Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed an isoelectric focusing procedure for resolving alkaline phosphatase (EC 3.1.3.1) isoenzymes and isoforms in serum. We use a thin-layer agarose gel film containing synthetic carrier ampholytes and a "separator" to flatten the pH gradient in the region of the isoenzyme and isoform isoelectric points. Sharp, highly resolved zones of enzyme activity are obtained by limiting diffusion; for this we rapidly couple the released product, 1-naphthol, to a diazonium salt, which forms a colored precipitate at the site of activity. We have resolved and identified 12 zones of alkaline phosphatase activity in the serum of ostensibly healthy persons within a wide age range. Theoretically, three basic isoenzymes are produced from independent gene loci: intestinal, placental, and nonspecific tissue alkaline phosphatase. The other zones of activity may be isoforms.
 ...
PMID:Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing. 369 Aug 36

A two site electrochemical enzyme immunoassay for thyrotropin (TSH) has been developed. This assay is based on the use of an immobilized capture antibody and a biotinylated second antibody. Detection is achieved via avidin labelled with alkaline phosphatase. The substrate 1-naphthyl phosphate was used and the product 1-naphthol was detected at disposable screen-printed carbon 8-electrode combs using specially designed instrumentation.
 ...
PMID:Avidin-biotin based electrochemical immunoassay for thyrotropin. 830 26

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compared as enzyme substrates for an amperometric milk progesterone biosensor utilising progesterone-conjugated alkaline phosphatase in a competitive immunoassay format. Cyclic voltammetry of the corresponding hydrolysis products, 4-aminophenol and 1-naphthol, at the surface of screen-printed carbon base transducers, uncoated or coated with anti-progesterone monoclonal antibody (mAb) showed well-defined anodic responses for both species, with the more sensitive being 4-aminophenol. Scan rate studies produced evidence that surface mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, toward the electrode surface. This was supported by computer simulation for the electrochemical rate constant (khet) using 4-aminophenol, which gave values at uncoated and mAb-coated electrodes of 6.5 x 10(-4) and 3.0 x 10(-4) cm s-1, respectively. The applied potential for oxidation of 4-aminophenol was 230 mV lower than for 1-naphthol. Nevertheless, by operating below +400 mV versus a saturated calomel reference electrode, it was possible to obtain a chronoamperometric signal for 1-naphthol in the absence of electrochemical interference from milk. Using mAb-coated SPCEs, calibration curves were obtained for progesterone in oestrus whole cow's milk spiked with standard concentrations over the range 0-50 ng/ml, using either 4-APP or 1NP as enzyme substrate. Precision values for triplicate sensors were 5.3-18.3% for 4-APP and 4.1-12.4% for 1-NP. An assay of real whole milk samples from different cows at various stages of the oestrus cycle produced correlations against a commercial EIA of r = 0.840 and 0.946 for 4-APP and 1-NP, respectively, 1-NP possesses the advantages over 4-APP of being inexpensive, easy to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From these observations, it is concluded that 1-NP is the preferred substrate for use with our proposed milk progesterone biosensor.
 ...
PMID:A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows' milk. 1045 17

Hereditary neurodegenerative diseases are connected with the expansion of trinucleotide repetitive sequences in genomic DNA. Molecular diagnosis of these diseases is based on the determination of the triplet repeat length. Currently used methods involve PCR amplification followed by electrophoretic determination of the amplicon size. We propose a novel electrochemical technique based on hybridization of target DNA (tDNA) immobilized at magnetic beads with a reporter probe (RP) complementary to the triplet repeats (12 units per RP). The biotin-labeled RP is detected via an enzyme-linked electrochemical assay involving binding of streptavidin-alkaline phosphatase conjugate and transformation of electroinactive 1-naphthyl phosphate to electroactive 1-naphthol. Pyrimidine residues within sequences flanking the homopurine (GAA)n repeat in tDNA are premodified with osmium tetroxide, 2,2'-bipyridine (Os,bipy), introducing electroactive labels in tDNA. The length of the triplet expansion is calculated from the ratio of the intensities of electrochemical signals of hybridized RP/tDNA-Os,bipy. The normalized signal increases linearly with the repeat length between 0 and about 200 triplet units, allowing for discrimination between normal, premutated, and mutated alleles. Application of this method for the detection of the asymptomatic heterozygous carrier of expanded alleles is demonstrated.
 ...
PMID:Electrochemical detection of DNA triplet repeat expansion. 1516 Dec 63

Hydroquinone diphosphate (HQDP) was synthesized and compared to phenyl phosphate (PP) and 1-naphthyl phosphate (NP) as a substrate for alkaline phosphatase (AP) under electrochemical immunoassay (EIA) conditions. Voltammetric and amperometric experiments showed that electrochemical oxidation of hydroquinone (HQ), which is the AP hydrolysis product of HQDP, did not produce electrode passivation, even with repeated biosensor use. In contrast, phenol and 1-naphthol, the hydrolysis products of PP and NP, respectively, were shown to be irreversibly oxidized on the electrode surfaces, and produced rapid electrode passivation, resulting in complete loss of electrode signal. When employed as AP substrate in an iridium oxide based EIA, HQDP produced significantly larger amperometric responses (117 microA/cm2) compared to PP (31 microA/cm2) and NP (27 microA/cm2). The results presented in this paper show that HQDP is an attractive alternative to commonly used AP substrates such as NP and PP. The substrate shows excellent hydrolytic stability, produces larger amperometric responses (than PP or NP), and does not produce sensor passivation.
 ...
PMID:Hydroquinone diphosphate: an alkaline phosphatase substrate that does not produce electrode fouling in electrochemical immunoassays. 1530 32

A new approach to the modification of carbon nanotubes with biomolecules for the development of nanoscale biosensors is presented. Alkaline phosphatase was immobilized on the surface of multi-wall carbon nanotubes utilizing a layer-by-layer methodology. Carbon nanotubes were incubated with streptavidin, resulting in the formation of a protein layer on the surface of the nanotubes. Biotinylated alkaline phosphatase was then allowed to bind to streptavidin, anchoring the sensing protein onto the surface. Electrochemical biosensors were constructed by using carbon nanotubes compacted into pellets. 1-Naphthyl phosphate, which is hydrolyzed by alkaline phosphatase to the electroactive 1-naphthol, was used as a substrate. Electrodes constructed in this manner were observed to generate an electrochemical signal that was a function of substrate concentration.
 ...
PMID:Protein immobilization on carbon nanotubes through a molecular adapter. 1551 93

A comparative study of enzymatic and non-enzymatic labels combined with luminescence detection, developed for immunosensing of pesticide residues (carbaryl, 1-naphthol, irgarol 1051) in organic media, is presented. Peroxidase and alkaline phosphatase enzymes with fluorogenic (3-p-hydroxyphenylpropanoic acid) and luminogenic (AMPPD derivative) substrates, respectively, were assessed as enzymatic markers. As an alternative, terbium(III) chelate, with time-resolved fluorescence detection, was evaluated as a non-enzymatic label. The best sensitivity was achieved by use of alkaline phosphatase in an immunocomplex capture assay format (I (50) values 0.06, 0.27, and 7.45 microg L(-1) in buffer, 1:1 methanol-buffer, and methanol, respectively). Results were also good (I (50) 1.00 and 6.30 microg L(-1) for water and aqueous-organic mixture, respectively) for Tb(III) chelate in an immobilized conjugate assay format. Use of alkaline phosphatase label to measure carbaryl (100 ng L(-1)) in different spiked river water samples, after solid-phase extraction and analyte elution with an ethyl acetate-methanol mixture, resulted in recoveries ranging from 81 to 98%, with acceptable precision (CV 4-14%, n=4).
 ...
PMID:Immunosensors for pollutants working in organic media. Study of performances of different tracers with luminescent detection. 1650 55

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.
 ...
PMID:Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila. 1747 3

In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to concentrations of the mouse IgG down to 1 ng mL(-1), with a linear range of 3-200 ng mL(-1) (S.D.<2; n=3), and very low non-specific adsorption.
 ...
PMID:Investigation of the enzyme hydrolysis products of the substrates of alkaline phosphatase in electrochemical immunosensing. 1858 1

We report herein the amperometric immunosensor for antibodies to Plasmodium falciparum histidine rich protein-2 (PfHRP-2). Screen-printed electrodes (SPEs) were modified with alumina sol-gel (Al(2)O(3) sol-gel) derived film and gold nanoparticles i.e. AuNPs/Al(2)O(3)sol-gel/SPE. A thin film was formed by dripping Al(2)O(3) sol on SPE followed by electrochemical deposition of gold nanoparticles (AuNPs). The modified SPEs were characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM-EDAX), Raman spectra and voltammetric experiments. Antibodies in rabbit serum sample were allowed to react with the PfHRP-2 protein that was immobilized on the modified SPE to form antigen-antibody immune complex (PfHRP-2/anti-PfHRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometry. The performances of the developed immunosensor and Dot-ELISA were tested against different dilutions of hyper immune serum (rabbit anti-PfHRP-2). Dot ELISA and the developed immunosensor (AuNPs/Al(2)O(3)sol-gel/SPE) results for the hyper immune serum containing anti-PfHRP-2 were distinctly positive when diluted upto 8 times (1 : 12800 dilution) and 11 times (1 : 102400 dilution), respectively. The developed immunosensor was applied for antibodies to PfHRP-2 in human clinical samples.
 ...
PMID:Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modified screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2. 2017 18


1 2 3 4 Next >>