Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Cryptosporidium parvum in water has been developed. The target molecule was a 121-nucleotide sequence from the C. parvum heat shock protein 70 (hsp70 mRNA from U71181 gene). This analyte offers the possibility of distinguishing dead from live oocysts. The assay involves covalent attachment of a primary DNA probe via its 5'-amine-terminus to self-assembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181 gene for the mRNA), by hybridization to a 20-base complementary sequence on the target (at sequence 1063-1082). A secondary DNA probe labeled with alkaline phosphatase (AP) was then hybridized to base sequence 1039-1062 on the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution, in which gold-coated silicon wafer modified with the complete assembly of the assay components was incubated, is linear with concentration of the target (5-50 microg/mL, where P1 and P2-AP concentrations are 50 microg/mL). A detection limit of 2 microg/mL (or 146 nM) of the DNA target was obtained. Cross-reactivity tests showed high selectivity for heat-shocked C. parvum. No signal was obtained for either the synthetic DNA for hsp70 of Campylobacter lari, Escherichia coli, Giardia lamblia, Salmonella typhimurium, and Listeria monocytogenes or for the products of heat-shocked whole organisms of E. coli, G. lamblia, Staphylococcus aureus, and Cryptosporidium muris.
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PMID:Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA. 1457 58

We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.
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PMID:Enzyme-amplified electrochemical detection of DNA using electrocatalysis of ferrocenyl-tethered dendrimer. 1458 3

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.
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PMID:Electric chips for rapid detection and quantification of nucleic acids. 1468 37

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1(0)P, which was of two different sequences we call 1(0)P(a) and 1(0)P(b)) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1(0)P(a), 47 bases were complementary to T1 and T2 at 543-590, while on 1(0)P(b), 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.
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PMID:Small-volume detection of Plasmodium falciparum CSP gene using a 50-microm-diameter cavity with self-contained electrochemistry. 1647 3

The two articles in this series are dedicated to bioaffinity electrodes with in situ detection of the product of the enzyme label after recognition by its conjugate immobilized on the electrode. Part 1 was devoted to direct electrochemical detection, whereas the present contribution deals with homogeneous chemical and enzymatic amplification of the primary electrochemical signal. The theoretical relationships that are established for these modes of amplification are applied to the avidin-biotin recognition in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichloro-phenyl phosphate as substrate, generating 2,6-dichloro-4-aminophenol as electrochemically active product. Chemical amplification then results from the addition of NADH, which reduces the 2,6-dichloro-quinonimine resulting from the electrochemical oxidation of 2,6-dichloro-4-aminophenol. An increased amplification is obtained when the reduction of 2,6-dichloro-quinonimine involves diaphorase in solution with NADH as substrate. The excellent agreement between theoretical predictions and experimental data required a detailed theoretical analysis and the independent determination of the key kinetic parameters of the system. The theoretical analysis was extended to monolayer and multilayered films of auxiliary enzyme as well as to electrochemical amplification by means of closely spaced dual electrodes so as to offer a rational comparative panorama of the amplification capabilities of the various possible strategies. Confinement of the profile of the product, and/or its oxidized form, in the vicinity the electrode surface appears as a key parameter of amplification.
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PMID:Theory and practice of enzyme bioaffinity electrodes. Chemical, enzymatic, and electrochemical amplification of in situ product detection. 1849 54

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.
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PMID:Investigation of the enzyme hydrolysis products of the substrates of alkaline phosphatase in electrochemical immunosensing. 1858 1

Three-dimensional flow-through electrodes were fabricated using superporous agarose (SPA) and reticulated vitreous carbon (RVC) composite materials that were suitable as a platform for sandwich assays. These SPA-RVC composite electrodes were fabricated by fitting a SPA-RVC composite cylinder inside a graphite tube and subsequently fixing the graphite tube onto a polypropylene micropipette tip. The electrode design allows for ease in reagent/washing steps involved in sandwich assay protocols and could easily be made portable. The electrode materials were characterized with respect to pore-size distribution, total free volume, ligament and bulk densities of the RVC, and physical structural characteristics. Coulometric detection of redox molecules such as K(3)Fe(CN)(6) and 4-aminophenol was possible using SPA-RVC electrodes by the trapping of these redox molecules inside the SPA-RVC electrodes. Avidin affinity molecules were covalently immobilized onto the SPA matrix inside the RVC electrodes by periodate-activation followed by reductive amination. The amount of avidin immobilized inside the SPA-RVC electrodes was (5+/-0.06)x10(-11) mol, which was determined by saturating the avidin sites with biotinylated fluorescein (b-fluo) and subsequently determining the amount of immobilized b-fluo via a standard addition method using fluorescence spectroscopy. Non-specific binding of labeled enzymes such as biotinylated alkaline phosphatase (b-ALP) onto the SPA-RVC electrodes without avidin capture sites was determined to be less than 1% compared to the specific binding of b-ALP on avidinylated SPA-RVC electrodes.
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PMID:Preparation and characterization of superporous agarose-reticulated vitreous carbon electrodes as platforms for electrochemical bioassays. 1860 32

We report on the use of flow-through electrodes fabricated from a composite of superporous agarose (SPA) and reticulated vitreous carbon (RVC) for carrying out sandwich bioassays via a model sandwich assay scheme. The flow-through design of the SPA-RVC electrodes allows for ease in solution handling with the use of micropipettors while allowing sandwich assays to be performed on the SPA matrix inside the RVC. A sandwich bioassay was devised for detecting biotinylated bovine serum albumin (b-BSA) as a proof-of-concept scheme to demonstrate applicability of SPA-RVC electrodes to carry out sandwich assays. In this bioassay scheme, SPA-RVC electrodes with avidin molecules immobilized on the SPA matrix were incubated with low quantities of b-BSA followed by incubation with avidinylated alkaline phosphatase (av-ALP). This construct creates a sandwich bioassay whereby b-BSA is sandwiched between the two avidin complexes. Av-ALP labels captured on the bound b-BSA catalytically hydrolyze conversion of 4-aminophenylphosphate (PAPP) to electrochemically active 4-aminophenol (PAP) which is then voltammetrically detected inside the RVC. The lower concentration detection limit for b-BSA was 0.32+/-0.1 ng mL(-1) and the absolute detection limit was 32+/-10 pg. Non-specific binding of av-ALP enzyme labels onto the avidin-activated SPA-RVC electrodes was low. Catalytic generation of PAP by non-specifically bound av-ALP occurs at a rate less than 2% of that for PAP generation by av-ALP in [(SPA-av)-(b-BSA-b)-(av-ALP)] sandwich constructs.
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PMID:Superporous agarose--reticulated vitreous carbon electrodes for electrochemical sandwich bioassays. 1892 7

The detection of an antibody against nucleoprotein (NP) of the avian influenza virus (AIV) was attempted by an electrochemical immunoassay by combining a secondary antibody-alkaline phosphatase conjugate and 4-aminophenylphosphate. Released 4-aminophenol was quantitated electrochemically, and the current derived from oxidation of the hydrolysis product increased linearly over a wide primary antibody concentration range (10 - 1000 ng/ml). The detection limit of this electrochemical immunoassay for the antibody against the NP of AIV was found to be 10 ng/ml.
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PMID:Detection of an antibody to avian influenza virus by an electrochemical immunoassay (eELISA). 1907 74

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.
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PMID:Electrochemical detection of epidermal growth factor receptors on a single living cell surface by scanning electrochemical microscopy. 1933 33


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