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Karyakin, Arkady Arkadevich. Enzyme electrodes using polymer semiconductors and inorganic polycrystals: abstract of thesis. ... Doctor of Chemical Sciences: 02.00.15 / Moscow State University. - Moscow, 1996. - 33 p.: ill. RSL OD, 9 96-4/634-2
Introduction to the work
Relevance of the problem, The proposed dissertation work is devoted to the ways of coupling electrode and enzymatic reactions. By the term “conjugation” the author means that the electrochemical reaction occurs in response to an act of biological recognition, which in this work is considered to be an enzymatic reaction. According to the generally accepted classification, enzyme electrodes are divided into three groups. The enzyme's active site can exchange electrons directly with the electrode material, as occurs in third-generation enzyme electrodes. Second generation enzyme electrodes are based on the use of diffusionally mobile or immobilized mediators for this purpose. Until now, the improvement of first-generation biosensors operating on the principle of oxidation-reduction of a conjugated substrate or enzymatic reaction product has not lost its relevance. In the proposed work, all three types of enzyme electrodes will be considered.
Currently, the requirements of clinical diagnostics, environmental protection and various industrial fields are driving the search for cheap, specific and rapid analysis methods. Electrochemical biosensors meet these requirements perfectly. The simplicity of the recording device and the specificity of biological recognition, coupled with high rates of catalysis, provide biological sensors with priority in bioanalytical chemistry. It is not without reason that just a few years after the discovery of the first biosensor, it was accepted for mass production by Yellow Springs Instruments. The success of another biosensor, the personal glucose detector, can be illustrated by the following figures: production, which began as a modest company in 1987, reached a turnover of half a billion US dollars per year in just seven years.
Not surprisingly, the proposed work also focuses on enzyme-based electroanalytical devices. The formulation of some problems actually arose from the need to improve existing biosensors.
From a practical point of view, it is important to note the use of enzyme electrodes also for the development of fuel cells and biospecific electrosynthesis systems. And, if the task of creating biofuel elements has somewhat lost its relevance over the past ten years, having shifted geographically to the countries of the Middle and Southeast Asia, then the problems of bioelectrosynthesis still have to be solved, perhaps in the near future. From a future technology perspective, electrode-enzyme reaction coupling systems may find unexpected applications as input/output devices in biological computers.
As it seemed when formulating the problem, such a study should be
is devoted to the application of knowledge accumulated by modern electrochemistry for the purposes of bioelectrocatalysis. However, the operating conditions of biological catalysts dictate their own requirements for the properties of modified electrodes. Thus, when performing this work, the author had to solve the actual electrochemical problems. The most striking examples include the prolongation of the redox activity of polyaniline to the range of physiological pH and the study of a new group of electrochemically active polymers obtained by electropolymerization of redox indicators of the azine series.
The purpose of the work there was a search for new ways to couple enzymatic and electrochemical reactions for the development of enzyme electrodes of the first, second and third generations using polymer semiconductor films and inorganic polycrystals. The development of enzyme electrodes was planned mainly for reasons of creating new, more advanced electroanalytical systems.
Scientific novelty. The proposed dissertation covers all existing types of coupling of electrode and enzymatic reactions. Beginning with the phenomenon of direct bioelectrocatalysis, research then moves into the application of conductive polymers and inorganic polycrystals to create first and second generation enzyme electrodes.
The dissertation work lays the foundations of several scientific directions. The phenomenon of bioelectrocatalysis by hydrogenases has formed the basis of numerous works in this area. Perhaps, what is still original is the comparison of the mechanisms of action of the enzyme in homogeneous and electrochemical modes. The proposed molecular mechanism of action of hydrogenases allowed the author to formulate a hypothesis about the inclusion of enzymes in direct bioelectrocatalysis through the mechanism of direct exchange of electrons between the active center of the enzyme and the electrode.
An independent area was the study of electropolymerization of azine dyes, which are mediators of bioelectrochemical reactions. The study of the structure of a new group of polymers and optimization of the conditions for their electrosynthesis have resulted in an independent scientific direction. The resulting polymers retained the properties of the original monomers, being a form of immobilization of mediators on the electrodes, and at the same time exhibited new unconventional properties. In particular, polymeric azines turned out to be effective electrocatalysts for the regeneration of cofactors, which made it possible to create dehydrogenase electrodes based on them.
Fundamental for the fundamental and applied electrochemistry of conducting polymers was the synthesis of self-doped polyaniline, which is electrochemically active in neutral and alkaline aqueous solutions. Using a self-doped polymer as an example, it was possible to trace the properties of polyaniline at high pH values. When moving from
It was proposed to create potentiometric biosensors based on yulianiline. In addition to the technological advantages of using a conductive polymer as a sensing element, the resulting biosensors had much higher sensitivity compared to known systems.
The proposed work contains a priority for the use of inorganic
ulicrystals of Prussian Blue for biosensor purposes. Managed to synthesize
Shock catalyst for selective reduction of hydrogen peroxide, insensitive
: oxygen in a wide range of potentials. This solved the age-old problem
imperometric biosensors - interfering influence of reducing agents. 4
Finally, the undoubted successful results achieved in this work include the optimization of enzyme immobilization on the surface of modified electrodes. The proposed method for the formation of enzyme-containing membranes made it possible to significantly increase the stability of biological catalysts.
Practical value consists primarily of creating new types of enzyme electrodes suitable for a variety of applications.
First generation enzyme electrodes based on Prussian Blue were developed for use in electroanalytical systems. Replacing platinum with an electrode modified with an inorganic polycrystal not only reduces the cost of the biosensor. Due to their high sorption activity, catalysts based on platinum group utalls can be poisoned by a large number of low molecular weight compounds, including thiols, sulfides, etc., which is not typical for electrocatalysts based on Prussian Blue. Due to the multilayer structure of the latter on modified electrodes, it is possible to achieve the highest current densities of hydrogen peroxide reduction in comparison with known electrocatalytic systems. Using a glucose biosensor based on Prussian Blue, the high sensitivity and selectivity of the sensors, which meet the requirements of non-invasive diagnostics, were demonstrated.
Synthesis of an electrocatalyst for the reduction of hydrogen peroxide, insensitive to oxygen, based on Prussian Blue, can significantly reduce the potential of the indicator electrode, which makes the sensor response independent of the presence of reducing agents such as ascorbate and paracetamol and thus allows us to solve the most important problem of amperometric biosensors based on oxidases . The use of the developed electrode as a detector in a flow injection system increases the speed of analysis. In addition to the demonstrated analysis of gluten-
4 goats and ethanol, a similar biosensor can be made to analyze any substance in the presence of the appropriate oxidase. Among the practically important substances that can be analyzed in this way are cholesterol, glycerol, amino acids, and galactose. Areas of application for biosensors based on Prussian Blue are clinical diagnostics and some areas of the food industry.
An important practical result is the development of potentiometric biosensors based on polyaniline. The use of the latter as a pH transducer makes it possible to increase the sensitivity of biosensors. The polyaniline-based glucose enzyme electrode had a three to four times higher response compared to a glucose-sensitive field-effect transistor. The detection limit of organophosphorus substances with a polyaniline-based biosensor was 10-7 m, which is lower than for known potentiometric systems (10/5 * 10 _ 6 M). Potentiometric biosensors based on polyaniline can be used in clinical diagnostics for the analysis of the same glucose, as well as bound cholesterol, triacylglycerides, etc. It is possible to use potentiometric biosensors based on polyaniline for environmental protection.
The creation of dehydrogenase electrodes opens up great opportunities for electroanalytical purposes, since the enzymes of this group number more than 500 names and catalyze the transformation of a wide variety of substances. Electropolymerization is a method of immobilizing mediators used in bioelectrocatalytic reactions on an electrode. The resulting modified electrodes are more efficient electrocatalysts and exhibit tens of times higher operational stability. The use of polymeric azines makes it possible to create biosensors for both oxidizing and reducing substrates of dehydrogenases, since the electrochemical regeneration of the NAD + /NADH cofactor can be carried out in any direction. Along with cofactor-dependent ones, short-lived reagent-free biosensors based on dehydrogenases have been developed.
Dehydrogenase electrodes, along with a reagent-free hydrogen enzyme electrode, can also be used to create biofuel cells.
The method of immobilizing enzymes into water-insoluble polyelectrolytes from water-alcohol mixtures with a high content of organic solvent has practical value. Nation enzyme-containing membranes have high stability and good adhesion to the surface of modified electrodes. In addition, such membranes are biocompatible.
Finally, the developed modified electrodes based on self-doped polyaniline, polymer azines, Prussian Blue and films requiring anodic and cathodic initiation can find application along with biotechnology.
5 chemical and in other areas of electrochemistry.
Research methods. The work used electrochemical and kinetic methods in modes that provide maximum information content. In kinetic studies, the concentration of the substrate or product of the enzymatic reaction was controlled spectrophotometrically or polarographically. Kinetic analysis was carried out using both initial reaction rates and full kinetics. To simplify the kinetic analysis, a generalized form of writing the rate equation for unbranched catalytic reactions in a stationary mode was proposed. Electrochemical studies were based on the methods of stationary polarization curves and cyclic voltammetry. The electrochemical impedance method was also used. Electropolymerization and electrodeposition were carried out in totenciodynamic and potentiostatic modes. To study electrochemical kinetics, it was necessary to use the rotating disk electrode method. The developed chemical and biological sensors were studied in sperometry modes at a constant potential of the indicator electrode and potentiometry. To analyze the structure of polymer azines, methods of spectroelectrochemistry and infrared spectroscopy were used. In order to increase the speed of analysis, a flow-injection installation was assembled with an electrochemical cell of the wall-jet type, which ensures an advantageous hydrodynamic mode of the indicator electrode.
Approbation of work. The results of the work were presented at Russian and international conferences: International Symposium on the Molecular Biology of Hydrogenases (Szeged, 1985), III All-Union Conference "Chemical Sensors" (Leningrad, 989), International Symposium on Bioanalytical Methods (Prague, 1990), International Congress "Sensors and Information Converters" (Yalta, 1991), International Conference "Biotechnology in Great Britain" (Leeds, 1991), Russian-German Meetings on Biosensors (Moscow, 1992, Munster, 1993), VII All-Union Imposium on Engineering Enzymology (Moscow, 1992), International scientific school on biosensor materials (Pushchino, 1994), seminar on the electrochemistry of conducting polymers at the Institute of Electrochemistry named after. A.N. Frumkin RAS (Moscow, 1995), International Meeting on the Electrochemistry of Electroactive Polymer Coatings, /VEEPF "95 (Moscow, 1995), IX International Conference "Eurosensors and Ransducers"95" (Stockholm, 1995), III International Meeting "Biosensor Systems for Industrial applications" (Lund, 1995), International Conference 5iocatalysis-95" (Suzdal, 1995), V International Symposium "Kinetics in Chalytic Chemistry" (Moscow, 1995), at a meeting of the electrochemical societies of Portugal and Spain (Apgarve, 1995), at I International Symposium on Biosensors of the Gran Pacific Region (Wollongong, 1995), at the International Meeting on
multifunctional polymers and thin polymer systems (Wollongong, 1996), at the VI International Conference on Electroanalysis "ESEAC96" (Durham, 1996).
Publications. Based on the dissertation materials, 41 printed works have been published, and an author's certificate has been received.
Structure and scope of work. The dissertation is a manuscript consisting of 12 chapters, introduction and conclusion, as well as conclusions and a list of cited literature (347 titles). The volume of the dissertation is 383 pages, including 76 figures and 8 tables.
JOURNAL OF ANALYTICAL CHEMISTRY, 2009, volume 64, no. 12, p. 1322-1323
ANNIVERSARY A.A. KARYAKIN
On December 9, 2009, Arkady Arkadyevich Karyakin, Doctor of Chemical Sciences, Professor and Head of the Laboratory of Electrochemical Methods of the Department of Analytical Chemistry of Moscow State University, celebrates his 50th anniversary. M.V. Lomonosov (MSU).
A.A. Karyakin was born in Moscow into a family of chemists. His father, Arkady Vasilyevich Karyakin, was a professor and head of a laboratory at the Institute of Geochemistry and Analytical Chemistry named after. Vernadsky Academy of Sciences of the USSR. After graduating with honors from the Faculty of Chemistry of Moscow State University in 1981, A. A. Karyakin continued to work at the faculty, rising from assistant to professor. In 1985 he defended his PhD thesis in the specialty “kinetics and catalysis” on the topic: “Chemical and electrochemical kinetics of the action of the enzyme hydrogenase”, and in 1996 he defended his doctoral dissertation in the same specialty on the topic “Enzyme electrodes based on semiconductor polymers and inorganic polycrystals".
His scientific interests are wide and varied. The main priority of activity, formed at the Department of Chemical Enzymology and implemented at the Department of Analytical Chemistry, is the development and application of new methods of electrochemical analysis using catalytic systems based on inorganic polycrystals, conducting polymers and biomolecules. Among the works carried out under the leadership of Arkady Arkadyevich, one can highlight the development of electrochemical sensors for the determination of hydrogen peroxide, which have record characteristics, as well as the construction on their basis of biosensors using enzymes of the oxidases class. He has authority in this field, both in the domestic scientific community and abroad. Research continues successfully, resulting in the development of sensors for T/U monitoring of human metabolites, systems for clinical analysis and food quality control. Being one of the pioneers in the
ANNIVERSARY A.A. KARYAKIN
field of direct bioelectrocatalysis, A.A. Karjakin continues the study of hydrogen enzyme electrodes based on hydrogenases, which he began even before defending his first dissertation. He developed fuel cells based on enzymes that have extreme current characteristics and function in a bacterial environment.
Under the leadership of Arkady Arkadyevich, 8 candidate dissertations were successfully defended, he published 4 monographs, together with his colleagues - 9 reviews, over 70 original articles, received 3 patents, and made many reports. He is a member of the editorial boards of the scientific journals Electroanalysis, Electrochemistry Communications and Talanta. Arkady Arkadyevich is actively developing international cooperation with leading scientific teams abroad. Among colleagues and friends of A.A. Karjakin widely known scientists from Sweden, Germany, Italy, USA and others
countries Research conducted under the direction of A.A. Karyakin, are supported by Russian and European scientific foundations. He is a member of two dissertation councils at the Faculty of Chemistry of Moscow State University.
Arkady Arkadyevich is engaged in classical singing. He is a member of the vocal studio at the Central House of Scientists of the Russian Academy of Sciences, led by People's Artist of the USSR Z.L. Sotkelava, enjoys horse riding and skiing. He is always friendly, actively collaborates with specialists in various fields of science, and enjoys authority among his colleagues and students.
Colleagues and friends, the editorial board of the Journal of Analytical Chemistry cordially congratulate Arkady Arkadyevich on his anniversary and wish him health and great creative success in his scientific and pedagogical activities.
JOURNAL OF ANALYTICAL CHEMISTRY volume 64< 12 2009
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The invention relates to a method for preparing a highly stable sensor element for hydrogen peroxide and can be used in analytical chemistry, clinical diagnostics, environmental monitoring, and in various fields of industry. The method involves stabilizing Prussian blue with nickel hexacyanoferrate. In this case, sequential deposition of Prussian blue and nickel hexacyanoferrate is carried out. The method makes it possible to create sensors with high sensitivity, selectivity, and good reproducibility of the current signal, i.e. with high stability. 1 salary f-ly, 2 ill.
Drawings for RF patent 2442976
The invention relates to a method for preparing a sensitive element of a sensor for hydrogen peroxide. In particular, to a method for stabilizing Prussian blue, which is an electrocatalyst for the reduction of hydrogen peroxide, with nickel hexacyanoferrate.
The determination of hydrogen peroxide is an important analytical task for clinical diagnostics, environmental monitoring and various industrial applications. Its content must be determined in groundwater and atmospheric precipitation, where it ends up as a result of emissions from industry and nuclear power plants, as well as in the food industry.
Today, the most effective sensing element for the determination of hydrogen peroxide is Prussian blue—iron(III) hexacyanoferrate(II). Inert electrodes (platinum, gold, glassy carbon) modified with Prussian blue are widely used in the design of hydrogen peroxide sensors and biosensors containing immobilized oxidases as a biosensitive element.
When the Prussian blue film interacts with the determined hydrogen peroxide, the latter decomposes to the hydroxide ion OH - . At low concentrations of hydrogen peroxide, its effect on the properties of the sensor is insignificant. However, during continuous measurements, a significant amount of hydroxide ions can be formed, which leads to the gradual dissolution of the Prussian blue coating from the electrode surface. To carry out continuous monitoring of hydrogen peroxide content, sensors are required that, along with high sensitivity and selectivity, have good reproducibility of the current signal, that is, they have high stability.
The essence of the invention is as follows:
A method has been proposed for joint deposition of a sensitive element (Prussian blue) and a stabilizer (nickel hexacyanoferrate) onto the surface of an electrode to produce a highly stable sensor for hydrogen peroxide;
A method has been proposed for sequential deposition of a sensitive element (Prussian blue) and a stabilizer (nickel hexacyanoferrate) onto the surface of an electrode to produce a highly stable sensor for hydrogen peroxide.
Electrochemical method of joint deposition of Prussian blue and nickel hexacyanoferrate on the surface of an electrode
Joint electrodeposition of nickel hexacyanoferrate and Prussian blue was carried out in potentiodynamic mode, when the potential applied to the working electrode was swept from 0 to +0.75 V, the potential sweep rate was 50-100 mV/s, for 5-20 cycles. The synthesis was carried out in a three-electrode cell containing a working electrode, a silver chloride reference electrode, and a glassy carbon auxiliary electrode. The growth solution contained 1 mM K3 and x mM NiCl2 and (1-x) mM FeCl3 (x from 0.1 to 0.9) in a background electrolyte of 0.1 M KCl, 0.1 M HCl.
Then the electrodes were cycled in the potential range from 0 to +1 V in a background electrolyte of 0.1 M KCl, 0.1 M HCl at a potential sweep rate of 40 mV/sec for 20 cycles. After which the electrodes were subjected to heat treatment at 100°C for 1 hour and cooled to room temperature.
Figure 1 shows a comparison of the current versus time dependences in a constant flow of 1·10 -3 M H 2 O 2 for sensors with sensitive elements based on Prussian blue and Prussian blue stabilized with nickel hexacyanoferrate by co-precipitation from salt solutions. For a mixed coating, it was possible to reduce the inactivation constant of the catalytic coating by almost an order of magnitude - it was 5·10 -3 min -1 compared to 45·10 -3 min -1 for Prussian blue. In the mode of constant flow of hydrogen peroxide to the electrode surface in 20 minutes, a sensor with a stabilized sensing element loses less than 10% of the initial signal value, while a sensor based on Prussian blue loses more than 35% of the signal value in 10 minutes.
Electrochemical method of sequential deposition of Prussian blue and nickel hexacyanoferrate onto the surface of an electrode
Sequential electrosynthesis of catalytic layers of Prussian blue and stabilizing layers of nickel hexacyanoferrate was carried out in various three-electrode cells. One of the cells contained a growth solution for the synthesis of nickel hexacyanoferrate: 1 mM K3 and 1 mM NiCl2 in a background electrolyte of 0.1 M KCl, 0.1 M HCl. The second cell contained a solution for the electrosynthesis of Prussian blue; salt concentrations were varied in the range of 0.5-4 mM for both FeCl 3 and K 3 . Electrochemical deposition of a nickel hexacyanoferrate coating was carried out in potentiodynamic mode, with a potential sweep from 0 to +0.75 V, the potential sweep rate was 50-100 mV/s, for 1-5 cycles. Electrodeposition of Prussian blue was carried out in potentiodynamic mode, with a potential sweep from +0.4 to +0.75 V, the potential sweep rate was 10-20 mV/s, for 1-5 cycles. After deposition of one of the compounds, the electrode was rinsed with distilled water and transferred to another cell for subsequent deposition of another compound. The total number of layers in the sensitive element of the sensor ranged from 2 to 20.
The stages of electrode processing after completion of electrosynthesis are similar to those described in example 1.
From Figure 2 it is clear that for a sensor with a sensing element based on a Prussian blue coating stabilized with nickel hexacyanoferrate by sequential electrodeposition, the signal is stable for 1 hour or more, while in the case of a sensor with an unstabilized sensing element, more than 35 are lost in 10 minutes % of the initial signal value. It was possible to reduce the inactivation constant of the catalytic coating of Prussian blue, stabilized with nickel hexacyanoferrate by sequential electrodeposition, by four orders of magnitude: for it the constant was 5·10 -6 min -1 , while for Prussian blue it was 4.5-10· -2 min -1.
All characteristics of the sensors were obtained from experiments carried out in flow-injection testing mode in phosphate buffer (0.1 M KCl, 0.1 M KH 2 PO 4, pH = 6.0). The flow rate of the buffer solution is 0.25 ml/min. Operating potential 0 V rel. Ag/AgCl/1 M KCl.
Literature
1. Arkady A. Karyakin, Prussian Blue and Its Analogues: Electrochemistry and Analytical Applications. Electroanalysis (2001), 13, 813-19.
CLAIM
1. A method for preparing a sensitive element of a sensor for hydrogen peroxide, characterized in that, in order to increase the stability of the sensitive element, Prussian blue is stabilized with nickel hexacyanoferrate.
2. A method for preparing a sensitive element according to claim 1, characterized in that to increase the stability of the sensitive element, sequential deposition of Prussian blue and nickel hexacyanoferrate is used.