Catalytically Synthesized Prussian Blue Nanoparticles Defeating

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Catalytically synthesized Prussian Blue nanoparticles defeating natural enzyme peroxidase Maria A. Komkova, Elena E. Karyakina, and Arkady A. Karyakin J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05223 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Journal of the American Chemical Society

Catalytically synthesized Prussian Blue nanoparticles defeating natural enzyme peroxidase Maria A. Komkova, Elena E. Karyakina, Arkady A. Karyakin* Chemistry faculty of M.V. Lomonosov Moscow State University, 119991, Moscow, Russia ABSTRACT: We synthesized Prussian Blue (PB) nanoparticles through catalytic reaction involving hydrogen peroxide (H2O2) activation. The resulting nanoparticles display the size-dependent catalytic rate constants in H2O2 reduction, which are significantly improved compared to natural enzyme peroxidase: for PB nanoparticles 200 nm in diameter the turnover number is 300 times higher, for 570 nm diameter nanoparticles – four orders of magnitude higher. Comparing to the known peroxidase-like nanozymes, the advantages of the reported PB nanoparticles are their true enzymatic properties: (i) enzymatic specificity (an absence of oxidase-like activity) and (ii) an ability to operate in physiological solutions. The ultra-high activity and enzymatic specificity of the catalytically synthesized PB nanoparticles together with high stability and low cost, obviously peculiar to noble metal free inorganic materials, would allow to substitute natural and recombinant peroxidases in biotechnology and analytical sciences.

INTRODUCTION Peroxidase is one of the oldest enzymes known; among those who first worked with it was the founder of Russian Institute of Biochemistry, A.N. Bach 1. In modern biotechnology peroxidase is apparently the most widely used enzyme. On one hand, a search in Web of Science for immuno-sensors (assays) with peroxidase as a label returns almost 2500 citations. On the other hand, it is the key enzyme to detect hydrogen peroxide including biosensing purposes. Not surprisingly, there is an increasing interest in mimicking peroxidase activity. First attempts dealt with porphyrins modelling haem, the prosthetic group in the peroxidase active site 2-5. Glutathione peroxidase activity was mimicked using seleno-organic compounds 6-8. However, real analytical applications have started only after the discovery of nanoparticles with peroxidase-like activity 9, later referred to as nanozymes 10. The highest catalytic peroxidase-like activity, not, however, reaching catalytic properties of the enzyme, was reported for nanoparticles containing iron (oxide 9, 11 or sulfide 12) or platinum (or other noble metal) 13-18. Unusual kinetic evaluation was reported for V2O5 nanowires when dimensions in µMole per minute were substituted by Mole per second improving reaction rate by 8 orders of magnitude 19. Catalytic activity comparable with peroxidase was reported for graphene oxide 20 (our estimations). The main disadvantage of the reported metal oxide and noble metal based nanozymes is their low specificity, usually possessing oxidase-like and catalase-like activities in addition 11, 14, 16, 21-23. Moreover, in physiological solutions (pH 7.0 – 7.5), most often used for bioanalytical applications, such nanozymes become catalytically inactive 9, 17, 24. Earlier 25-26 we’ve discovered ferric hexacyanoferrate,

or Prussian Blue (PB) as the most advantageous electrocatalyst for hydrogen peroxide reduction 27-28. In neutral media favorable for bioanalytical applications the advantages of Prussian Blue modified electrodes over the conventionally used platinum are: (i) three orders of magnitude higher activity in H2O2 reduction and oxidation in terms of the 1000 times higher electrochemical rate constants 29 and (ii) three orders of magnitude higher selectivity in hydrogen peroxide reduction relative to oxygen reduction 30-31. Moreover, as we’ve shown recently the catalysis of H2O2 reduction is an exclusive property of Prussian Blue; non-iron hexacyanoferrates, even iron triad-mates ones (cobalt and nickel hexacyanoferrates), are catalytically inactive 32. Not surprisingly, Prussian Blue nanostructures were reported to mimic the peroxidase enzyme 33-36. However, reaction rate constants are evaluated only in 34, reporting in reality the activity, which is several orders of magnitude lower compared to the enzyme peroxidase (see below). We report on Prussian Blue nanoparticles synthesized at their highest catalytic activity in course of reduction of ferricyanide, [Fe(CN)6]3−, and ferric ions, Fe3+ mixture either by hydrogen peroxide, or by conducting polymer forming organic molecules. Catalytic constants of the reported PB nanoparticles 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.

EXPERIMENTAL Materials Experiments were carried out in Milli-Q water (18.2

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MΩcm). Inorganic salts, hydrogen peroxide (30% solution), aniline hydrochloride, 3,3′,5,5′tetramethylbenzidine (TMB), o-dianisidine, citric acid and organic solvents were obtained from Sigma-Aldrich (USA) or Reachim (Russia) at the highest purity. Iron(III) in 0.1 M HNO3 standard (№7765-2000, Ecoanalytica, Russia) was used for ICP-MS measurements. Horseradish peroxidase (EC 1.11.1.7), type VI, RZ 2.9 (lyophilized powder, activity ≥250 pyrogallol units/mg, Sigma definition) was purchased from Sigma Aldrich (USA). Planar screen printed three-electrode sensor structures contained carbon working electrode (Ø = 1.8 mm) and planar H2O2 sensors with the working electrode modified with Prussian Blue film were produced by Ltd Rusens (Russia). Equipment Eppendorf MiniSpin centrifuge (Germany) was used for nanoparticles separation. Ultrasonication of PB NPs suspensions was carried out in PSB-Gals (Russia) ultrasonic bath. Dynamic light scattering was performed with Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK). X-ray powder diffraction experiment was performed with the use of a HUBER G670 Guinier camera (CuKα radiation, λ= 1.54059 Å). UV/Vis measurements in transmission mode were carried out using Lambda 950 Spectrophotometer (Perkin Elmer, USA). Renishaw InVia Raman microscope (Renishaw, UK) was used for Raman spectra recording. Scanning electron microscope Supra 50 VP LEO and analytical transmission electron microscope Carl Zeiss Libra 200MC (both Carl Zeiss, Germany) were used to visualize nanoparticles. Element analysis was carried out using ICP mass spectrometer Elan DRC II (Perkin Elmer, USA). Electrochemical investigations were carried out using PalmSens 3 potentiostat (PalmSens BV, the Netherlands). Methods Synthesis of PB nanoparticles was carried out in 1:1 mixture of FeCl3 and K3[Fe(CN)6] (4-100 mM) dissolved in 0.1 M KCl with 0.1 M HCl solution (pH 1.2) under vigorous stirring or ultrasonication. Prussian Blue deposition was initiated reductant addition to a final concentration of 10 – 200 mM. After 0.5 – 60 min (depending on the precipitation rate) the resulting mixture was centrifuged at 13000 r.p.m. during 1 min, dark blue precipitate was redispersed into 0.1 M KCl/0.1 M HCl solution; the procedure of centrifugation-redispersion was repeated 57 times. The obtained nanoparticles were stored at pH 1.2 and ultrasonicated prior to use. For electrochemical characterization a 2 µl drop of suspension containing PB nanoparticles (0.05-0.5 nM) was cast onto the working electrode surface of planar sensor structures and dried at a room temperature during 24 hours. Concentrations of iron atoms by ICP-MS were determined according to a multi-point standard curve covering the range of analyte concentrations. For X-ray powder diffraction experiment PB nanoparticles were washed with 0.1 M HCl and dried at room temperature.

Kinetics measurements were carried out at room temperature in 1.4 ml cuvette in 750 µl reaction mixture in citrate-phosphate buffer, pH 2.0-7.4.

RESULTS AND DISCUSSION Synthesis and characterization For Prussian Blue based nanozymes we’ve chosen the experimental conditions similar to those allowing us to synthesize the electrocatalyst, 1000 times more active than platinum in neutral media 29, 31. Hence, the synthetic procedure mimicking electrodeposition, has been expected to produce highly catalytically active nanoparticles. Accordingly, the synthesis has to be carried out reducing the mixture of ferricyanide, [Fe(CN)6]3−, and ferric ions, Fe3+, forming FeIII[FeIII(CN)6] complex 37, rather than by conventional mixing of iron salt and hexacyanoferrate with different oxidation states of iron atoms used for previously reported PB nanoparticles 33-36. Reduction of ferricianide – ferric ions mixture results in more regular structure of the hexacyanoferrate film, providing significant improvements both in activity and in stability of the electrocatalyst 29-30, 38-39. Accordingly, a crucial point is a choice of reducing agent for ferricianide – ferric ions mixture. Since Prussian Blue is the redox electrocatalyst, its catalytic activity is provided by an ability of its oxidized and reduced forms to, respectively, oxidize or reduce hydrogen peroxide. Prussian Blue is known to be similarly highly active electrocatalyst in H2O2 reduction and in its oxidation 26. Hence, synthesis of the highly active catalyst for one reaction (H2O2 oxidation) would provide its improved catalytic activity also in H2O2 reduction. From this point of view the best reductant for ferricianide – ferric ions mixture would be hydrogen peroxide. Indeed, since precipitation of Prussian Blue in this case requires oxidation of H2O2, the most catalytically active structures would have the highest growth rate and thus would grow predominantly. As a result the most catalytically active structures, also in H2O2 reduction, would be synthesized. Such procedure can be referred to as catalytic one. Accordingly, nanoparticles of ferric hexacyanoferrate were synthesized reducing the one-to-one mixture of ferricyanide, [Fe(CN)6]3−, and ferric ions, Fe3+, by hydrogen peroxide upon stirring. The size of nanoparticles is dependent on concentrations of precursors (figure S1, Supporting Information): concentrated growing solutions result in smaller nanoparticles. This is, however, valid for precursor concentrations below 70 – 80 mM. Figure 1a displays the two typical dynamic light scattering (DLS) distribution profiles fit to the log-normal distribution function. Both scanning and transmission electron microscopies (SEM and TEM) have been used to confirm DLS experiments (figure 1b). The evaluated value of the mean has been chosen to characterize the average diameter for each distribution. Stirring conditions also affect the size: the smallest nanoparticles are synthesized in an ultrasonic bath (figure 1a, right).


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A

B

C

Figure 1. (A) Hydrodynamic diameters from the dynamic light scattering fit to the log-normal distribution: Ø=78 nm (left) and Ø=42 nm (right). (B) SEM and TEM (inset) images corresponding to the average DLS diameter of ≈ 40 nm. (C) X-ray powder diffraction pattern, normalized to the intensity of the (200) reflection; vertical lines record 73-0687 (JCPDS). X-ray powder diffraction pattern of the synthesized nanoparticles (figure 1c) confirms they are of crystalline structure typical for Prussian Blue. Indeed, the reflexes from the corresponding record (73-0687) from JCPDS database, shown in figure 1b as vertical lines, perfectly match the diffraction pattern. As shown in 40, the reflection pattern from the record 73-0687 (JCPDS database) is valid for all forms of Prussian Blue. Molar extinction coefficient of the synthesized Prussian Blue nanoparticles has been determined at the wavelength of maximum absorbance known for this material (700 nm 41). Figure S2 (Supporting Information) displays the absorbance of a suspension of nanoparticles as a function of total concentration of iron atoms determined by means of ICP MS. The dependence is linear intersecting the origin. Considering the cubic structure of Prussian Blue 42 and thus assuming that the unit cell contains eight iron atoms, the molar extinction coefficient calculated from the slope in figure S2 is of 4.85104 M-1 cm-1. Obviously, except for hydrogen peroxide, a number of organic compounds can serve as reductants for the mixture of ferricyanide and ferric ions promoting synthesis of Prussian Blue nanoparticles. As we’ve shown in 43 discovering the open circuit deposition of the electrocatalyst, among the best reductants are organic molecules able to form conducting polymers upon oxidation. Reduction with aniline led to the highest stability and catalytic activity of the resulted Prussian Blue modified electrodes 43. Accordingly, except for hydrogen peroxide, also aniline has been used as the reducing agent for mixture of ferricyanide and ferric ions. Raman spectrum of the resulted nanoparticles (figure S3, Supporting Information) contains specific peaks as of polyaniline, as of Prussian Blue. Hence, the nanoparticles synthesized this way can be referred to as composite ones (PB-PAn). Catalytic activity Peroxidase activity of nanoparticles and the enzyme has been investigated oxidizing 3,3’,5,5’-tetramethylbenzidine (TMB), apparently the fastest substrate of the enzyme peroxidase, by hydrogen peroxide. Since absorption of the TMB˙+ cation radical is masked by the huge band of Prussian Blue (figure S4, Supporting Infor-

mation), catalytic activity has been monitored at 450 nm, the adsorption band of the fully oxidized form of the mediator: TMB2+. Accordingly, in course of the reaction the two-electron oxidation of the mediator occurs. Michaelis-Menten equation 44 is known as the general equation for initial reaction rate in steady-state enzyme kinetics:

v=

k cat [ E ]0 [ S ]0 K M + [ S ]0

, where Km is Michaelis constant, kcat is catalytic rate constant (or turnover number), [E]o and [S]o are initial enzyme and substrate concentrations. Initial reaction rate has been found to be linear function of the concentration of Prussian Blue nanoparticles; the corresponding straight line intersects the origin (figure S5, Supporting Information) confirming the validity of the Michaelis-Menten equation. For kinetic investigations the nanoparticles concentration of approximately 0.01 nM has been chosen, which is 5 times lower, than the upper limit of the linear range in figure S5. Figures 2a, b display the dependences of the initial reaction rates, catalyzed by catalytically synthesized nanoparticles, on concentrations of H2O2 and TMB. At nonzero H2O2 concentrations the dependence of the reaction rate on TMB concentration obeys the Michaelis-Menten equation (figure 2a, solid lines). This clearly shows peroxidase-like activity of the synthesized nanoparticles. Most importantly, in the absence of hydrogen peroxide no oxidation of TMB has been registered in its entire concentration range (figure 2a). On the contrary, after injection of nanoparticles the absorbance at 450 nm slightly decreases. Hence, the obtained PB nanoparticles do not display oxidase-like activity (reduction of molecular oxygen). An absence of oxidase-like activity of the catalytically synthesized PB nanoparticles has been confirmed using o-dianisidine as a substrate. Similarly, without hydrogen peroxide the only decrease of absorbance, characterizing its oxidized form, has been registered (figure S6, Supporting information). As mentioned, all previously reported metal oxide and noble metal based nanozymes aimed to mimic enzyme peroxidase also display oxidase-like activity 11, 14, 16,



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A

B

C

Figure 2. (A, B) The dependences of the initial reaction rate on concentrations of TMB (A) and H2O2 (B); (A) [H2O2]: ▲ – 0.0 mM, ○ – 0.2 mM, ● – 0.5 mM, □ – 1.0 mM, ■ – 2.0 mM; (B) [TMB]: ○ – 1 µM, ● – 2 µM, □ – 5 µM, ■ – 10 µM; 0.011 nM of PB nanoparticles; (C) The dependence of kcat for TMB, [H2O2] = 2 mM, on the sizes of nanoparticles, synthesized: (■, ▲ , □) – by mixing [Fe(CN)6]4- and Fe3+: in water (■), in buffer pH 5.0 (▲), in 0.1 M KCl/0.1 M HCl (□), (●,○) – reducing mixture of [Fe(CN)6]3- and Fe3+ with H2O2 (●) or aniline (○ ); pH 5.0. Dash line – kcat for peroxidase under similar conditions (λ = 450 nm). 21-23. Since the enzymes peroxidases do not have oxidase nanoparticles diameter. Catalytic constants displayed in activity themselves, the only candidates among figure 2c are evaluated for TMB as substrate, and thus nanozymes to mimic this enzyme are Prussian Blue nathey are dependent on the rate of H2O2 activation. For noparticles synthesized in course of the present work. catalytically synthesized nanoparticles the linear regression line in double logarithmic plots exceeds the slope of The dependence of the reaction rate on H2O2 concen2.5 (figure 2c). This indicates that hydrogen peroxide tration is linear; the corresponding straight line interpenetrates the bulk of nanoparticles. The latter conclusects the origin (figure 2b). The linearity has been tested sion is in agreement with the observation that H2O2 is in a wide range prolonged over almost three orders of able to penetrate Prussian Blue films 29-30. magnitude of concentration up to 5 mM (figure 2b, inset). Further increase in H2O2 concentration is not plauSurprisingly, for nanoparticles synthesized by reduction of [Fe(CN)6]3− and Fe3+ mixture by both H2O2 and sible because of diffusion constrains causing deviation from linearity even of the current responses for Prussian aniline the turnover numbers size dependence belongs to Blue based nano-electrode arrays 45-46. The absence of the same line (figure 2c). This indicates that the presence of polyaniline does not affect the diffusion of H2O2. saturation in the reaction rate – concentration plots (figure 2b) indicates that activation of hydrogen peroxide by As mentioned, the reported nanozymes with peroxithe catalytically synthesized PB nanoparticles occurs dase-like activity have never reached catalytic properties much faster as compared to the other elementary steps. of the natural enzyme 9, 11-18. Hence, it is enough to preThe observed linear dependence of the reaction rate sent the advantages of the synthesized PB nanoparticles on H2O2 concentration indicating ultra-fast activation of over the natural peroxidase. Catalytic constant of the natural horse radish peroxidase evaluated similarly (λ = the substrate confirms that catalytic properties of PB 450 nm) with TMB as substrate is presented in figure 2c nanoparticles are advantageous over all reported peroxas horizontal dash line. Kinetic curves for the enzyme idase-like nanozymes displaying Michaelis-Menten type peroxidase in similar conditions are shown in figure S7, dependence 9, 11-12, 14, 16-17. Moreover, even for the enzyme Supporting information). As seen, all PB nanoparticles peroxidase itself such linearity has never been observed synthesized by reduction of [Fe(CN)6]3− and Fe3+ mixture starting from B. Chance 47-49. Involving the enzymes peroxidases in bioelectrocatalysis with fast electron tunneldisplay higher catalytic constant as compared to the nating between the electrode and the enzyme active site, ural enzyme peroxidase. As seen in figure 2c, the resultwhen it is possible to keep the latter in fully reduced ing nanoparticles display the size-dependent catalytic state, does not improve the case: the upper linearity limrate constants in H2O2 reduction, which are significantly its of the current-concentration dependences for moderimproved compared to natural enzyme peroxidase: for ately rough electrodes are always below 1 mM 50. Hence, PB nanoparticles 200 nm in diameter the turnover numconcerning substrate (hydrogen peroxide) activation, ber is 300 times higher, for ø=570 nm nanoparticles – peroxidase type catalysis of the catalytically synthesized more than 6500 times higher. PB nanoparticles is advantageous even over the natural Since the described kinetic investigation has been enzyme. carried out considering initial reaction rates, it is imThe size dependence of nanozyme activities have portant to address operational stability of the catalyticalnever been investigated, particularly for peroxidase ly synthesized PB nanoparticles. As found, full kinetic mimicking catalysis (see 10, 51-52 for review), except for the curves are linearized in plots of integral form of Michaecurious report that smaller nanoparticles had higher catlis-Menten equation (Walker-Schmidt plots) indicating alytic activity 9. For PB nanoparticles the size dependthere is no inactivation of the catalyst in course of the ence of their activity is displayed in figure 2c. As exreaction (figure S8, Supporting Information). pected, catalytic constant is increased with the raise of


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A

B

C

Figure 3. (A) Turnover number for TMB ([H2O2]=2 mM) pH dependence for PB nanoparticles (ø=80 nm) () and enzyme peroxidase ();. (B) Current responses of PB nanoparticles, ø=70 nm (), and PB film () modified electrodes (0.7 nmol·cm-2 both) in batch upon stirring; pH 6.0, E=0.00 V. (C) Storage stability of PB NPs colloidal solution in 0.1 M KCl/0.1M HCl, kcat/KM for H2O2 ([TMB] = 0.05 mM) measured at pH 5.0; citrate-phosphate (A, C) or phosphate with 0.1 M KCl (B) buffer. It is important to compare catalytic properties of the reactions. Indeed, TMB2+|TMB° redox potential is insynthesized PB nanoparticles with the previously reportcreased as pH is decreased, whereas Prussian ed ones. As mentioned, the only article 34 presents the Blue|Prussian White redox potential is independent of data allowing to calculate catalytic rate constants. We the solution pH. note that true values of kcat for TMB as a substrate recalHigh catalytic activity of the catalytically synthesized culated from the corresponding figure in 34 are at the PB nanoparticles over a wide pH range, even in physiolevel of 2·103 s-1. Moreover, we show that in case of PB logical solutions would obviously provide their use for nanoparticles kcat for TMB is proportional to H2O2 conbiotechnological and analytical applications. centration (figure S9, Supporting information). Hence, Electrochemical properties in appropriate experimental conditions, also suitable for enzyme peroxidase ([H2O2] = 1 – 2 mM) the catalytic Electrochemical characterization of catalytically synrate constant reported in 34 is expected to be 100 times thesized PB nanoparticles has been carried out in order lower than kcat for both enzyme peroxidase and the to evaluate performance characteristics of the corresmallest catalytically synthesized PB nanoparticles (figsponding sensors. Simple drop-casting of the nanopartiure 2c). cles suspension with subsequent drying for 24 hours results in modified electrodes. The latter, similarly to conThe unexpectedly low catalytic activity of the previventional Prussian Blue film modified electrodes, in their ously reported PB nanoparticles accessed, moreover, in cyclic voltammograms display the two sets of peaks coracidic conditions (pH 3.5) 34 stimulated to investigate PB responding to Prussian Blue|Prusssian White and Berlin colloid synthesized conventionally by mixing [Fe(CN)6]4Green|Prussian Blue redox transitions (figure S10, Supwith Fe3+ ions. As seen in figure 2c, conventionally synporting Information). In the presence of hydrogen peroxthesized PB nanoparticles exhibit the size-dependence ide the cyclic voltammograms display the shapes peculiar similar to it for the catalytically synthesized nanopartito catalytic reactions indicating H2O2 oxidation by Berlin cles, but with an order of magnitude lower activity. Large Green and its reduction by Prussian White (figure S10, conventionally synthesized nanoparticles exhibit at pH Supporting Information). 5.0 the turnover number similar to it of the enzyme peroxidase. However, even for the smallest ones the cataAnalytical properties of PB nanoparticles modified lytic rate constant is an order of magnitude higher comelectrodes were investigated at constant potential (E = pared to the reported in 34. 0.00 V) providing hydrogen peroxide reduction. Figure 3b displays the corresponding current responses in batch For catalytically synthesized PB nanoparticles pH deregime upon stirring. As seen, linear calibration range is pendence of the turnover number is shown in figure 3a. prolonged over more than 3 orders of magnitude of hyAs seen for enzyme peroxidase, in the whole pH range drogen peroxide concentrations. Sensitivity evaluated as the turnover number evaluated in similar conditions is a slope of the calibration graph is of 0.85 AMcm-2. For significantly lower (figure 3a). comparison, figure 3b also displays chronoamperometry As mentioned, among disadvantages of the known of similar three-electrode structure modified with PB peroxidase-like nanozymes is the lack of their activity in film. As seen, for the latter the sensitivity is lower (0.65 physiological solutions 9, 17, 24. On the contrary, PB nano-2). The improved analytical performance characAMcm particles synthesized according to the proposed apteristics of the PB nanoparticles modified electrodes are proach are highly active even at pH 7.4, and kcat evaluatmost probably due to their increased roughness provided for TMB as a substrate at pH 7.4 is less than 1.5 times ing more efficient mass transfer compared to convenlower than in pH 5.0 used for kinetic investigations (figtional PB films. Alternatively, higher sensitivity of the ure 3a). A slight decrease of the nanoparticles activity in resulting sensors can be due to improved activity of the strong acidic solutions (pH 2.5) can be explained in catalytically synthesized PB nanoparticles. terms of increased potential difference between 2+ TMB |TMB° and Prussian Blue|Prussian White redox Stability



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Among the main advantages of the elaborated PB nanoparticles over the enzymes peroxidases is their stability. The nanoparticles can be annealed at 100 °C (during 1 hour) and re-dispersed without loss of their activity. Dried catalytically synthesized PB nanoparticles stored at a room temperature for a year displayed after redispersion their catalytic activity at the same level (± 10%). Stability study has been carried out for PB nanoparticles stored in acidic medium. As seen in figure 3c, within a year of storage at a room temperature the nanoparticles remain their catalytic performance characteristics at constant level.

CONCLUSIONS Kinetic peculiarities of the catalytically synthesized PB nanoparticles allow to conclude that they defeat natural enzyme peroxidase. Indeed, the initial reaction rate of hydrogen peroxide reduction, catalyzed by PB nanoparticles, is linearly dependent on H2O2 concentration, which has never been observed either for peroxidase-like nanozymes, or for natural peroxidase. This indicates that substrate (H2O2) activation by the reported PB nanoparticles occurs much faster even compared to the natural enzyme. Catalytic constants in H2O2 reduction for catalytically synthesized PB nanoparticles are nearly volume dependent (the slope of size dependence in double logarithmic plots exceeds 2.5) indicating that hydrogen peroxide penetrates the bulk of nanoparticles. Most importantly, for all sizes of PB nanoparticles the catalytic rate constants are higher, than for natural peroxidase; for PB nanoparticles 200 nm in diameter the turnover number is 300 times higher, for ø=570 nm nanoparticles – more than 6500 times higher. 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. Excellent storage stability of the catalytically synthesized PB nanoparticles (a year of storage at a room temperature both in dry state and in solution without any loss of activity), which is unreachable for enzymes, together with true enzymatic catalytic performance characteristics would obviously provide their leading role in biotechnology and analytical sciences, substituting natural and recombinant enzymes peroxidases. Among labels used in modern bioanalytical tools there are also colorimetric ones, like gold nanoparticles in immuno-chromatographic pregnancy test. We note that except for the extraordinary high peroxidase-like activity and selectivity, the reported nanoparticles are characterized by the extinction coefficient of 4.85104 M-1 cm-1 per PB unit cell. The ultra-high activity with high stability on one hand, and high extinction coefficient, on the other hand, would lead to wide application of the reported PB nanoparticles substituting both catalytic and colorimetric labels in biotechnology and analytical sciences.

ASSOCIATED CONTENT Supporting Information. Nanoparticles size dependence on the growing solution content, linear function of absorbance on concentration of iron atoms for PB nanoparticles, verification of the composite PB-polyaniline nanoparticles structure, UV-vis absorption spectra of reduced forms of TMB in the presence of PB nanoparticles, linearity of the reaction rate on concentration of PB nanoparticles, confirmation for the absence of oxidase-like activity with another reducing substrate, the dependencies of the initial reaction rate on concentration of both substrates for peroxidase, full kinetic curves in Walker-Schmidt plots for PB nanoparticles, kcat for TMB as a function of H2O2 concentration, cyclic voltammograms of planar electrode modified with PB nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Arkady A. Karyakin; e-mail: [email protected].

Author Contributions The manuscript was written through contributions of all authors.

Funding Sources Financial support through Russian Science Foundation grant # 16-13-00010 is greatly acknowledged.

ACKNOWLEDGMENT Authors thank Dr. Andrei A. Eliseev and Development Program of M.V. Lomonosov Moscow State University for TEM investigations.

ABBREVIATIONS PB, Prussian Blue; TMB, 3,3’,5,5’-tetramethylbenzidine.

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TOC graph 75x34mm (300 x 300 DPI)


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Catalytically Synthesized Prussian Blue Nanoparticles Defeating

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