Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.     

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。     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

Application of bioluminescence ATP measurement for evaluation of fungal viability of foxing spots on old documents

An adenosine triphosphate (ATP) bioluminescence‐based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish‐red stains, damaging the aesthetics of documents and their long‐term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold‐like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm², suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm². ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots. Copyright © 2012 John Wiley & Sons, Ltd.     

A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP

Cellular ATP is commonly determined as production of bioluminescence using a luciferin-luciferase reaction system. Before the measurement of bioluminescence, cellular ATP must first be extracted. Two commonly used extraction methods are: () Tris-borate buffer (pH 9.2) coupled with a heating process (to inactivate ATPase) and () perchloric acid followed by neutralization. However, we found that both Tris-borate buffer and perchloric acid interfered with the luciferin-luciferase system. Here, we report a convenient single-step boiling deionized water (DW) method for extracting cellular ATP to replace perchloric acid and Tris-borate buffer. We showed that the boiling DW method did not interfere with the bioluminescence and was effective in inhibiting ATPase. This improved method required no neutralization and dilution and thus was more convenient than the perchloric acid method. Unlike the Tris-borate/heating procedure, our method did not require a separate heating step because boiling DW effectively inhibited ATPase and thus accomplished the two missions in one step for both suspended and attached cells. The improved method was precise for both suspended cells and attached cells, when cell numbers were between 10(3) and 10(6). The method also was more sensitive than other methods because it required much fewer cells (10(4) to 10(5)) than other methods for ATP determination. Thus, this one-step method is suitable for routine assay of cellular ATP for both suspended and attached cells.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants

Aims:  The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain.
Methods and Results:  The reported assay, which is based on the BacTiter-Glo™ assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed.
Conclusions:  The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay.
Significance and Impact of the Study:  The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.     

Continuous-flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25°C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.     

ATP生物发光测定试剂研究进展

萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能。迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等。ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面。     

Rapid and direct microRNA quantification by an enzymatic luminescence assay

A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay uses miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated high cross-platform consistency between this assay and the TaqMan real-time polymerase chain reaction (PCR) assay with R(2)=0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than 1h. Compared with the real-time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.     

Development of a novel telomerase assay using the PPDK–luciferin–luciferase detection system

Telomerase participates in malignant transformation or immortalization of cells, and has attracted attention as an anticancer drug screening and diagnostic tumor marker. We developed a novel telomerase assay called the PPDK–luciferin–luciferase system bioluminescence assay (PLLBA) using pyruvate phosphate dikinase (PPDK). In this assay, pyrophosphate produced by the telomerase reaction and polymerase chain reaction (PCR) is converted to ATP by PPDK, and ATP is detected by the firefly luciferin–luciferase reaction. In this work, telomerase substrate was obtained in accordance with the telomeric repeat amplification protocol (TRAP). Telomerase‐positive (500 cells/assay), ‐inactive (heated for 10 min at 85 °C) and ‐negative (only Chaps lysis buffer) samples were used. As a result, the findings clearly showed that the signal‐to‐noise (S/N) ratio of the positive cells was 39.5. After the telomerase reaction and PCR, PLLBA was completed ~ 120 s later. A high level of reproducibility was obtained with ‐ coefficient of variation (CV) of 4.1% (positive cells). The detection limit for cells using telomerase was one cell per assay. This assay for telomerase activity was also shown to be adaptable to human cancer‐derived cell lines. Copyright © 2013 John Wiley & Sons, Ltd.     

An efficient method for quantitative determination of cellular ATP synthetic activity

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.     

Radiometric oil well assay for glucokinase in microscopic structures

Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet beta-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP:D-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H2O from D-[2-3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic beta-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the Km and the Hill number) in microscopic samples of tissue.     

Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

An indirect bioluminescence method for the quantitative measurement of polymorphonuclear cell chemotaxis

A method is presented which allows the quantification of the effects of chemotactic factors on polymorphonuclear leukocytes on the basis of a sensitive ATP measurement using bioluminescence. The assay measures those cells which have migrated through a commercial 3 μm filter system (Transwell?). The assay was tested under standardized conditions with different chemotactic agents (leukotriene B4 [LTB4], N-formyl-methionyl-leucyl-phenylalanine [FMLP], N-formyl-methionyl-leucyl-phenylalanine-methyl ester [M-FMLP]). Under appropriate conditions the migration of PMN-cells is time-dependent and linear for 60 minutes. Spontaneous migration of PMN cells is simultaneously quantified in a simple way, and the value obtained allows a determination of the actual chemotactic stiuation of the PMN cells. In healthy humans the spontaneous migration varied between 4.2% and 14.4% of the total number of PMN cells. An optimal chemotactic activity was detected at 10^?8/mol/I for FMLP and 10^?7 mol/l for M-FMLP in PMN leukocytes, which correlates with literature values. It was also found that in contrast to EDTA blood, heparinized blood lowers the ATP level of PMN cells (by about 50%) and therefore heparinized blood is not recommended for chemotactic experiments. This assay is a simple tool for quantification of the spontaneous migration, and the chemotactic response to specific factors and their inhibitors in particular for pharmacological experiments. In contrast to the ‘classical’ chemotactic assays this method also permits the simultaneous testing of the influence of chemotactic substances on cellular ATP levels.     

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。