ИСТИНА

В проекте предлагается разработать лактатные биосенсоры (второго поколения) с широким диапазоном линейности отклика на основе печатных электродных структур, в том числе модифицированных медиаторами и электрокатализаторами на стадии печати.

Compact electrochemical test systems, as well as (bio)sensor devices, are widely used in different fields of analysis, especially for medical purposes. As part of the transition to personalized medicine, high-tech healthcare and health-saving technologies (a priority area for the development of the Russian Scientific and Technical Complex), the development of easy-to-implement and operate express analysis methods is an important area of research. Electrochemical devices are one of the few medical devices available to any user for self-testing at home (for example, glucometers). In addition to glucose, lactate is an important metabolite, which is gaining interest by the year, especially in the field of medicine. Lactate serves as a marker of glycolysis, an anaerobic glucose metabolism, which makes it an important object of study for both clinical diagnostics and sports medicine. An elevated concentration of lactate serves as an indicator of oxidative stress in the body or in an organ. To date, interest in lactate is growing on the part of medicine, and devices for this purpose, in addition to laboratory clinical analyzers, are practically absent on the market. Despite the rapid development of continuous lactate monitoring, continuous sample flow, whether blood or non-invasively collected sweat, is difficult to achieve at home. Personal devices, similar to glucometers, will allow one to carry out express lactate analysis by themselves and do not require a large amount of a biosample. The actual task the project is aimed at is the creation of lactate (bio)sensors (test strips) for the analysis of invasively taken fluids (blood) and for non-invasive diagnostics (sweat analysis). The problem of direct determination of lactate in physiological fluids is the mismatch between the operating range of lactate biosensors and the content of this metabolite in the object of study. The linear range of most lactate biosensors is limited to 1 mmol/L, and therefore the main scientific task in the field of lactate detection is to expand the range of determined concentrations (limited by enzymatic kinetics) to the high values. Analysis of biosamples without preliminary sample preparation is a difficult task, firstly, from the point of view of the working range of sensors, which should be shifted to high concentrations, both for sweat (5–100 mM) and blood (0.5–20 mM) analysis, and second, because of matrix effects. A common method of solving the problem by forming additional diffusion-limiting membranes on top of the enzyme layer seems to be inappropriate, since it significantly complicates the manufacture of the biosensor and reduces the reproducibility of the sensor manufacturing technology. The simplest and inexpensive approach is the fabrication of electrode structures and biosensors by screen printing. The project proposes to increase the upper limit of the determined lactate concentrations by creating diffusion restrictions on the substrate and mediator without additional membranes. In the case of a charged substrate (lactate ion), the charge of the polyelectrolyte membrane affects not only the diffusion mobility of the mediator (immobilized on the electrode surface in the membrane or included in the material of the working electrode), but also the mobility of the analyte itself. An alternative way to shift the operating range of sensors for objects with a high content of lactate (sweat) can be enzyme-free sensors that function due to the specific interaction of polymeric aminophenylboronic acids with lactate hydroxyl groups. The development of enzyme-free methods for the detection of metabolites will make it possible to abandon the use of unstable biomolecules in the analysis, extend the life of sensors, and increase stability. The inclusion of a conductive polymer in the composition of graphite paste at the printing stage will simplify the procedure for manufacturing the sensor and eliminate the stage of electrochemical synthesis of the polymer. Implementation of the amperometric, potentiometric, and/or impedimetric method for detecting lactate on printed electrode structures without the participation of enzymes will expand the use of conductive polymers in analysis. In the course of the project implementation, it is planned to apply three approaches to the creation of lactate (bio)sensors: 1) co-immobilization of both the enzyme and the mediator in a polyelectrolyte membrane on the surface of printed electrodes, 2) modification of the working electrode with mediators and electrocatalysts at the printing stage with subsequent immobilization of the enzyme on top of such a volume-modified electrode; 3) development of enzyme-free sensors for lactate based on conductive polymers included in the electrode material, and methods for recording their signal. Thus, the proposed approaches to prolonging the range of lactate biosensors to the high concentrations are based on the study of electrostatic interactions between the analyte, mediator, and polymer matrix for enzyme immobilization. The project proposes to develop mediator biosensors (second generation), the signal of which is not limited by the oxygen concentration in the sample, which is a limitation for first generation biosensors based on platinum or Prussian blue. In addition, all the proposed approaches are designed to simplify the technological process of sensor production and include the stage of printing the electrode structure and its single droplet modification. In view of the inaccessibility and deterioration of the quality of imported materials, it is additionally planned to adapt available domestic graphite pastes for the manufacture of electrodes, which determines the industrial significance of the project results. To improve the properties of graphite pastes and membrane-forming mixtures, it is planned to test additives of carbon nanomaterials. It is known that carbon nanomaterials due to their properties - electrical conductivity, chemical and mechanical stability, as well as high surface area are applicable to improve the efficiency of electrochemical sensors.

В результате проекта будут получены: 1) Двухэлектродные и трехэлектродные сенсоры, изготавливаемые методом трафаретной печати, в том числе на основе паст, импрегнированных медиаторами, наночастицами катализаторов и проводящими полимерами на стадии печати. 2) Амперометрические биосенсоры и лактатные тест-полоски на основе электродов, модифицированных медиатором на стадии печати. 3) Медиаторные тест-полоски на лактат с расширенным линейным диапазоном на основе капельной модификации электродов мембранообразующей смесью, содержащей и фермент, и медиатор в растворе полимера. 4) Бесферментные электрохимические сенсоры на лактат, функционирующие за счет специфических взаимодействий с функционализированными проводящими полимерами.

Научная группа заявителей успешно развивает подходы к созданию высокостабильных и высокочувствительных биосенсоров, применимых для детектирования глюкозы и лактата в различных биологических жидкостях (кровь, пот, конденсат выдыхаемого воздуха), на основе электроактивных покрытий берлинской лазури, проводящих полимеров, медиаторов и электрополимеризованных красителей в качестве трансдьюсеров. В течение нескольких последних лет в научном коллективе заявителей ведутся работы по созданию тест-полосок для определения глюкозы. Достижения научного коллектива в области применения технологии трафаретной печати и создания (био)сенсоров на основе модифицированных углеродных паст подтверждают возможность адаптации новых углеродных паст отечественного производства для создания сенсоров и биосенсоров. Имеющиеся достижения в области применения различных электроаналитических методов (потенциометрических, амперометрических и импедиметрических), а также синтеза проводящих полимеров с «молекулярными отпечатками», подходящих для селективного определения лактата доказывает достижимость создания бесферментных биосенсоров для определения лактата в биологических средах.