Determination of adenosine disodium triphosphate using prulifloxacin-terbium(III) as a fluorescence probe by spectrofluorimetry

A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)–Tb³⁺ complex and adenosine disodium triphosphate has been studied by using UV–vis absorption and fluorescence spectra. Using prulifloxacin–Tb³⁺ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin–Tb³⁺ complex at λ = 545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 × 10⁻⁷ to 2.0 × 10⁻⁵ mol L⁻¹, and the detection limit (3 σ/k) is 1.7 × 10⁻⁸ mol L⁻¹. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin–Tb³⁺ complex by ATP was also discussed.     

离子色谱法测定三磷酸腺苷二钠制剂中主成分及有关物质的含量

建立了用于三磷酸腺苷二钠制剂中主成分及有关物质含量测定的离子色谱方法。采用IonPac AS11-HC色谱柱,以KOH溶液为淋洗液,梯度洗脱,流速为1.0 mL/min,进样10 μL,以Dionex AERS 500 4-mm抑制器的电导检测器检测,三磷酸腺苷二钠(ATP-Na₂)的含量按峰面积以外标法计算,二磷酸腺苷二钠(ADP-Na₂)及单磷酸腺苷二钠(AMP-Na₂)按加校正因子的主成分自身对照法计算,未知杂质按主成分自身对照法计算。ATP-Na₂、ADP-Na₂及AMP-Na₂的线性范围分别为0.000146~1.83 g/L、0.000484~1.51 g/L及0.000426~0.804 g/L,相关系数分别为0.9997、0.9996及0.9999;对照品溶液在24 h内的稳定性良好(峰面积RSD分别为1.3%、1.4%、2.5%);ATP-Na₂、ADP-Na₂、AMP-Na₂的方法定量限(S/N=10)分别为1.5 ng、4.8 ng、4.3 ng,检出限(S/N=3)分别为0.58 ng、1.21 ng、1.28 ng;ATP-Na₂在3个水平的加样回收率分别为96.50%、96.57%和96.77%。本方法适用于三磷酸腺苷二钠制剂的质量控制。     

基于与Ce3+相互作用共振光散射法测定三磷酸腺苷二钠

在Tris-HCl缓冲溶液中, 三磷酸腺苷二钠(ATP)与铈离子(Ce³⁺)相互作用产生很强的共振光散射(RLS)信号。实验表明,增强的散射信号与ATP的浓度在2.0~24 μmol/L范围内呈线性关系,据此建立了一种测定三磷酸腺苷的共振光散射法,检测限(S/N=3σ)为51.25 nmol/L。研究了共存物质的影响,用于临床三磷酸腺苷注射液的测定, RSD=2.29%。     

Determination of heparin using ciprofloxacin-Tb3+ as a fluorescence probe.

A new spectrofluorometric method was developed for the determination of trace amounts of heparin (Hep). Using ciprofloxacin (CIP)-terbium (Tb3+) as a fluorescent probe, in a buffer solution of pH 7.20, Hep can remarkably enhance the fluorescence intensity of the CIP-Tb3+ complex at lambda = 545 nm; also, the enhanced fluorescence intensity the Tb3+ ion is proportional to the concentration of Hep. The optimum conditions for the determination of Hep were also investigated. The dynamic range for the determination of Hep is 0.1 - 1.2 microg ml(-1) with a detection limit of 6.89 ng ml(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to assess Hep in biological samples. By the Rosenthanl graphic method, the association constant and binding numbers of heparin with the probe are 2.44 x 10(5) l mol(-1) and 19.7. Moreover, the enhancement mechanisms of the fluorescence intensity in the CIP-Tb3+ system and the CIP-Tb3+-Hep system have also been considered.     

Determination of ATP as a fluorescence probe with europium(III)-doxycycline.

A new spectrofluorimetric method has been developed for the determination of adenosine disodium triphosphate (ATP). We studied the interactions between the doxycycline (DC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-visible absorption and fluorescence spectra. Using doxycycline (DC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP could remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at lambda = 612 nm. The enhanced fluorescence intensity of the Eu3+ ion was in proportion to the concentration of ATP. The optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP were 1.00 x 10(-7) - 2.00 x 10(-6) mol L(-1) with detection limits of 4.07 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to the determination of ATP in samples. The mechanism of fluorescence enhancement between the doxycycline (DC)-Eu3+ complex and ATP was also studied.     

Determination of citrate in tablets and of oxytetracycline in serum using europium (III) luminescence

A direct method for the determination of citrate and oxytetracycline in samples containing complex matrices like tablets or serum has been developed using the luminescence of the ternary complex formed with Eu(III) ions. The triplet-state energy level of oxytetracycline (OxTc), the excitation maximum (412 nm) and the luminescence lifetime of Eu-OxTc (58 μs) were determined. A 17-fold luminescence enhancement at 615 nm occurs upon addition of citrate within a short 5-min incubation time at neutral pH. This is accompanied by a threefold increase of the luminescence decay time. The optimal conditions for determination of OxTc are equal concentrations of Eu (III) and citrate (C = 1 · 10^− 4 mol L^− 1) and pH 7.2. For determination of citrate, the optimal concentrations of Eu(III) and OxTc are 1 : 0.5 (C_Eu = 1 · 10^− 4 mol L^− 1, C_OxTc = 5 · 10^− 5 mol L^− 1) at pH 7.2. The linear range for determination of OxTc in serum is 0.25-250 μg mL^− 1, and for citrate in tablets from 0.5 to 10.0 μg mL^− 1 (2.3 · 10^− 6- 4 · 10^− 5 mol L^− 1). The detection limit was 0.1 μg mL^− 1 for OxTc and 0.2 μg mL^− 1 (1 · 10^-6 mol L^− 1) for citrate, respectively. A comparison of the new method with other methods for determination of citrate is given.     

Luminescent silver nanoclusters for efficient detection of adenosine triphosphate in a wide range of pH values

In this work, polymethacrylic acid (PMAA)-templated silver nanoclusters (Ag NCs) were developed as the fluorescent probe for the efficient and sensitive detection of adenosine triphosphate (ATP) in a wide range of pH values. The fluorescence intensity of the Ag NCs could keep stable with pH values ranging from 2.5 to 9.3. The detection of ATP was based on the quenching of the fluorescent Ag NCs in the presence of ATP. The fluorescence quenching of the Ag NCs with increasing ATP concentration was studied at pH 2.5, 4.5, 7.0 and 8.5 which involved a wide pH environment in body fluids. The limit of detection (LOD) for ATP was as low as 0.1 mmol/L in an acidic environment with pH of 2.5 and all the linear correlation coefficients were satisfactory under wide-span pH values from 2.5 to 8.5. In addition, the sensitive determination of ATP was also achieved by adding copper ions (Cu²⁺). The high selectivity and rapid detection process proved that the fluorescent probe had great potential to detect ATP in biological samples under different pH conditions.     

Luminescent silver nanoclusters for efficient detection of adenosine triphosphate in a wide range of pH values

In this work, polymethacrylic acid (PMAA)-templated silver nanoclusters (Ag NCs) were developed as the fluorescent probe for the efficient and sensitive detection of adenosine triphosphate (ATP) in a wide range of pH values. The fluorescence intensity of the Ag NCs could keep stable with pH values ranging from 2.5 to 9.3. The detection of ATP was based on the quenching of the fluorescent Ag NCs in the presence of ATP. The fluorescence quenching of the Ag NCs with increasing ATP concentration was studied at pH 2.5, 4.5, 7.0 and 8.5 which involved a wide pH environment in body fluids. The limit of detection (LOD) for ATP was as low as 0.1 mmol/L in an acidic environment with pH of 2.5 and all the linear correlation coefficients were satisfactory under wide-span pH values from 2.5 to 8.5. In addition, the sensitive determination of ATP was also achieved by adding copper ions (Cu²⁺). The high selectivity and rapid detection process proved that the fluorescent probe had great potential to detect ATP in biological samples under different pH conditions.     

Intracellular adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test.     

Real-time detection of food-borne bacterial adenosine triphosphate (ATP) using dielectrophoretic force and a bioluminescence sensor

A copper hexacyanoferrate nanostructure was prepared on the surface of a disposable pencil graphite electrode. The resulting electrode exhibits an excellent electrocatalytic activity for the oxidation of L-cysteine. Cyclic voltammetry and chronoamperometry were employed to characterize the response to L-cysteine that changes linearly in the concentration range from 1 to 13 μM, with a detection limit of 0.13 μM (at an SNR of 3). Typical features of the sensor include low cost, simple preparation, fast response, good stability, selectivity, and reproducibility. It was applied to the determination of L-cysteine in urine.     

Real-time colorimetric screening of endopeptidase inhibitors using adenosine triphosphate (ATP)-stabilized gold nanoparticles

The gold nanoparticles (AuNPs) that were stabilized with adenosine triphosphate (ATP) were stable over a wide range of pHs for the buffer, even in the presence of high concentrations of salt and protein. However, these stabilized AuNPs immediately aggregated when they were exposed to thiol-containing compounds, such as thiophenol. Endoprotease hydrolyzed the thioester bond in the CBZ-Phe-S-Ph substrate, and the hydrolyzed product (thiophenol) reacted with the AuNPs that were stabilized with ATP, causing them to aggregate, which in turn resulted in a visible color change in the AuNPs solution. This method enabled the real-time monitoring of the inhibition potencies of various endopeptidase inhibitors and the activity of endoprotease. This assay discriminated between the inhibition activities of various protease inhibitors for endoprotease on the basis of the color change of the assay solution.     

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.     

CdTe量子点荧光探针测定氯霉素含量的研究

在水溶液体系中制备出了具有高质量荧光性能,巯基乙酸(TGA)修饰的CdTe量子点(QDs),基于量子点与氯霉素混合后发生荧光猝灭作用,建立CdTe量子点作为荧光探针测定氯霉素的新方法。在Tris-HCl缓冲液(pH 7.00,0.10 mol·L-1)中,反应时间为10min时,氯霉素浓度在10～70μg·mL-1范围内与CdTe量子点的荧光猝灭程度呈良好的线性关系,相关系数为0.9981,检出限为0.799μg.mL-1。方法简便快速,灵敏度高,可用于实际样品中氯霉素的检测。     

Influence of morphology and topography on potentiometric response of magnesium and calcium sensitive PEDOT films doped with adenosine triphosphate (ATP)

Poly(3,4-ethylenedioxythiophene) (PEDOT) films doped with adenosine triphosphate (ATP) are used to study the biologically relevant competitive magnesium and calcium ion-exchange at ATP membrane sites. It is shown, by atomic force microscopy (AFM) and scanning electron microscopy (SEM), that the surface topography and morphology of the PEDOT-ATP films determines the quality of their potentiometric response. More smooth and less rough films result in better potentiometric characteristics, particularly in a faster response. The topography/morphology of the PEDOT-ATP films is influenced by conditions during electrodeposition (electrochemical method of deposition, pH, concentration of electrolytes) and post-deposition soaking (including net-time of soaking), as evidenced by X-ray photoelectron spectroscopy (XPS) and energy dispersive analysis of X-rays (EDAX).     

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.     

基于Ru(bpy)2+3/AuNPs/SWCNTs/腺苷适配体电化学发光生物传感器用于腺苷的检测

利用AuNPs/Nafion复合膜技术固定Ru(bpy)2+3,采用羧基化碳纳米管固定氨基化腺苷适配体,制备腺甘电化学发光生物传感器.采用循环伏安法和电化学发光法对传感器进行表征.结果表明,此传感器具有良好的稳定性和重现性.腺苷与传感器作用后,腺苷与其适配体形成G四面体结构,Ru(bpy)2+3的电化学发光强度降低.在最佳实验条件下,电化学发光强度降低量与腺苷浓度的负对数在1.0×10-11~1.0×10-7 mol/L范围内呈良好的线性关系,线性方程为ΔIECL=-890lgC-5050,检出限(S/N=3)为5.0 × 10-12 mol/L.对1.0 × 10-10 mol/L腺苷平行测定11次,相对标准偏差为2.7%.用于尿液中腺苷的测定,加标回收率在 97.1%~110.0%之间.     

Determination of salbutamol using R-phycoerythrin immobilized on eggshell membrane surface as a fluorescence probe

A fluorescence sensor was fabricated using R-phycoerythrin (R-PE) immobilized on eggshell membrane as the fluorescence probe, and salbutamol was determined based on the decrease in fluorescence intensity of R-phycoerythrin. The scanning electron and fluorescence micrographs showed the microstructure of the eggshell membrane and indicated that the R-PE was successfully immobilized on the eggshell membrane surface. The effects of some experimental parameters on the response of the biosensor were investigated in detail. The fluorescence sensor has a linear response to salbutamol concentrations ranging from 5.00 to 100 ng mL⁻¹. The detection limit for the salbutamol is 3.50 ng mL⁻¹ (S/N = 3). The reproducibility of fabricating the biosensors using six different membranes was good with a relative standard deviation (RSD) of 3.28%. The fluorescence sensor showed extremely good stability with a shelf life of at least 50 days and reversible response to salbutamol. Some common potential interferents showed little effect on the response of the salbutamol fluorescence sensor. The proposed method was successfully applied to the determination of the salbutamol in urine samples.     

稀土配合物Eu(DBM)3为发光体的有机电致发光器件

分别以稀土配合物为发光中心，以PPV、Alq3为空穴输送层和电子输送层制备了结构为ITO／PPV／PVK：PBD：Eu（DBM）3／Alq3／Al的电致发光器件，其中发射层由旋涂法形成，该器件的最大亮度为52cd·m－2，且具有很好的单色性。