Stabilization of alkaline phosphatase with Au@Ag2O nanoparticles

Here, we report that a conductive Au@Ag(2)O nanoparticle structure significantly enhances the stability of alkaline phosphatase (AlkP) in the presence of the inhibitors urea and l-phenylalanine (Phe). The enzyme/nanoparticle construct is prepared by associating the enzyme with citrate-capped Au particles, and then adding Ag(+). UV-vis and XPS spectroscopy and transmission electron microscopy confirm the core@shell structure. AlkP activity was quantified in the presence and absence of the two inhibitors using a time-resolved colorimetric assay. The results indicate that 21% of the initial active AlkP is incorporated into the nanoparticle structure. More importantly, however, the Au@Ag(2)O core@shell host reduces the inhibitory effect of urea and Phe by factors ranging from 3 to 12, depending on the inhibitor and its concentration, compared to the wild-type enzyme.     

Amperometric microcells for alkaline phosphatase assay

To develop simple electrochemical immunoassays, a screen printed amperometric microcell with graphite working and Ag/AgCl reference electrodes was tested for the determination of alkaline phosphatase enzyme (ALP) and anti-humanIgG conjugated ALP (alpha-hIgG-ALP) activity in 5-10 microl samples. To ensure reproducible, steady state conditions, the working electrode surface was coated with mass-transport controlling hydrogel layer. The kinetic response curves of the hydrogel coated electrodes were linear. In addition, the hydrogel layer reduced the nonspecific adsorption of the alpha-hIgG-ALP conjugate on the working electrode surface. The measurements were made in the range of 2 divided by 4000 mU ml(-1) enzyme activities using ascorbic acid 2-phosphate (AAP) as the enzyme substrate. AAP is commercially available, non-toxic and has excellent stability. The sensitivity of the determinations was about 71% of the sensitivity which could be achieved using p-aminophenylphosphate (PAPP), a not easily accessible and unstable enzyme substrate. The experimentally determined kinetic parameters of the ALP enzyme catalyzed reactions were the same with the bare and hydrogel layer coated electrodes.     

A ratiometric fluorescent probe for alkaline phosphatase with high sensitivity

Alkaline phosphatase(ALP)is one of essential biomarkers in mammalian tissue.Here we report a ratiometric probe for ALP,which is rationally designed and synthesized by employing ESIPT fluorophore N-(3-(benzo[d]thiazol-2-yl)-4-hydroxyphenyl)benzamide(BTHPB).The enzymatic dephosphorylation converts the probe to BTHPB,which exhibits a large spectral red-shift(120 nm),allowing extremely high sensitivity of ALP sensing at 0.004 mU/mL.The probe also shows excellent biocompatibility and has been applied for monitoring the endogenic ALP in living cells.     

Photometric and fluorimetric assay of alkaline phosphatase with new coumarin-derived substrates

Summary Two new coumarin-derived synthetic substrates for use in the direct and continuous kinetic assay of alkaline phosphatase are presented. They have been studied with respect to optimum pH (9.5) and rate of enzymatic hydrolysis (1.5–1.8 nmol/min at pH 9.5) by alkaline phosphatase from calf intestine. Detection limits were 0.0005 units/ml for the photometric assay, and 0.00001 units/ml for the fluorimetric one. The relatively longwave shifted absorption and emission maxima of the new substrates in addition to the large Stoke's shifts allow the determination of enzyme activities in a spectral range distinctly outside the intrinsic fluorescence of biological matter such as serum.     

Characterization of multi-phosphopeptides by muHPLC-ESI-MS/MS with alkaline phosphatase treatment

The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.     

Recent advances in fluorescence imaging of alkaline phosphatase

Phosphatase plays a vital important role in many biological functions due to the dephosphorylation serves varied roles in cellular regulation and signaling.Among the family of phosphatase,alkaline phosphatase(ALP) could act as crucial prognostic indicators for many diseases such as bone diseases and cancer.However,the detection of ALP is mainly limited to in vitro colorimetric method in clinic.Therefore,huge efforts have been paid on the fluorescence imaging that provides a reliable method to detect the real-time and in vivo changes of the level of ALP.In this review,we summarize recent advances in fluorescence imaging of phosphatase,mainly focused on ALP.The imaging probes of phosphatase are mainly classified according to their luminescence mechanisms.In the end,we assessed the challenges and future prospects of phosphatase probes.     

Monitoring yoctomole alkaline phosphatase by capillary electrophoresis with on-capillary catalysis-electrochemical detection

An electrophoretically mediated microanalysis method for detection of yoctomole (ymol) alkaline phosphatase (ALP) was developed by a combination of on-capillary enzyme-catalyzed reaction and electrochemical detection. In this method, ALP molecules were electrokinetically injected into a capillary of 10 μm i.d. and then electromigrated to the section of the capillary immersed in a warm water bath of 37 °C, where ALP reacted for a certain time with disodium phenyl phosphate as the enzyme substrate. ALP could be measured through determining the electroactive product phenol of the enzyme-catalyzed reaction by using electrochemical detection. The phenol concentration was proportional to the mass of ALP. As a catalyst, ALP was not consumed during the reaction, which provided amplification of signal with prolonged the reaction time. In order to enhance the signal-to-noise ratio, the detection end of the capillary was etched to a horn-shape and a single carbon fiber microcylinder electrode of 6 μm in diameter as the working electrode was inserted into the detection end of the capillary. Under these conditions, the mass of ALP as low as 1.2 × 10⁻²² mol (72 molecules) or 4.0 × 10⁻²³ mol (24 molecules) could be detected for the on-capillary reaction time of 15 min or 2 h.     

Enzymatic synthesis of phosphoric monoesters with alkaline phosphatase in reverse hydrolysis conditions

Title compounds were synthesized on a preparative scale using alkaline phosphatase, orthophosphoric monoester phosphohydrolase B.C. 3.1.3.1, in reverse hydrolysis conditions. Optimization for one of the 25 phosphoryl acceptors investigated (glycerol) shows that up to 55% synthesis yield can be obtained using a large excess of substrate, conditions in which the enzymatic activity remains high. From the results obtained with different phosphoryl group donors, phosphate, pyrophosphate and polyphosphates and with enzymes of different sources, it comes up that the best results are obtained with pyrophosphate and with the weakly purified calf intestine alkaline phosphatase. The extent of enzymatic hydrolysis of the donor can be reduced owing to the existence of two different pH optima for the two reactions, phosphorylation and hydrolysis. The synthesis can be also performed using inert co-solvents which allow to reduce the amount of acceptor used, as long as Zn⁺⁺ is added to the reaction medium. The results are discussed in terms of the catalytic mechanism of alkaline phosphatase.     

Amperometric sensor based on the alkaline phosphatase activity

The construction and response of an immobilized enzyme electrode as an amperometric sensor are described. Alkaline phosphatase was covalently bonded to a nylon filter mesh using glutaraldehyde and bovine serum albumin (BSA) as crosslinkers. This modified membrane was then attached to the surface of a glassy-carbon (GC) electrode. Substrate mass transport and enzymatic catalysis control were investigated in a rotating disk electrode. Various response characteristics and kinetic parameters were evaluated and are compared to those of a previously reported amperometric alkaline phosphatase electrode.     

A self-assembled conjugated micelle with improved sensitivity for monitoring alkaline phosphatase activity

An amphiphilic coumarin derivative which forms a π-extended micelle conformation 1 was designed and developed. The exciton efficiently migrates throughout the coumarin aggregates of 1, showing amplified fluorescence quenching in the presence of Cu²⁺ ions. The 1-Cu²⁺ complex displays a highly sensitive response to pyrophosphate (PPi), leading to 80% fluorescence recovery. The activity of alkaline phosphatase (ALP) was monitored by a real-time assay where a solution containing 1, Cu²⁺, and PPi in aqueous solution exhibits a sensitive turn-off fluorescence response to ALP.     

Analysing the output from primary screening

From a perspective of process knowledge and enhancement, the analysis of the results of biological screening should not be limited to the outcome of specific projects, but additionally encompass a process centric view. Summarising outcomes across multiple projects is a powerful tool to gain a greater understanding of biological screening that will also enable optimisation of the strategy for specific projects or target classes. We have analysed a set of 73,651 compounds with reproducible (confirmed) results from 63 high-throughput screening (HTS) campaigns to reveal the underlying trends in the population of active compounds. We have focused on the overall physico-chemical profile of compound populations derived from biological screening since the in vivo activity of drug molecules is the result of physico-chemical and structural properties of the compound.     

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.