Presence Of Adenosine Triphosphate Research Articles

Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate

In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6×10−10M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

Nonequilibrium structure and dynamics in a microscopic model of thin-film active gels.

In the presence of adenosine triphosphate, molecular motors generate active force dipoles that drive suspensions of protein filaments far from thermodynamic equilibrium, leading to exotic dynamics and pattern formation. Microscopic modeling can help to quantify the relationship between individual motors plus filaments to organization and dynamics on molecular and supramolecular length scales. Here, we present results of extensive numerical simulations of active gels where the motors and filaments are confined between two infinite parallel plates. Thermal fluctuations and excluded-volume interactions between filaments are included. A systematic variation of rates for motor motion, attachment, and detachment, including a differential detachment rate from filament ends, reveals a range of nonequilibrium behavior. Strong motor binding produces structured filament aggregates that we refer to as asters, bundles, or layers, whose stability depends on motor speed and differential end detachment. The gross features of the dependence of the observed structures on the motor rate and the filament concentration can be captured by a simple one-filament model. Loosely bound aggregates exhibit superdiffusive mass transport, where filament translocation scales with lag time with nonunique exponents that depend on motor kinetics. An empirical data collapse of filament speed as a function of motor speed and end detachment is found, suggesting a dimensional reduction of the relevant parameter space. We conclude by discussing the perspectives of microscopic modeling in the field of active gels.

Electrochemiluminescence aptasensor for adenosine triphosphate detection using host–guest recognition between metallocyclodextrin complex and aptamer

A sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed using host-guest recognition between a metallocyclodextrin complex, i.e., tris(bipyridine)ruthenium(II)-β-cyclodextrin [tris(bpyRu)-β-CD], and an ATP-binding aptamer. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition between tris(bpyRu)-β-CD and aptamer, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration was obtained in the range 10.0-0.05 nM, with a detection limit of 0.01 nM at the S/N ratio of 3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets.

A sensitive signal-off aptasensor for adenosine triphosphate based on the quenching of Ru(bpy)32+-doped silica nanoparticles electrochemiluminescence by ferrocene

In this paper, a novel and sensitive signal-off electrochemiluminescence (ECL) aptasensor was proposed for the detection of adenosine triphosphate (ATP) on a nanoporous gold (NPG) modified glassy carbon electrode. The NPG was easily synthesized by a selective dissolution of silver from silver/gold alloy in nitric acid. The ECL aptasensor consisted of two main elements: an ECL substrate, made by modifying the amino group functionalized Ru(bpy)32+-doped silica (Ru-silica), and an ECL intensity switch, which contains two probes, split from one aptamer. The first probe (ssDNA1) was functionalized with a thiol group at one end and attached to Ru-silica through the linking agent of N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. The other one (ssDNA2) is ferrocene-labeled probe, which was functionalized with ferrocene tag at the amino group functionalized end. We demonstrated that the ssDNA1/ATP/ssDNA2 sandwich composites layer could be formed in the presence of ATP, and lead to a quenching of Ru(bpy)32+. However, only a slight decrease could be observed in the absence of ATP. Under optimal conditions, the proposed biosensor provided a wide linear response range over 0.1pmolL−1–10.0nmolL−1 with a detection limit of 0.03pmolL−1.

Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

Sensitive Electrochemical Aptasensor by Coupling “Signal-on’’ and “Signal-off’’ Strategies

A simple electrochemical aptasensor for sensitive and selective determination of adenosine triphosphate (ATP) has been developed on the basis of a new dual-signaling amplification strategy. This aptasensor features both ''signal-on'' and ''signal-off'' elements. The ferrocene (Fc)-labeled aptamer probe (Fc-P) is designed to hybridize with the thiolated methylene blue (MB)-modified DNA probe (MB-P) on gold electrode to form rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer leads to the dissociation of the duplex DNA structure and thereby the release of the Fc-P from the sensing interface. The single-stranded MB-P could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such conformational changes result in the oxidation peak current of Fc decreases and that of MB increases, and the changes of dual signals are linear with the concentration of ATP. When "ΔI = ΔI(MB) + |ΔI(Fc)|" (ΔI(MB) and ΔI(Fc) are the change values of the oxidation peak currents of MB and Fc, respectively.) is used as the response signal for quantitative determination of ATP, the detection limit (1.9 nM) is much lower than that by using either MB-P or Fc-P alone. This new dual-signaling aptasensor is readily regenerated and shows good response toward the target. It will have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.

Aptamer-based electrochemical biosensor for detection of adenosine triphosphate using a nanoporous gold platform

In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4°C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Approaches for determining the effects of UV radiation on microorganisms in ballast water

To minimize the dispersal of aquatic nuisance species through shipping, ballast water can be treated to kill, remove, or inactivate organisms. Ultraviolet (UV) radiation is used in some ballast water management systems to address this goal. Because UV treatment renders cells nonviable (by sterilizing them, preventing reproduction) and does not necessarily kill organisms instantaneously, the efficacy of UV treatment has been verified historically by regrowth assays in which microorganisms are cultured (and thus, reproduce) under optimal growth conditions. Although regrowth assays are definitive, they are time consuming—lasting for days or weeks—and, importantly, are applicable only to organisms amenable to culturing. Furthermore, these estimates of cell concentrations are often accompanied by large error estimates. In this paper, several rapid alternatives to regrowth assays are described and evaluated. An ideal approach would shorten or simplify the analysis burden and, potentially, could be used for shipboard testing to determine compliance with national and international ballast water standards. Complicating this task is the requirement that compliance with the ballast water standards will be determined by quantifying the number of living organisms in ships’ ballast water, and while organisms may be living following UV treatment, they may not be viable (i.e., they may not reproduce). To address this dichotomy , alternatives to regrowth assays were categorized based upon the complexity of the analysis and the means used to determine the status of microorganisms (either as viable or living): 1. Instantaneous growth and cell replication, 2. Cell activity and metabolic rates, 3. Cell structural integrity, and 4. Biomolecule presence and status. With the suite of approaches currently available, it is not possible to determine the viability of organisms rapidly, that is, within minutes of collecting a ballast water sample. Measurements of the photosystem integrity via variable fluorescence and the presence of adenosine triphosphate (ATP) are currently the most promising for rapidly estimating concentrations of living cells in compliance testing of ballast water discharges; however, extensive validation is required to verify the applicability of these approaches for the complexity of real-world samples.

A Ligation Triggered Label-Free Fluorescent Assay for Adenosine-Triphosphate Based on Nicking Endonuclease Signal Amplification and Ligand Responsive G-Quadruplex Formation

A novel label-free fluorescent assay strategy was developed for sensitive and selective detection of adenosine-triphosphate (ATP) based on nicking endonuclease signal amplification (NESA) and N-Methyl mesoporphyrin (NMM)-responsive G-quadruplex formation. In this assay, the nick in the specially designed DNA duplex can be sealed by T4 DNA ligase in the presence of ATP. The following replication and strand displacement yield the duplex that contains the full recognition site for nicking endonuclease. Nicking enzyme's cutting the replicated DNA duplex results in a new replication site for polymerase and the continuous displacement of the nicked G-rich strand. The nicked strand can fold into G-quadruplex structure in the presence of K+ and selectively binds with NMM, yielding a significant fluorescence signal. Under the optimal conditions, the fluorescent assay strategy showed a dynamic response to ATP in the concentration range from 10 nM to 1000 nM and a detection limit of 0.8 nM, which compared favorably with most of the previous ATP assay methods. In addition, the proposed strategy exhibited superior selectivity toward ATP over other nucleosides and its analogues. The fluorescent assay was also applied in detecting ATP in serum with satisfactory results.

A highly selective turn-on ATP fluorescence sensor based on unmodified cysteamine capped CdS quantum dots

Unmodified cysteamine capped nanocrystalline cadmium sulfide quantum dots (Cys-CdS QDs) were demonstrated as a selective turn-on fluorescence sensor for sensing adenosine-5′-triphosphate (ATP) in aqueous solution for the first time. The fluorescence intensity of the Cys-CdS QDs was significantly enhanced in the presence of ATP. In addition, the fluorescence intensity of the Cys-CdS QDs increased when increasing ATP concentrations. On the other hand, other phosphate metabolites and other tested common anions did not significantly alter the fluorescence intensity of the Cys-CdS QDs. In addition, this sensor showed excellent discrimination of pyrophosphate (PPi) from ATP detection. The proposed sensor could efficiently be used for ATP sensing at very low concentration with LOD of 17μM with the linear working concentration range of 20–80μM. The feasibility of the proposed sensor for determining ATP in urine samples was also studied, and satisfactory results were obtained.

Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

Synthesis and Characterization of Hydroxyapatite Ceramics Organofunctionalized with ATP (Adenosine Triphosphate)

Adenosine triphosphate (ATP) is the main source of energy for cells. In this article we investigated, with a possible future finality in bone tissue engineering, the original association of ATP molecules and hydroxyapatite (HAp) fine powders. We have in particular inspected, in a preliminary way, the effects of the presence of ATP molecules on the physico-chemistry of apatite compounds obtained via co-precipitation. Hybrids were produced by association of increasing amounts of adenosine 5’-triphosphate (ATP) disodium hydrate salt with hydroxyapatite, leading to so-called “organoapatites”. The structure and composition of the polycrystalline hybrid compounds obtained were studied by XRD, FTIR, SEM and magic angle spinning (MAS) NMR techniques. The presence of ATP was found to alter the apatite crystallization process, leading to a departure from stoichiometry. MAS-NMR data attest to the presence of strong interactions between functional groups of ATP and apatite particles unveiling the obtainment of class II hybrids with potential future applications in cell-loaded bone scaffolds.

A label free exonuclease III-aided fluorescence assay for adenosine triphosphate based on graphene oxide and ligation reaction

A label free exonuclease III (Exo III)-aided fluorescence assay for adenosine triphosphate (ATP) was developed based on the ATP-dependent enzymatic reaction and graphene oxide (GO). This strategy relies on the principle that Exo III shows different cleavage capacity for a DNA substrate in the absence and presence of ATP and the preferential binding of GO to single-stranded DNA over double-stranded one. By combining the unique properties of SYBR Green I and GO adsorption, this sensor displays an improved sensitivity and a wide linear range within the ATP concentration from 1 nM to 200 nM with a low detection limit of 0.2 nM. The proposed method is simple, cost-effective and convenient, which might create a new methodology for developing a sensitive ATP biosensor.

Split Aptamer-Based Liquid Crystal Biosensor for ATP Assay

A novel liquid crystal (LC) biosensor based on the recombination of split aptamer chip was developed for the detection of adenosine triphosphate (ATP). One glass slide of the LC cell is first modified with the TEA/DMOAP mixed self-assembled monolayer (SAM) to ensure the homeotropic alignment of LC molecules and the black background of LC cell optical image under the crossed polarized light. The single-strand ATP aptamer is split into two fragments in this method. One of which, as capture probe, is covalently immobilized on the SAM, while another is used as the detection probe. In the presence of ATP, those two fragments will be combined with each other and exhibit the same role as the ATP aptamer. This binding event leads to a great enhancement in the optical signal of the LC biosensor due to the space size from small to big, which can effectively disrupt the orientational arrangement of LCs, resulting in the corresponding changes of optical images under the crossed polarized light. But in the absence of ATP, those two fragments can not be combined with each other, and as a result, there is no orientational response of LCs and the optical image under the crossed polarized light is still black. So the LC-based imaging method has a sensitive and clear distinction between positive and negative results. On the basis of such an inhibition mechanism the LC biosensor can be used as an effective way to realize the detection of ATP. When the concen- tration of analyte ATP below a critical value (10 nmol/L), there is no clear change in the optical image. The results showed that the detection limit of ATP is 10 nmol/L. This study provides a simple and sensitive ATP LC biosensing approach and offers effective strategies for the development of small molecules LC biosensors. Keywords liquid crystal; self-assembled film; biosensor; ATP; aptamer

ATP-Responsive Controlled Release System Using Aptamer-Functionalized Mesoporous Silica Nanoparticles

Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide, which plays a vital role in many biological processes, including muscle contraction, cells functioning, synthesis and degradation of important cellular compounds, and membrane transport. Thus, the development of ATP-responsive controlled release system for bioorganism application is very significative. Here, an original and facile ATP-responsive controlled release system consisting of mesoporous silica nanoparticles (MSN) functionalized with an aptamer as cap has been designed. In this system, the ATP aptamer was first hybridized with arm single-stranded DNA1 (arm ssDNA1) and arm single-stranded DNA2 (arm ssDNA2) to form the sandwich-type DNA structure and then grafted onto the MSN surface through click chemistry approach, resulting in blockage of pores and inhibition of guest molecules release. In the presence of ATP, the ATP aptamer combined with ATP and got away from the pore, leaving the arm ssDNA1 and ssDNA2 on the surface of MSN. The guest molecules can be released because single-stranded DNA is flexible. The release of the guest molecules from this system then can be triggered by the addition of ATP. As a proof-of-principle, Ru(bipy)(3)(2+) was selected as the guest molecules, and the ATP-responsive loading and release of Ru(bipy)(3)(2+) have been investigated. The results demonstrate that the system had excellent loading efficiency (215.0 μmol g(-1) SiO(2)) and the dye release percentage can reach 83.2% after treatment with 20 mM ATP for 7 h. Moreover, the ATP-responsive behavior shows high selectivity with ATP analogues. However, the leakage of Ru(bipy)(3)(2+) molecule is neglectable if ATP was not added, indicating an excellent capping efficiency. Interestingly, this system can respond not only to the commercial ATP but also to the ATP extracted from living cells. By the way, this system is also relatively stable in mouse serum solution at 37 °C. This proof of concept might promote the application of ATP-responsive devices and can also provide an idea to design various target-responsive systems using other aptamers as cap.

Fluorescence detection of adenosine triphosphate using smart probe

A novel fluorescent probe for adenosine triphosphate (ATP) assay based on DNA ligation is proposed in this article. This approach uses a novel smart probe, T4 DNA ligase, and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction and the ligation product restores the fluorescence of the smart probe. This method is very sensitive with a 0.5-nM limit of detection. Compared with current assay methods, the strategy is simpler, cheaper, and 40 times more sensitive.

Cationic conjugated polyelectrolyte-based sensitive fluorescence assay for adenosinetriphosphate and alkaline phosphatase

The cationic water-soluble poly(diethynylfluorene-co-diethynylcarbazole-co-diethynylbenzothiadiazole) (P1–P3) were synthesized and used for adenosine triphosphate (ATP) detection. The incorporation of diethynylbenzothiadiazole (DBT) into the polymer backbone offers dual emissive polyelectrolytes with both blue and red emission bands in the dilute solution resulting from incomplete intramolecular energy transfer. In the solution of P2, addition of ATP can quench the emission of fluorene-co-carbazole units of P2 more efficiently than that of DBT segments, which causes the fluorescent color change from blue to red. The asymmetric quenching of the blue and red emission bands in the presence of ATP might become a possible method for a real-time ATP detection. In addition, when ATP is cleaved into fragments by enzyme alkaline phosphatase (ALP), the blue emission of P2 can be recovered. Thus, it is possible to assay the enzyme activity by triggering the change in color and intensity of the emission of the solution of P2.

Oxidative Interaction between OxyHb and ATP: A Spectroscopic Study

The binding mode between oxyhemoglobin (oxyHb) and adenosine triphosphate (ATP) has been studied using absorption and fluorescence spectroscopy. OxyHb forms a ground state complex with ATP supported by five isosbestic points which appear in absorption spectra of oxyHb in buffer solution on addition of ATP. Moreover, the changes in absorption spectra suggest an oxidative interaction between the particular interacting systems. The binding constant has been determined from the quenching of fluorescence of oxyHb in the presence of a varied concentration of ATP, and that is 3.8 × 10(3) M(-1) at 25 °C. The negative changes in entropy and enthalpy indicate that the binding is enthalpy driven and the hydrogen bond and van der Waals (stacking) interactions play a major role. The oxygen affinity of oxyHb decreases with simultaneous formation of metHb in the presence of ATP. ATP-induced structural changes have been affirmed using both circular dichroism spectroscopy and synchronous fluorescence. A theoretical docking study gives the molecular details about the binding site of ATP in oxyHb.

Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate

The capability of silver nanoparticles (SNP) as redox tag in the construction of an electrochemical aptasensor for the detection of adenosine triphosphate (ATP) is investigated in the present manuscript. To construct the aptasensor, a well-known ATP binding aptamer (ABA) splits into two segments. The first amino-labeled segment of the aptamer was covalently immobilized on 3-mercaptopropionic acid modified gold electrode surface by the formation of carbodiimide bond. The second segment was modified by SNPs and associated with the first segment in the presence of ATP. The direct oxidation signal of SNPs is followed as the analytical signal to detect ATP. The sandwich assay shows a suitable signal gain and importantly, a good response time. The sensor can detect the concentrations of ATP as low as micromolar scales with a desirable stability under optimum conditions. Furthermore, analog nucleotides including GTP, UTP and CTP, do not show serious interferences and this sensor readily detects its target in a complex media such as human blood plasma.