Indirect Measurement of Yoctomole Alkaline Phosphatase by Capillary Electrophoresis with Electrochemical Detection

A method for indirectly detecting yoctomole (ymol) alkaline phosphatase was developed by capillary electrophoresis with electrochemical detection. In this method, disodium phenyl phosphate was used as the enzyme substrate and the product (phenol) of its hydrolysis reaction catalyzed by alkaline phosphatase was detected at the carbon fiber electrode. The optimum conditions of detection are 1.0×10^?2 mol/L Na₂B₄O₇ (pH 9.8) for the running buffer; 1.00×10^?3 mol/L disodium phenyl phosphate for the enzyme substrate; 20.0 kV for the separation voltage; 5 kV and 10 s for the injection voltage and injection time; 1.05 V (vs. saturated calomel electrode) for the detection potential and 10 min for the incubation time, respectively. In order to enhance the ratio of signal to noise, the shape and size of the working electrode, the shape of detection end of the capillary, and the capillary/electrode alignment method were studied in detail. When a single carbon fiber microcylinder electrode of 6 μm, a capillary of 10 μm ID with the etched detection end and the in‐capillary alignment were used, a ymol mass limit of detection for alkaline phosphatase was achieved.     

Rapid electrochemical assay for theophylline in whole blood based on the inhibition of bovine liver alkaline phosphatase

A disposable electrochemical test strip for determining clinically relevant concentrations of theophylline (0–300 μM) in whole blood is described, based on the generation of p-aminophenol from p-aminophenyl phosphate by the action of bovine liver alkaline phosphatase. Theophylline is an uncompetitive inhibitor of alkaline phosphatase and thus inhibits this process. The test strip consists of a screen-printed, carbon-based electrode system containing the enzyme and substrate in separate layers. Application of a 20-μl blood sample to the strip initiates the enzymic reaction, which will proceed to an extent that is inversely dependent on the amount of theophylline in the sample. After a 2-min incubation, the p-aminophenol generated is quantified by its electrochemical oxidation at + 150 mV (vs. Ag/AgCl) on the underlying carbon electrode. Caffeine and theobromine (0–1 mM), phenylalanine (< mM) and endogenous alkaline phosphatase (<2 U ml ^?1) do not interfere.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

Quantitation of phospholipase A2 and phospholipase C activity using alkaline phosphatase impregnated liposomes

Phospholipase A₂ and C activity was quantitated using liposomes impregnated with alkaline phosphatase. Release of alkaline phosphatase was dependent on phospholipase related hydrolysis of intact vesicles. Released alkaline phosphatase was quantitated after addition of its chromogenic substrate p-nitrophenyl phosphate. The lower limit of detectability for phospholipase A₂ and C activity was 0.5 unit/ml. These limits were 10-fold lower than a titrimetric method. Liposome destruction as measured by alkaline phosphatase release was calcium dependent and inhibited by 1 mM EDTA and 1 mM ZnSO₄. The assay was technically simple, generated same day results, and used automated enzyme-linked immunosorbent assay instrumentation.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

p-aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml^-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.     

Enzymatic determination of cadmium,zinc, and lead in plant materials

Prospects are outlined for using the following enzymes (native and immobilized on polyurethane foam) in the rapid and highly sensitive determination of cadmium, zinc, and lead ions in plant materials (wild grass, fresh pea, and grape): horseradish peroxidase and alkaline phosphatases isolated from chicken intestine and Greenland seal small intestine. The analytical ranges of the above metals are 1 × 10^–3?25, 7 × 10^?3?250, and 3 × 10^?2?67 mg/kg dry matter, respectively. The enzymatic determination procedures developed are based on the inhibiting effect of metal ions on the catalytic activity of peroxidase in the oxidation of o-dianisidine with hydrogen peroxide and alkaline phosphatases in the hydrolysis of p-nitrophenyl phosphate. The rates of enzymatic reactions were monitored spectrophotometrically or visually. In the analysis of plant extracts, their high acidity was diminished by choosing optimum dilution factors and pH values for test samples and the nature and concentration of a buffer solution. The interference of iron(III) was removed by introducing a 0.1 M tartaric acid solution into the indicator reaction. The accuracy of the results of the enzymatic determination of cadmium, zinc, and lead in plant materials was supported by atomic absorption spectrometry and anodic stripping voltammetry.     

Screening, Gene Sequencing and Characterising of Lipase for Methanolysis of Crude Palm Oil

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K _M of 0.75 mM and a V _max of 0.33 mM?min^?1 on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.     

An enzymatic method for determining magnesium

The effect of magnesium ions on the catalytic activity of alkaline phosphatases from three different sources in the reaction of p-nitrophenyl phosphate hydrolysis was studied. It was found that magnesium in concentrations of 0.6 ng/mL-20 µg/mL significantly activated alkaline phosphatase from chicken intestine; in higher concentrations (0.02–0.2 mg/mL), it weakly activated the enzyme from E. coli and had no effect on the catalytic activity of the enzyme from the small intestine of the Greenland seal. The strongest activating effect was observed in a Tris-HCl buffer solution at pH 9.8. The activating effect of magnesium on alkaline phosphatase from chicken intestine was used as the basis for developing a highly sensitive and selective enzymatic procedure for determining magnesium (0.6–6.0 ng/mL; RSD = 4% at c _min; n = 5) by spectrophotometrically monitoring the rate of enzymatic reaction. The developed procedure was applied to the determination of magnesium in urine.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 4, 2005, pp. 425–433.Original Russian Text Copyright © 2005 by Zhavoronkova, Muginova, Shekhovtsova.     

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

High-Sensitive Visually Controlled Membrane-Type Quantitation of NAD and Alkaline Phosphatase

Abstract

New high-sensitive visually controlled membrane-type analytical methods are proposed for quantitation of nicotineamide adenine dinucleotide and alkaline phosphatase in water solutions. The methods are based on using nitrocellulose membrane as a solid matrix on which the components of one-enzyme cofactor regeneration system are being immobilised by adsorption. In the presence of substances to be assayed, the end colored product is being adsorbed on the matrix as a result of enzymatic cyclic NAD/NADH regeneration in the active site of the matrix-bound alcohol dehydrogenase and some chemical successive reactions. Its colored intensity is a measure of the concentration of the analysed substances in solution. The general principle of NAD or alkaline phosphatase determination is successive immobilisation of separate components of the system (N-(6′-aminohexyl)salicylamide and horse liver alcohol dehydrogenase) on the matrix by adding their solutions to the wells of a specially designed cell with the membrane bottoms. In the case of alkaline phosphatase, the enzyme acted on NADPH as on a substrate. The reaction product, NAD was detected in the subsequent reaction of coenzyme regeneration. The other components of the amplifying system were added in substrate solutions at the stage of the alcohol dehydrogenase reaction. The lower detection limits for NAD and alkaline phosphatase were 3 × 10^?9 M and 1 × 10^?14 M respectively, the volume of the test sample ? 20 μl, the time of assay ? 5 min. The working concentration ranges were from 3 × 10^?9 to 1 × 10^?7 M and from 1 × 10^?14 to 1 × 10^?10 M levels for NAD(H) and alkaline phosphatase, respectively.     

Electrochemical derivatization of carbon surface by reduction of diazonium salts in situ generated from nitro precursors in aqueous solutions and electrocatalytic ability of the modified electrode toward hydrogen peroxide

The carbon electrode was covalently modified by electrochemical reduction of nitro precursor in the presence of NaNO₂ in aqueous solutions. The nitro precursor used is p-nitrophenyl phosphate, a well-known chromogenic substrate for the determination of acid and alkaline phosphatases. It is the first method for the covalent modification of carbon surface with a phosphate group. The modified electrode was characterized via cyclic voltammetry, electrochemical impedance spectroscopy, and X-ray photoelectron spectroscopy. It displays good electrocatalytic activity toward hydrogen peroxide reduction.     

Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase

Abstract

The results of a complete study of 8 substrates for acid and alkaline phosphatase indicated 7-hydroxycoumarin (umbelliferone) phosphate to be the best substrate for the analysis of these enzymes. Using this ester, from 10^?6 to 2 × 10^?2 units per ml. of alkaline phosphatase and 10^?5 to 0.06 units per ml. of acid phosphatase can be determined with an accuracy and precision of about 1.5%. Samples of serum as small as 1 μ;1. can be assayed. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification

An ultrasensitive electrochemical immunoassay method based on counting single magnetic nanobead (MNB) with combined amplification of nanobead and enzyme. Carcinoembryonic antigen (CEA) and MNB were initially immobilized on substrate at 1:1 molar ratio through sandwich immunoreactions. The MNBs were then labeled with alkaline phosphatase (AP) and continuously introduced to capillary with disodium phenyl phosphate (DPP). APs convert DPPs into a phenol zone around each moving MNB, i.e., one CEA. The phenol zones can be electrochemically detected as peaks and counted for CEA quantification. The detection limit for CEA is 5.0 × 10^?17 mol/L.     

微球载体固定化碱性磷酸酶的底物／产物吸附现象

微球载体固定化碱性磷酸酶的底物／产物吸附现象史国利，马建标，何炳林（南开大学高分子化学研究所，天津，３０００７１）关键词碱性磷酸酶，固定化，底物，产物，吸附在酶固定化研究中载体对底物和产物的吸附会造成载体内部和载体周围底物和产物的浓度不均一［１］，从...     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

A soluble form of phosphatase in Saccharomyces cerevisiae capable of converting farnesyl diphosphate into E,E-farnesol

After anion-exchange chromatography, the soluble fraction of a cell-free extract of Saccharomyces cerevisiae showed two phosphatase activity peaks when p-nitrophenyl phosphate (pNPP) was used as the substrate. However, only the second pNPP active peak demonstrated the ability to convert farnesyl diphosphate (FPP) into E,E-farnesol. N-terminal sequence analysis of the purified pNPP/FPP phosphatase revealed that it was a truncated form of alkaline phosphatase Pho8 lacking 62 amino acids from the N-terminus and was designated Pho8Δ62. Although other isoprenyl diphosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) could also be hydrolyzed by Pho8Δ62 to the corresponding alcohols, selectivity was observed among these substrates. The optimum pH was 7.0 for all three isoprenyl diphosphate substrates. Although lower hydrolytic activity was observed for FPP and GGPP at pH 6.0 and 8.5, hydrolysis of GPP was observed only at pH 7.0. Mg²⁺ and Mn²⁺ inhibited hydrolysis of FPP and GGPP, and GGPP was more sensitive to Mg²⁺ inhibition than FPP. The rate of FPP hydrolysis increased in the presence of Triton X-100.     

Determination of Rhodium in Biological Materials by Electrothermal Atomic Absorption Spectrometry with a Tungsten Tube Atomizer

Abstract

Electrothermal atomic absorption spectrometry (ETAAS) of rhodium with a tungsten tube atomizer has been investigated under optimum conditions (atomization temperature; 2230 C, purge gas; Ar 480 ml min^?1 + H₂ 20 ml min^?1, and pyrolysis temperature; 590 C). The absolute characteristic mass (the mass of element giving 0.0044 abs.) of rhodium by the atomizer was 86.5 pg and the detection limit was 16.5 ng ml^?1 (3S/N). The interferences caused by large amounts of interferents were evaluated. Al, Ca, Cu, Fe, K, Mg, Na, Pb and Zn severely interfered in the AA signal of rhodium. Ammonium phosphate, ascorbic acid, palladium nitrate, copper nitrate, lanthanum nitrate, thiocyanate and thiourea, well known as matrix modifiers were tested to eliminate the severe interferences. However, by the addition of these compounds, the rhodium signal was not recovered. The standard addition method was adapted for the determination of rhodium in biological materials. The recovery of spiked-rhodium in biological materials was in the range of 97.4 to 107%.     

A New Approach to the Enzymatic Determination of Magnesium,Calcium, and Barium

The effects of magnesium, calcium, and barium ions on the catalytic activity of alkaline phosphatase from the small intestine of the Greenland seal in the reaction of p-nitrophenyl phosphate hydrolysis in the presence of a number of complexones (ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, 1,2-cyclohexanediaminetetraacetic acid, and nitrilotrimethylenephosphonic acid) were studied. As a result, a new approach to the enzymatic determination of the above metals was developed. It consists in the use of the liberative effects of these metals on alkaline phosphatase preinhibited by nitrilotrimethylenephosphonic acid. Sensitive procedures for the enzymatic determination of magnesium, calcium, and barium ions were developed (c _min = 14 ng/mL, 24 ng/mL, and 0.8 µg/mL, respectively).__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 5, 2005, pp. 520–528.Original Russian Text Copyright © 2005 by Zhavoronkova, Muginova, Shekhovtsova.     

Enzyme Electrode for Amplification of NAD+/NADH Using Glycerol Dehydrogenase and Diaphorase with Amperometric Detection

Abstract

An enzyme electrode is made from a glassy carbon electrode covered with a gelatin membrane containing entrapped glycerol dehydrogenase (GDH) and diaphorase, and protected with a dialysis membrane. Based on amplification by the recycling reaction catalyzed by the two-enzyme systems, NAD⁺ and NADH can be determined with 800–1200 times higher sensitivity than for the same electrode in a substrate sensing mode when the flow rate was 0.08 ml/min. The detection limit was about 0.03 μM for NADH. The amplification factors were around 1000 for 0.08 ml/min, with quite large variations between electrodes. They had decreased to about 70% of the original value after 7 days. The biosensor is intended for detection in immunoassays with alkaline phosphatase as a marker.