Facile synthesis of Ag@Fe3O4/ZnO nanomaterial for label-free electrochemical detection of methemoglobin in anemic patients

Methemoglobinemia (MetHb, Fe3+) is a chronic disease arising from the unequal distribution of oxyhemoglobin (HbFe2+, OHb) in the blood circulatory system. The oxidation of standard oxyhemoglobin forms methemoglobin, causing cyanosis (skin bluish staining). Methemoglobin cannot bind the pulmonary gaseous ligands such as oxygen (O2) and carbon monoxide (CO). As an oxidizing agent, the biochemical approach (MetHb, Fe3+) is modified in vitro by sodium nitrite (NaNO2). The silver-doped iron zinc oxide (Ag@Fe3O4/ZnO) is hydrothermally synthesized and characterized by analytical and spectroscopic techniques for the electrochemical sensing of methemoglobin via cyclic voltammetry (CV). Detection parameters such as concentration, pH, scan rate, electrochemical active surface area (ECSA), and electrochemical impedance spectroscopy (EIS) are optimized. The linear limit of detection for Ag@Fe3O4/ZnO is 0.17 µM. The stability is determined by 100 cycles of CV and chronoamperometry for 40 h. The serum samples of anemia patients with different hemoglobin levels (Hb) are analyzed using Ag@Fe3O4/ZnO modified biosensor. The sensor's stability, selectivity, and response suggest its use in methemoglobinemia monitoring.

www.nature.com/scientificreports/ in patients with high levels of MetHb (10-15%) than with normal hemoglobin due to oxygen deficiency 2 . The clinical trials find that MetHb levels vary from 10 to 70%. An increase in [Fe 3+ ] concentration changes red blood cell (RBCs) color from red to brownish mud, indicating severe cyanosis and hypoxia 14 . Methylene blue (MB) is the first antidote cofactor for NADH reductase, which lowers chronological methemoglobinemia in respiratory and cardiac patients 15 .
Recent studies show that methemoglobin (MetHb) can be synthesized by a controlled reaction of hemoglobin (Hb) with potassium ferrocyanide [K 3 Fe(CN) 6 ] at specific temperatures and pressure. Redox reactions convert standard hemoglobin to methemoglobin. Hydrogen peroxide (H 2 O 2 ), hydrogen sulfide 16 (H 2 S), and sodium nitrite (NaNO 2 ) are the strong oxidizing agents catalyzing this reaction 17 . Several methods have been adopted for detecting hemoglobin and its components. Spectroscopic and separation techniques such as infrared (IR) spectroscopy, mass spectrometry (MS), fluorometry 18 , fluorescence spectroscopy, gas chromatography 19 (GC), and high-performance liquid chromatography (HPLC) are used for the hemoglobin detection. Specific gravity, colorimetry, electrochemical techniques, and Kurt electric resistance 20 are also employed for quantitative and qualitative analysis of methemoglobin.
The developments in electronic, wearable, and robotic technologies have brought variations in mechanical sensors 21 . Electrochemical sensors are inexpensive, durable, sensitive, simple, and portable 22,23 . They have specific mechanical stress/strain properties for biomolecule detection 24 . Advancements in electrical detections and fabricated pathways have a role in developing chemical and electrical composites as electrochemical sensors 25,26 . Electrochemical methods include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) 27 , chronoamperometry (CA), and differential pulse voltammetry (DPV) [28][29][30] .
Various electrochemical biosensors have been developed for hemoglobin detection. Voltammetric MXenebased two-dimensional (2D) transition metal carbides 31 , tin oxide (SnO 2 ) nanoparticles 32 , graphite carbon nitride (G-C 3 N 4 ), 33 platinum (Pt) doped iron phosphorus carbide (FeP-C) 34 , tellurium nanowires doped graphene oxide (TeNWs/GO) nanocomposite 35 , ticlopidine/titanium dioxide (Tic-TiO 2 ) nanoparticles 36 , boron-doped grapheme (B-GQDs) quantum dots 37 , and chiral nano-imprinted (Fe 3 O 4 /SiO 2 ) polymers 38 have been reported for the quantitative analysis of hemoglobin. MXene compositional variability, hydrophilicity, elevated metallic conductivities, and large surface make them efficient tools for analyte detection. Although, it faces some challenges during its synthetic process, i.e., no proper termination step and development of new etching layers. A less toxic and eco-friendly method must be introduced for MXene synthesis 31 . SnO 2 is utilized in different fields, including lithium-ion batteries and dye-sensitized solar cells, due to their high chemical stability and catalytic activity 32 . G-C 3 N 4 has been extensively employed as an electrochemical chemosensor and a water-splitting agent. Although it is a major limitation, it cannot be utilized alone because of its low conductivity. It is used with semiconductors, metal nanoparticles, carbon material, and metal ions 33 .
In this study, Ag@Fe 3 O 4 /ZnO (SIZO) nanomaterial is synthesized hydrothermally for the quantitative electrochemical sensing of methemoglobin (MetHb) in anemic patients. Methemoglobinemia (anemic) patients carry lower oxygen levels in a metabolic cycle, which leads to cyanosis. The (SIZO) modified electrode shows enhanced electro-catalytic activity and fast electron transfer for in vitro and in vivo electrochemical sensing of methemoglobin compared to other nanomaterials. By far, sensors have been reported to detect hemoglobin, and its link to anemia has been a concern. According to our literature survey, the relationship between sensors for detecting methemoglobin and anemia has never been reported. The fabricated nanocomposite will assist in detecting methemoglobin, making an earlier diagnosis of methemoglobinemia, further leading to cyanosis much easier. The electrochemical analysis of methemoglobin is accomplished in the blood samples of anemic patients. The novel silver-doped iron zinc oxide (SIZO) nanomaterial is being reported for the first time in the electrochemical sensing of methemoglobin (MetHb). Synthesis of silver nanoparticles. Silver nanoparticles (Ag-NPs) were prepared by hydrothermal method. Precursor mixtures were prepared in distilled water. 80 mL solution of 0.5 g AgNO 3 was heated at 60 °C and added to preheated 20 mL solution of (C 6 H 5 O 7 Na 3 ) and (C 6 H 8 O 7 ) at 60 °C and stirred for 20 min 39 . The mixture was transferred to a 100 mL TEFLON-lined hydrothermal autoclave and placed at 160 °C for 12 h (Eq. 1). The reaction products were washed and purified by deionized water under ultracentrifugation. The washed nanomaterial was dried at 65 °C for 3 days and stored for further use.

Synthesis of methemoglobin from oxyhemoglobin. A 200 mL PBS buffer of pH 7.4 was used to
adjust the HbFe 3+ MetHb. To synthesize MetHb, 3.6 g standard hemoglobin (HbFe 2+ OHb) was diluted in 200 mL PBS under constant stirring for 6 h 40 . The reactor vessel was adjusted with a suction pump to remove extra froth during the reaction, and the mixture was recirculated repeatedly. As shown in the mechanism, a 10 mL sodium nitrite (NaNO 2 ) solution (0.05 g/mL) was injected through a 10 mL syringe. After 4 h stirring and recirculating this solution, the dark brown color indicated the formation of MetHb, as shown in Fig. 1. The obtained methemoglobin solution was refrigerated at 4 °C for 1 week.
Electrochemical sensing of methemoglobin using Ag@Fe 3 O 4 /ZnO nanocomposite. The electrical conductivity and redox reaction of Ag@Fe 3 O 4 /ZnO was evaluated by CV. A potentiostat model (COR-RTEST-CS120) with Ag@Fe 3 O 4 /ZnO modified glassy carbon (GCE) as the working electrode, platinum (Pt) as a counter electrode, and Ag/AgCl as the reference electrode were used to determine the electrochemical reactions. GCE was polished with ethanol and water to avoid contamination. Ag@Fe 3 O 4 /ZnO NPs were dispersed in deionized water to obtain slurry for making reference electrodes. This slurry of Ag@Fe 3 O 4 /ZnO was deposited on GCE by micropipette and dried. HbFe 3+ MetHb solution was diluted in different PBS concentrations. All mentioned parameters were adjusted at room temperature.
Collection and analysis of anemic blood samples. Blood samples of anemic patients (major) were collected from Sahiwal Medical College, Sahiwal, Pakistan, with the prior approval of the Ethical Committee of Sahiwal Medical College Sahiwal Pakistan. All methods were carried out in accordance with relevant guidelines and regulations. The samples were collected from in K2-EDTA (BD-Vacutainers) 41 with their prior informed consent of volunteers and analyzed on potentiostat to determine the comparative aspects of methemoglobin in "vivo" and modified MetHb from standard hemoglobin in "vitro".

Results and discussion
Characterizations. UV-visible spectrophotometer (AQ7100APAC Thermo Fischer Scientific UK Spectrophotometer) is used to analyze Ag@Fe 3 O 4 /ZnO and MetHb/OHb at a wavelength ranging from 200 to 800 nm. UV-visible spectra of Ag@Fe 3 O 4 /ZnO and MetHb/OHb are shown in Fig. 2A,B, respectively. The absorption bands at 234 nm, 324 nm, 361 nm, and 461 nm indicate the nanocomposite formation. Figure 2B shows absorption bands at 395 nm, 409 nm, 550 nm, and 562 nm for MetHb/OHb. FTIR spectra of Ag@Fe 3 O 4 /ZnO and MetHb/OHb are shown in Fig. 2C,D, respectively, and are obtained by measuring transmittance from 4000 to 400 cm −1 on INVENIO FTIR Spectrophotometer Bruker Germany. The bands between 3500 and 2800 cm −1 represent OH and CH stretch. The sharp band at 2380 cm −1 and shoulder peak between 1600 and 1350 cm −1 indicate the stretching vibration of Ag NPs. The peaks from 1600 to 1700 cm −1 represent C=O and C-NH. The sharp peaks from 600 to 540 cm −1 represent pure metal and metallic oxides (FeO ZnO and Ag + ). MetHb/OHb shows a broad band from 3300 to 3000 cm −1 for the hydrogen bonding of the OH bond. The amide and (α, β) sheet bands are shown at 1600 and 1100 cm −1 . CMA in MetHb/OHb, shows a sharp band from 550 to 500 cm −1 , as reported in the literature 42 .
The prepared Ag-Fe 3 O 4 (Fig. 3A), ZnO (Fig. 3B), and Ag@Fe 3 O 4 /ZnO (Fig. 3C) exhibit rough surfaces. X-ray diffraction (XRD) analysis data of Ag@Fe 3 O 4 /ZnO is given in Fig. 3D. The crystalline structure of Ag@Fe 3 O 4 /ZnO     www.nature.com/scientificreports/ ECSA plays an essential role in the detection of the analyte. Increased ECSA provided more reactive sites for albumin to interact with fabricated material, increasing sensor response. The sole reason is the interaction between the electrode surface and analyte generating signal, which the sensor detects.
To change is quantified via the charge transfer coefficient (α), determined by the following equation:    44 . The scan rates vary between 10 to 70 mV/s in redox solution; the outcome is shown in Fig. 7A,B. The obtained surface area of Ag@Fe 3 O 4 /ZnO modified GCE is 0.0791 cm 2 , and for the unmodified GCE is 0.073 cm 2 .
Whereas the kinetic parameter is calculated from the following equation: X is the ΔEP is used to determine ψ as a function of ΔE P from the experimentally recorded voltammetry. Thus, the obtained value of the kinetic parameter (ψ) is 0.63. Transfer coefficient (α). The transfer coefficient is determined by employing the following equation:

The catalytic reaction rate constant (kcat).
here, R denotes the global gas constant (8.314 JK −1 mol −1 ), T denotes thermodynamic temperature (298.15 K), n denotes the number of electrons transferred in the rate-determining step, i.e., 2 and F refer to the faraday constant (96,485 C mol −1 ). The calculated transfer coefficient is found to be 0.71. The calculated Tafel slope (b) value is 0.24 mV/dec (Fig. 8).
Selectivity factor. The selectivity factor of the sensor is calculated by using the following equation: where Ci, Cj, i t, and K are the concentrations of the target analyte, the concentration of interfering species at 35 µM, total current response, and catalytic reaction rate constant, i.e., 1.06 × 10 -8 , respectively. Σk ij amp is the amperometric selectivity coefficient, a measure of the preference of the sensor for the analyte relative to the interferents (Fig. 9).
Σk ij amp is found to be 3.49.

Roughness factor (fr).
The fr is the ratio between peak current I p2 and surface area A 2 of material (Ag@ Fe 3 O 4 /ZnO) to the peak current I p1 and surface area A 1 of blank GCE as shown in Eq. (8). The given formula calculates the roughness factor:  www.nature.com/scientificreports/ The electrode dimensions and quantity of redox centers on the analyte surface determine the roughness factor's strength (fr). It is intended by I pa of ferrocyanide [Fe(CN) 6 ] 3−/4− through redox couple equaling to blank GCE. The electrode surface area ratio is equivalent to the oxidation ratio between two electrodes, representing the change in actual surface area. The actual dimensions of the electrode followed by an electrochemical pathway and redox cores existing on the surface are responsible for the fluctuation in fr 45 . The surface areas of Ag@Fe 3 O 4 / ZnO (A 2 ) and blank GCE (A 1 ) are 0.0791 cm 2 and 0.073 cm 2 , respectively, and the calculated fr is 1.08. MetHb molecules are bulky, which causes the steric hindrance toward charge transfer. The effect of pH is determined by applying impedance at sequential pH of 6.8. 7.0, 7.2, 7.4, 7.6, and 7.8, as shown in Fig. 11C. In the equivalent circuit diagram, R p is the electron transfer resistance on the surface of an electrochemical sensor, R s is solution resistance, and C dl is the component capacitance of the electrochemical sensor.

Electron transfer rate constant (k°) in heterogeneous phase. The electrochemical cells work in an
electro-catalytic solution under alternating current (AC) potential. Hence, EIS determines the charge distribution on electrodes by applying sinusoidal perturbation in continuous linear and semicircular segment circuits. The interfacial capacitance (CdI), Ohmic resistance (Rs), electron transfer resistance (Rct), and Warburg impedance (Zw) are the major components of a continuous circuit. The electron transfer resistance (Rct) detects electron transference in a redox reaction and is measured by semicircular diameter. Ag@Fe 3 O 4 /ZnO NPs enhance the electron transfer rate between electrodes due to excess conductivity and reliability. Linearity in signal is due to dispersion at lower frequencies. GCE

Determination of limit of detection (LOD).
The alternative, dependent derivative, i.e., the limit of detection (LOD), determines the kinetics and completion of the chemical reaction with actual concentration. LOD refers to the minimum concentration of the analytical sample, which can be distinguished by zero in an analyte. LOD varies with the influence of reaction conditions and the redox pH of analytical components. The following equations can determine LOD: here "s" represents the standard deviation, "m" is the slope, and the calculated LOD is 0.17 µM.

MetHb detection in blood serum and recovery analysis. Ag@Fe 3 O 4 /ZnO NPs show peculiar char-
acteristics as electro-catalyst. The oxidized form of "heme" (Fe 3+ ) in serum samples of methemoglobinemia patients is analyzed by a CV to verify the applicability of the modified Ag@Fe 3 O 4 /ZnO sensor. Blood samples (S1, S2, S3, and S4) are collected from anemic patients. The samples are diluted in a buffer of pH 7.4, and the recovery spike is denoted over the oxidative and reductive curves of cyclic voltammograms, as shown in Fig. 12. Samples show a maximum oxidation form of standard hemoglobin in anemic patients, which refers to anemia. The recovery percentage ranges from 88.8 to 97.7% of MetHb, as shown in Table 1. Oxidation and reduction signals accurately detect methemoglobin levels in anemic patients.