Introduction

In the realm of advanced research, the demand for novel functional materials, characterized by low toxicity, high performance, and easy preparation, has reached a critical level of urgency. A significant amount of time and resources is being devoted by scientists and engineers to research and develop materials that exhibit a broad spectrum of versatile functions. These materials are designed to meet the increasing demand for innovative solutions in various industries, from electronics and medicine to energy and environmental protection.

Recently, graphitic carbon nitride (CN) with two dimensional structure has received much consideration as a metal-free semiconductor and is rapidly becoming a chosen material in various applications [1]. The heptazine ring structure and the high degree of condensation enable carbon nitride to possess many advantages such as high surface area, extended catalytic durability, small bandgap, high mechanical stability and an attractive electronic structure [2]. The synthesis of carbon nitride is a one-step polymerization route from the cheap feedstocks like cyanamide [3], urea [4], thiourea [5], melamine [6] and dicyandiamide [7]. The construction of heterojunction structures has become an effective approach to improve the performance of a material [8]. Compared with single-component, heterojunction structures are beneficial to promote functional performance because of increased charge transfer through the junctions at the interfaces [9, 10].

The two-dimensional carbon nitride serves as a perfect stabilization platform for the uniform dispersion of nanomaterials without any agglomeration. A dendrite-structured α-Fe2O3-CN was reported for carbon dioxide reduction and found that the synergy between both the components has enhanced the reduction performance considerably [11]. Manganese oxide (α-MnO2) supported carbon nitride was synthesized by precipitation, followed by calcination method. The composite system was applied as an electrocatalyst for the chemical hydrogenation of CO2. The synergetic effect of both MnO2 and carbon nitride facilitate the hydrogenation reaction with a good yield of formic acid [12]. The composite system of Cu2O-carbon nitride was synthesized using a hydrothermal route and was reported for electrochemical hydrogen evolution reaction [13]. Layer structured carbon nitride with high surface area attract significant attention as electrode materials in renewable energy storage devices. Hybrid system of Zn2GeO4 nanoparticles and ultrathin CN layer exhibited high reversible capacity and excellent rate capability that produced the system suitable for battery application [14]. Graphitic carbon nitride incorporated MnO2 and SnO2 systems exhibited symmetric supercapacitor application. The specific capacitance obtained for MnO2-CN and SnO2-CN composite electrodes were 174 and 64 F.g−1, respectively, as compared to the bare CN electrode 50 F.g−1 [15].

Titania (TiO2) is an abundantly occurring metal oxide with bandgap of 3.2 eV acts as a UV-active photocatalyst. The composite system of TiO2 nanotube and carbon nitride showed superior photocatalytic activity than the individuals [16]. In another report, TiO2-CN system, synthesized by a thermal transformation route, exhibited the degradation of methylene blue and reduction of chromium (VI) ions. The improved photocatalytic activity of the hybrid system was due to the formation of a synergistic heterojunction that enabled a faster electron transfer at the interface between CN and TiO2 [17]. Enhanced catalytic performance of TiO2-CN system for the photocatalytic oxidation of formaldehyde was reported because of the efficient space separation of photo-induced charge carriers [18]. A sensor, based on two-dimensional CuO-CN nanocomposite, fabricated by a pyrolysis technique using melamine and cupric acetate monohydrate as precursors, was reported for electrochemical detection of dopamine. The sensitivity of the sensor was attributed due to electron transfer process, where the hybrid system (CuO-CN) acted as an electron donor and the oxidized dopamine functions as an electron acceptor [19]. Graphitic carbon nitride based nanomaterials have appeared as widely applied hybrid materials that attracted interest to the scientific community due to their chemical stability, easy synthesis method, cost-effectiveness and biocompatibility. The abundance of functional amine groups and high surface area have inspired the use of carbon nitride as emerging electrochemical sensing platforms.

In this work, we report the fabrication of carbon nitride supported titanium dioxide composite system (TOCN) using a high temperature route. The hybrid system was characterized by different analytical techniques and extracted the microscopic, surface and optical properties. The synthesized system was applied as a catalyst for the electrochemical detection of epinephrine, a neurotransmitter and neuroendocrine hormone. The epinephrine detection was performed under various types of devices. The formation of oxygen vacancies in titanium dioxide facilitated the oxidation of epinephrine. This was due to the presence of oxygen-deficient sites, which caused an unbalanced electronic structure, resulting in a rapid electron transfer between the electrode and analyte.

Experimental

Materials and Methods

Titanium isopropoxide, urea, epinephrine hydrochloride, glucose, tyrosine, urea, aminobutyric acid, valine and glutamic acid were received as analytical grade and used without further purification. All other chemicals and solvents were used as received.

Material Synthesis

Synthesis of Titanium Dioxide

Titanium dioxide nanoparticles were synthesized by using a hydrothermal route according to previously reported literature [20]. In a typical synthesis process, 0.1 M of titanium isopropoxide was diluted in 20 ml of iso-propanol under continuous stirring condition and then transferred into an autoclave at 150 °C for 3 h. The solid mass was washed with deionized water, dried under vacuum at 80 °C, and subsequently calcined at 450 °C for 6 h.

Synthesis of Titanium Dioxide-Carbon Nitride (TOCN) Composite System

Preformed titanium oxide and urea (1:5 ratio) were uniformly mixed and transferred to a quartz made reaction chamber. The reaction chamber was kept in a furnace at 500 °C for 4 h. At the end of the reaction, the product was collected and characterized to determine the microscopic and structural properties. The synthesized material was applied as a catalyst for the electrocatalytic detection of epinephrine. In a separate experiment, pure carbon nitride was synthesized using urea as a precursor under above mentioned reaction condition.

Electrode Preparation

The working electrode was prepared as per the following protocol. For a typical electrode preparation method, 10 mg of synthesized material (TOCN) was thoroughly grinded in presence of ethyl acetate (500 µL) to produce a slurry. A nafion solution (20 µL) was added to the slurry as a binder. A bare glassy carbon electrode (3 mm in diameter) was polished with alumina powder (0.05 µm) on the abrasive paper and rinsed with absolute ethanol and deionized water, respectively. The prepared slurry was deposited on a polished glassy carbon electrode under a heating lamp for 2 h and used as a working electrode.

Material Characterization

Microscopy study was performed using JEOL (JEM-2100) transmission electron microscope (TEM) equipped with a LaB6 source. The X-ray diffraction (XRD) patterns were recorded on a Shimadzu XD-3A X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å) within the diffraction angle (2θ) range from 20º to 80º. X-ray photoelectron spectra (XPS) were collected from Perkin-Elmer PHI 560 ESCA/SAM. Fourier transform infrared spectroscopy (FTIR) spectra were collected utilizing a Shimadzu IRAffinity-1. Electrochemical studies were carried out with a Bio-Logic, SP-200, potentiostat connected to a data controller. A three-electrode system was used in the experiment with a glassy carbon electrode (GCE) as the working electrode. The Ag–AgCl electrode and a Pt-electrode were used as the reference and counter electrodes, respectively. Electrochemical experiments were performed in 10 mM of phosphate buffered saline (PBS)solution, serving as the electrolyte.

Result and Discussion

The X-ray diffraction pattern (black line) of the synthesized graphitic carbon nitride (CN) supported titanium dioxide nanoparticle is shown in the Fig. 1A, within 2θ range from 20° to 80°. The overall diffraction pattern is indexed according to the anatase phase of titanium dioxide, which belongs to tetragonal structure with the space group of I41/amd (JCPDS: 894,921, blue bar). The inset figure shows the unit cell structure of titanium dioxide, projected along a-axis. The unit cell structure was constructed using the lattice parameter of a = b = 3.777 Å, c = 9.501 Å and α = β = γ = 90°. The peaks positioned at 21.5° and 26.55° belongs to (100) and (002) plane, respectively and matched according to the hexagonal phase of carbon nitride (JCPDS: 871526). The transmission electron microscopy images, Fig. 1A and B, with different magnifications, show the titanium dioxide particle (dark spots) within the graphitic carbon nitride network.

Fig. 1
figure 1

A X-ray diffraction pattern (black line) of the graphitic carbon nitride supported titanium dioxide, with the unit cell representation of titanium dioxide (projected along a-axis). B and C TEM images of graphitic carbon nitride supported titanium dioxide under different magnifications. Dark spots indicate the presence of titanium dioxide nanoparticles. The inset figure C shows the selected area electron diffraction pattern

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To identify the formation of carbon nitride (CN) during the synthesis of TOCN, an infrared analysis technique was performed. Figure 2 shows the Fourier-transform infrared (FTIR) spectra of CN and TOCN, which suggests the existence of condensed aromatic carbon–nitrogen heterocycles. In the CN sample, the overall feature in the IR-spectrum is identical to those are already reported in the literature [21]. The vibrational bands at 1623 and 1555 cm−1 are attributed to C = N stretching, while the bands at 1407, 1321 and 1236 cm−1 correspond to aromatic C − N stretching mode. All the above mentioned signatures are also visible in the TOCN sample, indicates the formation of carbon nitride. The triazine ring mode, correspond to condensed carbon–nitrogen heterocycles, detected at 804 cm−1 for both the samples with different intensities [21, 22]. In the IR signal, the broad bands within the range of 3000–3500 cm−1 are also visible for both the samples (CN and TOCN) correspond to the stretching modes of the amine groups, such as, − NH2 or = NH.

Fig. 2
figure 2

Fourier-transform infrared spectra of CN and TOCN

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The X-ray photoelectron spectroscopy (XPS) technique was applied to analyse the elemental composition of the carbon nitride supported titanium dioxide (TOCN) system. Figure 3A shows the survey scan within the ranges from 0 to 900 eV and the spectrum displayed the presence of elements, such as Ti, O, C, and N. A high resolution deconvoluted spectrum of Ti 2p is illustrated in Fig. 3B. In this spectrum, the doublet Ti 2p3/2 and Ti 2p1/2 with the binding energy values 458.6 and 464.8 eV was noticed, originated from spin orbit-splitting, consistent with Ti4+ in TiO2 lattice [23]. In the deconvoluted spectrum, the appearance of another peak at the binding energy 461.1 eV correspond to Ti3+ [24], indicate the presence of titanium ion with mixed valence state. Broad (~ 6 eV) O 1s spectrum, Fig. 3C, was deconvoluted with Gaussian peak fit, which produced two components centred at 530.5 and 531.6 eV. The peak, positioned at 530.5 eV, attributed to the surface lattice oxygen of TiO2, whereas the higher binding energy peak at 531.6 eV ascribed as oxygen defects in the matrix of metal oxide, related to oxygen vacancies [25, 26]. In general, four approaches have been adopted to create oxygen vacancies in the structure, (a) control over the synthesis, (b) atom substitution, (c) surface modification (through doping) and (d) in situ surface treatment [27]. In the current synthesis approach, during the formation of carbon nitride at high temperature, in presence of TiO2, oxygen vacancies were created at the matrix of metal oxide system. High resolution C 1s spectrum, Fig. 3D, deconvoluted into main intensity peak positioned at 288.4 eV, originated from N – C = N in the triazine unit and a diffuse peak at 284.8 eV is related to the C–C bond in the graphitic carbon nitride system [28]. The deconvoluted spectra of N 1s, Fig. 3E, shows two prominent peaks at ~ 398.8 eV and ~ 400.4 eV, originated from the nitrogen of triazine ring and primary amine (-NH2) group of carbon nitride matrix [29].

Fig. 3
figure 3

A The XPS survey spectrum of carbon nitride supported titanium dioxide (TOCN) system. High resolution deconvoluted spectrum of Ti 2p B, O 1s C, C 1s D and N 1s E

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Application of TOCN for the Electrochemical Detection of Epinephrine

Using a Conventional Electrochemical Cell

For the electrochemical detection of epinephrine, the cyclic voltammetry (CV) technique was applied in the presence of PBS electrolyte (6.0 mL) under the scan rate of 50 mVs−1 within the voltage range from -0.1 to 0.8 V. To study the electrochemical detection of epinephrine, we have used both titanium dioxide and TOCN as a working electrode modifier. Figure 4A shows the cyclic voltammograms for bare GCE in absence of epinephrine and the corresponding voltammogram (a) shows the current value of 0.17 µA at 0.28 V. An increased current value 0.47 µA at 0.28 V was observed for bare GCE in presence of epinephrine (20 µM), voltammogram (b), indicates the oxidation of the analyte on the electrode surface. It is also important to mention that titanium dioxide modified GCE did not exhibit considerable enhancement of current response in presence of epinephrine (20 µM), voltammogram (c), whereas TOCN modified GCE displayed a well-defined peak with the current value of 0.9 µA at 0.28 V in presence of epinephrine (20 µM), indicated improved catalytic oxidation of the analyte. In the TOCN sample, the formation of oxygen vacancy, as evidenced by XPS study, could be responsible for the improved electrocatalytic oxidation of epinephrine. It is well documented in the literature that the creation of oxygen vacancy has recognized as an effective strategy to prepare high-performance catalysts for the oxidation of organic compounds, photoelectrochemical reactions, supercapacitor application and water oxidation because the oxygen-deficient sites can lead to an unbalanced electronic structure, resulting in rapid electron transfer [25,26,27, 30, 31]. The oxygen vacancies serve as shallow donors that effectively enhanced electronic and photoelectrochemical properties of Fe2O3 nanostructures [32,33,34]. Similarly, oxygen vacancies in α-Fe2O3 nanorods improved its donor density that facilitate the electrochemical performance for supercapacitor application [25].

Fig. 4
figure 4

A Cyclic voltammograms for bare GCE in the absence of epinephrine (curve ‘a’), bare GCE in presence of epinephrine, 20 µM (curve ‘b’), titanium dioxide modified GCE in presence of epinephrine, 20 µM (curve ‘c’) and TOCN modified GCE in presence of epinephrine, 20 µM (curve ‘d’), B Cyclic voltammograms for TOCN modified GCE with increasing concentration of epinephrine, C Square-wave voltammograms for TOCN modified GCE with increasing concentration of Epinephrine

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Figure 4B shows a steady increase of current values with increasing concentration of epinephrine for the TOCN modified GCE electrode. In absence of epinephrine, the current value 0.40 µA at 0.28 V was noticed. The maximum current value was reached to 1.3 µA for the epinephrine concentration 50 μM with a prominent anodic peak at 0.28 V. The increase of anodic peak current, with increasing epinephrine concentration, indicate enhanced electron transfer through the defect sites of titanium dioxide. The sensitivity value for the detection of epinephrine using cyclic voltametric technique was obtained 0.255 μA.μM−1.cm−2. The detection of epinephrine was further verified using square-wave voltammetry technique, which is considered as the second generation of pulse technique with higher sensitivity and rapid measurement [35]. Similar results were also observed in the square wave voltammetry technique, Fig. 4C, where the increase of current value was noticed with increasing epinephrine concentration with the sensitivity value of 2.26 μA.μM−1.cm−2.

The chronoamperometric response of the TOCN modified electrode towards the subsequent addition of epinephrine was further assessed under the optimized experimental condition. Figure 5A, shows the amperometric response (current as a function of time) of epinephrine at 0.25 V. As documented in the figure, the amperometric signal showed a good linear correlation and a stepwise increase of current value for every addition of epinephrine (10 µM), within the range from 10 μM to 100 μM in every 40 s time interval. The calibration plot of current response with varying concentration of epinephrine is displayed in Fig. 5A, inset. The regression coefficient and sensitivity values, within the time interval from 200 to 660 s, are 0.99822 and 3.1196 μAμM−1.cm−2, respectively. The TOCN modified electrode showed tolerance towards other analytes including glucose, tyrosine, urea, aminobutyric acid, valine and glutamic acid, 50 µM each, Fig. 5B. The absence of a substantial current alteration upon the introduction of the aforementioned analytes signifies the selectivity of TOCN towards epinephrine.

Fig. 5
figure 5

A Amperometric response of TOCN modified GCE on successive addition of 10 μL of epinephrine in every 40 s time interval with an applied potential of 0.25 V. Inset: the calibration plot of current response with varying epinephrine concentrations. B The addition of interferences species (glucose, tyrosine, urea, aminobutyric acid, valine and glutamic acid, 50 μL each) show the irresponsive nature towards the amperometric current signal

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Electrochemical Impedance Characteristics of the TOCN Modified Electrode

Figure 6, main panel, illustrates the Nyquist plots that depict the electrochemical impedance characteristics of the TOCN based electrode before and after the exposure to epinephrine within the range from 200 kHz to 100 mHz. The electrochemical impedance spectroscopy exhibited two distinct regions. The higher frequency region displayed a semicircular shape, governed by electrode reaction kinetics (charge transfer) process. On the other hand, the lower frequency range exhibited a linear characteristics, suggesting a diffusion-controlled process. The diameter of the semicircle represents the charge transfer resistance that provide the insight of electron transfer kinetics at the electrode–electrolyte interface during the electrochemical process. The electrochemical impedance analysis revealed a significant changes in charge transfer resistance value, which was 162 kΩ (before the addition of epinephrine) and decreased to 56.35 kΩ (after the addition of epinephrine), indicating a facile approach of epinephrine on the surface of TOCN system. The Nyquist plots are modelled according to the equivalent circuit diagram, Fig. 6, inset, and the summarized values of the fitting parameters are shown in Table S1, supplementary information. The diagram constant of two components (R2Q2 and R3Q3) connected in series combination. The addition of epinephrine causes a significant shift in the semicircular arc pattern. Such a behavior is associated with the faster charge transfer process between electrode and electrolyte interface and that leads to decrease in the resistance values (R1, R2 and R3) of the device. The R1, R2, and R3 represent equivalent series resistance, charge transfer resistance and leakage resistance, respectively. The low-frequency region deviates from the ideal capacitive trend and could be attributed to the leakage resistance (R3) in parallel with the leakage capacitance (Q3). Ion diffusion mechanism between electrode and electrolyte interface leads to Warburg impedance (W), which is connected in series with R3.

Fig. 6
figure 6

Electrochemical impedance spectra of TOCN modified GCE before and after the addition of epinephrine within the frequency range from 1.0 MHz to 1.0 mHz

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Detection of Epinephrine Using a Custom Made Electrochemical Device

A conducting graphitic pencil was applied to draw the working electrode (WE) and counter electrode (CE), 1 mm × 15 mm, each, 10 mm apart, on a piece of paper, supported on a glass slide. A thin-line was drawn in between WE and CW electrodes using silver paint, acted as a reference electrode (RE). A thin film of the synthesized material (TOCN) was deposited on the WE. 100 µL of PBS solution was deposited on the device to connect the working, counter, and reference electrodes, Fig. 7A. Figure 7B displayed the cyclic voltammogram of different concentrations of epinephrine within the range from 0.5 to 160 µM. Figure 7C shows the graphical representation of current as a function of epinephrine concentration. The calibration plot, Fig. 7C, inset, shows the current response with varying concentration of epinephrine. The sensitivity of the device was achieved 0.1122 μAμM−1.cm−2 within the range from 10 to 160 µM (analyte concentration). Chronoamperometric study was performed by adding 10 µM epinephrine under different time interval between 100 to 1200 s, Fig. 7D. Sharp increase of current responses were noticed after every addition of the epinephrine (10 µM), whereas no significant changes in current value was observed during the addition of analytes like glucose, tyrosine, urea, aminobutyric acid, valine and glutamic acid, 20 µM each, indicates selectivity of the TOCN catalyst for epinephrine detection in custom made device.

Fig. 7
figure 7

A Schematic diagram of the custom made device. B Cyclic voltammogram of different concentrations of epinephrine (0.0–160 µM). C Graphical representation of current as a function of epinephrine concentration (0.5–160 µM). Inset: the calibration plot of current response with varying epinephrine concentrations. D The addition of interferences species (glucose, tyrosine, urea, aminobutyric acid, valine and glutamic acid, 20 μM each) show the irresponsive nature towards the amperometric current signal

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Detection of Epinephrine in Pharmaceutical Sample

To verify the practical applicability of the custom-made device, it was utilized to determine the concentration of epinephrine in commercial injection, Epipen®. The as fabricated electrochemical cell was introduced with the epinephrine injection solution with the concentrations of 10 µM, 20 µM, and 30 µM. The cyclic voltammogram of the device was recorded and the results have been shown in the Table 1. The results showed good performance of the electrocatalyst that emphasized the reliability of the device to detect the samples in an accurate manner.

Table 1 Selective determination of epinephrine in pharmaceutical sample
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Detection of Epinephrine Using a Screen Printed Electrode

Screen printed electrodes are widely recognized for their significance in the field of electrochemistry. The major advantages of the screen printed electrodes are cost-effectiveness and adaptability for miniaturization. Screen printed electrodes have extensive use in point-of-care diagnostics, which makes them indispensable in healthcare and pharmaceutical applications [36,37,38]. The electrochemical detection of epinephrine was further performed on a screen printed electrode (SPE), Metrohm, DropSen DRP-C110, using TOCN as a catalyst. Figure 8 displays the cyclic voltammograms of the TOCN modified screen printed electrode for various concentrations of epinephrine. An increase in current value was noticed with addition of epinephrine. The voltammograms shows a steady current response from 1 to 40 µM and subsequently a saturation point was reached, as evidenced by the voltammogram. It is also important to mention that the custom made device is more sensitive for wider range of epinephrine detection as compared with the conventional electrochemical cell and commercial screen printed electrode.

Fig. 8
figure 8

Cyclic voltammograms of different concentrations of epinephrine (0.0–60 µM) using a screen printed electrode

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A comparison table shows the electrochemical recognition of epinephrine under different catalytic conditions, Table 2, [39,40,41,42,43,44,45,46,47].

Table 2 Comparison of epinephrine detection under different catalytic conditions
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Conclusion

Here we report a simple method to create the oxygen vacancy in titanium dioxide by applying an oxygen vacancy defect engineering strategy to prepare a high-performance catalyst for the efficient electrochemical recognition of epinephrine. The catalyst, carbon nitride supported titanium dioxide nanoparticle, was synthesized by applying a high temperature route. The X-ray photoelectron spectroscopy analysis predicted the formation of oxygen vacancies in the matrix of titanium dioxide nanoparticle. The oxygen-deficient sites led to the creation of an unbalanced electronic structure, which, in turn, caused rapid electron transfer and facilitated the oxidation of epinephrine. The synthesized hybrid system presented the sensitivity values of 0.255 and 2.26 μAμM−1 cm−1 for the electrochemical detection of epinephrine using cyclic voltammetric and square wave voltammetric techniques, respectively, applying glassy carbon as working electrodes. A custom made device was also fabricated based on hybrid catalyst to evaluate the sensing performance of epinephrine in pharmaceutical sample. The findings mentioned above suggest that carbon nitride-supported titanium dioxide has the potential to serve as a catalyst for the electrochemical detection of epinephrine.