Extraction Of ATP Research Articles

Application of BactTiter-Glo ATP bioluminescence assay for Mycobacterium tuberculosis detection

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a global health threat, necessitating faster and more accessible diagnostic methods. This study investigates critical parameters in the application of a commercial ATP bioluminescence assay for the detection of MTB. MethodOur objective was to optimize the ATP bioluminescence protocol using BacTiter-Glo™ for MTB, investigating the impact of varying volumes of MTB suspension and reagent on assay sensitivity, evaluating ATP extraction methods, establishing calibration curves, and elucidating strain-specific responses to antimicrobial agents. ResultsATP extraction methods showed no significant improvement over controls. Calibration curves revealed a linear correlation between relative light units (RLU) and colony-forming units (CFU/mL), establishing low detection limits. Antimicrobial testing demonstrated strain-specific responses aligning with susceptibility and resistance patterns. ConclusionOur findings contribute to refining ATP bioluminescence protocols for enhanced MTB detection and susceptibility testing. Further refinements and validation efforts are warranted, holding promise for more efficient diagnostic platforms in the future.

Analysis of Phenolic Compounds in Nicotiana tabacum Based on Aqueous Two-Phase Extraction Coupled with Liquid Chromatography

Background: Phenolic compounds in Nicotiana tabacum L. have anti-tumor, antibacterial, anti-inflammatory, and other pharmacological effects. Therefore, either to optimize the utilization of plant resources or to reduce the physiological harm caused by cigarette, it is necessary to separate and analyze the phenolic compounds in Nicotiana tabacum. 
 Aim: To develop a protocol for extraction and analysis of phenolic compounds in Nicotiana tabacum. The method based on aqueous two-phase extraction (ATP) should provide a solid experimental basis for future large-scale medical industrial extraction.
 Results: Five of phenolic compounds in Nicotiana tabacum including catechol, caffeic acid, coumarin, rutin, and quercetin were extracted with the developed ATP method and analyzed by the optimized liquid chromatography method simultaneously. In the concentration range of 10-200 µg/mL, the linear correlation coefficients of calibration curve for five compounds were all greater than 0.999. The developed ATP extraction method employed 70% acetonitrile water solution as the extractant. The ammonium sulphate was selected as the preferred phase separation salt. The extraction efficiencies of five phenolic compounds were all more than 90%. In concrete sample, the concentrations of five compounds were determined based on acetonitrile/salt ATPE system,
 Conclusion: Analysis of phenolic compounds in Nicotiana tabacum was achieved based on acetonitrile/ammonium sulphate ATP extraction coupled with liquid chromatography. The developed ATP extraction protocol employed 70% acetonitrile water solution as the extractant. The ammonium sulphate was selected as the preferred phase separation salt. The accuracy and precision were satisfied. The protocol developed in this study is expected to be applied in medical industry.

ATP bioluminescence-based method for screening of multiclass antibiotic residues in animal derived foods

In present study, a new ATP bioluminescence assay with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium was developed to screen multiclass antibiotic residues in animal derived foods. The development experiments includes the optimization on the usage of D-luciferin, bacterial ATP extraction condition, incubation condition of detection system and sample pre-treatment. Additionally, the validation experiments of sensitivity, specificity, accuracy, precision and stability were performed. This method can detect β-lactams (6), aminoglycosides (4), tetracyclines (4), sulfonamides (4), macrolides (4), lincosamides (1) and quinolones (3) in muscle and kidney of swine at or close to maximum residue limits (MRLs) established by European Union (EU). The false-positive rate was 0%, and the false negative rates were < 5%. The recovery rate of antibacterial drugs were between 60% and 120%, and the daily coefficients of variation were less than 15%, which meets the criteria in Commission Decision 2002/657/EC. The shelf life of the detection system was more than 5 months. Accordingly, the proposed ATP bioluminescence assay shows great potential for rapid, sensitive and high throughput detection of multiple antibiotic residues in animal foods, which can provide a preliminary screening informations of antibiotic residues for the following chemical confirmation experiments and expedite disposal decisions of unqualified food for enhanced food safety inspection.

Metal-enhanced bioluminescence by detergent stabilized Ag and Au nanoparticles

The assessment of microbial contamination is an important aspect of ensuring human food safety. One of the modern methods for the evaluation of microbial contamination is the estimation of the amount of ATP using firefly luciferase. In this case, the choice of an effective composition of the extraction buffer is crucial. In this study, we examined the influence of silver and gold nanoparticles on the firefly bioluminescent system during the ATP extraction process. It was found that gold nanoparticles stabilized with benzalkonium chloride and Triton X-100 enhanced bioluminescent system signal intensity due to metal-enhanced bioluminescence. Moreover, silver and gold nanoparticles could be used as extracting agents. So, using gold nanoparticles stabilized with BAC and Triton X-100 as ATP extraction agents with further detection by a bioluminescent system makes it possible to develop an ATP biosensor with higher sensitivity.

Laboratory experiment of ATP measurement using Mars soil simulant: as a method for extraterrestrial life detection

We present a laboratory experiment of ATP measurement using Mars soil simulant and Escherichia coli (E. coli) with a point of view for future application to searching extraterrestrial life. We used Mars Global Simulant MGS-1 (Exolith Lab) as soil simulant, added E. coli suspension to it, then the soil simulant with E. coli was dried. Various configurations of samples with different E. coli density, 1.75 × 102, 1.75 × 103, 1.75 × 104, 1.75 × 105, and 1.75 × 106 cells (g soil)−1, were prepared together with controls. ATP extraction reagent and luminescence reagent were added to the sample, and bioluminescence measurement was performed. The result suggests significant detection of ATP for samples with E. coli density used in this work. Similar experiments but without the soil simulant were carried out, and results with and without the soil simulant are compared. Based on the ATP measurement studied in this work, we discussed extraterrestrial life search, planetary protection, relation with the panspermia hypothesis, and also other applications.Graphical abstract

Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities

Mitochondrial function depends on the efficient import of proteins synthesized in the cytosol. When cells experience stress, the efficiency and faithfulness of the mitochondrial protein import machinery are compromised, leading to homeostatic imbalances and damage to the organelle. Yeast Msp1 (mitochondrial sorting of proteins 1) and mammalian ATAD1 (ATPase family AAA domain-containing 1) are orthologous AAA proteins that, fueled by ATP hydrolysis, recognize and extract mislocalized membrane proteins from the outer mitochondrial membrane. Msp1 also extracts proteins that have become stuck in the import channel. The extracted proteins are targeted for proteasome-dependent degradation or, in the case of mistargeted tail-anchored proteins, are given another chance to be routed correctly. In addition, ATAD1 is implicated in the regulation of synaptic plasticity, mediating the release of neurotransmitter receptors from postsynaptic scaffolds to allow their trafficking. Here we discuss how structural and functional specialization imparts the unique properties that allow Msp1/ATAD1 ATPases to fulfill these diverse functions and also highlight outstanding questions in the field.

Estimating Postmortem Interval by Bioluminescent Determination of ATP Content in the Muscle of Olive Flounder (Paralichthys olivaceus)

ATP content was determined by a luminometer for PMI of sliced raw fish within 24 h. Comparison of RLU by luminometer and HPLC-based ATP content had a high correlation of r2 = 0.96 to 0.98. 0.5 M phosphate buffer (pH 6.8) was selected as the optimal buffer for ATP extraction.

Ballast water compliance monitoring: A new application for ATP

The coming into force of the USCG ballast water regulations and the IMO ballast water management convention resulted in the development of several technologies approved for the treatment of ballast water. To ensure compliance of these technologies, the development of rapid and robust analysis methods was necessary.In collaboration with the SGS Group (Switzerland) and LuminUltra (Canada), Aqua-tools (France) has developed an innovative Ballast Water Treatment Monitoring (BWTM) kit for rapid onboard testing. The affordable kit provides results in less than 1h, is easy to use and durable ensuring that the ballast water treatment system on the ship is fully compliant with the discharge standards upon arrival in port.The core of this method is a combination of high-quality reagents (lysis solution and ATP 2G Luminase™ enzyme) not inhibited by salinity and a patented fast homogenizing method for ATP extraction developed for a higher ATP recovery from zooplankton and phytoplankton. Compared to traditional analysis methods, the BWTM Kit provides fast and accurate results for all three fractions of microorganisms (≥50μm, ≥10÷<50μm and bacteria). Preliminary tests carried out in cooperation with SGS showed that the proposed method was able to detect onboard the efficiency of the treatment systems used. Compliance limits were established for all size fractions and a correlation between the standard methods (microscopy, plate count, MPN) and ATP was evaluated. The BWTM kit can provide a fast indication of compliance or gross exceedance. The rare borderline cases, when encountered, of course require additional confirmation.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

ABSTRACTMammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.

Development of an ATP measurement method suitable for xenobiotic treatment activated sludge biomass.

Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge's degradation of a xenobiotic, any disadvantageous effect on the sludge's performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R(2) of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD=0.00045mg/L, a LOQ=0.0015mg/L for HPLC measurement of ATP, a MDL=0.46mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4±2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have.

Комплекс препаратов тестостерона, АТФ и экстракта миокарда в качестве коррекционной схемы при экспериментальной дилатационной кардиомиопатии

Комплекс препаратов тестостерона, АТФ и экстракта миокарда в качестве коррекционной схемы при экспериментальной дилатационной кардиомиопатии

Evaluation of ATP content in hair bulbs in human scalp.

The content of ATP in scalp hair bulbs in humans was measured in the hair roots from 15 healthy volunteers. Light and electron microscopy confirmed the presence of outer and an inner root sheaths in the root of pulled out anagen hair. Incubation of samples in buffer solution led to extraction of ATP, which was measured by the chemiluminescent method. Mechanic disintegration of hair bulbs and their freezing-defrosting did not increase ATP output. The results of microscopy indicated that ATP extraction procedure was associated with separation of the outer radical sheath from the inner one without impairing the structure of the inner sheath. The mean content of ATP was 12 ± 2 pmol per bulb. The use of pulled out hair bulbs for ATP measurements simplified the procedure as involved no surgical removal of follicles.

Novel Reversible Zn2+-Assisted Biological Phosphate “Turn-On” Probing through Stable Aryl-hydrazone Salicylaldimine Conjugation That Attenuates Ligand Hydrolysis

A novel reversible zinc(II) chemosensing ensemble (2·Zn(2+)) allows for selective "turn-on" fluorescence sensing of ATP and PPi in aqueous media (detection limits: 2.4 and 1.0 μM, respectively) giving selective binding patterns: ATP ∼ PPi > ADP ≫ AMP > monophosphates ≈ remaining ions tested. The conjugated hydrazone [C═N-NH-R] resists hydrolysis considerably, compared to the imine [C═N-CH2-R, pyridin-2-ylmethanamine] functionality, and generalizes to other chemosensing efforts. Prerequisite Zn(2+)·[O(phenol)N(imine)N(pyr)] binding is selective, as determined by UV-vis and NMR spectroscopy; ATP or PPi extracts Zn(2+) to regenerate the ligand-fluorophore conjugate (PPi: turn-on, 512 nm; detection limit, 1.0 μM). Crystallography, 2-D NMR spectroscopy, and DFT determinations (B3LYP/631g*) support the nature of compound 2. 2-Hydrazinyl-pyridine-salicylaldehyde conjugation is unknown, as such; a paucity of chemosensing-Zn(2+) binding reports underscores the novelty of this modifiable dual cation/anion detection platform. A combined theoretical and experimental approach reported here allows us to determine both the potential uniqueness as well as drawbacks of this novel conjugation.

Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.

Abstract It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay's ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties (&amp;lt;50% recovery). This new, homogeneous 3D-assay format requires minimal sample manipulation, a brief incubation period (generally less than 30 min), and exhibits excellent sensitivity. It should be amenable to both low- and high-throughput applications and accurately report drug sensitivities of cells assayed in both 2D and 3D culture models. Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

AbstractThe assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

A revised protocol for efficient ATP extraction from diluted boar spermatozoa

A revised protocol for efficient ATP extraction from diluted boar spermatozoa

Estimation of total bacteria on mango surface by using ATP bioluminescence

This paper presents a rapid ATP assay for monitoring the total bacterial load associated with mango fruit surface. Conventional methods such as total plate count for the determination of total microbial counts are time consuming and prone to contamination resulting in low accuracy. Thus the need for quick and accurate methods for determination of total microbial count has led to development of many rapid techniques such as ATP bioluminescence. In this study the potential of ATP bioluminescence method for the determination of total bacterial count on mango surface has been evaluated using two types of luminescence techniques based on fluorescence spectroscopy and bioluminescence. Various mechanical and chemical extraction processes were employed for the extraction of ATP from bacterial cells and the best extraction was achieved with 0.03M TCA. Minimum level of detection of ATP using fluorescence spectrophotometer and bioluminometer was found to be 10−8M and 10−13M, respectively. This ATP concentration was correlated with the number of bacterial cells present on mango surface. The minimum level of detection of bacterial cells in fluorescence spectroscopy and bioluminescence was found to be 107cfu/ml and 102cfu/ml, respectively.

Design of Bioluminescence Sensor System for Rapid Detection of Bacteria in Samples without Cultivation

Based on the principle of ATP bioluminescent reaction, we design a portable bioluminescence sensor system to detect bacteria in samples rapidly, which included an optical sensing cell, a flow injection unit and a center processing unit. A fast ATP extraction technique was developed by using trichloro acetic acid as extraction reagent Experimental results showed that the intracellular ATP could be released from bacterial cells sufficiently within 5 min by TCA In the presence of Lucifer ace the reaction between ATP and lucifer5in would generate light and the luminescence intensity RLU was proportional to the amount of bacteria existed in the sample. The dynamic range of the sensor system is 10-107 CFU /ml. The linear regression coefficient was 0.976, and the detection limit was 10CFU /ml. The whole testing time needed was about 10 min. Compared with the plate count method, bacteria concentration in samples could be detected rapidly by using the portable sensor system without cultivation.

Rapid detection of bacteria without cultivation with a portable bioluminescence sensor system

Based on the principle of ATP bioluminescence reaction, we designed a portable bioluminescence sensor system to detect bacteria in samples rapidly, which included an optical sensing cell, a flow injection unit and a center processing unit. A fast ATP extraction technique was developed by using trichloroacetic acid (TCA) as extraction reagent. Experimental results showed that the intracellular ATP could be released from bacterial cells sufficiently within 5 min by TCA. In the presence of luciferase, the reaction between ATP and luciferin would generate light and the luminescence intensity (RLU) was proportional to the amount of bacteria exsited in the sample. The dynamic range of the sensor system ranged from 10 - 107 CFU/ml. The linear regression coefficient was 0.976, and the detection limit was 10 CFU/ml. The whole testing time needed was about 10 min. Compared with the plate count method, bacteria concentration in samples could be detected rapidly by using this portable sensor system without cultivation. Key words: ATP, portable, bioluminescence sensor system, trichloroacetic acid, luminescence intensity.

生物発光法による抗らい菌活性測定法の開発

Antimicrobial susceptibility testing of Mycobacterium leprae by non-radioactive bioluminescence assay was developed. Optimization of the assay conditions such as temperature and time for ATP extraction, bacteria dose, preparation of bacteria suspension and pH of culture medium was carried out using M. leprae Thai 53 strain. Samples of bacterial suspension of M. leprae were first treated with filamentous cell treatment reagent at room temperature for 30 minutes and ATP was extracted from the leprosy bacilli by heating at 60 degrees for five minutes. Luciferin luciferase was added to the extract after cooling to room temperature followed by measurement of relative light units (RLU) of each sample using a luminometer. The concentrations of the drugs used for the evaluation of antimicrobial activities of rifampin (RFP), clofazimine (CLF), ofloxacin (OFLX) and clarithromycin (CAM) were 0.125, 0.50, 2.0 and 8.0 microg/ml respectively. Middlebrook 7H9 broth medium was used (pH6.6) as the basal medium and the bacilli were cultivated at 32 C for 0-14 days. ATP was extracted from 0.1 ml of culture suspension and inhibition of the luminescent activity was calculated. The results were compared to that obtained by radio-active CO2 detection system, Buddemeyer method which is commonly used for measuring anti-M. leprae activity. There was a good correlation between the results obtained by ATP method on the tenth day of culture and the results obtained by Buddemeyer method on the seventh day of culture. ATP method may be useful for the determination of drug susceptibility ofM. leprae.