Simultaneous determination of orthophosphate and total phosphates (inorganic phosphates plus purine nucleotides) using a bioamperometric flow-injection system made up by a 16-way switching valve

Orthophosphate and total phosphates (inorganic phosphates plus purine nucleotides) can be determined simultaneously in a novel flow-injection system made up by a 16-way switching valve with two sample loops, acid phosphatase (AcP) immobilized reactor and a delay coil needed to separate two peaks corresponding to two sample portions injected simultaneously. An orthophosphate enzyme electrode with a hybrid membrane of trienzyme film and poly(1,2-diaminobenzene) film was used to selectively detect both the endogenous orthophosphate and orthophosphate generated enzymatically into the AcP immobilized reactor, without any interferences from electroactive species, such as ascorbate and urate. Because two sample portions passed through the flow line with different residence time, two peaks were obtained. The first peak corresponded selectively to orthophosphate and the second peak to the total of inorganic phosphates and purine nucleotides. The maximum currents of both peaks were linearly related to the concentration of orthophosphate and total phosphates (as orthophosphate) in the range 5×10⁻⁷-8×10⁻⁴ M, respectively; 30 samples per hour can be processed with an R.S.D. <2.5%.     

Rapid measurement of fish freshness indices by an amperometric flow-injection system with a 16-way switching valve and immobilized enzyme reactors

A novel flow-injection system is proposed for the rapid measurement of the fish freshness indices K₁ and K₂: K₁=[([HxR+[Hx])×100/([IMP]+[HxR]+[Hx])] and K₂=[[Hx]×100/([HxR]+[Hx])], where [IMP], [HxR] and [Hx] are inosine-5′-monophosphate, inosine and hypoxanthine concentrations, respectively. For the estimation of index K₁, 5′-nucleotidase immobilized reactor and nucleoside phosphorylase (NP)/xanthine oxidase (XO) coimmobilized reactor were incorporated in series in the flow-injection line made up by a 16-way switching valve with two sample loops. For the estimation of index K₂, NP and XO immobilized reactors were also incorporated in the similar flow-line. Two sample portions passed through the flow-line with different residence times so that two peaks were obtained. The first and second peaks obtained in the K₁-determining system corresponded to the total of HxR and Hx and the total of Hx, HxR and IMP, respectively. Similarly, the first and second peaks obtained in the K₂-determining system corresponded to Hx and the total of Hx and HxR, respectively. Therefore, the indices K₁ and K₂ can be estimated by

where i₁ and i₂ present the peak current of the first and second peaks, respectively, and f the ratio of the peak currents of the first and second peaks for a Hx standard solution. A sea bream was selected as a model fish and it was stored at 4 °C after death. A good correlation was found between the index K₁ and the storage time over a period of 400 h, with a correlation coefficient of 0.992, but no correlation between the index K₂ and the storage time. The measurements could be performed at a rate of 22 samples per hour with satisfactory precision (0.6–1.2% R.S.D.), without calibration for each species, accurate weighing of fish meat and any interferences in fish meat.     

On-line microdialysis assay of l-lactate and pyruvate in vitro and in vivo by a flow-injection system with a dual enzyme electrode

A flow-injection biosensor system with an on-line microdialysis sampling is proposed for the simultaneous assay of l-lactate and pyruvate in serum and rat brain. The dialysate collected in the sample loop by perfusing Ringer’s solution through the microdialysis probe is automatically injected into the flow-injection line with a dual enzyme electrode arranged in parallel for the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film to two sensing parts, which respond selectively to l-lactate and pyruvate, respectively, without any cross-reactivity. Both the sensing parts respond linearly to the concentrations of both analytes between 0.01 and 5 mM, without any interference from oxidizable species and low-molecular weight proteins present in the dialysate. The proposed flow-injection analysis (FIA) method can be successfully applied to the simultaneous in vitro and in vivo assays of both analytes in serum and rat brain, respectively. The system can be automatically processed at an analytical speed of 19 dialysates h⁻¹ over a period of 5 h.