Poly(pyrrole‐co‐pyrrole‐2‐carboxylic acid)/Pyruvate Oxidase Based Biosensor for Phosphate: Determination of the Potential, and Application in Streams

Abstract

A biosensor based on conductive poly(pyrrole‐co‐pyrrole‐2‐carboxylic acid) [Poly(Py‐co‐PyCOOH)] copolymer film coated gold electrode was developed for the quantitative phosphate determination. Enzyme pyruvate oxidase was immobilized chemically via the functional carboxylated groups of the copolymer. The potential to be applied which is deficiency of phosphate biosensor studies for precise phosphate detection was clarified by using differential pulse voltammetry technique. Performance of the sensing ability of the biosensor was improved by optimizing cofactor/cosubstrate concentrations, polymeric film density and pH. The biosensor showed a linearity up to phosphate concentration of 5 mM, operational stability with a relative standard deviation (RSD) of 0.07 % (n=7) and accuracy of 101 % at −0.15 V (vs. Ag/AgCl). Detection limit (LOD) and sensitivity were calculated to be 13.3 μM and 5.4 μA mM−1 cm−2, respectively by preserving 50 % of its initial response at the end of 30 days. It's performance was tested to determine phosphate concentrations in two streams of Zonguldak City in Turkey. Accuracy of phosphate measurement in stream water was found to be 91 %.

A Novel Flow‐injection Rhodium Nanoparticles Modified Phosphate Biosensor and its Operation in Artificial Urine

“…The carrier solution was composed of 20 mM pH 7.0 oxygenated citrate buffer containing 0.7 mg mL À 1 of LiClO 4 used as supporting electrolyte, 1 mM of magnesium (Mg + 2 ), 0.5 mM of pyruvate, 50 μM of TPP and 5 μM of FAD. Concentrations of Mg + 2 , pyruvate, TPP and FAD cofactors were optimized in our previous study [9]. After a stable background current was reached, analytes were injected into the flow-injection system using the injection valve and amperometric measurments were monitored online on the computer screen of the potentiostat.…”

“…Pyruvate oxidase, which belongs to the family of oxidoreductases, is an enzyme that catalyzes the oxidative decarboxylation of pyruvate, phosphate and oxygen to acetyl phosphate, carbon dioxide and hydrogen peroxide (equation 1) in the presence of cofactors such as FAD, TPP and Mg + 2 . TPP and FAD have an important place in the overall enzymatic reaction mechanism in presence of Mg + 2 [9]. The reaction mechanism proceeds as follows: After the ionization of the TPP C2-H, pyruvate reacts with the C2 carbanion in the active site, leading to the formation of 2-lactyl-TPP.…”

“…Meanwhile, the formed 2-hydroxyethyl-TPP is oxidized by FAD, leading to the formation of acetyl-TPP and two-electron reduced FADH 2 . The reduced form of FAD (FADH 2 ) is re-oxidized with molecular oxygen to form hydrogen peroxide and FAD, then acetyl-TPP is split apart phosphorolytically to acetyl phosphate and TPP C2 carbanion [9,32,33]. Rhodium nanoparticles acted as mediator for the electrocatalytic reduction of H 2 O 2 .…”

“…Even though the phosphate concentration of urine is measured by colorimetric analysis, the commonly used method is spectrophotometric at present [1,6]. These conventional methods have certain disadvantages such as tedious, time consuming, the use of health hazardous carcinogenic chemicals, requiring expertise and expensive instrumentation [4,9]. An alternative approach to the conventional method to overcome these shortcomings is biosensors with high selectivity and specificity to measure phosphate directly.…”

“…Interest in the development of phosphate biosensors for use in different related fields such as biological, environmental, food and agricultural has been continuing since the first report published by Guilbault and Nanjo in 1975 [7,10]. Although researchers have developed singleenzyme biosensors [9,[11][12][13], bi-enzyme biosensors [14][15][16][17], tri enzyme biosensors [18,19] for the over four decades, the simple, cost effective, more efficient and preferred one is pyruvate oxidase based single-enzyme phosphate biosensors [9]. Pyruvate oxidase catalyzes the oxidative decarboxylation of pyruvate, phosphate and oxygen to acetyl phosphate, carbon dioxide and hydrogen peroxide in the presence of cofactors such as flavin adenine dinucleotide (FAD), thiamine pyrophosphate chloride (TPP) and magnesium (Mg + 2 ).…”