INFORMATION ABOUT PROJECT,
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Project titleNanozymes "artificial peroxidase" for medical diagnostics and therapy
Project LeadKaryakin Arkady
AffiliationFederal State Budgetary Educational Institution of Higher Education Lomonosov Moscow State University,
|2019 - 2021
|extension for 2022 - 2023
PROJECT EXTENSION CARD
Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-205 - Analytical chemistry
Keywordsnanosymes, nanoparticles, Prussian Blue, nanostructured catalysts, transition metal hexacyanoferrates, electroanalysis, immunosensors, DNA- (RNA-)sensors
The project is aimed to develop the scientific background for substitution of natural enzymes peroxidases in diagnostics (immuno-enzyme assays, DNA- (RNA-) sensors) and for applying peroxidase activity in therapy, using for this aim nanozymes “artificial peroxidase”. Immuno- and DNA- (RNA-) sensors as well as affinity based analytical kits comprise an important part of vital activity for modern society since they are used in a variety of areas from medicine to agriculture. Hence, current project, which deals with improvement of such analytical systems and devices targets the important challenges of the modern society. For signal generation these devices use labels, among which one of the leading positions belongs to the enzyme peroxidase. Novel rapidly growing field of modern science is synthesis of nanozymes – nanoparticles with enzyme-like activity. The term “nanozyme” is apparently not more than five years old. The most progressive and actual application of nanozymes is to substitute natural enzymes in various fields of science and technology allowing to avoid the most important problems of the enzymes: their inherent instability and relatively high cost. Nanozymes obviously require advanced catalysts. International scientific priority of the Principal Investigator (leader of the proposed project) for already 25 years is the Prussian Blue based most advantageous (thousands times more active and selective than platinum) electrocatalyst for hydrogen peroxide reduction, the very reaction catalyzed by the enzyme peroxidase. The project will be based on the one of the results of the finalizing RSF grant # 16-13-00010: catalytic synthesis of Prussian Blue nanoparticles and elaboration on it basis the nanozymes “artificial peroxidase”; the corresponding article is just issued in the most prestigious chemical journal: M.A. Komkova, E.E. Karyakina, A.A. Karyakin. Journal of the American Chemical Society 140 (2018) 11302, IF = 14.3. This provides not only scientific novelty, but also an international priority of the proposed investigations. Catalytic rate constants of the nanozymes are up to 4 orders of magnitude higher as compared to the natural peroxidase. Except for the activity defeating even the natural enzyme, the advantages of the reported PB nanoparticles over the known peroxidase-like nanozymes are: (i) enzymatic specificity (an absence of oxidase-like activity) and (ii) an ability to operate in physiological solutions, similarly to the enzymes. The elaborated nanozymes do not contain noble metals, thus their cost is negligible compared to the enzymes. Nanozymes are characterized by stability peculiar to inorganic materials, which is unreachable for the enzymes. For elaboration of scientific background for substitution of natural enzymes peroxidases in diagnostics and for applying peroxidase activity in therapy the following tasks are planned: - investigation of nanozymes “artificial peroxidase” by means of physical and physico-chemical methods, a discovery of structure-function relationships; - investigation of catalytic properties of nanozymes “artificial peroxidase”, and their mechanism of action in hydrogen peroxide reduction; - synthesis of composite core-shell nanoparticles for stabilization of Prussian Blue based nanozymes with nickel hexacyanoferrate shell; - synthesis of ultra-small nanozymes with dimensions comparable to the enzymes (4-6 nm); - electrochemical synthesis of nanozymes “artificial peroxidase”; - deposition of nanozymes “artificial peroxidase” onto the electrode surfaces and investigation of electrocatalytic and analytical properties of the resulting electrodes; - synthesis of nanozymes with functional groups for covalent linking with biomolecules; - entrapment of nanozymes “artificial peroxidase” in liposomes in order to elaborate anti-inflammatory drugs; - synthesis of conjugates of nanozymes “artificial peroxidase” with antibodies, DNA (RNA) for immuno- and DNA- (RNA-) sensors; - elaboration of immuno- and DNA- (RNA-) sensors on the basis of nanozymes “artificial peroxidase” and electrochemical platforms, suitable for “point of care” diagnostics. Complexity of the proposed tasks is illustrated by using of various physical and physico-chemical methods, as well as by involvement in the project different scientific areas: nanotechnology (synthesis and investigation of nanoparticles), catalysis (catalytic synthesis, enzyme-like activity of nanoparticles), biochemistry and molecular biology (synthesis of nanoparticle conjugates with antibodies and DNA, RNA), analytical chemistry and medical diagnostics (immuno-enzyme test systems, DNA- (RNA-) sensors and electroanalytical systems on their basis).
The project will be resulted in novel synthetic methods and new nanozymes “artificial peroxidase” on their basis: stabilized ones for operation in basic solutions, ultra-small nanozymes comparable in dimensions with the enzymes, ones containing functional groups for covalent linking to biomolecules. The knowledge of the catalytic mechanism would obviously allow, on the one hand, to choose the optimal conditions for the nanozymes action, and, on the other hand, to find the ways for further improvement of their catalytic activity. Conjugates of nanozymes with biomolecules: antibodies, DNA, RNA will be synthesized. For anti-inflammatory therapy liposomes containing nanozymes “artificial peroxidase”: as catalytically synthesized ones, as composite core-shell nanoparticles with the stabilization layer of nickel hexacyanoferrate, will be made. All this will allow to elaborate in the end of the project the electrochemical immuno- and DNA- (RNA-) sensors and analytical systems suitable for “point of care” diagnostics. Considering importance of the results of the project, we note that the use of enzymes for analysis of biological liquids is commonly based on their high specificity. For example, glucose oxidase transforms the only free glucose; either its isomers or glucose containing oligo-saccharides are not oxidized. Such specificity for organic substrate for nanozymes is hardly expectable. On the other hand, for applications as labels the enzymes have to be highly active and specific only towards the oxidizer, for which peroxidase is the most valuable example. Hence, the nanozymes “artificial peroxidase” are the first ones, and most probably, the only nanozymes suitable and highly required for medical diagnostics. Scientific value of the project is also provided by the development of the new rapidly growing field: the term “nanozyme” is apparently not more than 5 years old. We note that synthesized in (M.A. Komkova, E.E. Karyakina, A.A. Karyakin. Journal of the American Chemical Society 140 (2018) 11302) nanozymes “artificial peroxidase” are the first nanoparticles with true enzymatic properties: catalytic activity, which is even several orders of magnitude higher compared to peroxidase, and selectivity of this enzyme (an absence of oxidase-like activity – reduction of molecular oxygen). The project is multidisciplinary: its results are important for various scientific fields: nanotechnology, catalysis, biochemistry and molecular biology, analytical chemistry, and the mentioned medical diagnostics. The social importance of the project is provided mainly by the development of diagnostic systems. Immuno- and DNA- (RNA-) sensors as well as affinity based analytical kits in a variety of areas from medicine to agriculture. Hence, current project, which deals with improvement of such analytical systems and devices targets the important challenges of the modern society.
Annotation of the results obtained in 2021
I. Physico-chemical and catalytic properties of ultra-small nanozymes. A comprehensive investigation of ultra-small nanozymes (d = 4-5 nm), synthesized in reversed micelles during the previous stages of the project, has been carried out. The investigation involves physico-chemical methods (optical and electrochemical). As found, synthesis of nanozymes in reversed micelles at hydration degree of 10 allows to result in nanoparticles with diameter less than 8 nm. Raman spectra of nanozymes, synthesized in reversed micelles, contain peaks, which correspond to the Prussian Blue (PB) structure. Wide peaks at 1500 cm-1 in the spectra of composite nanoparticles PB-polyaniline most probably corresponds to the structure of conductive polymer. We note that there are no peaks, which correspond to surfactant forming reversed micelles. This undoubtedly proves complete surfactant removal during nanozymes purification. Ultra-small nanozymes have been immobilized onto the electrode surface by adsorption. Cyclic voltammograms display a couple of peaks at a potential of 0.12 V (vs. Ag|AgCl), which correspond to Prussian Blue/Prussian White redox transition. Electrodes with immobilized ultra-small nanozymes have been applied in amperometric detection of hydrogen peroxide. Small diameter of the electrocatalyst nanoparticles provides high sensitivity in H2O2 detection (0.74 ± 0.09 А∙М-1∙cm-2). Catalytic properties of ultra-small Prussian Blue nanozymes were investigated in reversed micelles. As shown, an initial rate of hydrogen peroxide reduction as a function of the hydration degree displays a maximum in the latter range from 7 to 10, which corresponds to the nanozymes synthesis conditions. An initial reaction rate displays a hyperbolic dependence on the reducing substrate (pyrogallol, guaiacol) concentration, which is typical for enzyme kinetics. The apparent catalytic rate constants found from these dependencies, are linear functions of hydrogen peroxide concentration. From the slopes of the obtained linear dependencies it is possible to evaluate the bimolecular rate constant of the second, rate-limiting stage (k2). As found the rate constants k2 are of 63 M-1s-1 for pyrogallol, and of 0.83 M-1s-1 for guaiacol. For ultra-small nanozymes synthesized in reversed micelles the slope of an apparent catalytic rate constant dependence on the nanoparticles diameter in double logarithmic plots reaches the value of 2.6. This indicates that catalytic process occurs not only at the surface, but also in the bulk of nanoparticles. The apparent catalytic constant values for nanoparticles synthesized in aqueous phase and in reversed micelles belong to the same dependence. Hence, catalytic properties of ultra-small nanozymes can be referred to as typical for catalytically synthesized Prussian Blue nanoparticles. 2. Investigation of hybrid nanoparticles, based on nanozymes and si-RNA entrapped in liposomes, for decrease of reactive oxygen species content in cells During the last state of the project the elaboration of the anti-tumor drugs has been carried out. The presence of Prussian Blue in hybrid nanoparticles should provide the decrease of the level of reactive oxygen species (ROS) in damaged cells, as well as provide an increase of RNA interference efficiency by preventing siRNA from degradation. As found, Prussian Blue nanoparticles are able to accumulate in mice hepatocyte cells (AML12). It is important that the nanozymes even in their high concentration (up to 0.1 mM PB) do not affect the mitochondrial activity of cells and do not display cytotoxicity. Flow-through cytometry with dichlorofluorescein indicated that after injection of nanozymes in cell culture the significant (for 70%) decreased of the ROS level is observed. The possibility for joint entrapment of siRNA and catalytically synthesized Prussian Blue nanoparticles of different sizes (30 nm and 120 nm) in liposomes is shown. According to dynamic light scattering data, the diameter of hybrid nanoparticles is approximately 100 – 140 nm, which is determined by the size of lipid shell. An entrapment of nanozymes in liposomes is confirmed by means of electron cryo microscopy. An increase of Prussian Blue nanoparticles zeta-potential after formulation also indicates an entrapment of nanozymes in liposomes. The kinetics of hydrogen peroxide reduction catalyzed by nanoparticles in hybrid nanoparticles, has been investigated. An apparent catalytic rate constant for nanozymes entrapped in liposomes is decreased as expected, and the Michaelis constant is increased, as compared to those for nanozymes in solution. However, the decrease of catalytic activity is not significant. Moreover, it is expected that being delivered to the target cells, the hybrid nanoparticles would lose their lipid shell releasing both nanozymes and siRNA directly to cytoplasm. Thus the synthesized hybrid nanoparticles display high catalytic activity in hydrogen peroxide reduction and have a potential for the dual action therapy. As expected such anti-inflammatory drugs would substantially decrease the ROS level and improve an efficiency of RNA interference. 3. Synthesis of nanozyme-oligonucleotide and nanozyme-antobody bio-conjugates, and investigation of nanozymes (electro)catalytic properties in their structure For application in immuno-chromatography we synthesized nanozymes of different size as well as composite nanoparticles PB-polyaniline (also with the entrapment of anthranilic and aminobenzenesulfonic acids), PB-polyethylene dioxithiophene (PEDOT), PB-azidomethyl-PEDOT and their conjugates. As found, independently of the particles size (30 or 100 nm), surface potential (-25 mV or 4 mV), and the use of surfactants (polyethylene glycol, proteins), no movement of nanoparticles and their conjugates with oligonucleotides along nitrocellulose membrane has been observed. Hence, we have to conclude that it is not possible to substitute gold/silver nanoparticles in immuno-chromatography tests. In order to elaborate the reagentless electrochemical DNA sensors at the Second stage of the Project Prussian Blue nanoparticles functionalized by azido-groups have been synthesized. In course of the last stage of the Project an efficiency of the single-valence copper as a catalyst according to Meldal and Sharpless (CuAAC or Cu-catalyzed azide-alkyne cycloaddition) has been shown. As shown, the catalyst allows to increase both the conjugation efficiency and, as a result, the sensitivity of labelled oligonucleotide detection (5 – 10 times). Fluorimetry method has been used to control the number of oligonucleotide onto nanozyme surface. For future study the conjugates with 10-20 nucleotides per nanozyme have been chosen. Electrocatalytic activity of conjugates in hydrogen peroxide reduction has been studied after their immobilization onto the electrode surface. As found, the limiting the sensitivity in case of conjugates (0.41 А∙М-1∙cm-2) is only slightly lower than that for the case of nanoparticles PB-azidomethyl-PEDOT (0.45 А∙М-1∙cm-2). An apparent catalytic rate constant of hydrogen peroxide reduction in the presence of catechol for bioconjugates (3∙104 s-1) is less than 2 times lower than that for free nanozymes labels on the basis of PB-azidomethyl-PEDOT (5.5∙104 s-1). At the same time the Michaelis constant is increased almost twice (up to 7 mM), which can be explained in terms of steric constrains for substrate diffusion to the nanozymes surface in conjugate. For synthesis of conjugates with antibodies the nanozymes with amino- and carboxyl groups have been chosen. The antibodies of European rabbit (Oryctolagus cuniculus) to campagnol (Apodemus agrarius) immunoglobulins, as well as donkeys (Equus asinus) antibodies to European rabbit immunoglobulins have been used. Bioconjugation has been carried out with dicyclohexyl carbodiimide. 4. Elaboration and investigation of electroanalytical systems based on DNA(RNA)-sensors/immunosensors using bioconjugates nanozyme-oligonucleotide/nanozyme-antibody The surface of carbon screen-printed electrodes has been modified in course of electropolymerization of azidomethyl-EDOT; final film thickness is from 50 nm to 300 nm. The fact that azido-substituents remain in the polymer is confirmed by incomplete internal reflection spectroscopy in infrared region. Redox transitions of the known mediators (ferri-/ferrocyanide) on the surface of azidomethyl-PEDOT are characterized by the significantly higher electrochemical rate constants as compared to blank electrodes. For elaboration of DNA- and immunosensors oligonucleotides were immobilized on the surface as azidomethyl-PEDOT, as nanozymes by click-reaction, antibodies were conjugated with nanozymes using carbodiimide, on carbon surfaces both antigens and oligonucleotides were simply adsorbed. The label (nanozymes) has been registered by direct (mediator-free) hydrogen peroxide electroreduction. As found, specific interaction through hybridization of complementary DNA results in an order of magnitude increased sensitivity of the corresponding hydrogen peroxide sensor. Calibration graphs for HULC oligonucleotides conjugated with nanozymes have been recorded. For the electrode modified with azidomethyl-PEDOT, which decrease non-specific adsorption, the lowest detectable concentration reached 0.1 nM. Thus, the elaborated approach is suitable for real DNA-sensors. Operation of the latter is expected to be based on hybridization of both surface immobilized and conjugated with nanozyme oligonucleotides with different parts of the target DNA. Such hybridization would obviously lead to immobilization of the electrocatalytic label on the electrode surface. Similarly, the nanozymes electrocatalytic activity in the absence (non-specific sorption) and in the presence of immobilized antibodies has been registered. Most probably, due to shielding by protein molecules the direct electrocatalysis in this case has been found as low-effective. Accordingly, the mediator (catechol) has been added to the system. As found, the specific binding leads to 20-fold increase of the corresponding peroxide sensor sensitivity, thus, opening an opportunity for elaboration of real immunosensors on this basis. Concluding, high electrocatalytic activity of the elaborated nanozymes, as well as a possibility for their covalent linking to biomolecules (oligonucleotides and DNA) provide elaboration of prototypes of electrochemical immuno- and DNA sensors, which would allow express detection of target analytes in their sub-nanomolar concentration. 5. Review preparation and plenary lectures. Project leader was invited to contribute with a review to Microchimica Acta (IF = 5.83, Q1), in which the results of the current project have been reviewed; the review is in press. Project leader delivered plenary lectures during two prestigious international Congresses. 6. Publications in highly ranked international journals. During the third stage five papers on project results have been published in Q1 journals: Journal of Physical Chemistry Letters (2) (IF = 6.475, Nature Index), Electrochimica Acta (IF = 6.9), Electrochemistry Communications (IF = 4.724), Dalton Transactions (IF = 4.39). 7. Participation scientific conferences. Project results have been presented at International and All-Russian scientific conferences: The Eighteenth International Symposium on Electroanalytical Chemistry (18th ISEAC), Changchun, China, XXVIth International Symposium on Bioelectrochemistry and Bioenergetics, Cluj-Napoca, Romania, The 10th International Workshop on Surface Modification for Chemical and Biochemical Sensing, Warsaw, Poland, European Biosensor Symposium “EBS 2021 online”, Wildau, Germany. Students participated International scientific conference “Lomonosov-2021”. Electroanalytical symposium ESEAC-20 and Frumkin Symposium mentioned in the project plan, have been moved to 2021 because of COVID-19. 8. Participation of students in scientific competitions Project team members have been awarded for their victories during scientific competitions. 9. Defense of course and diploma projects by MSU Chemistry faculty students Under supervision of the Project team members carried out and defended with an excellent mark 8 diploma projects in analytical chemistry. Among them 4 were especially highly ranked by Attestation Committee. During the third year 4 course project on analytical and physical chemistry were defended.
1. A.A. Karyakin Glucose biosensors for clinical and personal use Electrochemistry Communications, v. 125, p. 106973 (year - 2021) https://doi.org/10.1016/j.elecom.2021.106973
2. E.V. Karpova, E.V. Shcherbacheva, M.A. Komkova, A.A. Eliseev, A.A. Karyakin Core–Shell Nanozymes “Artificial Peroxidase”: Stability with Superior Catalytic Properties The Journal of Physical Chemistry Letters, v. 12, p. 5547–5551 (year - 2021) https://doi.org/10.1021/acs.jpclett.1c01200
3. M.A. Komkova, K.R. Vetoshev, E.A. Andreev, A.A. Karyakin Flow-electrochemical synthesis of Prussian Blue based nanozyme ‘artificial peroxidase’ Dalton Transactions, v. 50, p.11385-11389 (year - 2021) https://doi.org/10.1039/D1DT02107A
4. M.A. Komkova, O.A. Ibragimova, E.E. Karyakina, A.A. Karyakin Catalytic Pathway of Nanozyme “Artificial Peroxidase” with 100-Fold Greater Bimolecular Rate Constants Compared to Those of the Enzyme Journal of Physical Chemistry Letters, v. 12, p. 171-176 (year - 2021) https://doi.org/10.1021/acs.jpclett.0c03014
5. V.N. Nikitina, E.D. Maksimova, M.D. Zavolskova, A.A. Karyakin Flow injection amperometry as an alternative to potentiometry for solid contact ion-selective membrane-based electrodes Electrochimica Acta, v. 377, p. 138074 (year - 2021) https://doi.org/10.1016/j.electacta.2021.138074
Annotation of the results obtained in 2019
1. Investigation of catalytic peculiarities and the mechanism of action of Prussia Blue based nanozymes. For investigation of the nanozymes mechanism of action the formal kinetic approach similarly to enzyme kinetics has been used. The reaction of nanozyme catalyzed hydrogen peroxide reduction was monitored controlling spectrophotometrically either a decrease in concentration of the reduced form of the second substrate or an accumulation of its oxidized form. The kinetics of hydrogen peroxide reduction catalyzed by the synthesized Prussian blue based nanozymes has been investigated with the most widely used substrates of the enzyme peroxidase. The mechanism of hydrogen peroxide reduction catalyzed by Prussian blue based nanozymes is represented by the two-stage reaction scheme. At the first stage the nanozymes reacts with the reducing substrate, which occurs without formation of a nanozyme-substrate complex via bi-molecular mechanism. Oxidation of the resulting Prussian White (the reduced form of Prussian Blue) by hydrogen peroxide at the second stage occurs irreversibly. The ratio of the forward and the backward reaction constants at the first stage is determined by the difference between redox potentials of the mediator and the Prussian Blue|Prussian White redox couple. Concluding, the nanozymes based on catalytically synthesized Prussian Blue nanoparticles can be referred to as low-potential “artificial peroxidase” (≈0.15 V, Ag|AgCl), up to 200 times in terms of the catalytic rate constant more active than the natural enzyme even in the presence of the relatively high-potential reducing substrates. 2. Stabilization of nanozymes: synthesis of composite core-shell nanoparticles with the catalytically active core (iron hexacyanoferrate) and stabilization shell (nickel hexacyanoferrate). For synthesis of stabilized nanozymes the catalytic core (Prussian Blue) has been covered by the shell of nickel hexacyanoferrate, the material which is isostructural to iron hexacyanoferrate. Despite nickel hexacyanoferrate is catalytically inactive, it is much more stable both mechanically and chemically compared to Prussian Blue. The synthetic protocol for stabilized nanozymes has been elaborated. After incubation in the growing solution containing both nickel and hexacyanoferrate salts the diameter of Prussian Blue nanoparticles is increased. The resulting nanoparticles sizes, evaluated by dynamic light scattering, are between 40-50 nm and 100 nm. The sizes are independently confirmed by the transmission electron microscopy data. Optimal protocol for the synthesis of stabilized nanozymes involves catalytic synthesis of Prussian Blue nanoparticles of ≈40 nm in diameter followed by their covering with the shell of nickel hexacyanoferrate to the final diameter of ≈80 nm. The stability of the resulting composite nanoparticles in neutral medium (pH 7.4), characterized by the corresponding inactivation constant, has been an order of magnitude increased. By modification of the working electrode area with the stabilized nanozymes the hydrogen peroxide sensors have been elaborated; their sensitivity and operational stability have been determined. The investigations in this direction will be completed during the second year of the project. 3. Synthesis of ultra-small nanozymes “artificial peroxidase” with the enzyme-level dimensions (4-6 nm). Synthesis of ultra-small Prussian Blue nanoparticles with dimensions 4-5 nm has been carried out in three-phase systems water/octane/surfactant by reducing mixture of ferricyanide and iron (III) chloride by aniline. Size distribution of the resulting ultra-small Prussian Blue nanoparticles has been investigated by means of both dynamic light scattering and transmission electron microscopy. The average diameter of the nanoparticles is of 4.4±0.3 nm. The ultra-small Prussian Blue nanoparticles have been extracted from the reversed micelles medium and stabilized in aqueous phosphate-citrate buffer solution (pH 5.0) by entrapment in micelles of Triton X-100. Catalytic activity of the Prussian Blue based nanozymes has been investigated; the ratio of catalytic constant to Michaelis constant (kcat/KM): 0.36 10-3 M-1s-1 for tetramethylbenzidine and 0.65 M-1s-1 for H2O2. The investigations also will be completed during the second year of the project. 4. Electrochemical synthesis of nanozymes “artificial peroxidase” using flow-through electrodes. For tunable electrochemical synthesis of Prussian blue based nanozymes the electrochemical flow-through wall-jet cell has been elaborated. The cell allowed integration of the electrodes on the basis of graphite felt. Potentiostatic synthesis of Prussian Blue nanoparticles in the elaborated cell has been realized, the nanoparticles sizes are determined by the applied potential. Electrochemical synthesis of nanoparticles provides higher reproducibility of the material as compared to the open circuit deposition. On the other hand, electrochemical synthesis is easily scalable and suitable for mass-production of electroactive nanoparticles. The electrochemically synthesized Prussian Blue nanoparticles are characterized by narrow size distribution. For all electrochemically synthesized nanoparticles their catalytic activity in hydrogen peroxide reduction by tetramethylbenzidine is higher than that for the enzyme horse radish peroxidase. For instance, nanozymes 135 nm in diameter the catalytic rate constant (kcat) is of 7500 s-1, which is 35 times higher than for peroxidase. 5. Deposition of nanozymes “artificial peroxidase” onto the electrodes and investigation of their catalytic properties in comparison with the conventional Prussian Blue modified electrodes. The immobilized Prussian Blue based nanozymes on the electrode surfaces are stable and are characterized by high electroactivity. Redox reactions involve the whole thickness of the layer; the fraction of electroactive material is determined by the dimensions of nanoparticles and in course of increasing of the latter is decreased exponentially approaching 25%. The proposed protocol for nanoparticles immobilization onto the electrode surface is simple and allows to control the morphology of the electrode covering controlling the dimensions of the deposited nanoparticles. Surface morphology of the Prussian Blue nanozymes based sensors provides, most probably due to its improved roughness, the 30% higher sensitivity of the resulting sensors to hydrogen peroxide as compared to the sensors on the basis of conventional Prussian Blue films. The response time for Prussian Blue nanozymes based sensors and biosensors does not exceed 10 s, the linear calibration range encounters 4 orders of magnitude of H2O2 concentrations: from 2·10-7 M to 1·10-3 М. Sensitivity of the lactate biosensor in batch mode is of 210 ± 20 мА·М-1·см-2, exceeding two times the sensitivity of similar sensor based on conventional Prussian Blue film. The results are published: Vokhmyanina Darya V., Andreeva Ksenia D., Komkova Maria A., Karyakina Elena E., Karyakin Arkady A.‘Artificial peroxidase’ nanozyme – enzyme based lactate biosensor. Talanta, 2020, 208, 120393 DOI: 10.1016/j.talanta.2019.120393 6. Investigation of nanozymes “artificial peroxidase” by means of physical and physico-chemical methods. Phase composition of the catalytically synthesized Prussian Blue nanoparticles has been confirmed by X-ray powder diffraction pattern, Mossbauer and Raman spectra. The catalyst synthesized by reduction of polymer-forming organic materials is composed of Prussian Blue and conducting polymer. Granulometric analysis of catalytically synthesized Prussian Blue nanoparticles has been carried out by means of dynamic light scattering, scanning electron microscopy and transmission electron microscopy; the results of all three methods are in a good agreement. Prussian Blue nanoparticles are of submicro- and nanodimensions; the elaborated catalytic synthesis allows to determine their diameter. The hydration of Prussian Blue nanoparticles was evaluated by means of thermogravimetry. The high extinction coefficient (ε=4.85·104 М-1·см-1) of the catalytically synthesized Prussian Blue nanoparticles, which was determined coupling spectroscopy with ICP-MS) allows to consider them also as colorimetric labels in immunosensors and DNA-/RNA- sondes. 7. A review of literature sources. On the basis of materials of the ongoing project, as well as of RSF project successfully completed in 2018, as well as on application of the Prussain Blue based electrocatalyst the authors are applying for a review in Uspekhi khimii (Succeeds in chemistry), Q1, entitled “Prussian Blue: from the advanced electrocatalyst of hydrogen peroxide reduction to nanozymes defeating natural enzyme peroxidase” (for publication in 2020-21). 8. In addition to the Plan for the first year of the Project the flow-injection amperometric readout approach as an alternative to conventional potentiometry has been elaborated for detection of non-electroactive ions and even neutral molecules. Since Prussian Blue is electroactive material which capture or release cations from the solution bulk for charge compensation, it is highly attractive also to use it as a sensor for alkali metal cations. For detection of non-electroactive ions as an alternative to conventional potentiometry the constant potential amperometry in flow-injection mode is proposed. Sensor response is a couple of the oppositely directed current peaks (cathodic and anodic ones) in flow-injection mode and one peak in constant flow mode. Thorough investigation has shown that analytically valuable in flow-injection mode is the first sharp peak. Analytical performance characteristics of the amperometric mode are advantageous over the conventional potentiometric one: for example, the signal-to-noise ration of the FIA dc amperometry is 20-25 times improved. The results are published: Zavolskova Marina D., Nikitina Vita N., Maksimova Ekaterina D., Karyakina Elena E., Karyakin Arkady A. Constant Potential Amperometric Flow-Injection Analysis of Ions and Neutral Molecules Transduced by Electroactive (Conductive) Polymers Analytical Chemistry, 2019, 91, № 12, 7495-7499 DOI 10.1021/acs.analchem.9b00934 9. Prepared for publication 5 papers for Q1 journals, the two of them are already published. 10. Prepared for defense the PhD dissertation by Karpova E.V. (the defense is scheduled for December, 18th 2019). Grant team members participated 5 international conferences and symposiums in Russian Federation and abroad, as well as in exhibitions. Young grant team members participated several scientific competitions are have won awards and scholarships.
1. Vokhmyanina Darya V., Andreeva Ksenia D., Komkova Maria A., Karyakina Elena E., Karyakin Arkady A. ‘Artificial peroxidase’ nanozyme – enzyme based lactate biosensor Talanta, 208, с. 120393 (year - 2020) https://doi.org/10.1016/j.talanta.2019.120393
2. Zavolskova Marina D., Nikitina Vita N., Maksimova Ekaterina D., Karyakina Elena E., Karyakin Arkady A. Constant Potential Amperometric Flow-Injection Analysis of Ions and Neutral Molecules Transduced by Electroactive (Conductive) Polymers Analytical Chemistry, 91, № 12, с. 7495-7499 (year - 2019) https://doi.org/10.1021/acs.analchem.9b00934
3. Andreeva K.D., Vokhmyanina D.V., Karyakin A.A. Stabilized lactate biosensors based on composite alkoxysilane-Nafion membranes Proceedings of the 15th International Students Conference “Modern Analytical Chemistry”Prague, 19—20 September 2019 edited by Karel Nesměrák. -- 1st edition. -- Prague : Faculty of Science, Charles University, 2019. - 285, pp.155-159 (year - 2019)
4. Korolev A.I. Стабилизированный биосенсор на основе берлинской лазури для определения концентрации глюкозы в биологических жидкостяхдоклад XXIX Менделеевская школа-конференция молодых ученых, г. Иваново, Ивановский государственный химико-технологический университет ,Россия, Россия, 21-27 апреля 2019, стр.58 (year - 2019)
5. Nikitina V.N., Zavolskova M.D., Karyakin A.A. Flow-injection dc amperometry as an alternative to potentiometry for electroactive polymer supported sensors Matrafured 2019 International Conference on Chemical Sensors Будапешт, Венгрия, 2019. — P. 37–37., стр.37 (year - 2019)
6. Shavokshina V.A., Andreev E.A., Karyakin A.A. Sensing element based on poly(3,4-(1-azidomethylethylene)dioxythiophene) as electroactive layer of electrochemical DNA sensors Proceedings of the 15th ISC Modern Analytical Chemistry ▪ Prague 2019 Edited by Karel Nesměrák, Faculty of Science, Charles University, Prague, pp.8-14 (year - 2019)
7. Zavolskova M.D., Nikitina V.N., Karyakin A.A. DC amperometric flow-injection analysis of ions and neutral molecules transduced by electroactive polymers Proceedings of the 15th International Students Conference “Modern Analytical Chemistry”Prague, 19—20 September 2019 edited by Karel Nesměrák. -- 1st edition. -- Prague : Faculty of Science, Charles University, 2019. - 285, pp.167-173 (year - 2019)
Annotation of the results obtained in 2020
1. Screening of organic compounds as reductants of the ferric (iron III) – ferricyanide mixture for synthesis of nanozymes. Nanozyme synthesis via reduction of the ferric-ferricyanide mixture by organic compounds. The synthesis of Prussian Blue nanoparticles using precursors of conducting polymers (pyrrole, aniline, thiophene and their derivatives) as reductants of the ferric-ferrricyanide mixture has been carried out. Entrapment of conducting polymers in Prussian Blue nanoparticles except functionalization causes significant stabilization of the catalyst. The highest operational stability has been registered for nanozymes based on self-doped polyaniline (the polymer of 3-aminophenylboronic acid), which is significantly higher it not only for pure Prussian Blue nanoparticles, but also for Prussian Blue films. Among the most convenient functional groups for conjugation with biomolecules is azide (modification by click-reaction of azide-alkyne cycloaddition). For azide functionalization the nanozymes have been synthesized using azidomethyl EDOT (azidomethyl ethylenedioxythiophene) as a reductant of the ferricyanide of iron (III). The fact that Prussian Blue nanoparticles are indeed azide-functionalized has been confirmed using Raman spectroscopy. 2. Final optimization of nanozyme synthesis, which were elaborated during the first year of the project (electrochemical, core-shell, ultrasmall dimensions). 2.1 Final optimization of electrochemical synthesis of nanozymes “artificial peroxidase” has been carried out. The dimensions of nanoparticles, which have been synthesized in a flow-through wall-jet cell, are mainly determined by concentration of precursors and the value of reduction potential. Both an increase of the precursor salts concentration and a decrease of electrode potential cause a decrease of the nanoparticles diameter (down to 50-60 nm). Final optimization of the synthetic conditions allowed synthesis of nanozymes with catalytic rate constant, which was 200 times higher than kcat for horse radish peroxidase. Being simple and reagentless, solely electrochemical approach allows to control nanoparticles size during their synthesis, which is extremely important for further commercial (biotechnology) applications. 2.2 Stabilized core-shell nanozymes have been synthesized by immersion of Prussian Blue (PB) nanoparticles in solutions containing nickel (Ni2+) ions and ferrocyanide ([Fe(CN)6]3‒) for covering with nickel hexacyanoferrate (Ni-HCF) shell. Sodium hexametaphosphate has been used as a stabilizer of the resulting core-shell nanoparticles. Stability of core-shell nanozymes at neutral pH values, which has been characterized by the optical density at 700 nm (Prussian Blue), according to the corresponding inactivation constant, has been improved by an order of magnitude; particularly, for the nanozymes of 120 nm in diameter kin (PB) = 2.4∙10-3 s-1, kin (PB-Ni-HCF) = 3.1∙10-4 s-1. The corresponding hydrogen peroxide sensors (method of their preparation on the basis of nanoparticles was elaborated during the first year of the project) in case of core-shell nanozymes are also characterized by the significantly improved operational stability. In hard conditions of continuous monitoring of 1 mM of hydrogen peroxide sensors based on PB-Ni-HCF core-shell nanoparticles are characterized by both 3 times higher half-inactivation time and 5 times lower inactivation constant as compared to the sensors on the basis of Prussian Blue nanoparticles. 2.3 Optimization of synthetic conditions for ultra-small Prussian Blue based nanozymes in reversed micelles (octane/AOT/water) has been carried out. The catalytic synthesis has also been used. For this aim micelles system containing iron (III) hexacyanoferrate and iron (III) chloride was mixed with the micelles system containing the reductant – aniline. Dynamic light scattering experiments give the mean diameter of nanoparticles in micelles system of 4.7±0.5 nm, which has been confirmed by transmission electron microscopy. Ultrasmall nanozymes (7 nm in diameter) in aqueous solution can be obtained in course of precipitation with acetone of the micelles nanoparticle system and re-suspending them in phosphate-citrate buffer solution, pH 5. 3. Investigation of the synthesized nanozymes by physical and physico-chemical methods. Phase composition of Prussian Blue nanozymes stabilized by nickel hexacyanoferrate (core-shell type nanoparticles) have been investigated using ICP MS. For samples with the high content of nickel hexacyanoferrate the ratio Fe:Ni tends to unity. According to transmission electron microscopy data the samples with low and high content of nickel hexacyanoferrate consist of low-crystallinity grains with average diameter of 7 – 20 nm. For both samples the grains are aggregated in larger particles with diameter of 40 - 150 nm. According to the local X-ray spectrometry data the Fe to Ni ratio for stabilized core-shell nanoparticles is of 13, when the diameter is 40 nm, and of 3 for nanoparticles of 155 nm in diameter. We suppose that synthesis of core-shell nanoparticles occurs as via sorption of nickel hexacyanoferrate on the surface of Prussian Blue nanoparticles, as in course of aggregations of the latter with nanoparticles of nickel hexacyanoferrate being formed in the reaction mixture. Large (in diameter) nanoparticles with high content of nickel hexacyanoferrate seem to be agglomerates of Prussian Blue nanoparticles covered by thin layer of Ni-HCF and nickel hexacyanoferrate nanoparticles. 4. Investigation of catalytic properties of the resulting nanozymes. The dependence of the initial reaction rate on substrate concentrations almost in all cases obeys the Michaelis function. Integral kinetics (analysis of the whole kinetic curve) points to the slow release of the product (oxidized form of the reducing substrate) from the nanozymes. Kinetic mechanism of the nanozymes “artificial peroxidase” action involves the three following stages: complexation with the reducing substrate, oxidation of the complex as formed by hydrogen peroxide (irreversible catalytic stage) and release of the oxidized substrate from the nanozymes. Catalytic constants for low-potential substrates: catechol, pyrrogallol and ferrocyanide are, respectively 2.6±0.1∙107 М-1s-1, 1.3±0.1∙108 М-1s-1 и 1.9±0.1∙108 М-1s-1, being higher than the rate constant of hydrogen peroxide interaction with the active site of the enzymes peroxidases (1–2∙107 М-1s-1). However, for peroxidases the rate-limiting is the reaction of the Compound II with electron-donor substrate, which is at least one order of magnitude slower. Hence, the rate-limiting step of hydrogen peroxide reduction catalyzed by the nanozymes is at least 100 times faster compared to it for enzymes peroxidases. For core-shell nanozymes the catalytic rate constant is approximately 2-2.5 times lower. However, whereas for pure Prussian Blue nanoparticles its value in neutral solutions is decreased by 50% during first 5 hours, the core-shell nanozymes retain in similar conditions 80% of its value during 50 hours. 5. Organic peroxides as substrates for nanozymes “artificial peroxidase”. Catalytic activity of Prussian Blue nanoparticles is decreased in a raw of peroxides: hydrogen peroxide – urea peroxide – methyl ethyl ketone peroxide – t-butyl hydroperoxide – kumol hydroperoxide. As found, for small peroxides (hydrogen and urea peroxides) the catalytic reaction occurs in the bulk of nanoparticles. On the contrary, large peroxides (t-butyl hydroperoxide and kumol hydroperoxide) are reduced only on the surface of nanozymes. Also it has been found that the nanozymes are not only more catalytically active than the enzymes peroxidases, but also they are more selective to hydrogen peroxide. For nanoparticles (d = 68 nm) the nanozyme selectivity coefficients are 4 times higher than those for enzyme horse radish peroxidase. Thus the elaborated nanozymes can be used in enzyme-free systems for peroxides detection, as well as in biochemical analysis. 6. Entrapment of nanozymes “artificial peroxidase” in liposomes for elaboration of anti-inflammatory drugs. A possibility for simultaneous entrapment of catalytically synthesized Prussian Blue nanoparticles (50 nm in diameter) and siRNA in liposomes has been investigted. According to the dynamic light scattering data, the diameter of the hybrid nanoparticles is approximately 100 nm. Entrapment of nanoparticles in lipid layer partially blocks transport of chemical substances. Simultaneously the catalytic activity of nanozymes is decreased by less than 30%. As found, catalytically synthesized Prussian Blue nanoparticles, as well as core-shell Prussian Blue nanoparticles stabilized by nickel hexacyanoferrates, do not display any cytotoxicity. Preliminary experiments have shown a possibility for accumulation of nanoparticles in AML12 cells and, as a result, a decrease of reactive oxygen species concentration. 7. Synthesis of nanozyme conjugates with DNA. In order to elaborate reagentless DNA/RNA sensors it was proposed to use Prussian Blue nanoparticles functionalized by azide groups allowing bio-conjugation by click-reaction of azide-alkyne cycloaddition. Taking gene HULC as an example with the use of fluorescent labels it has been found that in the resulting conjugates one nanozyme is linked with 10-15 oligonucleotide fragments. Nanozymes zeta potentials confirm bio-conjugation. Bio-conjugates with different distances from the catalytic label “artificial peroxidase” to 5’ end (from 5 to 70 nucleotides) have been synthesized. It has been shown that nanozymes retain their electroactivity in bio-conjugates. This provides a background for using of nanozymes “artificial peroxidase” as redox/electrocatalytic labels. 8. Review preparation. Project team members were invited to write a chapter in the special issue of Comprehensive Inorganic Chemistry III (Elsevier). 9. Patent search and patent issuing. The patent for invention entitled “Biosensor on the basis of polyalcoxisilane membranes with improved sensitivity” was issued; № 2 731 411, September, 2; 2020. 10. Publications in highly ranked international journals. During the second year of the project 4 papers have been published in Q1 journals. Prepared for publication 3 papers: on the mechanism of nanozymes action, on solely electrochemical synthesis of nanozymes and on superstable core-shell nanozymes. 11. Participation in scientific conferences. Scientific results of the project have been reported during 3 International and Russian scientific conferences including 71st Annual Meeting of the International Society of Electrochemistry (Belgrad, Serbia). Electroanalytical symposium ESEAC-20 and Frumkin Symposium mentioned in the project plan, have been moved to 2021 because of COVID-19. 12. Participation of students in scientific competitions. Project team members have been awarded for their victories during scientific competitions.
1. E.V. Karpova, A.I. Laptev, E.A. Andreev, E.E. Karyakina, A.A. Karyakin Relationship Between Sweat and Blood Lactate Levels During Exhaustive Physical Exercise ChemElectroChem, т. 7, вып. 1, с.191-194 (year - 2020) https://doi.org/10.1002/celc.201901703
2. E.V. Karpova, E.E. Karyakina, A.A. Karyakin Wearable non-invasive monitors of diabetes and hypoxia through continuous analysis of sweat Talanta, том: 215, номер статьи: 120922 (year - 2020) https://doi.org/10.1016/j.talanta.2020.120922
3. M.A. Komkova, A.A. Zarochintsev, E.E. Karyakina, A.A. Karyakin Electrochemical and sensing properties of Prussian Blue based nanozymes “artificial peroxidase” Journal of Electroanalytical Chemistry, том 872, номер статьи: 114048 (year - 2020) https://doi.org/10.1016/j.jelechem.2020.114048
4. E.V. Karpova, A.A. Karyakin Noninvasive monitoring of diabetes and hypoxia by wearable flow-through biosensors Current Opinion in Electrochemistry, том. 23, October 2020, стр.16-20 (year - 2020) https://doi.org/10.1016/j.coelec.2020.02.018
5. - Биосенсор на основе мембран полиалкоксисилана с повышенным коэффициентом чувствительности -, 2 731 411 (year - )