Green sonochemical synthesis and fabrication of cubic MnFe2O4 electrocatalyst decorated carbon nitride nanohybrid for neurotransmitter detection in serum samples.

The binary nanomaterials and graphitic carbon based hybrid has been developed as an important porous nanomaterial for fabricating electrode with applications in non-enzymatic (bio) sensors. We report a fast synthesis of bimetal oxide particles of nano-sized manganese ferrite (MnFe2O4) decorated on graphitic carbon nitride (GCN) via a high-intensity ultrasonic irradiation method for C (30 kHz and 70 W/cm2). The nanocomposites were analyzed by powder X-ray diffraction, XPS, EDS, TEM to ascertain the effects of synthesis parameters on structure, and morphology. The MnFe2O4/GCN modified electrode demonstrated superior electrocatalytic activity toward the neurotransmitter (5-hydroxytryptamine) detection with a high peak intensity at +0.21 V. The appealing application of the MnFe2O4/GCN/GCE as neurotransmitter sensors is presented and a possible sensing mechanism is analyzed. The constructed electrochemical sensor for the detection of 5-hydroxytryptamine (STN) showed a wide working range (0.1-522.6 μM), high sensitivity (19.377 μA μM-1 cm-2), and nano-molar detection limit (3.1 nM). Moreover, it is worth noting that the MnFe2O4/GCN not only enhanced activity and also promoted the electron transfer rate towards STN detection. The proposed sensor was analyzed for its real-time applications to the detection of STN in rat brain serum, and human blood serum in good satisfactory results was obtained. The results showed promising reproducibility, repeatability, and high stability for neurotransmitter detection in biological samples.

Introduction

Nanomaterials synthetic techniques are developing on greener and nontoxic aspects which eliminate and minimize the use of toxic solvents and hazardous sources [1], [2], [3]. Advance greener methods involve the use of sonochemical, electro-deposition, microwave, spin coating, supercritical solvents, and biosynthesis [4], [5]. In particularly, sonochemical reaction and methods based route of composite synthesis is an advanced and highly exploring area, due to their advantages such as low temperature, simplicity, low cost, and diverse applicability [6], [7]. A Sonochemical reaction arises from acoustic cavitation, which are involves the growth, formation and collapse of bubbles in a reaction liquid and create pressure and temperature followed by high rate of production [8], [9], [10]. The nanomaterials and composites are often responsible for various size and shape to selective synthesis based on solvent. Current report is sonochemical reactions based green synthesis and characterization of bimetal oxide (MnFe2O4) nanomaterials converted graphitic carbon nitride composite. Manganese iron oxide synthesis requires additional calcination of the obtained precursors formed by sonochemical reactions [11], [12], [13]. Since, pH points does not necessitate a play role in the nanocomposite preparation. Collisions of the bimetal oxide nanoparticles improved by shock-waves instigated and the process helped to get nanocomposite by transient cavitation’s and energy barrier for the evolution of ferrite-based materials [14], [15]. Moreover, the calcination process is one of the important methods to get pure product and it influences the properties of nanomaterials such as particle size, coercivity, porosity and magnetization properties [17], [18]. In this work mainly focused on the synthesis of the composite based on basic concept of sonochemical reactions and its application toward the electrochemical detection by various methods. Thus, sonochemical methods has many applications in materials sciences, life sciences and medicine department. In addition, the synthesis of biometal oxide-based composite using sonochemical and ultrasound irradiation provides nano-sized metal oxide particles with high surface area and its results in high electrocatalytic activity.

5-Hydroxytryptamine (serotonin; STN) is an important biogenic amine and neurotransmitter in the mammalian CNS (central nervous system) [19], [20]. The oxidative of STN is involved in various biological functions, enzymatic and nonenzymatic processes in biological system and it is associated with a variety of biological processes and illnesses [20], [21]. Mainly, the control of the nervous system also one of the roles and function [22], [23]. STN is more responsible for controlling the biological processes such as the brain functions, blood pressure, mydriasis, and lipolysis. In addition, the use of STN in the pharmacological treatment of many kind of diseases (anaphylaxis, hypertension, cardiac arrest, bronchial asthma and others) [23], [24]. Therefore, STN monitoring in human body is more important due to their functions. Our studies of electrochemical method and behavior can reveal their STN monitoring function. Some of the diseases are associated with the STN level have health problems, weight loss and gain, digestive problems, insomnia, and other problems. Moreover, recent studies and analysis confirmed that there is a relationship between main disorder (Parkinson's disease, Alzheimer's disease) based on levels of STN [25], [26]. The use of STN concentrations in human body is varied based on the age and body conditions. Therefore, the monitoring of STN concentration in medical aspects during the therapeutic treatment is more important. These are the aspects detection and determination of the STN, need to develop an effective modified (bio) sensor for real time applications. Moreover, simple and robust detection of STN concentrations is main focusing in this work based on the sonochemically developed nanomaterials. The detection should be monitorable over a wide linear range of the STN level and accurate of the modified sensor. Furthermore, the efficient performance with lowest detection limit of the modified electrode and sensor is another focus of this work. The nanocomposite modified sensor should explore a large and longtime stability with reproducibility of the fabrication devices.

Herein, the bimetal oxide nanoparticles decorated graphitic carbon nitride composite/GCE was used for the determination of STN. A glassy carbon electrode (GCE) consists of a transducer. The well fabricated MnFe2O4/GCN/GCE showed a low limit of detection, high current, a wide linear range, fast detection, reproducible, low cost and valuable detection method. In this work, the electrocatalytic properties and abilities of MnFe2O4/GCN electrodes was developed and their performances towards the electrochemical detection of STN have been analyzed by many electrochemical and sonochemical methods (Scheme 1).

Scheme 1

Green and sonochemical synthesis of MnFe2O4/GCN nanocomposite and the electrochemical detection of neurotransmitter in biological samples.

Experimental section

Materials

Iron(III) chloride (99.9%), manganese sulfate monohydrate (97.0%), monosodium phosphate (NaH2PO4; 98.0%), and ethanol (99.8%) were purchased from Sigma-Aldrich. Phosphate buffer solution (PB and 0.05 M) was prepared by mixing the stock solution of monosodium phosphate and disodium phosphate. Aqueous solutions were prepared with DD water. More information’s were given in supporting file (S1).

Facile synthesis of MnFe2O4/C3N4

In the sonochemical synthesis of MnFe2O4/GCN, 1 mol of iron(III) chloride (FeCl3) and 3 mol of manganese sulfate monohydrate (MnSO4·H2O) were added in a 100 mL beaker containing 55 mL of DD water under stirring process by magnetic stirrer for 30 min. 3.5 g of potassium hydroxide (KOH) was added into the beaker and the stirring was continued at room temperature for 15 min. Then, the reaction solution was moving to high-intensity ultrasonic irradiation method for sonochemical reaction (30 kHz/70 W; model: UZ SONOPULS HD 2070) for 30 min. After the sonochemical reaction, the nanomaterial precipitate moves to washing process by centrifuged with DD water several times and then dried in oven at 75 °C. The obtained powder was calcinated at 450 °C for 2 h. Furthermore, 10 mg of MnFe2O4 and 5 mg of GCN (synthesis of GCN is given in supporting information) were added into 10 mL DD water (20 mL vial). Then, the vial was ultrasonicated for 30 min at room temperature, there is formation of MnFe2O4/GCN by indirect ultrasound process under ultrasonic cavitation. All formed composite due to ultrasound cavitation process to form MnFe2O4/GCN (Scheme 2).

Scheme 2

Sonochemical synthesis of MnFe2O4/C3N4 composite and its applications.

Fabrication of MnFe2O4/GCN modified electrode

Prior to modify the surface of GCE with MnFe2O4/GCN, 0.03 µm alumina powder was used to polish electrode surface and washed with DD water and ethanol. Then, washed GCE was dried at electric oven (55 °C) for 15 min. Then, optimized concentration of 8 µL (2 mg/mL) of the MnFe2O4/GCN solution was drop casted on the GCE surface. The MnFe2O4/GCN/GCE was dried electric oven (55 °C) for 15 min. Similar procedure were applied to makes MnFe2O4/GCE and GCN/GCE. Finally, all modified and unmodified electrodes were developed for electrochemical applications towards neurotransmitter (5-hydroxytryptamine) detection.

Preparation of the solution

A stock standard solution of STN concentrations (1, 3, 5 mM) were prepared every day in DD water and also 0.05 M of PB was prepared. For real time analysis, rat brain serum and human blood serum is used without STN lab concentration. Then, 2 mg. mL−1 of STN was diluted with serum samples and obtain the final concentration. Then, different range of spiked analysis is focusing and plot the calibration curve. The anodic peak current was measured using DPV method under the optimized conditions and the standard addition method is used to determine level of accuracy and RSD towards STN in the biological samples.

Result and discussion

Morphological and elemental analysis

The morphological features of the as-synthesized MnFe2O4/GCN nanocomposite were analyzed by TEM. The TEM image in Fig. 1A–C illustrates the decoration and incorporation of MnFe2O4 particles on the wrinkled sheet structures of carbon nitride. TEM analysis was shows clearly, the MnFe2O4 particles nano-sized. Fig. 1C presents the TEM image of the MnFe2O4/GCN and the image shows that the carbon nitride sheets are decorated with small spherical MnFe2O4 nanoparticles. The mean nanoparticle size (Fig. 1B) of MnFe2O4 is 216 nm. The presence of manganese, iron, oxygen, carbon, and nitrogen in the MnFe2O4/GCN composite was detected by EDS as shown in Fig. 1E. Quantitative analysis were given in inset Fig. 1E. It was used to investigate weight percentage of the element present in the composite.

Fig. 1

(A-C), TEM analysis of the MnFe2O4/GCN nanocomposite. (D), TEM image of graphitic carbon nitride. EDS and quantitative analysis of the MnFe2O4/GCN nanocomposite.

XRD analysis of the MnFe2O4/GCN

The XRD analysis are more important studies for nanomaterials development. Mainly, XRD analysis of the MnFe2O4/GCN composite and MnFe2O4 particles were conducted using powder XRD analysis. Fig. 2A shows the XRD patterns of the synthesized MnFe2O4/GCN composite and MnFe2O4 particles. In the XRD patterns of MnFe2O4/GCN and MnFe2O4, the presence of the peaks at 2θ = 29.98, 35.31, 42.97, 53.24, 57.06 and 63.25°, which correspond to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) planes of cubic MnFe2O4 (JCPDS 74-2403) was observed [27], [28]. For the pure graphitic carbon nitride, two diffraction peaks related to the (1 0 0) and (0 0 2) planes were observed in the composite [29], [30]. In the XRD pattern of the nanocomposite, all peaks that are characteristic XRD peaks of bimetal oxide of cubic MnFe2O4. This indicated that during the formation of MnFe2O4/GCN composites via the sonochemical and ultrasound assistant method. Furthermore, crystal structure of (sonochemically synthesized) MnFe2O4 nanoparticle was analyzed in Fig. 2B–D. Space group of the cubic crystal structure is Fd-3m and unit cell volume is 617.38 Å3. Fig. 2B–D, shows three type of models like ball-stick (B), space filling (C), and polyhedral based on the cubic MnFe2O4 particles. Further details were given in Scheme S1 and S2 and supporting file (SI).

Fig. 2

(A), XRD analysis of the MnFe2O4 and MnFe2O4/GCN nanocomposite. Crystal structure analysis of ball-stick (B), space filling (C), and polyhedral model of the cubic MnFe2O4 particles.

XRD analysis of the MnFe2O4/GCN composite

The chemical compositions of the MnFe2O4/GCN have been investigated with XPS (Fig. 3A–F). Fig. 3A, XPS survey spectrum shows the C1s, N1s, O1s, Mn 2p and Fe 2p elements and the major compositions in the synthesized MnFe2O4/GCN nanocomposite [31], [32]. For the XPS spectra of Mn 2p (Fig. 3B), peaks at 642.1 eV and 653 eV is attributed to Mn 2p3/2 and Mn 2p1/2, respectively [32]. Then, the XPS spectra of Fe 2p (Fig. 3C), peaks at 711.5 eV and 725.1 eV is attributed to Fe 2p3/2 and Fe 2p1/2, respectively [32]. Fig. 2D reveals that the O 1s peak can be split into three peaks at 530.4 eV, 532.5 eV, and 534.2 eV corresponding to the binding energies of lattice oxygen and chemisorbed oxygen molecules in MnFe2O4 particles [32], [33]. The high-resolution C 1s spectra included three peaks located in Fig. 3E, and those peaks are corresponding to CC, C—C, and C—N bonds [34], [35]. The XPS high-resolution N 1s spectra included four peaks located in Fig. 3F, and those peaks are corresponding to pyridinic carbon, pyrrolic carbon, graphitic carbon, and N–oxide bonds in the composite [36].

Fig. 3

(A), XPS survey of the MnFe2O4/GCN nanocomposite. (B-F), High-resolution XPS analysis of the elements in the MnFe2O4/GCN composite.

Electrochemical detection of neurotransmitter (Serotonin)

The favorable electrochemical properties of the electrode coated with MnFe2O4/GCN point to a possible use in a sensor, which is proven here for detection of STN. Fig. 4 shows a prominent oxidation peak for cyclic voltammogram of the MnFe2O4/GCN/GCE in 30 µM STN in a PB (pH 7.0). Compared to MnFe2O4/GCE, GCN/GCE and bare GCE, the MnFe2O4/GCN composite modified GCE has high intensity. Due to the synergetic effect of GCN sheets and MnFe2O4 nanoparticles. The STN electrochemical behavior has been proposed to occur via the electrochemical electron transfer mechanism as shown in Scheme 3. The first step involves STN oxidation to STN-quinone, which reacts chemically generating quinone compound [23], [37]. It is a two electron transfer reaction peaks should be observed at pH = 7.0. Fig. 4A shows two well-defined peaks owing to STN detection using the MnFe2O4/GCN/GCE. The modified electrode sensing without any fouling effect and it was confirmed by different concentration effect in Fig. 4B. The electrochemical process at the MnFe2O4/GCN interface is diffusion controlled, as demonstrated in subsidiary experiments in which the scan rate was varied from 20 to 200 mV.s−1 (Fig. 4C). Fig. 3D, shows that anodic peak currents increased linearly with the square root of the scan rate. Moreover, different pH analysis was performed and optimized the electrolyte conditions. Based on the electrochemical analysis of the pH studies, pH 7.0 is more suitable and stable electrolyte for electrochemical detection of biological analyte. In our studies also proved this case in Fig. S1. Therefore, all electrochemical studies were analyzed in 0.05 M PB (pH 7.0).

Fig. 4

(A) CVs obtained at unmodified (a), MnFe2O4/GCE (b), GCN/GCE (c), and MnFe2O4/GCN/GCE (d) in 0.05 M PB (pH 7.0) containing 30 µM serotonin. (B) CVs obtained at MnFe2O4/GCN/GCE in PB (pH 7.0) containing serotonin (10 to 50 µM). (C) CVs of MnFe2O4/GCN modified electrode in PB (pH 7) containing 30 µM serotonin at different scan rates (20 to 200 mV/s) and (D) Square root of scan rate (V/s)1/2 vs. peak currents (µA).

Scheme 3

Mechanistic pathway of serotonin oxidation at MnFe2O4/GCN nanocomposite film modified GCE.

DPV analysis towards serotonin oxidation at MnFe2O4/GCN modified electrode

DPV analysis of the STN sensor is more important based on sensitivity, limit of detection, and linear range. The electrocatalytic performance of the STN sensor was optimized in electrolyte and potential ranges (Fig. 5A). To optimize the condition of the experiments, the obtained the electrochemical conditions were 0.05 M PB and wide potential range −0.1 to 0.4 V. DPV response of the modified sensor with different additions and concentrations of STN on MnFe2O4/GCN modified GCE (Fig. 5A). The DPV peaks were linear increased with STN low concentrations, 100 nM to 552.6 μM (Fig. 6B). Based on the DPV responses, calibration plot was obtained and the regression equation calculated as Ipa (μA) = 0.3488x + 0.9753C (STN/μM) with R2 = 0.9937. Moreover, the detection limit of the modified sensor was calculated as 0.0031 μM. In addition, the sensitivity of the sensor is 19.377 μA μM−1 cm−2. Mainly, the MnFe2O4/GCN/GCE sensor performances was compared with previously published other STN sensors is given in Table 1. It showing that MnFe2O4/GCN modified sensor exhibits outstanding catalytic performances towards STN oxidation.

Fig. 5

(A) DPV response of serotonin based on MnFe2O4/GCN modified electrode for different additions of serotonin into PB (pH 7). (B) [serotonin]/µM vs. current (µA). (C) Selectivity of MnFe2O4/GCN modified electrodes towards biological analytes and (D) Stability analysis of the nanocomposite modified electrode.

Fig. 6

(A) DPV response of serotonin at MnFe2O4/GCN modified electrode for repeatability test. (B) reproducibility test based on MnFe2O4/GCN modified electrodes towards serotonin.

Table 1

Current sensor performances at MnFe2O4/GCN film modified electrode with previously reported electrodes.

Electrode	LOD (µM)	Sensitivity (µA µM−1cm−2)	Linear range/(µM)	Ref.
GR-FeTSPc/CFME	0.02	2.15	0.05–60	[38]
rGO-Ag2Se	29.6	0.5	0.1–15	[39]
Fe3O4–CNT–poly(BCG)	0.08	1.72	0.5–100.0	[40]
PDMS@cZIF	0.03	2.45	1.0–60	[41]
MnO2-GR	0.01	4.92	0.1–400	[42]
SWCNT	32	0.919	0.1–100	[43]
Carbon sphere	700	1.71	40–350	[44]
Reduced graphene	32	5.26	1.0–100	[45]
MWCNT/CHT	50	8.16	0.05–16	[46]
MWCNT/Ni/Zn	118	5.38	0.006–62.8	[47]
CNT/CHT/p-ABSA	8	4.26	0.1–522.6	[48]
MnFe2O4/GCN	0.0031	19.377	0.025–909.12	This work

GR-FeTSPc = graphene-iron-tetrasulfophthalocyanine, rGO-Ag reduced graphene oxide-silver selenide, Fe Fe3O4-multiwalled carbon nanotubes- poly (bromocresol green), CDP-Choline/MCPE = citicoline sodium modified carbon paste electrode, PDMS@cZIF = polydimethylsiloxane@carbonized ZIF-67@ZIF-8, MnO MnO2-graphene, CNT/CHT = multiwalled carbon nanotubes/chitosan, CNT/CHT/p-ABSA) = multiwalled carbon nanotubes, chitosan and poly(p-aminobenzenesulfonate).

Anti-interference ability of the MnFe2O4/GCN modified electrode is highly significant parameter in the modified electrochemical applications in human blood serum. Some potential interferences are analyzed based on optimized condition such as ascorbic acid (AA), caffeic acid (CA), methionine (MH), dopamine (DA), methyl nicotinate (MN), uric acid (UA), H2O2, sodium ions (Na+), potassium ions (K+), and STN (30 μM). The anti-interference property of the MnFe2O4/GCN electrode is tested and obtained results were evaluated and given in Fig. 5C. Fig. 5C, exploring that the anodic current response increase adding STN and then, negligible peak responses can be obtained after adding other interference chemicals. Therefore, these experiments are verified the anti-interference ability of MnFe2O4/GCN/GCE. Its high useful for the practical applications in biological samples. Further, the long-time stability of the MnFe2O4/GCN/GCE as a STN sensor is evaluated. Then, the DPV peak based analysis is given in Fig. 5D and it shows that the modified electrode and experiment conditions can keep 94.22% of the original current intensity after 15th days (based on MnFe2O4/GCN). Therefore, the obtained results were indicating outstanding stability and performance of the designed MnFe2O4/GCN modified GCE towards STN sensor.

The repeatability analysis of the MnFe2O4/GCN modified sensor was evaluated with addition of 30 μM STN at 0.05 M PB (pH 7.0) as well. In Fig. 6A, shows that the DPV response and the repeated measurements, which reveals repeatability test of the MnFe2O4/GCN proposed sensor. Moreover, the reproducibility analysis, DPV peaks were tested by sensing of 30 μM STN with different MnFe2O4/GCN modified electrodes based on the optimized condition (Section 3.4). Mainly, the above experiments indicated from Fig. 6B, the relative standard deviation of the sensor is 2.96% and it exploring valuable reproducibility of the MnFe2O4/GCN modified electrochemical sensor.

Practicality analysis in biological samples

The MnFe2O4/GCN electrode was further used for the determination of 5, 10, and 20 μM of the target analyte STN spiked in biological samples, such as rat brain serum and human blood serum samples (Figs. S2 and S3). All three samples presented obvious DPV responses of STN. The average recovery recorded from three parallel experiments and the recoveries were 97.8, 98.1, and 98.05% for rat brain serum and 98.4, 97.9, and 95.65% for human brain serum, suggesting the reliable reproducibility of STN detection with the MnFe2O4/GCN electrode and its potential for practical application. The real samples analyses of STN detection in biological samples were presented in Table 2. By altering the MnFe2O4 and graphitic carbon nitride contents in the composite, optimal modified sensors with the excellent electrocatalytic performances were obtained.

Table 2

Real sample analysis in biological samples at MnFe2O4/GCN/GCE (n = 3).

Samples	Added (µM)	Obtained (µM)	Accuracy (%)	RSD* (%)
Rat brain serum	0	0	–	–
	5.0	4.89	97.80	3.12
	10.0	9.81	98.10	3.08
	20.0	19.61	98.05	2.76
Human Blood serum	0	0	–	–
	5.0	4.92	98.40	3.62
	10.0	9.79	97.90	2.98
	20.0	19.13	95.65	2.73

Conclusions

In summary, we have reported the preparation of MnFe2O4 nanosphere decorated graphitic carbon nitride-based nanocomposites as an active electrode material for high-performance of neurotransmitter detection in biological samples. Owing to the synergistic effect originating from the intimate coexistence of MnFe2O4 nanospheres on the GCN surface, the MnFe2O4/GCN nanocomposite shows a large electrochemical surface area with good electron transfer rate. A sensor based on MnFe2O4/GCN as the electrode demonstrates the electrochemical detection of STN with an excellent detection limit. The MnFe2O4/GCN modified device demonstrates its stability and reproducibility performances and also exhibits a practicality of the modified sensor. To the best of our knowledge, this is the first time the use of MnFe2O4/GCN as a STN sensor in electrochemical detection has been reported. The demonstration of the practical application of this sensor suggests that the MnFe2O4/GCN nanocomposite is a very promising modified electrode material for high-performance non-enzymatic sensors.

CRediT authorship contribution statement

Mohamed S. Elshikh: Methodology. Tse-Wei Chen: Methodology, Formal analysis. G. Mani: Writing - original draft, Resources. Shen-Ming Chen: . Po-Jui Huang: Formal analysis. M. Ajmal Ali: Funding acquisition. Fahad M. Al-Hemaid: Resources, Methodology. Amal M. Al-Mohaimeed: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.