Electroanalytic system based on accumulative column biosensor for determination of low lactate concentrations

FIELD: chemistry.

SUBSTANCE: system consists of two peristaltic pumps, a loop-containing injector, a flow amperometric cell with includes a biosensor, a potentiostat. As the biosensor, the electroanalytic system contains a lactate biosensor. The injector loop includes an accumulative column.

EFFECT: increase of selectivity and sensitivity of determination, reduction of the limit of lactate detection.

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The technical field to which the invention relates

The invention relates to electroanalytical systems. Electroanalytical system includes a flow detector type wall-jet (operating in continuous flow mode) with an integrated high-performance the lactate biosensor belonging to the class of electrochemical biosensors, as well as a column for sorption concentration. Concentration is based on ion exchange.

The level of technology

Lactate is one of the most important chemical compounds from the point of view of clinical diagnostics. It is a marker of glycolysis, anaerobic metabolism. leading to the death of tissues in the body. Sports medicine considers the monitoring of lactate as a means of determining the so-called "lactate threshold" [Faude, O., W. Kindermann, T. Meyer. Lactate threshold concepts: how valid are they? Sports Medicine 39 (2009) 469-90], [H. Heck, R. Beneke. 30 years of lactate thresholds - what remains to be done? Deutsche Zeitschrift Fur Sportmedizin 59 (2008) 297-302) showing the level of fitness of the athlete. Lactate as a product of fermentation [S. Q. Liu. Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations. International Journal of Food Microbiology 83 (2003) 115-31] may serve as a marker of natural food products.

Not surprisingly, the lactate-sensitive electrodes were among the first of biosensors [D. L. Williams, A. R. Doig, A. Korosi. Electrochemical-enzymatic analysis ofblood glucose and lactate. Analytical Chemistry 42 (1970) 118-21]. Subsequently, we have developed a large number lactate biosensors [I. Karube, T. Matsunaga, N. Teraoka, S. Suzuki. Microbioassay of phenylalanine in blood sera with a lactate electrode. Analytica Chemie 119 Acta (1980) 271-6], |M. Mascini, D. Moscone, G. Palleschi. A lactate electrode with lactate oxidase immobilized on nylon net for blood-serum samples in flow systems. Analytica Chemie Acta 157 (1984) 45-51], [F. Mizutani, K. Sasakl, Y. Shlmura. Sequential determination of l-lactate and lactate dehydrogenase with immobilized enzyme electrode. Analytical Chemistry 55 (1983) 35-8], [J. G. Schindler, M. Vongulich. L-lactate flow-through electrode with immobilized lactate oxidase. Fresenius Zeitschrift Fur Analytische Chemie 308 (1981) 434-6], [D. L. Wang, A. Heller. Miniaturized flexible amperometric lactate probe. Analytical Chemistry 65 (1993) 1069-73], including the last one with better analytical characteristics [I. E. Yashina, A. V. Borisova, E. E. Karyakina. O. I. Shchegolikhina, M. Y. Vagin, D. A. Sakharov, A. G. Tonevitsky, A. A. Karyakin. Sol-gel immobilization of lactate oxidase from organic solvent: advanced Toward the lactate biosensor. Analytical Chemistry 82 (2010) 1601-4]. However, for all known systems of the lower boundary of the designated content was not less than several micromoles. This, in particular, is not sufficient for non-invasive analysis of collected exhaled breath condensate, the lactate level in which, as reported in an article by E. M. Marek et al., is in the range of units of micromoles in a liter [E. M. Marek, J. Volke, I. Hawener, P. Platen, K. Muckenhoff, W. Marek. Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects. Journal of Breath Research 4 (2010)].

In order to improve the analytical characteristics of the methods of analysis, namely the lowering of the detection limit�tion and increase the sensitivity and selectivity of widely used methods of concentration, based, in particular, ion exchange, when the designated substance is captured by a suitable sorbent in the cartridge or column, and then desorbed and determined in decorate [Q. Jia, X. Kong, W. Zhou, L. Bi Flow-injection on-line preconcentration with an ion-exchange resin coupled with microwave plasma torch atomic emission spectrometry for the determination of trace rare earth elements. Microchemistry 89 (2008) 82-87]. Such methods are simple hardware design, low cost and possibility of automation.

Analog of the invention is a system flow-injection analysis (FIA) based on a lactate biosensor with the best analytical characteristics described in [I. E. Yashina, A. V. Borisova, E. E. Karyakina, O. I. Shchegolikhina, M. Y. Vagin, D. A. Sakharov, A. G. Tonevitsky, A. A. Karyakin. Sol-gel immobilization of lactate oxidase from organic solvent: advanced Toward the lactate biosensor. Analytical Chemistry 82 (2010) 1601-4]. This electroanalytical system based biosensor consists of two peristaltic pumps, loop injector, flow-through amperometric cell type wall-jet with integrated biosensor potentiostat.

As already mentioned, existing lactate electroanalytical biosensors and systems based on them do not have sufficient sensitivity for non-invasive diagnostics and, in particular, analysis of exhaled breath condensate. Furthermore, hydrogen peroxide, is a mediator of the majority of biosensors, including those described in the prior art, �goes into the composition of many real objects and can distort the results of the determination of low concentrations of substances. Thus, there is also the need to improve the selectivity of the analysis.

Disclosure of invention

The problem solved by the authors of the present invention is the development of electroanalytical system for determination of lactate, which allows to determine low concentrations of lactate. The technical result of the invention consists in increasing the selectivity and detection sensitivity, lower limit of detection of lactate. The proposed system allows the non-invasive determination of the concentration of lactate, for example, when using as the test sample of sweat or urine. Said technical result is achieved due to the fact that in the injection loop included the concentrating column.

The proposed electroanalytical invention is a flow-injection system, including a system of pumping liquids containing a loop injector, flow-through electrochemical cell integrated with the lactate biosensor, the potentiostat and the Registrar of the signal, characterized in that to increase the sensitivity and selectivity of the analysis in a loop injector included the concentrating column.

Despite the apparent simplicity of the system, a combination of sorption of the concentrating column with the biosensor was represented extremely complex task�. The complexity of the development of the present invention is that, on the one hand, ion exchange little selective, and lactate is a pretty small hydrophilic molecule, so pick a selective method of concentration for him is extremely difficult. On the other hand, lactate biosensor sensitive to any changes in ionic strength. For the development of electroanalytical system and overcoming these difficulties is carefully selected scheme of the laboratory setup and the experimental conditions. Namely, we have studied the use of multiple ion-exchange sorbents; found that the most effective sorption of lactate is observed when the ion-exchange sorbents, well able to hold lactate, in particular sorbent Strata SAX silica gel grafted with Quaternary ammonium groups.

According to the present invention can be optimized volume column for concentration, because if you select too large speaker problems arise at the stage of sorption, and the use of small, low degree of extraction. The same can be optimized composition desorbers solution for quantitative sorption of lactate, which complicates the determination of lactate using biosensors. Private embodiment has been optimized volume passed�solution at all stages, the flow rate, column length 10 mm, diameter 2 mm.

The proposed electroanalytical system with preliminary concentration is shown schematically in the Figure. 1. It consists of pumping the liquid (which can be used two peristaltic pump, injector, flow-through electrochemical cell included with the lactate biosensor connected to the potentiostat and the Registrar of the signal. In a loop injector is enabled column for concentration. Private embodiment of the present invention, the electrochemical cell can be used amperometric flow cell. As a lactate biosensor can be used, for example, a planar electrode modified Prussian blue with immobilized enzyme lackadasical.

According to the present invention as a Registrar of the signal may be any device that provides a read signal, in particular a personal computer.

Electroanalytical system works as follows. In the experiment, the peristaltic pump (2) continuously circulates through the system supporting electrolyte from the vessel (9). When the handle position of the injector (3) in the "download" peristaltic pump (1) pump analyze Rast�the PR from the vessel (7) through the concentrating column (6), included in the loop of the injector (3), wherein the lactate from the test solution are adsorbed by aminoalkanoic filling column (6). After the aliquot of the test solution passes through the concentrating column (6) and go to the drain (8), the pen injector is rotated into the "injection", with a peristaltic pump (2) supporting electrolyte circulates from the vessel (9) through the concentrating column (6). At this stage, the desorption of lactate, which in concentrated towards the source of the analyzed solution form is pumped to lactate biosensors in flow-through amperometric cell (4). The potentiostat (5) registers a peak at amperograms.

The invention is illustrated by the following examples.

Brief description of the drawings

Figure 1 schematically shows a private version of the proposed electroanalytical system with preliminary concentration. It consists of two peristaltic pumps (1) and (2) of the injector (3), flow-through amperometric cell included with the lactate biosensor (4) connected to a personal computer potentiostat (5). In a loop injector (3) is enabled column for the concentration (6). Other positions on the drawing: (7) - a vessel for the test solution; and (8) - drain; (9) a vessel of the supporting electrolyte, which also serve as decor�youseo solution.

Figure 2 presents the calibration dependence of the analytical signal from the concentration of lactate in the sample for electroanalytical system based on the lactate biosensor concentration without it.

The implementation of the invention

Example 1. Determination of lactate in the system flow-injection analysis with preliminary concentration.

The experiments were carried out in the system of PIA in phosphate buffer (0.1 M KCl, 0.05 M KH2PO4, pH 6.0). The flow rate of buffer solution of 0.40 ml/min operating voltage 0.0 Rel. HSA (silver chloride electrode).

The calibration dependence of the analytical signal from the concentration of lactate in the sample for electroanalytical system based on the lactate biosensor concentration and without it is shown in Fig.2. It is seen that the system PIA concentration provides increased sensitivity 50 times and lower detection limit by more than an order.

According to available data, the concentration of hydrogen peroxide in exhaled breath condensate (SSC) does not exceed 5,0×10-3M.

Example 2. To increase the selectivity (for example, shaped sweat test).

The experiments were carried out in the system of PIA in phosphate buffer (0.1 M KCl, 0.05 M KH2PO4, pH 6.0). The flow rate of buffer solution of 0.40 ml/min operating voltage 0.0 Rel. HSA.

To increase the selectivity (for example, the sweat test sample)The analyzed sampleThe dilution timeMethodWith lactate, MSweat500PIA (P=0.95, n=4)(3.4±0,1)×10-2Sweat + 2×10-4MH2O2(4.6±0,3)×10-1Sweat25000PIA+conc-e (R=0.95, n=3)(3.3±0,5)×10-2Sweat + 2×10-3MH2O2(3.4±0,6)×10-2

As can be seen from the table, hydrogen peroxide, even in large numbers does not interfere with the determination of lactate in the system PIA concentration.

Thus, achieved the claimed technical effect (increase the selectivity and detection sensitivity, lower detection limit).

1. Electroanalytical flow-injection system, including a system of pumping liquids containing a loop injector, flow-through electrochemical cell with integrated La�tatnam the biosensor, the potentiostat and the Registrar of the signal, characterized in that to increase the sensitivity and selectivity of the analysis in a loop injector included the concentrating column with ion exchange sorbent, well able to hold lactate.

2. Electroanalytical flow-injection system according to claim 1, characterized by the fact that as the sorbent used is ion exchange sorbent Strata SAX silica gel grafted with Quaternary ammonium groups.



 

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