Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline

An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser-induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate-filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2'-bibenzothiazol]-6-hydroxy-benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.     

Spectrophotometric enzyme-amplified immunoassay for thyroid stimulating hormone.

Thyroid stimulating hormone (TSH) regulates the function of the thyroid gland. Its determination at low concentrations in serum is useful in the diagnosis of hyperthyroidism. In this paper, it is detected using a spectrophotometric enzyme-amplified immunoassay. The reporter enzyme is alkaline phosphatase and its substrate is flavin adenine dinucleotide phosphate (FADP). Reaction with alkaline phosphatase converts FADP into flavin adenine dinucleotide (FAD), which, unlike FADP, re-activates apo-D-amino acid oxidase (apo-AOD). Re-activation of apo-AOD allows the product of the reporter enzyme to be amplified. The lower limit of detection for TSH by this method is 0.06 microU cm-3. This compares with 0.54 microU cm-3 for an identical assay in which p-nitrophenyl phosphate was the substrate for alkaline phosphatase. Contaminating alkaline phosphatase was removed from the reagents by affinity chromatography.     

Nanostructured Screen Printed Graphite Electrode for the Development of a Novel Electrochemical Genosensor

The aim of this work is the preparation of DNA‐sensing architectures based on gold nanoparticles (AuNPs) in conjunction with an enzyme‐amplified detection to improve the analytical properties of genosensor. In order to assess the utility of study as DNA‐sensing devices, a thiolated DNA capture probe sequence was immobilized on the gold nanoparticle modified surface. After labeling of the biotinylated hybrid with a streptavidin‐alkaline phosphatase conjugate, the electrochemical detection of the enzymatic product was performed on the surface of a disposable electrode. Two different enzymatic substrates to detect the hybridization event were studied. In the first case, the enzyme catalyzed the hydrolysis of α‐naphthyl phosphate; the product is electroactive and has been detected by means of differential pulse voltammetry (DPV). In the second one, the enzyme catalyzed the precipitation of an insoluble and insulating product on the sensing interface. In this case, the electrochemical transduction of the hybridization process was performed by electrochemical impedance spectroscopy (EIS).     

The measurement of alkaline phosphatase at nanomolar concentration within 70 s using a disposable microelectrochemical transistor

We report a new approach to the measurement of alkaline phosphatase concentration based on the use of a disposable poly(aniline) microelectrochemical transistor. The measurement is carried out in a two cell configuration in which the poly(aniline) microelectrochemical transistor operates in acid solution and is connected to the alkaline buffer solution containing the analyte by a salt bridge. Disposable microelectrochemical transistors were reproducibly fabricated by electrochemical deposition of poly(aniline) onto photolithographically fabricated gold microband arrays. Using these devices alkaline phosphatase was detected by employing p-aminophenyl phosphate as the substrate for the enzyme and using glucose and glucose oxidase to recycle the p-aminophenol generated upon enzyme catalysed hydrolysis of the phosphate. Recycling the p-aminophenol with glucose and glucose oxidase amplified the detection of alkaline phosphatase approximately tenfold. Using this approach we obtain linear calibration curves for alkaline phosphatase up to 5 nM within 70 s on single use devices.     

A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection

In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.     

Preparation of gold nanoparticles on eggshell membrane and their biosensing application

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25 ± 7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl₄ without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl₄) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH ≤3.0 and ≥7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO_x) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO_x. The linear response range of the glucose biosensor is 20 μM to 0.80 mM glucose with a detection limit of 17 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.     

An Automated Spectrophotometric Flow Injection Assay of Alkaline Phosphatase

Abstract

Assay of alkaline phosphatase using an automated flow injection manifold is described. The detector used is a spectrophotometer. The measurement is based on the rate of formation of p-nitrophenol at 405 nm, from the substrate p-nitropheny1 phosphate. The linear range, precision and accuracy of the technique for the assay of the enzyme is given. The advantages of the approach to assay of plasma enzymes are also discussed.     

Solid-supported single- and multi-enzyme amplified assays for clostridiopeptidase A

A rapid and sensitive spectrophotometric assay for Clostridium histolyticum clostridiopeptidase A (collagenase) was accomplished by measuring the activity of an alkaline phosphatase indicator enzyme released into solution from insoluble, covalently linked alkaline phosphatase indicator enzyme released into solution from activity of the alkaline phosphatase was monitored spectrophotometrically using either p-nitrophenyl phosphate as substrate or more sensitively by a signal amplification system consisting of NAD⁺, alcohol dehydrogenase, diaphorase and INT-Violet. Under the reaction conditions the amount of indicator enzyme produced is directly proportional to the concentration of collagenase. With p-nitrophenyl phosphate as substrate the magnitude of the signal was 0.003 abs. min^?1 per 100 ng ml^?1 collagenase whereas with the multienzyme amplification system it was 0.035 abs. min^?1, i.e. approximately as 12-fold increase. The method consists in first incubating the substrate with the bacterial collagenase for 20 min, then up to 96 samples of the released alkaline phosphatase can be analysed in 2 min using a microtitre plate reader run in the kinetic mode.     

Biomimetic fibronectin/mineral and osteogenic growth peptide/mineral composites synthesized on calcium phosphate thin films

Composites of fibronectin/mineral and osteogenic growth peptide/mineral were synthesized on calcium phosphate coated substrates immersed in Dulbecco's phosphate-buffered saline solution containing biomolecules. The kinetics of coprecipitation for two biomolecules was similar, and the biomolecules participated in the formation of the crystal latticework and influenced the mineral structure and composition.     

Synthesis of gold and silver nanoparticles using purified URAK

This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV-visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 μg/ml.     

Biocatalytic Self-Assembly Cascades

The properties of supramolecular materials are dictated by both kinetic and thermodynamic aspects, providing opportunities to dynamically regulate morphology and function. Herein, we demonstrate time-dependent regulation of supramolecular self-assembly by connected, kinetically competing enzymatic reactions. Starting from Fmoc-tyrosine phosphate and phenylalanine amide in the presence of an amidase and phosphatase, four distinct self-assembling molecules may be formed which each give rise to distinct morphologies (spheres, fibers, tubes/tapes and sheets). By varying the sequence or ratio in which the enzymes are added to mixtures of precursors, these structures can be (transiently) accessed and interconverted. The approach provides insights into dynamic self-assembly using competing pathways that may aid the design of soft nanostructures with tunable dynamic properties and life times.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium.

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Rational nanoconjugation improves biocatalytic performance of enzymes: aldol addition catalyzed by immobilized rhamnulose-1-phosphate aldolase

Gold nanoparticles (AuNPs) are attractive materials for the immobilization of enzymes due to several advantages such as high enzyme loading, absence of internal diffusion limitations, and Brownian motion in solution, compared to the conventional immobilization onto porous macroscopic supports. The affinity of AuNPs to different groups present at the protein surface enables direct enzyme binding to the nanoparticle without the need of any coupling agent. Enzyme activity and stability appear to be improved when the biocatalyst is immobilized onto AuNPs. Rhamnulose-1-phosphate aldolase (RhuA) was selected as model enzyme for the immobilization onto AuNPs. The enzyme loading was characterized by four different techniques: surface plasmon resonance (SPR) shift and intensity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). AuNPs-RhuA complexes were further applied as biocatalyst of the aldol addition reaction between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal during two reaction cycles. In these conditions, an improved reaction yield and selectivity, together with a fourfold activity enhancement were observed, as compared to soluble RhuA.     

Synthesis and biological activity of N-sulfonylphosphoramidates: probing the electrostatic preferences of alkaline phosphatase.

N-Sulfonylphosphoramidates have been synthesized to investigate the electrostatic requirements for binding to alkaline phosphatase. Alkyl- and aryl N-benzylated sulfonamides were phosphorylated with bromophosphates or synthesized via phosphoramidite chemistry in moderate yields (44-77%.) The resulting tribenzylated N-sulfonylphosphoramidates may be deprotected in one step to give the free acids in quantitative yields. Physical data of N-sulfonylphosphoramidates, including pK(a)'s and stability toward hydrolysis, were determined. Inhibition data suggests that AP does not bind trianionic N-sulfonylphosphoramidates better than dianionic N-sulfonylphosphoramidates, although N-sulfonylphosphoramidates are bound tighter than N-phenylphosphoramidate. k(cat) for the hydrolysis of N-sulfonylphosphoramidates by bovine and E. coli alkaline phosphatases is 10-60% that of p-nitrophenyl phosphate.     

Non-equivalence of D- and L-trehalose in stabilising alkaline phosphatase against freeze-drying and thermal stress. Is chiral recognition involved?

Comparison of the ability of the enantiomeric forms of trehalose to stabilise alkaline phosphatase towards dehydration and heat showed that natural D-trehalose is superior to L-trehalose, although both disaccharides provide some protection for the enzyme. The result of this novel experiment suggests a chiral differentiation between carbohydrate and protein and thus lends support for the water replacement hypothesis of solute-based stabilisation of biomolecules, but the non-crystallinity and the physical form of the L-isomer may also be a key factor.     

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.     

A simple visual method for DNA detection based on the formation of gold nanoparticles

A simple visual method for DNA detection during the formation of gold nanoparticles (AuNPs) was developed based on different electrostatic properties of single strand DNA (ssDNA) and double strand DNA (dsDNA). Since the ssDNA is easy to bind to AuNPs due to its exposed bases which could prevent salt-induced aggregation of AuNPs. The dsDNA always present negative charge because its negatively charged phosphate backbone is exposed. In this case, the dsDNA could disturb the adsorption between dsDNA and AuNPs and result in non-aggregation of AuNPs. After hybridization, chloroauric acid and ascorbic acid were added to the mixture solution, and the solution changed to red immediately and turned to purple in 10 min in the present of target DNA. TEM results confirmed that the change of color stemed from aggregation of AuNPs. In order to obtain accurate results by naked eye, the DNA detection assay should be conducted under pH 7.0.     

Synthesis, radiolabeling and in vivo biodistribution of diethylstilbestrol phosphate derivative (DES-P)

Diethylstilbestrol (DES) is a well known, nonsteroidal estrogen with high affinity for the estrogen receptor (ER). Today DES is used to treat breast and prostate cancers. A phosphate derivative of DES [Diethylstilbestrol diphosphate (DES-P)] which is specific to tumor cells consisting alkaline phosphatase enzyme was synthesized and labeled with ^99mTc using tin chloride as reducing agent. In vivo biological activity of ^99mTc labeled diethylstilbestrol phosphate compound (^99mTc-DES-P) was examined by biodistribution studies on Wistar Albino rats. Statistical evaluation was performed using SPSS 13 program. The percentage (%) radiolabeling yield of ^99mTc-DES-P and quality control studies were done by Thin Layer Radio Chromatography (TLRC). Results showed that, ^99mTc-DES-P may be proposed as an imaging agent for ER enriched tumors such as uterus and prostate and their metastases in bones.     

A simple visual method for DNA detection based on the formation of gold nanoparticles

A simple visual method for DNA detection during the formation of gold nanoparticles (AuNPs) was developed based on different electrostatic properties of single strand DNA (ssDNA) and double strand DNA (dsDNA). It could identify target DNA in 10 min.     

Electrochemical Sensing Platforms for HER2‐ECD Breast Cancer Biomarker Detection

Screening and early diagnosis are crucial to increase the success of cancer patients’ treatments and improve the survival rate. To contribute to this success, distinct electrochemical immunosensing platforms were developed for the analysis of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2‐ECD) through sandwich assays on nanomaterial‐modified screen‐printed carbon electrodes (SPCEs). The most promising platforms showed to be SPCEs modified with (i) gold nanoparticles (AuNPs) and (ii) multiwalled carbon nanotubes combined with AuNPs. The antibody‐antigen interaction was detected using a secondary antibody labelled with alkaline phosphatase and 3‐indoxyl phosphate and silver ions as the enzymatic substrate. The electrochemical signal of the enzymatically generated metallic silver was recorded by linear sweep voltammetry. Under the optimized conditions, linear calibration plots were obtained between 7.5 and 50 ng/mL and the total assay time was 2 h 20 min, achieving LODs of 0.16 ng/mL (SPCE‐MWCNT/AuNP) and 8.5 ng/mL (SPCE‐AuNP), which are well below the established cut‐off value of 15 ng/mL for this cancer biomarker.