applied our method successfully on well‐known species such as Aspergillus flavus, A. niger, A. fumigatus, A. versicolor, Neosartorya fischeri, Eurotium chevalieri, Penicillium chrysogenum, Chaetomium globosum and Ulocladium spp. The results suggest that the ATP bioluminescence assay provides a sensitive and time‐saving method for detecting viable fungal spores. The validity of the procedure was also tested on spores killed by steam and on spores treated with ethylene oxide. We showed that EC determination could be used for a rapid control of the effectiveness of a disinfection process performed with ethylene oxide. Copyright © 2003 John Wiley & Sons, Ltd.     

Using ATP bioluminescence technique for monitoring microbial activity in sludge

This study demonstrates, for the first time, that the presence of suspended solids in waste‐activated sludge interferes with adenosine triphosphate (ATP) bioluminescence tests. The sludge subject to acid/alkaline treatment represented the test sample. Without consideration of the effect of solid concentrations, one would erroneously estimate the density levels of heterotrophic bacteria in the sludge using ATP data. A light blockage model was proposed to evaluate the luminescence reading without the interference of suspended solids. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 469–474, 2001.     

Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.     

New bioluminescence pyrophosphate assay for high‐sensitivity detection of food‐borne pathogens

Traditional methods of identifying food‐borne pathogens are time consuming and laborious, so innovative methods for their rapid identification must be developed. Testing for bioluminescence pyrophosphate is a convenient and fast method of detecting pathogens without complex equipment. However, the sensitivity of the method is not as high as that of other methods, and it has a very high detection limit. In this study, the method was optimized to improve its sensitivity. The shortcomings of the method were first identified and corrected using dATPαS instead of dATP for the polymerase chain reaction (PCR), therefore reducing the background signal. Also, when the DNA template extracted from the food‐borne pathogens was purified, the new bioluminescence pyrophosphate assay had a limit of detection of <10 copy/μl or 10 colony‐forming units/ml, and its sensitivity was higher than that of fluorescent real‐time quantitative PCR. Moreover, a single copy of a food‐borne pathogen could be detected when a single DNA template was included in the PCR. Salmonella was detected in and isolated from 60 samples of broiler chicken, and the accuracy of the results was verified using a culture method (GB 4789.4–2010). These results showed that the new bioluminescence pyrophosphate assay has the advantages of an intuitive detection process, convenient operation, and rapid measurements. Therefore, it can be used for the rapid detection of pathogenic bacteria and probiotics in various fields.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads）,通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面,制备固相腺苷酸双磷酸酶（Beads-apyrase）,用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。     

Substrate-stimulated oxygen consumption by isolated rat brain microvessels in the presence and absence of ATP

We report here properties of isolated brain microvessels such as the rate of oxygen consumption with different substrates; the permeabilizing effect of added ATP is studied. With the isolation procedure presented the cerebral endothelium has a metabolic activity comparable to that reported in the literature. The respiratory rate of the microvessels is not affected by the addition of ATP, whereas it is significantly increased by addition of succinate and -chetoglutarate. The exposure of the isolated brain capillaries to ATP, in a Ca²⁺-free medium, increases the uptake of 6-carboxyfluorescein. This may be due to pores opened by ATP in the endothelial cell membrane in the absence of divalent cations.     

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

We have developed a bioluminescence‐based non‐destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)‐targeted copepod luciferase (GLuc‐KDEL), respectively, by using multi‐integrase mouse artificial chromosome (MI‐MAC) vector. We have demonstrated that the time‐dependent concentration response curves of ELuc luminescence intensity and WST‐1 assay, and GLuc‐KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant‐treated ELuc‐ and GLuc‐KDEL‐expressing HepG2 stable cell lines. We have clarified that the increase of GLuc‐KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc‐KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage‐associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc‐KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc‐KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

ATP生物荧光法快速检测化妆品中菌落总数的研究

本文从快速检测的必要性出发,介绍了ATP生物荧光法及其原理,并针对某款化妆品通过一系列实验建立了一种快速检测菌落总数的方法。将该方法用于CTFA(美国化妆品香料香精协会,Cosmetic Toiletry and Fragrance Association)实验中,并通过与平板计数法结果进行比对,发现该方法可以有效的运用于样品细菌总数的快速检测。     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.