Co²⁺ ion-doped MnO₂ electrode for microfluidic H₂O₂ biosensors: formation, characterization, and catalytic performance

Abstract

H₂O₂ serves as a vital signalling molecule within biological systems and presents potential as a disease biomarker. Consequently, the development of sensitive and accurate sensors for its measurement within microfluidic systems is of significant importance. This article focuses on the development of a working electrode for an electrochemical microfluidic system designed for the quantitative determination of hydrogen peroxide. Here, we show that a MnO₂-based catalytic material can be improved by adding Co²⁺ ions. By changing the Co²⁺ concentration, an electrode material with improved catalytic performance was formed. The results of amperometric measurements of catalytic activity show a substantial increase in sensitivity and an expanded range of H₂O₂ concentrations that can be quantified using the new electrode. The characteristics of the MnO₂-modified Co²⁺ electrode were thoroughly examined by SEM, XPS. The combination of previously accumulated material and new data offers insights into the underlying reasons for the high catalytic activity of MnO₂, supported by quantum-chemical calculations. This research lays the groundwork for a method utilizing MnO₂ as a working electrode for detecting the concentration of H₂O₂ within a microfluidic cell. During experiments, a stop-flow protocol is employed, enabling data collection from coulometric measurements of the electrochemical processes occurring on the electrode within a limited measurement timeframe.

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1. Introduction

Microfluidic systems as an analytical tool are a relatively recent development, but their use opens up great prospects.^1–3 In the development of electrochemical microfluidic biosensors, the formation of working electrodes is of utmost importance. These electrodes should provide sensitivity and selectivity to the sensor, but another important characteristic, especially in microfluidic systems, is mechanical strength.^4–6 Therefore, electrodes with a catalyst layer are developed individually for specific analytes. The aim of this research is to develop a microfluidic biosensor for the determination of H₂O₂ in biological samples. Such sensors are of crucial importance and in high demand for a great number of applications. H₂O₂ is an anchor substance in the body's antioxidant system: peroxide compounds are intermediates or end products in almost all transformations.^7,8 Consequently, H₂O₂ can be used as a marker in biosensors for studying cancer tissue,^9–13 diseases of various organs,^14–17 etc. In Ukraine, since the beginning of the war, such studies are unfortunately in demand, in such topics as the influence of polytrauma on the development of oxidative stress in people exposed to blast injuries.

In most studies, Au nanoparticles are used as a catalyst for the detection of H₂O₂,^18–22 and the much cheaper MnO₂ (a specific catalyst for the decomposition of H₂O₂) is used much less frequently.^23,24 In our previous work, we developed an electrode that performed very well for glucose detection in microfluidic systems. This electrode was based on MnO₂ electrochemically deposited on the surface of ITO under conditions selected to exhibit maximum catalytic properties for high selectivity and sensitivity for hydrogen peroxide detection. In this study, we aim to improve the catalytic properties of MnO₂ by doping the oxide with Co²⁺ ions. The method of oxide doping is quite widespread,^25–27 but despite this, there are only a few works on MnO₂ doping with cobalt ions,^28,29 although one might have expected otherwise since the cobalt atom is very close in size to the manganese atom.

Investigating the reasons for the influence of doping on the properties of MnO₂ is also of interest. Most studies agree that doping affects defects in the crystal lattice of MnO₂.³⁰ We will try to consider this effect from the point of view of changes in the oxygen-containing compounds in the sediment composition compared to pure MnO₂. To do this, we use XPS, SEM, and quantum chemical modelling to analyse a set of electrodes characterised by different contents of Co²⁺ ions.

The final part of this research is the creation of a microfluidic chip where the developed electrode was utilised to determine H₂O₂ concentrations.

2. Experimental

2.1. Reagents and chemicals

All materials and chemicals were purchased commercially and used in the form in which they were received. Manganese sulphate, cobalt sulphate and sodium sulphate for electrolytes preparation, sodium dihydrogen phosphate dihydrate and sodium hydrogen phosphate dihydrate for preparation of phosphate buffer (PB) (0.1 M, pH 7), and hydrogen peroxide (30%) were supplied by Sigma Aldrich. All solutions were prepared with ultra-pure water (18.2 MΩ cm) from a Sartorius Arium Comfort I system. ITO-coated glass was obtained from Biotain Crystal Co., Limited, with the following characteristics: resistance 6÷8 Ohm sq⁻¹, ITO coating thickness ∼185 nm, transmittance >84%.

2.2. Fabrication of MnO₂ films on the surface of ITO

Glass substrates were sonicated for 20 min in acetone, then in ethanol, washed with deionised water and dried with an Ar stream. A hole laser cut in Kapton tape defined the working area of the electrode of 0.69 cm². The electrochemical formation of the electrodes’ functional layer was carried out by the methods of cyclic voltammetry (CV) with the region of scanned potentials from 1.0 V to 1.8 V. As electrolytes, we used water solutions of 0.005 M MnSO₄, 0.5 M Na₂SO₄ with pH 1 (the pH was adjusted by adding sulfuric acid) and added CoSO₄ in concentrations 0.01 M, 0.05 M and 0.1 M. The scan rate was 50 mV s⁻¹. All electrochemical experiments were performed using a potentiostat/galvanostat BioLogic, model SP-300 in a conventional three-electrode glass cell. An Ag/AgCl/3 M KCl electrode was used as reference and a Pt mesh as counter electrode. Each potential reported in this paper is given against Ag/AgCl/3 M KCl at a laboratory temperature of 25 ± 1 °C. After electrodeposition, the dioxide films were dried at 60 °C or 90 °C in air for 15 h. Initial tests were performed with a Pt working electrode, which is a platinum disk with a diameter of 4 mm sealed in Teflon. The electrode surface was prepared as follows: mechanical polishing, rinsing with deionized water, and oxidation in a “piranha” solution (a mixture of concentrated sulfuric acid and hydrogen peroxide) for 5 min.

2.3. Analytical procedures for determination of H₂O₂

The electrochemical measurements were all carried out at a temperature of 37 °C (the temperature was maintained in a water bath). The cyclic voltammograms of the MnO₂/ITO electrode were scanned from 0.2–0.9 V in 0.1 M PB. The scan rate was 100 mV s⁻¹.

The catalytic activity of the MnO₂/ITO electrodes was determined using chronoamperometry in 0.1 M PB. The amperometric response was measured at an applied potential of 0.45 V vs. Ag/AgCl/3 M KCl during repeated injections of hydrogen peroxide.

2.4. Physical characterization of MnO₂/ITO electrodes

The Microstructure and morphology of the MnO₂/ITO samples were investigated using a FEI Nova NanoSEM 450 scanning electron microscope.

Electrochemical impedance spectroscopy (EIS) was measured at OCP in 5 mM [Fe(CN)₆]^3−/4− in the frequency range of 0.25 to 100 000 Hz with an amplitude of 5 mV.

X-ray photoelectron spectroscopic (XPS) measurements were performed using a PHI 5000 VersaProbe (ULVAC-PHI) spectrometer with monochromatic Al Kα radiation (h = 1486.6 eV) from an X-ray source operating at 100 µm spot size, 25 W and 15 kV. The high-resolution (HR) XPS spectra were collected with a hemispherical analyser at a pass energy of 117.4 and an energy step size of 0.1 eV. The X-ray beam was incident at the sample surface at an angle of 45° with respect to the surface normal, and the analyser axis was located at 45° with respect to the surface. The CasaXPS software was used to evaluate the XPS data. Deconvolution of all HR XPS spectra was performed using a Shirley background and a Gaussian peak shape with 30% Lorentzian character. The measured binding energies (BE) for individual elements were corrected with respect to the C 1s carbon peak at 284.8 eV.

2.5. Quantum chemical calculations

Quantum chemical modelling was performed using non-empirical methods of the WinGAMESS program.^31–35 Cluster systems were calculated using the spin-unrestricted Hartree–Fock method. The central metal atom was described by the ECP basis, and the ligand atoms by the 6-311G basis. The hybrid B3LYP method of density functional theory was used, which included five functionals: Becke, Slater, and Hartree–Fock exchange functionals, as well as LYP and VWN5 correlation functionals. In the simulation process, the studied ions were optimised in the environment of the first solvation shell and the energies of the optimised complexes were calculated. Then, the energies were refined taking into account solvation using the polarisation continuum model.

Since it makes sense to compare the electronic energies of different structures only if they contain the same type of atoms, when calculating the energy of complexes with different geometries of the first hydrate shell, we considered cluster systems of the composition [Mn^z+(H₂O)_n]·xH₂O under the condition that (n + x) = const. In this structure, n water molecules are directly connected to the central atom of the aqua complex, and the remaining x water molecules are at some distance, which excludes their chemical interaction with the metal ions. The cluster structure with the smallest sum of the energies of the metal aqua complex [Mn^z+(H₂O)_n] and the energies x of the water molecules moved to the outer sphere was considered to be the most energetically favourable.

2.6. Microfluidic testing. Fabrication of the microfluidic system

The PDMS chip is a T-shaped channel with two inputs. The channels are 250 microns high and 500 microns wide. Fluid manipulations were controlled by a Nemesys pump controlled using dedicated computer software. The PDMS chip was connected to the ITO glass with a 2 mm wide working electrode formed on it and with a graphite counter electrode at a distance of 2 mm from the working electrode. A silver chloride electrode was connected between them as a reference electrode. All measurements were carried out and recorded using a potentiostat, the characteristics of which are given in section 2.2.

3. Results and discussion

3.1. Electrodeposition of doped Co²⁺ MnO₂ on ITO

The formation of precipitates was carried out based on the experience of precipitation of pure manganese dioxide from a sulphate electrolyte (Fig. 1d).

Fig. 1 (a) CV obtained by polarizing the Pt electrode in an electrolyte of the composition 0.5 M Na₂SO₄ + 0.1 M CoSO₄, pH1; electrodeposition of MnO₂ by CV method from solution of 0.005 M MnSO₄ + 0.5 M Na₂SO₄ electrolyte, pH1 in the presence of (b) 0.01 M CoSO₄, (c) 0.1 M CoSO₄, (d) without additives. Scan rate 50 mV s⁻¹.

In our previous work³⁶ the MnO₂ coating with the best catalytic and mechanical properties was formed by cyclic voltammetry (CV) in a solution with pH 1 and subsequently dried at 60 °C for 12 hours. These settings were selected as the starting point for the new electrode formation. The pristine MnO₂ electrode is hereafter referred to as pH1_CV_60 and serves as a reference for comparison with the Co²⁺-doped samples throughout this study. To obtain precipitates doped with Co²⁺ ions, sulphuric acid electrolytes were prepared with the addition of cobalt sulphate in concentrations from 0.01 M to 0.1 M. The quantities are significant, but it should be taken into account that it is unlikely that positively charged ions will move to the positively charged anode during electrophoresis; most likely, the doping mechanism is somewhat different.

Fig. 1a shows the CV dependence in a solution where only Co²⁺ ions are present. No reaction is observed on the platinum anode, which means that all the Ox-Red reactions in Fig. 1b–d are associated with the oxidation of the Mn²⁺ ions. Fig. 1b and c show the process of MnO₂ coating formation in the presence of 0.01 M and 0.1 M Co²⁺ ions in solutions, respectively. The influence of the doping agent on the deposition process can be analysed by comparison with the dependence in Fig. 1d (precipitation of pure manganese dioxide). We observe a shift in the electrooxidation potential of Mn²⁺ by 0.5 V in the negative direction, which we associate with the influence of Co²⁺ ions. Based on the following studies, it can be assumed that Co²⁺ ions affect the structure and composition of the complex, in which divalent manganese is oxidized at the anode. Perhaps it is as part of such complexes that cobalt is delivered to the surface of the electrode, where it is incorporated into the forming deposit. The impact of cobalt ions on the nucleation and initial stages of manganese dioxide electrodeposition remains a subject of significant interest and warrants further investigation.

For catalytic studies, a set of sediments was prepared using different precipitation conditions. The differences are in the content of cobalt ions in the electrolyte and the number of CV cycles, resulting in different thicknesses of the sediment. The names of the electrodes used in the remainder of the article, with the corresponding deposition conditions, are summarised in Table 1.

Table 1 Conditions of precipitation formation

Name of sample	Common conditions of electrodeposition electrolyte: 0.5 M Na2SO4, 0.005 M MnSO4, pH1, with the addition of CoSO4
	Concentration of Co^2+, M	Number of CV cycles
0.01Co_20	0.01	20
0.01Co_30	0.01	30
0.01Co_40	0.01	40
0.01Co_50	0.01	50
0.05Co_20	0.05	20
0.05Co_30	0.05	30
0.05Co_40	0.05	40
0.05Co_50	0.05	50
0.1Co_20	0.1	20
0.1Co_30	0.1	30
0.1Co_40	0.1	40
0.1Co_50	0.1	50
pH1_CV_60	—	30

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3.2. Characterization of electrodes

X-ray photoelectron spectroscopy (XPS) measurements were carried out to determine the presence of Co²⁺ ions in the sediment of MnO₂. We also investigated the influence of the electrolyte composition on the content and composition of the resulting catalysts from the point of view of manganese forms. The following three coatings were selected: 0.01Co_30; 0.05Co_30; 0.1Co_30 (the description of precipitation formation is given in section 3.1). The composition of electrolytes differed only in the concentration of cobalt ions.

The obtained XPS spectra are shown in Fig. 2. We used studies^37–40 that provide detailed descriptions of the interpretation of the XPS spectra of MnO₂ sediments. The Mn 2p spectrum provides information that we indeed have a precipitate of manganese dioxide through the value of spin energy separation, which is equal to the distance between the Mn 2p_3/2 and Mn 2p_1/2 signals. The areas of the peaks at 641.9 eV and 644.9 eV of the Mn 2p_3/2 signal allow us to calculate the ratio of the Mn³⁺ and Mn⁴⁺ forms. Particularly interesting results were obtained from the analysis of the O 1s spectrum. We identify the peak at 529.6 eV as the presence of Mn–O–Mn bonds, and the peak at 531.3 eV corresponds to the presence of Mn–O–H bonds. The Co 2p2 spectrum confirms the presence of Co²⁺ ions in the resulting precipitates. The amount of cobalt ions we present as the content in the sediment relative to all manganese ions. The results are given in Table 2. We also added the results of XPS analysis of pure MnO₂, which was deposited under the same conditions. This way we can see the effect of doping on the catalyst composition.

Fig. 2 Mn 2p, O 1s, Co 2p XPS spectra of (1a, 1b) pH1_CV_60; (2a, 2b, 2c) 0.01Co_30; (3a, 3b, 3c) 0.05Co_30; (4a, 4b, 4c) 0.1Co_30. For details, see the main text.

Table 2 The XPS spectra interpretation

Precipitate	Mn 2p spectrum, spin energy separation, eV	Mn^3+, %	Mn^4+, %	Mn–O–Mn, %	Mn–O–H, %	Co^2+, %
Pristine MnO2 (pH1_CV_60)	11.7	67.9	32.09	65.53	18.62	—
0.01Co_30	11.7	47	52.9	26.23	65.68	2.3
0.05Co_30	11.6	63.9	36	10.99	74.36	2.4
0.1Co_30	11.8	54.3	45.7	4.95	88.84	6.8

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For reference, data for the pristine MnO₂ electrode (labelled ‘pH1_CV_60’ in section 3.1) are included. From the data in the table, we can conclude that Co²⁺ ions are indeed present in the structure of the formed MnO₂-based coating. However, their content does not linearly depend on the concentration in the solution. We see a sharp increase at 0.1 M Co²⁺ concentration in the electrolyte.

The ratio of Mn³⁺/Mn⁴⁺ forms changes in the direction of increasing the content of manganese in its tetravalent form. The increase in catalytic activity due to the increase in the Mn⁴⁺ content agrees with the conclusions from our previous publication.³⁶ Also, the data shows that with the addition of Co²⁺ ions into the sediment, the amount of Mn–O–H bonds increases sharply. Such significant changes in the sediment composition can also influence the increase in the catalytic activity of the electrodes.

Electrochemical impedance spectroscopy (EIS) was performed on selected samples, with the corresponding data and parameters provided in Fig. 3 and Table 3, respectively.

Fig. 3 Electrochemical impedance spectroscopy was performed on the (1) pH1_CV_60, (2) 0.01Co_30, (3) 0.05Co_30, (4) 0.01Co_30, (5) 0.01Co_50. For details, see main text.

Table 3 The values of charge-transfer resistance, R₂, for the corresponding precipitation in Fig. 3

Electrode	Pristine MnO2 (pH1_CV_60) (1)	0.01Co_30 (2)	0.05Co_30 (3)	0.1Co_30 (4)	0.1Co_50 (5)
R2, Ohm	87.11	123.8	131.1	238.2	708.6

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Charge transfer kinetics within the film were elucidated using the R₁ + Q₂/(R₂ + W₂) equivalent circuit. The parameters include uncompensated resistance R₁, charge-transfer resistance R₂, a constant phase element Q₂, and the Warburg impedance W₂.

A comparative analysis of the data presented in Fig. 3 and Table 3 reveals a clear correlation between the electrode composition and its electrochemical properties. For reference, data for the pristine MnO₂ electrode (labelled ‘pH1_CV_60’ in section 3.1) are included. As shown in Table 2, an increase in the Co²⁺ dopant concentration leads to a significant reduction in electrode conductivity. This trend is attributed to the formation of the MnOOH phase during the doping process, as indirectly supported by the XPS analysis. Furthermore, comparing samples 0.1Co_30 and 0.1Co_50 shows that increasing the film thickness further diminishes conductivity, which is expected to influence the subsequent electrocatalytic performance.

The morphology of the sediments were studied by scanning electron microscopy. Fig. 4 shows precipitates doped with Co²⁺, electrodeposited in solutions with different cobalt ion contents. Fig. 4g and h show the morphology of pure MnO₂ and are given for comparison. All precipitates were formed during 30 cycles of CV at the same pH of the electrolyte and the same concentration of Mn²⁺ ions.

Fig. 4 SEM images of MnO₂ films deposited under conditions: (a and b) 0.01Co_30, (c and d) 0.05Co_30, (e and f) 0.1Co_30, (g, h) pH1_CV_60. For details, see the main text.

First of all, it is clear that the doped coatings are very similar. The structure is branched like the branches of trees. When compared with pure MnO₂, the structure is more branched, but the branches are smaller. We also observe an increase in the density of sediments; the structure of the ITO is not visible through the layer of manganese dioxide. At lower magnification we note the uniformity of the coating. We observe the integrity of the sediment, there are no cracks, which was typical for pure MnO₂. As was the case with pure MnO₂, XRD (SI) shows that the deposited film is amorphous, without clear peaks from manganese oxide.³⁶

When cobalt ions are added to the electrolyte, the resulting deposits become much denser, but the structure remains branched. The composition of the coating has undergone significant changes. The ions Co²⁺ were found in the structure of all sediments. The effect on the structure of MnO₂ is also noticeable: the number of hydroxyl groups associated with manganese has sharply increased (Mn–O–H bonds).

3.3. Analytical procedure for the determination of H₂O₂

Based on convincing data from the literature,^41,42 the mechanism of formation of an electrochemical signal during anodic polarisation of the working electrode is as follows. H₂O₂ decomposes into water and molecular oxygen when it comes into contact with MnO₂. The catalytic action is exerted by Mn⁴⁺, which is reduced to Mn³⁺. During anodic polarisation of the electrode, the Mn³⁺ will be electrochemically oxidized back to Mn⁴⁺, which can be measured as a faradaic current. Interpretation of the data from the catalytic studies will be grounded in this theory.

The catalytic effect of the precipitate and the potentials at which it is observed were investigated by comparing voltammograms recorded in the presence and absence of H₂O₂ (Fig. 5). The cyclic voltammograms in the presence of H₂O₂ (Fig. 5(2)) show a higher current than CVs in the absence of hydrogen peroxide (Fig. 5(1)) even at low potentials. This means that even a low anodic polarisation will provide an analytical signal.

Fig. 5 CVs of electrode 0.1Co_30 in the absence (1) and presence (2) of 2 mM H₂O₂ in PB. Scan rate 100 mV s⁻¹.

Chronoamperometry was chosen as the main method for studying the catalytic activity in determining H₂O₂ for a set of 12 electrodes. The method is indicative and convenient in interpretation. 0.45 V vs. Ag/AgCl/3 M KCl was chosen as a favourable potential at which the analytical signal is recorded. At this potential, we record sufficient catalytic activity, but avoid excess noise from the evolved gaseous products. Additionally, low values of electrode polarisation are desirable when studying biological objects, due to the presence of potentially interfering reactions.

Fig. 6 shows the I–t dependences of all electrodes with H₂O₂ injections in such a way that the concentration of peroxide in the electrolyte changes stepwise according to the following scheme: 0.1 mM → 0.2 mM → 0.5 mM → 1 mM → 2 mM → 3 mM → ⋯ → n mM. The high level of noise seen in Fig. 5 is due to the release of gaseous products from the decomposition of H₂O₂ while testing the electrodes, as described above.

Fig. 6 Amperometric I–t curveson successive injection of H₂O₂ into the stirring PB at the applied potential of 0.45 V vs. Ag/AgCl/3 M KCl of MnO₂ dopped with Co²⁺ electrodes: (a1) 0.01Co_20, (a2) 0.01Co_30, (a3) 0.01Co_40, (a4) 0.01Co_50, (b1) 0.05Co_20, (b2) 0.05Co_30, (b3) 0.05Co_40, (b4) 0.05Co_50, (c1) 0.1Co_20, (c2) 0.1Co_30, (c3) 0.1Co_40, (c4) 0.1Co_50. Note that vertical scales are different.

For clarity, the results are divided into three groups: each Fig. 6a, b and c shows chronoamperograms of electrodes formed with the same concentration of Co²⁺ ions and differ in sediment thickness. Thus, it is immediately clear that electrodes deposited with 20 to 30 CV cycles exhibit the highest activity. The activity of the catalysts decreases sharply at 40 and 50 CV cycles. This decrease in MnO₂ catalytic activity is a consequence of the relatively poor electrical conductivity of the material The I–t dependencies of the best representatives of each group are converted into an amperometric response to H₂O₂ concentration and are compared with each other and with a pristine MnO₂ electrode (labelled ‘pH1_CV_60’ in section 3.1) (Fig. 7).

Fig. 7 Relationship of the amperometric response vs. the H₂O₂ concentration.

All catalysts doped with Co²⁺ ions show a sharp increase in the range of detectable H₂O₂ concentrations compared to pure MnO₂.

Another important parameter of such a dependence is its linearity. In this case, we have a calibration plot of electrocatalytic current versus H₂O₂ concentration. Pure MnO₂ shows a linear relationship with the concentration of H₂O₂ from 0.1 mM to 3 mM. Electrode 0.01Co_20 – from 0.1 mM to 4 mM, electrode 0.05Co_30 – from 0.1 mM to 6 mM, electrode 0.1Co_30 – from 0.1 mM to 9 mM. When selecting the electrode with the best performance, we distinguish 0.1Co_30 with the widest range of the calibration plot. Such a great improvement in the catalytic properties of the sediment, from the point of view of all the above studies, can be associated with a sharp increase in the Mn–O–H groups and their participation in the decomposition of hydrogen peroxide. We are going to consider changes in the electrode structure during the electrode transition. For this, we use the capabilities of quantum chemical modelling.

3.4. Quantum chemical computations

The purpose of the calculations was to study the oxidation reaction of Mn³⁺ to Mn⁴⁺, which is responsible for the electrocatalysis of the material. Section 2.5 provides references showing good correlation between calculated data and experimental data for Mn complexes.

As is known from previous studies,⁴³ manganese dioxide contains Mn³⁺ in hydrolysed form. It is in these forms that we find the presence of the Mn–O–H bonds that attracted attention in the XPS study. All possible clusters were analysed and the actual charge distributions in them were calculated, and the spatial particles were constructed. The results are shown in Table 4.

Table 4 Distribution of charges on manganese atoms and ligands in clusters [Mn (OH)_x(H₂O)_n]^z+ (H₂O)_m and [Mn O(OH)(H₂O)₂] + (H₂O)₂

Clusters	q(Mn)	q(∑OH)	q(O)	q(∑H2O)
[Mn(OH)(H2O)5]^2+	+1937	−0362	—	+0425
[Mn(OH)2(H2O)4]^+	+1751	−0996	—	+0244
[Mn(OH)3(H2O)2]^0(H2O)	+1500	−1607	—	+0127
[MnO(OH)(H2O)2]^0(H2O)2	+1342	−0561	−0829	+0048
[Mn(OH)(H2O)5]^3+	+2121	+0076	—	+0801
[Mn(OH)2(H2O)4]^2+	+1819	−0341	—	+0522
[Mn(OH)3(H2O)2]^+(H2O)	+1589	−0832	—	+0244
[MnO(OH)(H2O)2]^+(H2O)2	+1617	−0414	−0469	+0266

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From Table 4, which shows the charge levels in trivalent manganese complexes, we can conclude that in reality, the Manganese atom does not carry a charge of +3, but rather the charge is redistributed among the oxygen-containing clusters around the central atom. Therefore, the hydrolysed form of Mn³⁺ is natural. And it is precisely in such complexes that a large presence of Mn–O–H bonds is observed.

The next stage of calculations is the analysis of energy changes associated with the transition of an electron during the oxidation of trivalent manganese to the tetravalent state and back. This is actually the reaction that provides electrocatalysis. Possible reactions of electron transition from hydrolysed trivalent manganese are presented in Table 5. There you can see the energy characteristics of electron transition and the calculated standard electrode potentials of these reactions.

Table 5 Values of standard Red-Ox potentials of one-electron oxidation of hydrolysed forms of manganese(III) aquacomplexes

No.	Reaction	ΔE, kJ mol^−1	φ^0, V (standard hydrogen potential)
1	[Mn(OH)(H2O)5]^2+ = [Mn(OH)(H2O)5]^3+ + e	775 791	2.90
2	[Mn(OH)2(H2O)4]^+ = [Mn(OH)2(H2O)4]^2+ + e	692 902	2.15
3	[Mn(OH)3(H2O)3]^0 = [Mn(OH)3(H2O)2]^+(H2O) + e	577 246	1.11
4	[Mn(OH)3(H2O)2]^0(H2O) = [Mn(OH)3(H2O)2]^+(H2O) + e	602 132	1.33
5	[MnO(OH)(H2O)2]^0(H2O)2 = [MnO(OH)(H2O)2]^+(H2O)2 + e	512 457	0.53

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Most likely reaction number 5 occurs. The standard electrode transition potential is very low, which correlates with our catalytic measurements. We conclude that trivalent manganese in the electrolytic film MnO_x is present in the form of the compound [MnO(OH)(H₂O)₂]. Removing water and the OH group from the structure showed that such a particle could not be calculated, which demonstrates the importance of the presence of these groups in the structure of the sediment. Calculations also indicate that other particle geometries are possible, but the electrode transition potential increases sharply in this case. In Fig. 8 shows the structures of [MnO(OH)(H₂O)₂] in the original and, accordingly, in the oxidized state.

Fig. 8 Spatial structure of particles: a – [MnO(OH)(H₂O)₂]⁰; b – [MnO(OH)(H₂O)₂]⁺.

As can be seen, the removal of an electron from [MnO(OH)(H₂O)₂] does not lead to a significant reorganization of its structure. The Mn–OH bonds remain unchanged and stable: the internuclear distances in them are 1.99 Å and 2.01 Å in the molecular form and 1.99 Å and 1.99 Å in the ionized form. A slight displacement of Manganese and Oxygen nuclei is observed only in Mn–O and Mn–O–H bonds. In the molecular form, they are 1.73 Å and 1.88 Å, respectively, and in the ionized form, 1.65 Å and 1.74 Å. The valence angle O–Mn–O–H also changes little: from 163.30 in the molecular form to 152.20 in the ionized form. The above listed indicates the absence of major structural and chemical transformations in the redox system [MnO(OH)(H₂O)₂]/[MnO(OH)(H₂O)₂]⁺. Which makes it possible to use in objects in which rapid charge cycling is assumed.

Thus, it can be concluded that the more Mn–O–H bonds present in the precipitate, the greater the probability of the formation of a hydrolysed structure of trivalent manganese [MnO(OH)(H₂O)₂] a certain geometry. Then the reactions of the Mn³⁺/Mn⁴⁺ transformations will occur easily and quickly at minimal electrode polarization potentials. This is reflected in the excellent catalytic performance of the MnO₂ electrode.

3.5. Measurements in a microfluidic system

The developed MnO₂-based electrode with the best performance was implemented as a working electrode in an amperometric microfluidic device for determining the concentration of H₂O₂ in a solution. The creation of the microfluidic cell is described in detail in section 2.6 and is also visually represented in the Fig. 9. The T-shaped channels and two inlets allow manipulation with two working solutions. One of them was PB with H₂O₂ of a given concentration, and the second was pure PB, which was used to wash the channels between receiving signals. In this system, with a very small working electrode area and microamounts of electrolyte, the amplification of the analytical signal is achieved due to the hydrodynamic effect of analyte delivery. H₂O₂ concentrations of the order of µM were measured. However, one drawback of the electrode was noticed. Despite the low speed of liquid movement (20 µl s⁻¹), the MnO₂ layer was quickly washed off during measurements. This is because Mn³⁺ compounds are soluble in water. Thus, despite the high sensitivity, the signal would not reach a steady-state, and a calibration curve could not be established.

Fig. 9 Microfluidic cell with MnO₂-based working electrode in work.

Therefore, an alternative scheme for working with MnO₂ electrodes needed to be developed. We stop the flow of electrolyte with H₂O₂ when it hits the electrode and measure the charge (q) passing through the electrode while hydrogen peroxide decomposes. This charge is related to the amount of decomposed H₂O₂ through Faraday's first law:

m = M × q/F × n,

where m is the mass of the manganese that took part in the Ox-Red reaction, proportional to the decomposed hydrogen peroxide, M is the molar mass manganese dioxide, q is the total electric charge passed through the electrolyte (in Coulombs), n is the number of electrons involved in the reaction and F is Faraday's constant, approximately 96 485 Coulombs per mol.

For specific electrolysis M, F, n are constants; thus, the value of q is equivalent to m. In the case of constant-current electrolysis,

q = It

where I is the current, t is the time of process.

The Fig. 10 shows the signal that we received. The PB solution flows through the channel to point 1. From point 1 to point 2 a solution containing hydrogen peroxide flows. From point 2 to point 3, the movement of solutions in the system stops. From point 3, pure PB flows and washes the cell channels, and the process can be repeated. The charge (q) is measured as the area under the chronoamperogram curve from point 2 to point 3.

Fig. 10 I–t dependence obtained in the microfluidic system. Analyte 20 µM H₂O₂ in 0.1 M PB.

Based on our experience, time periods should be selected in accordance with the object of study. In this investigation, the optimal program was as follows: PB flow for 200 s, then inclusion of H₂O₂ in phosphate buffer for 30 s, stopping the flow for the reaction to proceed for 300 s. The flow rate is always 20 µl s⁻¹. In the process of developing the methodology, 4 electrodes were used and 6 concentrations of hydrogen peroxide were measured on them: 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM. As a result, calibration curves are considered as the dependence of the charge recorded on the working electrode on the concentration of H₂O₂.

The electrodes were evaluated in terms of the reproducibility of successive measurements on them. Referring to Fig. 10, then the first measurement spans from 200 s to 500 s, the second measurement is from 750 s to 1050 s, and the third measurement is from 1300 s to 1600 s. Results for one of the electrodes are shown in Fig. 11. The first measurement can often be unreproducible as shown by a large variation and missing data points. Dependencies 2 and 3 are quite reproducible and have a correlation coefficient of about 0.99. When processing the results. For this reason, the second measurement was used in the analyses.

Fig. 11 Dependence of charge recorded on the working electrode on the concentration of H₂O₂: 1 – first measurement, 2 – second measurement, 3 – third measurement.

Fig. 12 shows the calibration curves for four electrodes. Several series of measurements (6 concentrations of hydrogen peroxide) were carried out on all electrodes; the figure shows an example of two series of measurements for the fourth electrode.

Fig. 12 Dependence of charge recorded on the working electrode on the concentration of H₂O₂: 1 – first electrode, 2 – second electrode, 3 – third electrode, 4_1 –first measurements on the fourth electrode, 4_2 – second measurements on the fourth electrode.

All electrodes showed the possibility of constructing a calibration curve with a correlation coefficient from 0.96 to 0.99. However, we note that the reproducibility between electrodes is insufficient. In effect, this means that before using the electrode, a separate calibration should be carried out. But, as the examples 4_1 and 4_2 show, very high reproducibility is seen from measurement to measurement, which implies that this is a manufacturing issue, rather than a fundamental limitation of the MnO₂ electrodes.

This study showed that a highly sensitive electrode for a microfluidic cell was developed for quantitative measurement of H₂O₂ at concentrations as low as 5 μM.

The proposed MnO₂ electrode may exhibit cross-sensitivity to common interferents (e.g., ascorbic and uric acids), potentially compromising its performance in clinical settings. To ensure selectivity in such a case, either mathematical compensation or a Nafion permselective membrane can be employed. The latter acts as a size-exclusion barrier, preventing larger molecules from reaching the electrode surface while allowing the diffusion of H₂O₂.⁴⁴

4. Conclusion

Doping MnO₂ with Co²⁺ ions is a simple procedure, but it significantly improves the catalytic properties of this catalyst. We combined physical methods for studying the composition of sediments and their morphology with quantum chemical modelling of the basic catalytic reaction. The results indicate that due to the increase in the hydrolysed forms of manganese (Mn–O–H bonds), the range of determination of H₂O₂ concentrations relative to pure MnO₂ increases several times. In systematic catalytic studies in a macroscopic cell with stirred solution, the electrode 0.1Co_30 with the highest content of Co²⁺ ions and Mn–O–H bonds in the composition showed mechanical strength, a stable range of determination of hydrogen peroxide concentrations from 0.1 mM to 9 mM. For comparison, the range of detectable H₂O₂ concentrations when using an electrode with pure MnO₂ is from 0.1 mM to 3 mM.

Testing the 0.1Co_30 sample with the best characteristics in a microfluidic system showed the high sensitivity of the catalyst. Stable determination of H₂O₂ concentration 5 µM. The measurement mechanism we have developed takes into account the characteristics of the electrode composition to ensure stability of the electrode during repeated measurements and ensures sufficiently long-term operation with a large number of analysed samples.

We would like to add that after use the MnO₂ layer is easily removed and the ITO base can be used again. Our experience shows that ITO glass can be use this way for at least 6 months with a reprecipitation frequency of once every 1–2 weeks. This makes the analysis inexpensive and environmentally friendly.

Author contributions

VP: conceptualization, formal analysis, investigation (electrochemical experiments), writing – original draft; MK investigation (XPS); GM: investigation (calculations); MJN: formal analysis, funding acquisition, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

Data associated with this paper have been made available at the RepOD repository, https://doi.org/10.18150/PUHR5I.⁴⁵

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6dt00434b.

Acknowledgements

This work was supported by the Norway Grants 2014–2021 via the National Centre for Research and Development (NOR/POLNOR/UPTURN_UA/0060/2019).

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