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40 AZERBAIJAN CHEMICAL JOURNAL № 4 2023 ISSN 2522-1841 (Online)
ISSN 0005-2531 (Print)
UDC 544.344; 577.1.08
CATALASE AND PEROXIDASE BIOMIMETIC SENSOR BASED ON Ag-ELECTRODE
N.N.Malikova, N.LAlizade, T.M.Nagiev
M.Nagiyev Institute of Catalysis and Inorganic Chemistry, Ministry of Science and Education
of the Republic of Azerbaijan
tnagiev@azeurotel.com
Received 27.02.2023 Accepted 31.03.2023
Research has been carried out on the development of a biomimetic sensor of the catalase and peroxidase type to determine trace amounts of peroxide hydrogen and low concentrations of ethyl alcohol in an aqueous solution. By potentiometric research, the physicochemical feature of the catalase and peroxidase biomimetic sensor was studied, the transducer of which was Ag. Rapid and accurate determination of hydrogen peroxide is very important, but no less important side of this issue is associated with the determination of trace amounts of C2H5OH in aqueous media of various origins. The need to develop express methods for determining the concentration of C2H5OH in aqueous solutions follows from the requirements for the quality of wine and alcohol products. As you know, the taste and drinking qualities of the product depend on the ratio of methyl and ethyl alcohols. In this work, biomimetic sensors of the catalase and peroxi-dase types based on an Ag electrode were developed for determining trace amounts of hydrogen peroxide and ethanol. Biomimetic sensors of the catalase and peroxidase types based on an Ag electrode have been developed for the determination of microquantities of hydrogen peroxide and ethyl alcohol. The applicability of a biomimetic sensor for determining trace concentrations of hydrogen peroxide, as well as for determining microquantities of ethyl alcohol in an aqueous solution with a low detection limit, high sensitivity, wide recognition range, high stability, and repeated use, is shown. The developed and synthesized biomi-metic sensor of the catalase and peroxidase types based on TPhPFe3+/Al2O3/Ag have high sensitivity, stability, and do not lose their activity for a long time.
Keywords: biomimetic, sensor, tetraphenylporphyrin of iron, catalase, smart material.
doi.org/10.32737/0005-2531-2023-4-40-47 Introduction
The requirements of modern analysis are sensitivity, selectivity, cheapness, simplicity and rapidity. Electrochemical sensors meet these requirements in the best possible way. They are simple, easy to use, and also allow continuous monitoring of key analytes, which is important for clinical diagnostics, industrial production and environmental monitoring.
The desire to increase the sensitivity and selectivity, as well as the durability of electrochemical sensors and biosensors, has generated a huge amount of research aimed at designing functional layers on the electrode surface. In addition, the limit of detection of an electrochemical sensor can be lowered by minimizing the size of the electrode, which changes the signal-to-noise ratio.
Bioselectors of biosensors, as is known, are enzymes, cells, antibodies, receptors, etc. Biosensors for enzymatic analysis are characterized by availability and low cost. Biosensors
have limitations associated with their high sensitivity to the external environment, short service life, high cost and multi-stage analysis.
In chemical sensors, the working element is a chemical compound, which allowed their use for a long time in any conditions.
In recent years, the creation of biome-metic sensors has been developed, in which bio-selectors are replaced by analogues of enzymes, which combines high sensitivity and stability. In works [1, 2], biomimetic sensors based on highly efficient iron-porphyrin-containing bio-mimetic catalysts were developed, which were used as a working element - selector.
In addition to the fact that hydrogen peroxide itself is an important analyte, its definition makes it possible to most effectively match biochemical and physicochemical reactions in the framework of creating first-generation biosensors based on oxidase enzymes (most often used in enzymatic biosensors).
Hydrogen peroxide can be determined by spectrophotometry, fluorimetry, and chemilumi-nescence. However, in accordance with the requirements of modern analysis (simplicity and rapidity of the method, the possibility of continuous determination in small volumes, miniaturization and low cost of equipment), preference is given to electrochemical methods.
Among the main electroanalytical systems for determining H2O2, the following can be distinguished: 1) sensors based on noble metals [3, 4]; 2) biosensors based on peroxidase [5, 6]; 3) sensors based on oxides or hexacyanofer-rates of transition metals [7, 8].
One of the important analytical tasks is the express determination of the content of lower alcohols. The determination is carried out in various technological processes, for example, in the pharmaceutical and chemical industries, in the fermentation of ethyl alcohol, in the quality control of alcoholic beverages. Traditional methods for the determination of alcohols are either expensive and characterized by a long analysis time, insufficient accuracy, or are laborious. Most often, the concentration of alcohols is determined using a hydrometer (alcohol meter) or a pycnometer, however, these methods are not selective, since the presence of salts, carbohydrates and other impurities in the solution distorts the measurement results. Gas chromatography, which is the standard method for evaluating alcohols, is considered expensive and requires skilled personnel. An important direction of research will be the development of an analysis method that would simplify and reduce the cost of the procedure for determining these components without loss of accuracy and specificity. A promising approach is the development of biosensor technology. To date, a significant number of biosensor models for the detection of ethanol and other aliphatic alcohols based on the use of enzymes have been described.
In works [9, 10], biosensors were studied and the results on the detection of lower alcohols were shown.
A test strip has been developed [11] for determining the content of ethyl alcohol in the blood by an electrochemical method using an
amperometric cell. The test strip includes a planar graphite electrode with a surface modified with manganese dioxide hydrosol and coated with an enzymatic mixture. Moreover, the enzymatic mixture contains Pichia pastoris or Hansenula polymorpha alcohol oxidase, detergent and high molecular weight alcohol. EFFECT: invention provides determination of ethyl alcohol content in blood by electrochemical method in the concentration range from 1 to 32 mM.
The authors of [12] prepared a new bio-mimetic peroxidase sensor for the adsorption of hematin (aHtin-GCE), an iron (III) porphyrin present in heme-containing proteins.
Peroxidase-like activity was studied in the presence of hydrogen peroxide (H2O2) using the chromogenic substrate 3,3',5,5'-tetramethylben-zidine. Nanozyme Co3(PO4)28H2O was synthesized in situ by a direct reaction between cobalt (II) and phosphate. The effect of pH, temperature, and incubation time on the conversion of tetra-methylbenzidine to oxidized volume (I) and phosphate catalyzed by Co3(PO4)28H2O in the presence of H2O2 was optimized [13].
Taking into account the valuable internal peroxidase activity, work [14] can find wide application in the field of sensors and biosensors for various applications. The peroxidase-like activity of N-doped TiO2 was evaluated. The results show that IL can stabilize nano-materials without changing their reactive surface chemical properties. In the future, the main characteristics of the surface of the nanoparti-cles are retained with improved stabilization and increased catalytic activity. Stabilized na-noparticles as a catalyst are subject to many problems in direct application. For example, such stabilized nanoparticles coated with fragments block the interaction of nanosurfaces.
In this study [15], hemin is used as a catalyst component, and other materials such as an organometallic framework and carbon nano-tubes are additionally used to mitigate the disadvantages of hemin. In this work, Hemin cMIL- 88- NH2/CNT as a catalyst for use in H2O2 sensors is more attractive and better than other heme proteins, and its catalytic activity is
well preserved even under harsh pH and temperature conditions for a long time.
Bioinspired molecular complexes that mimic the enzymatic catalysis of redox transformations offer a universal platform for the development of non-enzymatic mediator-free sensors with high sensitivity, selectivity, and reliability without the use of precious metals [16]. In this work [16] there was the manufacture and study of biomimetic sensors based on the electrocatalytic reduction of hydrogen peroxide and oxygen by a series of immobilized iron and manganese complexes with porphyrin macrocycles for the detection of hydrogen peroxide.
Another important problem for determining ethanol and acetone vapors in air is the use of sensors [17] with gas sensitive layers based on tin dioxide with nanoscale catalytic additives of palladium, platinum, antimony, and lanthanum. Determination of ethanol vapor is selective, because the optimal solution for which has not yet been proposed. Semiconductor sensors are widely used for the production of breathalyzers, but these devices cannot distinguish between ethanol vapor and acetone vapor. Therefore, ketosis can be mistaken for heavy drinking.
The solution of these problems is largely associated with the search for new materials and metal oxide structures with a developed surface containing nanocatalytic additives. Article [18] also proposes another solution related to the transition from the steady state regime. Semiconductor sensors traditionally used for operation in non-stationary modes allow pulsed heating and rapid cooling of the sensor to combine high gas catalytic activity and thus increase the response. Titanium dioxide nanoparticles doped with non-metals have been used due to their remarkable characteristics such as non-toxicity to biological systems, large surface area for perception, high accuracy, easy availability, easy preparation and low cost [18].
Previously, we have carried out successful work in this area. As a result of the studies of the biomimetic sensor for catalase and peroxidase activity, it was found that the electrode with TPhPFe3+/Al2O3 makes it possible to detect trace
concentrations of hydrogen peroxide in an aqueous solution [19-24].
In connection with the above, we used Ag as a transducer to prepare a biomimetic sensor.
3+
TPhPFe preliminarily adsorbed on Al2O3 was deposited on the Ag electrode by gluing (silver paste was used as the gluing material).
Experimental part
Experimental studies of the electrode potential of the catalase and peroxidase reaction as a function of time were carried out by the po-tentiometric method. The electrochemical setup was equipped with a magnetic stirrer to create an equilibrium solution. The background solution is bidistilled water. To determine the cata-lase activity, experiments were carried out in a reaction medium consisting of various concentrations of aqueous solutions of H2O2.
Figure 1 shows an infographic of a bio-mimetic sensor in an aqueous solution.
Results and discussion
On Figure 2 shows a graph of changes in e.m.f. systems depending on time at different concentrations of hydrogen peroxide for TPhPFe3+/Al2O3/Ag catalase biomimetic sensor. As is known [1, 2, 25, 26], as a result, cata-lase and electrochemical reactions proceed sequentially in the system, which can be represented as follows:
catalase reaction
TPhPFe3+/Al203/Si
2H202 -> 2H20 + 02 (1)
electrochemical reaction
02 + 4e" + 2H+ electrode > 20H" (2)
According to the literature, hydrogen peroxide is a weak dibasic acid. It can be assumed that, as a result of the catalase activity of the biomimetic sensor, the pH of the H2O2 solution should change due to reactions (1) and (2). Therefore, if the proposed reactions take place on a biomimetic sensor, then ultimately the pH of the solution should have a lower value than at the beginning of the experiment.
Analyte Smart material
v.? al2ü3 0 ai2o3
W* ■h • Z ° » » CK! Fe / \
N N-
J
al2o3 al2o3
Transducer
Electric signal
Fig. 1. Infographic
AE, mV
-0,14
-0,12 :
-0,1
-0,08
-0,06
-0,04
-0,02
8
t,sec.
Fig. 2. E.m.f. change systems versus time at low H2O2 concentrations for the TPhPFe +/Al2O3/Ag bio-
mimetic sensor.
1. Ag-electrode + silver paste
1. Ch2o2=1 wt.%
2. CH2O2 = 0,1 wt. %
4. CH2o2 = 10-4wt. %
5. CHz02 = 10-6wt. %
6. Coo2 = 10-8wt.%
1
0
0
2
4
6
Figure 3 shows the proposed mechanism of the catalase reaction on the surface of the biomimetic electrode.
Research continued on the development of a peroxidase biomimetic sensor for the determination of trace amounts of ethyl alcohol in an aqueous solution.
Figure 4 shows the results of potentio-metric studies to determine trace concentrations of С2Н5ОН in an aqueous solution. The studies were carried out as follows: first, the potential of bi-distilled water (background solution) was measured, then the required amount of C2H5OH was added and the change in potential was observed.
The experiments were carried out in a reaction medium consisting of 10-3 wt.%, 10-4 wt.% and 10-6 wt.% (Figures 4 and 5) of an aqueous solution of C2H5OH, and then 10-4 wt.% (Figure 4) and 10-6 wt.% (Figure 5) of an aqueous solution of H2O2.
Thus, in the electrochemical system under study, the biomimetic sensor in a water-alcohol solution had a limiting sensitivity threshold of 10-6wt% H2O2. It should be noted that the stability observed for the catalase bio-mimetic sensor is also characteristic of the pe-roxidase one.
Fig. 3. The proposed mechanism of the catalase biomimetic sensor in electrocatalytic mode.
20
40
60
80
t, sec
Fig. 4. E.m.f. change systems depending on time at low concentrations of С2Н5ОН for TPhPFe3+/Al2O3/Ag biomimetic sensor. C2H5OH - 10-6wt.% + Н2О2 - 10-4wt.%
1. C2H5OH - 10-4wt.% + Н2О2 - 10-4wt.%
2.
C2H5OH - 10-3wt.% + Н2О2 - 10-4wt.%
0
AE, mV. -175 -170 --165 --160 --155 -150 --145 --140
0
20
40
60
80
t,sec
Fig. 5. E.m.f. change systems depending on time at low concentrations of C2H5OH for
TPhPFe3+/Al2O3/Ag biomimetic sensor. 1. C2H5OH - 10-6wt.% + H2O2 - 10-6wt.%
2.
-2H5O
W
C2H5OH - 10-4wt.% + H2O2 - 10-6wt.%
3. C2H5OH - 10-3wt.% + H202 - 10-6wt.%
1202 I2O2 I2O2
3
Fig. 6. The proposed mechanism of the peroxidase biomimetic sensor in electrocatalytic mode.
According to the most probable mechanism of the catalase reaction, the peroxidase reaction (Figure 6) proceeds.
In the electrochemical system under study, along with the catalase reaction, the per-oxidase reaction also proceeds synchronously. These two coherently synchronized reactions interact with each other through the common
3+
intermediate TPhPFe /Al2O3, which is formed in both stages of these synchronized reactions.
It should be added that the proton transferred to the active center of the biomimetic electrode can be replaced by H+ from the volume of the reaction medium.
Possible ideas about the ways of realization of catalase and peroxidase reactions in the electrochemical mode are given by their mechanisms. The ratio of the rates of interaction of hydrogen peroxide and ethyl alcohol with the surface intermediate determines the ratio of the products formed in both reactions, oxygen and CH3CHO.
As noted above, the synthesized biomi-metic sensor showed high sensitivity and did not lose its activity for a long time under the influence of an oxidizing agent and its intermediates, as well as the end products of H2O2 decomposition and ethanol oxidation.
Conclusions
3+
Biomimetic sensors TPhPFe3+/Al2O3/Ag of the catalase and peroxidase types were prepared, which showed the lowest detection limit for hydrogen peroxide and ethyl alcohol in an aqueous solution.
The catalase biomimetic sensor develo-
3+
ped and synthesized by TPhPFe3+/Al2O3/Ag is active and makes it possible to determine trace concentrations of H2O2 and the maximum sensitivity to the concentration of H2O2 in an aque-
о
ous solution was 10- wt.%.
The synthesized biomimetic sensor TPhPFe3+/Al2O3/Ag of the peroxidase type has a limiting threshold of sensitivity to С2Н5ОН equal to 10-6 wt.%.
The developed and synthesized biome-metic sensors of the catalase and peroxidase
3+
types based on TPhPFe3+/Al2O3/Ag have high
sensitivity, stability, and did not lose their activity for a long time.
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Ag-ELEKTRODU ÜZORINDO KATALAZ VO PEROKSIDAZ TIPLI BIOMIMETIK SENSOR
N.N.Malikova, N.LOli-zada, T.M.Nagiyev
Sulu mahlulda hidrogen peroksidin va a§agi konsentrasiyali etil spirtinin iz miqdanni tayin etmak ügün katalaza va peroksidaz tipli biomimetik sensorun yaradilmasi üzarinda tadqiqatlar apanlmi§dir. Potensiometrik tadqiqatla katalaz va peroksidaz biomimetik sensorunun fiziki-kimyavi xüsusiyyati öyranilmi§, va transdüser kimi Ag olmu§dur. Hidrogen peroksidin süratli va daqiq tayini gox vacibdir, lakin bu masalanin heg da az vacib tarafi müxtalif man§ali sulu mühitlarda C2H5OH-nin iz miqdarinin tayini ila baglidir. Sulu mahlullarda C2H5OH konsentrasiyasini tayin etmak ügün ekspress üsullarin i§lanib hazirlanmasi ehtiyaci §arab va araq mahsullarinin keyfiyyatina dair talablardan irali galir. Malum oldugu kimi, mahsulun dadi va igma keyfiyyatlari metil va etil spirtlarinin nisbatindan asilidir. Bu i§da hidrogen peroksid va etanolun iz miqdarini tayin etmak ügün Ag elektrodu asasinda katalaz va peroksidaz tipli biomimetik sensor hazirlanmi§dir. Hidrogen peroksidin iz konsentrasiyalarini tayin etmak, elaca da a§agi a§karlama haddi, yüksak hassasliq, geni§ taninma diapazonu, yüksak sabitlik va takrar istifada ila sulu mahlulda etil spirtinin mikromiqdarlarini tayin etmak ügün biomimetik sensorun tatbiqi göstarilmi§dir TPhPFe3+/Al2O3/Ag asasinda hazirlanmi§ katalaz va peroksidaz tipli biomimetik sensor yüksak hassasliga, dayaniqliga malikdir va uzun müddat öz aktivliyini itirmir.
Agar sözlzr: biomimetik, sensor, katalaz, peroksidaz, d3mir tetrafenilporfirin, smart material.
