A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 muA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.     

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

High-sensitivity capillary electrophoresis method for monitoring purine nucleoside phosphorylase and adenosine deaminase reactions by a reversed electrode polarity switching mode

A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis-Menten constants (K(m)) for PNP and ADA and the inhibition constants (K(i)) for standard inhibitors determined with the new method were consistent with literature data.     

Extremely highly efficient on-line concentration and separation of gold nanoparticles using the reversed electrode polarity stacking mode and surfactant-modified capillary electrophoresis

In this study, gold nanoparticles (Au NPs) were separated using the reversed electrode polarity stacking mode (REPSM) of a capillary electrophoresis (CE) system for on-line enhancement prior to performing surfactant-modified CE separation. Under optimized conditions [running electrolyte buffer, sodium dodecyl sulfate (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (10 mM) at pH 10.0; applied voltage, 20 kV; operating temperature, 25°C; REPSM strategy for sample on-line concentration; REPSM applied prior to initializing separation], two parameters were varied to further enhance the concentration and separation of the Au NPs: (i) the rate of polarity switching (from -20 to +20 kV) between the REPSM and surfactant-modified CE separation modes and (ii) the length of the capillary column. At a polarity switching rate of 1333 kV min(-1) and a column length of ca. 83.5 cm, the resolution of the separation of a mixture of 5.3- and 40.1-nm Au NPs was greater than 19; in addition, the numbers of theoretical plates for the 5.3- and 40.1-nm-diameter Au NPs were greater than 15,000 and up to 1.15×10(7), respectively-the latter being extremely high. Thus, this CE-based method for separating Au NPs provided high performance in terms of separation resolution and the number of theoretical plates, both of which were improved by greater than fivefold relative to those published previously. Notably, the sensitivity enhancement factors for the 5.3- and 40.1-nm-diameter Au NPs were improved (by ca. 20- and 500-fold, respectively) relative to those obtained using conventional surfactant-modified CE separation.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

A transient enzymatic inhibition as an efficient tool for the discriminating bioluminescent analysis of three adenylic nucleotides with a fiberoptic sensor based on a compartmentalized tri-enzymatic sensing layer

A fiberoptic biosensor has been developed for the specific and alternate determination of ATP, ADP and AMP. The sensing layer is arranged by compartmentalizing the tri-enzyme sequence adenylate kinase – creatine kinase – firefly luciferase. The two kinases are covalently co-immobilized on a collagen membrane, whereas firefly luciferase is bound alone on a separate one. For the specific determination of each adenylic nucleotide, three particular reaction media are needed with which flow-injection analysis can be performed in the 2.5–2500 pmol for ATP, 10–2500 pmol for ADP and 25–5000 pmol for AMP linear ranges. For ten consecutive injections of 100 pmol ATP, 100 and 250 pmol ADP, the RSD were 3.5, 2.0 and 2.2%, respectively. When the three nucleotides are present simultaneously in the same sample, the transient inhibition of adenylate-kinase activity by adenosine 5′-monosulphate enables their specific and alternate measurement.     

A Fluorescence Sensor Array Based on Zinc(II)-Carboxyamidoquinolines: Toward Quantitative Detection of ATP**

The newly prepared fluorescent carboxyamidoquinolines ( 1 – 3 ) and their Zn(II) complexes ( Zn@1-Zn@3 ) were used to bind and sense various phosphate anions utilizing a relay mechanism, in which the Zn(II) ion migrates from the Zn@1-Zn@3 complexes to the phosphate, namely adenosine 5’-triphosphate (ATP) and pyrophosphate (PPi), a process accompanied by a dramatic change in fluorescence. Zn@1-Zn@3 assemblies interact with adenine nucleotide phosphates while displaying an analyte-specific response. This process was investigated using UV-vis, fluorescence, and NMR spectroscopy. It is shown that the different binding selectivity and the corresponding fluorescence response enable differentiation of adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), pyrophosphate (PPi), and phosphate (Pi). The cross-reactive nature of the carboxyamidoquinolines-Zn(II) sensors in conjunction with linear discriminant analysis (LDA) was utilized in a simple fluorescence chemosensor array that allows for the identification of ATP, ADP, PPi, and Pi from 8 other anions including adenosine 5’-monophosphate (AMP) with 100 % correct classification. Furthermore, the support vector machine algorithm, a machine learning method, allowed for highly accurate quantitation of ATP in the range of 5–100 μM concentration in unknown samples with error <2.5 %.     

Simultaneous separation and analysis of camptothecin alkaloids in real samples by large‐volume sample stacking in capillary electrophoresis

Large‐volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether‐β‐cyclodextrin and 20 mm ionic liquid 1‐ethyl‐3‐methyllimidazole l ‐lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 μg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.     

NAD-硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用

ＮＡＤ－硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用于世林,苗凤琴,杨朝霞,冯茹（北京化工大学应用化学系,北京,１０００２９）关键词烟酰胺腺嘌呤二核苷酸（ＮＡＤ）,亲和色谱,核苷酸烟酰胺腺嘌呤二核苷酸（ＮＡＤ,辅酶Ⅰ）是多种脱氢酶的辅酶,通...     

A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases

Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.     

Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.     

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

On‐line preconcentration and determination of six sulfonylurea herbicides in cereals by MEKC with large‐volume sample stacking and polarity switching

A new MEKC method with large‐volume sample stacking and polarity switching was developed for on‐line preconcentration and detection of sulfonylurea herbicide (SUH) residues in cereals, including nicosulfuron (NS), thifensulfuon (methyl) (TFM), tribenuron‐methly (TBM), sulfometuron‐methyl (SMM), pyrazosulfuron‐ethyl (PSE), and chlorimuron‐ethyl (CME). In order to achieve a high resolution and enrichment factor, several parameters were optimized, such as the pH of the running buffer, the concentration of the BGE and the SDS, the separate voltage, the sample size, the pH, and the electrolyte concentration of the sample. The optimal running buffer was composed of 30 mM borate and 80 mM SDS at pH 7.0. The borate concentration in the sample was 30 mM and the pH value of the sample was the same as that of the running buffer. The concentrating voltage and the separating voltage were –15 kV and 15 kV, respectively. The sample size was 1.455 kPa × 780 s (33.11 cm). Under the optimum conditions, for NS, TFM, TBM, SMM, PSE, and CME, the enrichment factors were 613, 642, 835, 570, 709, and 599; the LODs were 0.29–0.50 ng/g, 0.22–0.36 ng/g, 0.60–0.89 ng/g, 0.39–0.72 ng/g, 0.28–0.56 ng/g, and 0.31–0.57 ng/g; the LOQs of six SUHs were all 5 ng/g; the average recoveries of the spiked sample were 86.68–92.99%, 80.73–93.65%, 81.49–94.40%, 82.97–95.1%, 82.96–98.84%, and 80.41–92.94%, respectively.     

Determination of haloalkane dehalogenase activity by capillary zone electrophoresis

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.     

核苷磷酸盐对含双芘侧链荧光敏感器化合物的荧光猝灭与分子识别

合成了一种在水溶液中对核苷磷酸盐ATP,ADP,AMP阴离子具有识别能力的荧光化学敏感器分子---化合物1。通过检测化合物1在水溶液中芘激基缔合物荧光发射强度随核苷磷酸盐、腺嘌呤等的加入而引起的变化,求出不同核苷磷酸盐及腺嘌呤对芘激基缔合物荧光发射的猝灭常数,并进行了比较研究。利用荧光发射强度随不同核苷磷酸盐引入而发生的变化计算出化合物1对不同核苷磷酸盐进行配位的配位稳定常数。结果表明所合成的化合物1对ATP有着良好的识别力选择功能。此外,还利用分子力学的计算方法对化合物1及其与核苷磷酸盐形成配合物后的分子构象及几何尺寸作了计算,并结合实验结果进行了初步讨论。     

Electrochemical determination of pyrophosphate at nanomolar levels using a gold electrode covered with a cysteine nanofilm and based on competitive coordination of Cu(II) ion to cysteine and pyrophosphate

We are presenting an electrochemical method for the determination of pyrophosphate ions (PPi) that is based on the competitive coordination of Cu(II) ion to a nanofilm of cysteine (Cys) and dissolved PPi. Cys was immobilized on the surface of a gold electrode by self-assembly. The Cys-modified gold electrode was loaded with Cu(II) ion which is released from the surface on addition of a sample containing PPi. The sensor shows an unprecedented electrochemical response to PPi, and the reduction peak currents is linearly related to the logarithm of the concentration of PPi in the 100 nM to 10 mM range (with an R² or 0.982). The limit of detection is ~10 nM which is lower than the detection limits hitherto reported for PPi. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and common anions give a much weaker response. The method demonstrated here is simple, effective, highly sensitive, hardly interfered, and does not require the addition of a reagent. The method was applied to the determination of PPi in (spiked) serum samples.

Schematic illustration of the pyrophosphate sensing process.

     

Binding of AMP,ADP, and ATP Nucleotides by Polyammonium Macrocycles

The macrocyclic polyamines 4 – 6 , when protonated, bind strongly and selectively nucleotides (AMP, ADP, ATP) and pyrophosphate in aqueous solution. The stoichiometry of the complexes formed was determined by titration experiments followed by ³¹ P-NMR spectroscopy. Compounds 4 and 5 form 1:1 complexes with ATP, ADP, and pyrophosphate, whereas 6 forms complexes with ATP and ADP involving 2 nucleotides and 1 receptor molecule. The stability constants of these complexes have been determined by pH-metric measurements. At pH 7, both 5 and 6 give complexes of mainly the fully protonated species 5 . 6H ⁺ and 6 . 8H ⁺, whereas 4 yields predominantly complexes of 4 . 5H ⁺ and 4 . 4H ⁺.     

On‐line sample preconcentration by polarity switching in floating electrode‐integrated microchannel

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly‐dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)‐integrated straight‐channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530‐fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high‐pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode‐embedded channel, the mixture of AF488‐labeled proteins was also well concentrated and separated within 100 s.