Composite Interfaces

A number of biosensors have been developed for phosphate analysis particularly, concerning its negative impact within the environmental and biological systems. Enzymatic biosensors comprising either a single or multiple enzymatic system have been extensively used for the direct and indirect analysis of phosphate ions. Furthermore, some non-enzymatic biosensors, such as affinity-based biosensors, provide an alternative analytical approach with a higher selectivity. This article reviews the recent advances in the field of biosensor developed for phosphate estimation in clinical and environmental samples, concerning the techniques involved, and the sensitivity toward phosphate ions. The biosensors have been classified and discussed on the basis of the number of enzymes used to develop the analytical system, and a comparative analysis has been performed.

Sensors and Actuators B: Chemical, 1992

An enzymatic system for the determination of inorganic phosphate is described that is based on the sequentially acting enzymes nucleoside phosphorylase and xanthine oxidase. Here in dependence on the phosphate concentration inosine is phosphorylated while hypoxanthine is formed. In the second reaction hypoxanthine is oxidized under consumption of oxygen and formation of hydrogen peroxide. The enzymes have been membrane immobilized and fixed on a Clark-type oxygen electrode. The depletion of oxygen served as a measure for the phosphate concentration between 0.5 and 100 FM. Stabilization of xanthine oxidase is attained by decomposing hydrogen peroxide with catalase. The sensitivity has been increased 20-fold with analyte recycling by alkaline phosphatase. Thus nanomolar concentrations could be measured.

Applied Microbiology and Biotechnology, 2005

A screen-printed phosphate biosensor based on immobilized pyruvate oxidase (PyOD, E.C. 1.2.3.3) has been developed for monitoring phosphate concentrations in a sequencing batch reactor (SBR) system. The enzyme was immobilized by a nafion matrix and covered a poly (carbamoyl) sulfonate (PCS) hydrogel on a screen-printed electrode. PyOD consumes phosphate in the presence of pyruvate and oxygen and generates hydrogen peroxide (H 2 O 2 ), carbon dioxide and acetylphosphate. The electroactive H 2 O 2 , monitored at +420 mV vs Ag/AgCl, is generated in proportion to the concentration of phosphate. The sensor has a fast response time (2 s) and a short recovery period (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 μM to 625 μM phosphate with a detection limit of 3.6 μM. There was good agreement (R 2 =0.9848) between the commercial phosphate testing kit and the phosphate sensor in measurements of synthetic wastewater in a SBR system. This sensor maintained a high working stability (>85%) after 12 h of operation and involved a simple operation procedure. It therefore serves as a useful tool for rapid and accurate phosphate measurements in the SBR system and probably for process control.

Analytica Chimica Acta, 1995

A new, highly sensitive enzyme sensor was developed for the determination of inorganic phosphate using maltose phosphorylase, acid phosphatase, glucose oxidase and mutarotase. The combination of the first two enzymes generates two glucose molecules per reaction cycle and recycles one molecule of phosphate. Finally, the oxidation of glucose is catalysed by mutarotase and glucose oxidase. The four enzymes were coimmobilized on a regenerated cellulose membrane which was mounted on the tip of a platinum amperometric electrode for the detection of enzymatically formed hydrogen peroxide. Thus, a detection limit of lo-' M was obtained and the sensor response was linear in the range 0.1-l PM, which is relevant for the monitoring of water pollution.

Biosensors and Bioelectronics, 1991

Ah&met: A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at O-7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-l-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100~~ phosphate, with a minimum detectable concentration of 1*25@b1 phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.

Analytica Chimica Acta, 2001

A biosensor for phosphate detection has been fabricated using a mixture of maltose phosphorylase (MP), mutarotase (MR), and glucose oxidase (GOD) entrapped in an inorganic laponite clay. The response of the biosensors to phosphate and glucose additions was measured by potentiostating the modified electrodes at 0.6 V versus Ag/AgCl in order to oxidize the enzymatically generated hydrogen peroxide. The bioelectrochemical response of the biosensor for phosphate detection is strongly affected by the ratio of each enzyme in the enzyme-clay mixture. The optimum pH and temperature of the biosensor are 6.5 and 40 • C. Under these optimum conditions, the phosphate sensitivity and the linear range are 52.4 mA M −1 cm −2 and 1-50 M, respectively. The maximum response current of the biosensor is stable at least for 2 weeks, a 30% decay of initial activity was observed after 42 days at 4 • C.

2007

Alkaline Phosphatase was immobilized on aminated glass fiber disks by covalent bond in the vacuum process. In this procedure, amide bonds were formed between carboxyl groups on the enzyme and amino groups on the glass surface. 10% Glycidoxypropyle trimethoxysilane was the best coupling reagent which could help form bonds between carboxyl and amino group on the glass fiber disk. A 10% concentration of coupling reagent, pH 9.0 and 2 gram of silica were found to be the best conditions for coupling the enzyme over the glass surface showed the highest enzyme activity. The covalent attached immobilized enzyme not only retained its activity but also could be reused at least 4 times after washing without loss of enzyme activity. Immobilized enzyme showed nearly 16% loss of enzyme activity after the first trial. An average of 1.65 mg of reusable alkaline phosphatase was immobilized per gram of glass fiber. Phosphate elements were measured from water, raw milk and raw shrimp sample by the used of this alkaline phosphatase immobilized disk as a biosensor. Immobilized enzyme can converts substrate to product and then will converts it to a measurable signal. This study demonstrates the possibility of using such a glass disk for the development of biosensors application.

Biosensors and Bioelectronics, 2018

Despite the availability of numerous electroanalytical methods for phosphate quantification, practical implementation in point-of-use sensing remains virtually nonexistent because of interferences from sample matrices or from atmospheric O 2. In this work, phosphate determination is achieved by the purine nucleoside phosphorylase (PNP) catalyzed reaction of inosine and phosphate to produce hypoxanthine which is subsequently oxidized by xanthine oxidase (XOx), first to xanthine and then to uric acid. Both PNP and XOx are integrated in a redox active Os-complex modified polymer, which not only acts as supporting matrix for the bienzymatic system but also shuttles electrons from the hypoxanthine oxidation reaction to the electrode. The bienzymatic cascade in this second generation phosphate biosensor selectively delivers four electrons for each phosphate molecule present. We introduced an additional electrochemical process involving uric acid oxidation at the underlying electrode. This further enhances the anodic current (signal amplification) by two additional electrons per analyte molecule which mitigates the influence of electrochemical interferences from the sample matrix. Moreover, while the XOx catalyzed reaction is sensitive to O 2 , the uric acid production and therefore the delivery of electrons through the subsequent electrochemical process are independent of the presence of O 2. Consequently, the electrochemical process counterbalances the O 2 interferences, especially at low phosphate concentrations. Importantly, the electrochemical uric acid oxidation specifically reports on phosphate concentration since it originates from the product of the bienzymatic reactions. These advantageous properties make this bioelectrochemical-electrochemical cascade particularly promising for point-of-use phosphate measurements. ABBREVIATIONS PNP, purine nucleoside phosphorylase; XOx, xanthine oxidase; GCE, glassy carbon electrode; P-Os, poly(1-vinylimidazoleco-allylamine)-[Os(bpy) 2 Cl]Cl; PEGDGE, poly(ethylene glycol) diglycidyl ether

Biosensors and Bioelectronics, 2011

A label free biosensor for direct detection of inorganic phosphate based on potential-step capacitance measurements has been developed. The high-affinity Pho84 plasma membrane phosphate/proton symporter of Saccharomyces cerevisiae was used as a sensing element. Heterologously expressed and purified Pho84 protein was immobilized on a self-assembled monolayer (SAM) on a capacitance electrode. Changes in capacitance were recorded upon exposure to phosphate compared to the control substance, phosphate analogue methylphosphonate. Hence, even without the explicit use of lipid membranes, the Pho84 membrane protein could retain its capacity of selective substrate binding, with a phosphate detection limit in the range of the apparent in vivo K m. A linear increase in capacitance was monitored in the phosphate concentration range of 5-25 M. The analytical response of the capacitive biosensor is in agreement with that the transporter undergoes significant conformational changes upon exposure to inorganic phosphate, while exposure to the analogue only causes minor responses.

Biosensors and Bioelectronics, 2006

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2 :5 ,2-terthiophene-3-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s −1. The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H 2 O 2 by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 M and 100 M and the detection limit was determined to be about 0.3 M. The response time of the biosensors was about 6 s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.