Phosphate Sensor Research Articles

High-throughput assay for profiling the substrate specificity of Rab GTPase-activating proteins.

Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can in principle be applied to any GAP-GTPase system.

High Sensitive and Selective Hydrogen Phosphate ISFET Based on Polyvinyl Chloride Membrane

The present work describes the preparation and characterization of polyvinyl chloride membrane doped with methyl-salin ionophore. This electrode was successfully used for selectivity and high sensibility of monohydroxiphosphate in aqueous solution. 1 μM detection limit of mono-hydrogen phosphate was obtained by using dibutyl phthalate and o-nitrophenyl octyl ether-poly vinyl chloride membrane in dynamic solution. The co-existence of a large excess of salts did not interfere with the detection in case of onitrophenyl octyl ether-poly vinyl chloride membrane that maked this selective electrode more adequate for the monohydrogen detection. Also, a highly selective and sensitive dihydrogen phosphate sensor was fabricated by autoassembly of polyvinyl chloride into silicium nitride gate of ISFET.

Application of Layered Double Hydroxide Membranes as Phosphate-Sensitive Electrodes

GRAPHICAL ABSTRACT

A disposable cobalt-based phosphate sensor based on screen printing technology

Screen printing is a promising technology because of its simplicity, low-cost, high reproducibility, and efficiency in large-scale production. In this work, a cobalt-based phosphate sensor was successfully fabricated using the screen printing technology for the determination of phosphate concentration in the aqueous solution. The disposable sensor consists of a fully integrated cobalt (Co) electrode, which is a layer of carbon conductive ink (C) physically doped with Co powder, and Ag/AgCl reference electrode. The SEM images show that the morphology of the Co electrode changes after exposure to the phosphate solution, indicating that the expendable reaction exists during the measurement. At the Co/C ratio of 1:99, the cobalt-based phosphate sensor shows phosphate-selective potential response in the range of 10-4 to 10-1 mol/L, yielding a detection limit of 1×10-5 mol/L and a slope of over 30 mV/decade in acidic solution (pH 4.5) for H2PO4-. The proposed screen-printed sensor also exhibited significant reproducibility with a small repeated sensing deviation (i.e., relative standard deviation (R.S.D.) of 0.5%) on a single sensor and a small electrode-to-electrode deviation (i.e., R.S.D. < 3.2%). The recovery study of H2PO4- in real wastewater samples gave values from 95.4% to 101.8%, confirming its application potential in the measurement of phosphate in real samples. Apart from its high selectivity, sensitivity, and stability comparable with a conventional bulk Co-wire electrode, the proposed phosphate sensor still yields many other advantages, such as low price, compactness, ease of use, and the possibility of integration with other analytical devices such as flow injection analysis.

The human response to acute enteral and parenteral phosphate loads.

The human response to acute phosphate (PO4) loading is poorly characterized, and it is unknown whether an intestinal phosphate sensor mechanism exists. Here, we characterized the human mineral and endocrine response to parenteral and duodenal acute phosphate loads. Healthy human participants underwent 36 hours of intravenous (IV; 1.15 [low dose] and 2.30 [high dose] mmol of PO4/kg per 24 hours) or duodenal (1.53 mmol of PO4/kg per 24 hours) neutral sodium PO4 loading. Control experiments used equimolar NaCl loads. Maximum PO4 urinary excretory responses occurred between 12 and 24 hours and were similar for low-dose IV and duodenal infusion. Hyperphosphatemic responses were also temporally and quantitatively similar for low-dose IV and duodenal PO4 infusion. Fractional renal PO4 clearance increased approximately 6-fold (high-dose IV group) and 4-fold (low-dose IV and duodenal groups), and significant reductions in plasma PO4 concentrations relative to peak values occurred by 36 hours, despite persistent PO4 loading. After cessation of loading, frank hypophosphatemia occurred. The earliest phosphaturic response occurred after plasma PO4 and parathyroid hormone concentrations increased. Plasma fibroblast growth factor-23 concentration increased after the onset of phosphaturia, followed by a decrease in plasma 1,25(OH)2D levels; α-Klotho levels did not change. Contrary to results in rodents, we found no evidence for intestinal-specific phosphaturic control mechanisms in humans. Complete urinary phosphate recovery in the IV loading groups provides evidence against any important extrarenal response to acute PO4 loads.

A potentiometric cobalt-based phosphate sensor based on screen-printing technology

A potentiometric cobalt-based screen-printing sensor was fabricated by electroplating cobalt on the surface of a screen-printing electrode as the sensitive layer for the determination of dihydrogenphosphate (H2PO4−) in wastewater samples. The electrochemical performance of this sensor was fully examined to determine its detection calibration, detection limit, response time, selectivity, and interference with pH, various ions, and dissolved oxygen (DO). The cobalt-based phosphate sensor showed a phosphate-selective potential response in the range of 10−5 mol·L−1 to 10−1 mol·L−1, yielding a detection limit of 3.16 × 10−6 mol·L−1and a slope of −37.51 mV·decade−1 in an acidic solution (pH 4.0) of H2PO4−. DO and pH were found to interfere with sensor responses to phosphate. Ultimately, the performance of the sensor was validated for detecting wastewater samples from the Xiaojiahe Wastewater Treatment Plant against the standard spectrophotometric methods for H2PO4− analysis. The discrepancy between the two methods was generally ±5% (relative standard deviation). Aside from its high selectivity, sensitivity, and stability, which are comparable with conventional bulk Co-wire sensors, the proposed phosphate sensor presents many other advantages, such as low price, compactness, ease of use, and the possibility of integration with other analytical devices, such as flow injectors.

Phosphate wasting and fibroblast growth factor-23

The purpose of this study is to review the regulation of phosphate and recent progress in fibroblast growth factor-23 (FGF-23), a key phosphate regulatory hormone. Phosphate is required for mineralization of bone, muscle strength and a host of biologic functions. Phosphate is sensed by bone that responds with secretion of FGF-23. The major action of FGF-23 is to stimulate phosphaturia. Feedback loops between FGF-23, 1,25-dihydroxyvitamin D3 and parathyroid hormone maintain phosphate homeostasis. Information about FGF-23 has accumulated from studies in patients with oncogenic osteomalacia and inherited disorders of phosphate wasting rickets that explains the pathophysiology. Exciting new discoveries have highlighted FGF-23 as an independent risk factor for cardiovascular disease in patients with chronic kidney disease. The phosphate sensor triggering FGF-23 production remains to be identified. Derangements in FGF-23 production, half-life or downstream response are responsible for several disorders of phosphate wasting, rickets and oncogenic osteomalacia. Very high levels of FGF-23 in renal failure are an independent risk for cardiovascular disease.

Facile Fabrication of Zirconia Modified Screen‐Printed Carbon Electrodes for Electrochemical Sensing of Phosphate

AbstractWe report here a facile method to immobilize zirconia nanoparticles on a disposable screen‐printed carbon electrode (designated as ZrO2‐SPCE) for phosphate sensor application. Simply by ultrasonicating a bare SPCE in a ZrO2 slurry, ZrO2 nanoparticles can be immobilized effectively on the electrode surface as verified by surface characterization evidences. Using ferricyanide as a redox probe, an increase in the charge transfer resistance (Rct) of ferricyanide upon adsorption of phosphate on ZrO2 is used for the determination of phosphate. This ZrO2‐SPCE phosphate sensor shows a wide linear range up to 1 mM and a detection limit of 1.69 µM (S/N=3). Practical applicability of the ZrO2‐SPCE is demonstrated by detecting phosphate content in human blood samples.

Application of biopolymer produced from metabolic engineered Acinetobacter sp. for the development of phosphate optoelectronic sensor

In this study, we report the development of a phosphate sensor by exploiting phosphate-binding capability of Acinetobacter sp. biopolymer. An engineered strain of Acinetobacter sp. overproducing biopolymer was selected by Tn5 mutagenesis. The mutant showed a five-fold increase in biopolymer production and an increase in expression of intracellular biopolymer synthetic enzymes, phosphoglucomutase and glucosyl transferase. Physical and chemical analysis of biopolymer revealed a porous, compact surface morphology and a polysaccharide structure with predominance of uronic acid residues. Cellulose acetate membranes precoated with purified biopolymer were exposed to phosphate solutions and membrane-bound phosphate was determined by stannous chloride method. The colored membranes were analyzed by a LED (690 nm)/photodiode detection system. The phosphate concentration was estimated by converting the amperometric signal to phosphate concentrations from a standard plot. In conclusion, we anticipate that the developed sensor could be used for on-site phosphate analysis due to its compactness, portability, low detection levels (1 μg L−1), and low power consumption.

Electrochemical response of titania, zirconia, and alumina electrodes to phosphate adsorption

Low-range (nanomolar), on-site sensing of phosphate concentrations in water is yet to be available, as current methods trade off sensitivity for processing time. To aid in the development of an electrochemical phosphate sensor that satisfies these needs, metal oxides were tested for sensitivity to the phosphate analyte. Titania (TiO2), zirconia (ZrO2) and alumina (γ-Al2O3) coated electrodes produced via sol–gel methods were compared as sensing materials for this purpose, as these materials strongly adsorb phosphate. Perturbations of cyclic voltammograms and electrochemical impedance spectra indicated the effects of phosphate addition over the pH range of 6.00–8.75. Potential cycling of the titania and zirconia electrodes produced cathodic features that showed competition between the phosphate ion and OH−. Both lower pH and greater phosphate concentrations increased capacitance for all metal oxides tested. Titania and zirconia electrodes produced a lin–log relationship between the phase of impedance and phosphate concentration, and the relationship proved to be definitive for the porous titania electrodes over the full pH range tested. Titania also showed lesser memory effects with respect to the signal coming from phosphate adsorption than did that of zirconia. Overall, the titania-coated electrode is better poised for purposes of developing a sensor for phosphate.

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the "safety belt" conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.

Phosphate Sensitive Electrodes in Model Studies for Phosphorylation and Dephosphorylation Reactions

Highly selective and sensitive phosphate sensors have been fabricated by constructing a solid membrane disk consisting of variable mixtures of aluminium powder (Al), aluminium phosphate (AlPO4), and powdered copper (Cu). Both binary and ternary electrode systems were produced. The ternary membranes exhibited greater selectivity over a wide range of concentrations. The ternary electrode with the composition 25% AlPO4, 25% Cu, and 50% Al was selected as our preferred electrode for the study. Complexation and dephosphorylation reactions of model compounds which mimic enzyme and substrates were monitored. The results showed that the complexation reactions are fast and diffusion controlled.

Novel switchable sensor for phosphate based on the distance-dependant fluorescence coupling of cysteine-capped cadmium sulfide quantum dots and silver nanoparticles

A novel switchable sensor was developed for the determination of phosphate based on Ce(3+) induced aggregation and phosphate triggered disaggregation of cysteine (Cys)-capped CdS quantum dots (QDs) and silver nanoparticles (AgNPs). The rare earth metal Ce(3+) could aggregate a mixture of QDs and AgNPs, which induced electron or energy transfer between CdS QDs and AgNPs and serious fluorescence quenching. However, phosphate dissociated the formed aggregation of CdS QDs and AgNPs, restoring the enhanced fluorescence of Cys-capped CdS triggered by AgNPs. Although, CdS QDs alone could also be used to detect phosphate through the aggregation-disaggregation mechanism adjusted by Ce(3+) and phosphate. It was found that the distance-dependent interaction between AgNPs and CdS QDs driven by Ce(3+) and phosphate could lead to enhanced quenching or enhancement of the fluorescence of Cys-capped CdS to form a more sensitive detection system for phosphate. The developed method was applied in the detection of phosphate in real water samples with acceptable and satisfactory results.

Phosphate as a sensor and signaling molecule

In prokaryotes and eukaryotes inorganic phosphate plays a vital role in many cellular and biological processes. Maintenance of proper phosphate homeostasis is therefore essential and any deviation from that state can lead to several acute and chronic disease states. In order to maintain physiological levels, a tightly regulated phosphate sensing and signaling mechanism needs to exist. The earliest and best characterized mechanisms of phosphate sensing and signaling have been described in yeast and bacteria involving the Pho regulon. The Pho regulon has been shown to function in a coordinated fashion in order to meet the cellular needs of the organism. Studies have also shown that the protein kinase A (PKA) signaling pathway is involved in phosphate metabolism. In eukaryotes, due to the complexity of the regulatory mechanisms and involvement of several key regulators of phosphate secreted from different organs, it has been difficult to identify the phosphate sensor. Nonetheless the crosstalk between organs in response to phosphate provides strong evidence to support the existence of such a mechanism. This review will focus on this evidence and highlight the parallels tha exist with the Pho regulon. We will focus on the kidney-arathyroid, kidney-intestinal, parathyroid-intestinal, kidney-bone, and finally the parathyroid-bone axes.

Effects of pH on Phosphate Adsorption to Hematite as Studied with Cyclic Voltammetry and Electrochemical Impedance Spectroscopy

Nanoporous hematite (α-Fe2O3) coated electrodes were applied for the investigation of the electrochemical effects of pH on adsorption of the phosphate ion to iron oxide in concentrations commonly found in natural waters. The hematite electrode was tested with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods in NaClO4 solution and in 10−8 – 10−4 M phosphate over the pH range 6.00 – 8.75. In cyclic voltammograms, the current features attributed to specific adsorption of H+ or OH− were accentuated by phosphate addition. Impedance studies indicated that hematite behaved like a blocking electrode with deviations to blocking behavior occurring at higher pH and with increased phosphate. Resultant spectra were modeled with an analog electric circuit with a resistor in series with three parallel lines consisting of resistive, capacitive, and constant phase elements (CPEs) highlighting processes over sections of the applied EIS frequency range. This analysis of the phosphate-hematite system may assist with the production of an electrochemical sensor for phosphate at environmentally relevant pH.

Time-dependent, reversible, oxaborole inhibition of Escherichia coli leucyl-tRNA synthetase measured with a continuous fluorescence assay

Enzyme assays for the catalytic activity of aminoacyl-tRNA synthetases generally measure the incorporation of radioactive amino acids into tRNA. Such assays are necessarily discontinuous. Leucyl-tRNA synthetase has recently gained attention as the target of novel antimicrobial compounds based on the oxaborole scaffold, examples of which have been shown to have slow binding and dissociation kinetics. Investigations of the kinetics of inhibition by these compounds would be facilitated by a continuous assay of leucyl-tRNA synthetase catalysis. Here we report a continuous fluorescence intensity-based assay for leucyl-tRNA synthetase in which the pyrophosphate product is converted to phosphate, which is detected with nanomolar sensitivity by a phosphate sensor protein. This assay was used to measure the time constants for the slow onset of inhibition and long residence time of an oxaborole-based inhibitor.

An inorganic phosphate (Pi) sensor triggers ‘turn-on’ fluorescence response by removal of a Cu2+ ion from a Cu2+-ligand sensor: determination of Pi in biological samples

A fluorescent ligand that displays a high selectivity for Cu2+ has been synthesized. On complexation with Cu2+, the fluorescence of the ligand is quenched. Inorganic phosphate ions decomplex Cu2+ displaying a fluorescence enhancement that can even be seen with naked eyes. The method was successfully used in quantitative determination of inorganic phosphates in serum, urine, and saliva samples.

A multicomponent sugar phosphate sensor system specifically induced in Bacillus cereus during infection of the insect gut

Using a previously developed Bacillus cereus in vivo expression technology (IVET) promoter trap system, we showed that spsA, a gene of unknown function, was specifically expressed in the larval gut during infection. Search for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) as an activation signal. Analysis of the spsA-related 5-gene cluster indicated that SpsA is part of a new sugar phosphate sensor system composed of a 2-component system (TCS) encoded by spsR and spsK, and 2 additional downstream genes, spsB and spsC. In B. cereus, American Type Culture Collection (ATCC) 14579, spsRK, and spsABC are separate transcriptional units, of which only spsABC was activated by extracellular G6P. lacZ transcriptional fusions tested in mutant and complemented strains showed that SpsRK, SpsA, and SpsB are essential for the transcription of spsABC. Deletion mutant analysis showed that SpsC is essential for the G6P uptake. gfp-transcriptional fusions showed that these genes are required for host-activated expression, as well. This sugar phosphate sensor and transport system is found in pathogenic Bacillus group and Clostridia bacteria and may be important for host adaptation. Our findings provide new insights into the function of 2-component sensor systems in host-pathogen interactions, specifically in the gut.

New solid state phosphate sensitive electrodes for routine environmental monitoring

Highly selective and sensitive phosphate sensors have been fabricated by constructing a solid membrane disk consisting of variable mixtures of aluminium powder (Al), aluminium phosphate (AlPO4) and powdered copper (Cu). Both binary and ternary electrode systems are produced. The ternary membranes exhibit greater selectivity over a wide range of concentrations. The ternary electrode with the composition 25% AlPO4, 25% Cu and 50% Al was selected as our preferred electrode. The newly fabricated ternary membrane phosphate selective electrodes exhibited linear potential response in the concentration range of 1.0 × 10?1 to 1.0 × 10?6 mol L?1. The electrodes also exhibit a fast response time of <60 s. Their detection limit is lower than 1.0 × 10?6 mol L?1. The unique feature of the described electrodes is their ability to maintain a steady and reproducible response in the absence of an ionic strength control. The electrodes have a long lifetime and can be stored in air when not in use.

Light triggered detection of aminophenyl phosphate with a quantum dot based enzyme electrode

An electrochemical sensor for p-aminophenyl phosphate (pAPP) is reported. It is based on the electrochemical conversion of 4-aminophenol (4AP) at a quantum dot (QD) modified electrode under illumination. Without illumination no electron transfer and thus no oxidation of 4AP can occur. pAPP as substrate is converted by the enzyme alkaline phosphatase (ALP) to generate 4AP as a product. The QDs are coupled via 1,4-benzenedithiol (BDT) linkage to the surface of a gold electrode and thus allow potential-controlled photocurrent generation. The photocurrent is modified by the enzyme reaction providing access to the substrate detection. In order to develop a photobioelectrochemical sensor the enzyme is immobilized on top of the photo-switchable layer of the QDs. Immobilization of ALP is required for the potential possibility of spatially resolved measurements. Geometries with immobilized ALP are compared versus having the ALP in solution. Data indicate that functional immobilization with layer-by-layer assembly is possible. Enzymatic activity of ALP and thus the photocurrent can be described by Michaelis- Menten kinetics. pAPP is detected as proof of principle investigation within the range of 25 μM - 1 mM.