In situ green synthesis of the nanocomposites of MnO₂/graphene as an oxidase mimic for sensitive colorimetric and electrochemical dual-mode biosensing

Abstract

Herein we report the colorimetry and an electrochemical for the determination of dopamine (DA) by using MnO₂ nanoparticles and graphene nanosheets composite (MnO₂@G) that display oxidase mimicking property. MnO₂@G could directly oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into a blue product (oxTMB) without extra oxidants such as H₂O₂. Nevertheless, the presence of DA will inhibit the TMB oxidation due to the presence of the competitive reaction of MnO₂@G and DA, giving a product color change from blue to colorless. A colorimetric assay for detect the concentration of DA was worked out according to this finding. Response is linear in the 0.1 to 15 μM DA concentration range, and the detection limit is 0.14 μM. Wider detection range is achieved in an electrochemical method which is due to the pronounced electrocatalytic activity of MnO₂@G. The MnO₂@G was modified on the surface of the glassy carbon electrode in order to fabricate one type electrochemical sensor. The sensor achieves a wide detection two linear ranges from 0.4 to 70 μM, with the detection limit of 1.16 μM. The detection of DA in real serum sample proved that the nanozyme based on MnO₂@G could be developed into a colorimetry and electrochemical dual-readout sensing platform.

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1. Introduction

In spite of highly efficient catalytic ability and excellent selectivity, natural enzymes are associated with many disadvantages, such as high-cost extraction, easy deterioration and poor stability, which restrict their applications.¹ To overcome these shortages, enzyme-mimetic nanomaterials (nanozymes), with low-cost and highly stable substitutes, have been developed and applied in a variety of fields.^2,3 Over the past few years, nanozymes are utilized in many applications, such as sensing, catalysis, imaging, therapeutics, pollutant removal and beyond.^2,4–6 Especially in the sensing field, nanozymes have been found intensively useful for biochemical detection, environmental monitoring and food analysis.^7–9 Xu et al. reported a KCl-doped lignin carbon dots nanozymes with peroxidase-like activity and applied the nanomaterial for colorimetric detection of glutathione.¹⁰ Ren et al. designed a tunable structure defect strategy to regulate the nanozyme activity of MOFs for the online electrochemical detection of uric acid.¹¹ Ge et al. developed a colorimetric assay for quantitative malathion detection based on the oxidase-mimicking activity of Fe–N/C SAzymes, and the detection technology was successfully applied in environmental and food samples.¹² Han et al. recently designed 2D Fe–Mn bimetallic nanozyme (Dex-FeMnzyme) with highly oxidase-like activity for the total antioxidant capacity (TAC) assay, Dex-FeMnzyme showed satisfactory sensitivity and convenient colorimetric biosensor TAC.¹³ Nowadays, although colorimetric or electrochemical sensors based on nanozyme have been used to detect different analytes, there are still some shortcomings for some nanozymes, such as low catalytic activity, poor stability or single form of detection.^14,15 Dual-mode detection methods gradually becomes a new focus, because they not only provide more than one signal output mode, but also offer good accuracy and diversified results.^16,17 Therefore, there is an urgent need to prepare a multifunctional nanozyme with both intrinsic enzyme-like catalytic activity and electrocatalytic activity, and use it to construct a dual-mode sensing platform for colorimetric and electrochemical sensors.

Currently, it is reported that nanozymes are broadly classified according to the composition of nanomaterials, including precious metals (gold, silver, platinum, palladium, etc.), metal oxides and sulfides (MnO₂, CeO₂, MoS₂, VS₂, etc.), carbon-based nanomaterials (fullerenes, carbon nanotubes, carbon nanodots, graphene, etc.), metal–organic frameworks (MOFs) and their derivatives, etc.^1,18–25 Among these nanozymes, graphene materials/composites are attracting increasing attention because of their excellent electron transfer capability and synergistic effect.^26–28 Numerous graphene-based nanozymes have been developed and mainly depend on the following two strategies. The first one is functionalizing graphene, in which graphene materials are modified with functional groups to improve catalytic activity. For example, Song et al. prepared carboxyl-modified graphene oxide with peroxidase-like activity by covalently grafting polyethylene glycol onto the graphene oxide of chemically activated surfaces and edges.²⁹ Liu et al. recently reported a kind of peroxidase-like nanozymes, prepared by reducing and functionalizing graphene oxide by Ganoderma polysaccharide.³⁰ The other one is preparing graphene-based composites, in which their enzyme-mimetic activity can be further enhanced. For instance, Guo et al. have developed a nanozyme of peroxidase-like activity by depositing PtCu bimetallic nanoparticle on poly-styrene-sulfonate-functionalized graphene.³¹ Wang et al. prepared a composite with oxidase-like activity consisting of NiCo₂S₄ and reduced graphene oxide (rGO) via a hydrothermal process.³² Song et al. demonstrated that fluoride capped V₆O₁₃-rGO nanocomposites exhibited high oxidase mimetic activity for the first time.³³ Nevertheless, most of these nanozymes may still encounter with some problems like low catalysis activity, complicate synthesis and high cost, hindering their further application. Therefore, it is of great interest to develop new graphene-based nanozymes with easy preparation, high catalytic activity and low cost. Although it has been reported that graphene-based composites are used for sensing different analytes, their material synthesis steps are complicated.³⁴ In addition, there are few studies on the simultaneous use of MnO₂@G composites with nanozymes activity for the construction of colorimetric and electrochemical sensors.

In this work, an in situ green synthesis method is proposed, and a novel composite constructed by MnO₂ nanoparticles and graphene nanosheets (MnO₂@G) is prepared via a modified Hummers' method and the following after-treatment. In detail, the impurities of Mn²⁺ ions in crude graphene dispersion are oxidized by additional MnO₄⁻ to in situ generate MnO₂ nanoparticles on or between graphene sheets. Therefore, the MnO₂@G were obtained facilely and economically. Moreover, a novel colorimetric and electrochemical dual-channel sensor for the determination of dopamine (DA) is developed by utilizing MnO₂@G as an oxidase mimic. MnO₂@G can directly catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to convert colorless TMB to blue oxidized production (oxTMB), showing a superior oxidase activity. Under the optimized conditions, the concentration of DA was quantitatively determined by the UV-vis absorbance (652 nm) of oxidized TMB. Meanwhile, it is noted that MnO₂@G modified electrodes possess excellent electrocatalytic activity to the oxidation of DA. The MnO₂@G-based electrochemical sensor shows a wider detection range toward DA. Moreover, the colorimetric and electrochemical dual-mode sensor based on MnO₂@G was constructed for the detection of DA with limit of detection (LOD) of 0.14 μmol L⁻¹ and 1.16 μmol L⁻¹, respectively (Scheme 1).

Scheme 1 Colorimetric and electrochemical assay for DA.

2. Experimental section

2.1 Materials and instruments

Graphite powders (325 mesh), dopamine (DA), 3,3′,5,5′-tetramethylbenzidine (TMB), L-glycine (Gly) and other amino acid was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China); the potassium permanganate (KMnO₄), sulfuric acid (purity: 98%, H₂SO₄), potassium hydroxide (KOH), ethanol, N,N-dimethylformamide (DMF), hydrogen peroxide (purity: 30%, H₂O₂) and other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); the concentrations of KH₂PO₄–Na₂HPO₄ and CH₃COOH–CH₃COONa were 0.1 M and 0.2 M, respectively, for the preparation of a PBS and NaAc-Hac buffer solution. The ultrapure water was used as experimental water throughout the experiment.

UV-vis spectra were performed by a Shimadzu UV-2550 UV Visible spectrophotometer (Shimadzu Co., Kyoto, Japan). Electrochemical experiments were performed using a CHI-660E electrochemical workstation (Chenhua Apparatus Co., Shanghai, China). The scanning electron microscopy (SEM) images were obtained using a scanning electron microscope (Quanta FEG 250 FEI, USA). Transmission electron microscopy (FEI Tecnai F20, FEI, USA) was used to record the high-resolution TEM images. The crystal structures of the samples were characterized by X-ray diffraction (XRD-7000, Shimadzu, Japan). X-Ray Photoelectron Spectroscopy (XPS) was recorded by Kratos AXIS Supra spectrometer (Ultima IV, Rigaku Corporation, Japan).

2.2 Synthesis of MnO₂@G

The graphene was prepared from graphite powder using an improved Hummers' method.^35,36 Firstly, 5 g graphite powder was put into 150 mL H₂SO₄, and then 15 g KMnO₄ was added to the reaction system under ice condition slowly, followed by stirring for 0.5 h. Successively, the reaction system was transferred to a 40 °C water bath and stirred for about 1 h. After that, 500 mL H₂O and sufficient H₂O₂ were added to turn the color of the solution from dark brown to yellow. Then, KOH was slowly added into the solution and turned the pH of the solution to 7 ± 1 with agitation. Finally, 10 g KMnO₄ were added again and sufficiently stirred for 4 h at 85 °C to proceed comproportionation reaction of Mn element. The obtained products were filtrated and then washed with H₂O. After drying under 80 °C and successively thermal treatment under 120 °C to reduce graphene MnO₂@G was obtained.

2.3 Fabrication of the enzymatic-like modified electrode

The glassy carbon electrode (GCE) was polished on a polishing cloth with 0.05 μm alumina slurry, successively ultrasonically cleaned in ethanol and ultra-pure water respectively and then dried in room temperature before use. The MnO₂@G modified GCE (named MnO₂@G/GCE) was fabricated by dispersing the MnO₂@G in DMF (10 mg mL⁻¹) and drop casting 3 μL of this solution on the GCE surface followed by drying at infrared lamp for 15 minutes.

2.4 Oxidase-like activity of MnO₂@G

By examining the relative catalytic activity (defined as the ratio of the absorbance at the target point to the maximum absorbance) of the MnO₂@G at various temperatures (10–60 °C), pH (3.0–8.0), and response time (1–60 minutes), the optimum reaction conditions for the colorimetric system were found. To assess the oxidase-like activity of MnO₂@G, TMB was chosen as the chromogenic substrate. Typically, MnO₂@G (1.0 mg mL⁻¹, 15 μL) and the TMB (10 mM, 35 μL) were added to NaAc-HAc buffer solutions (0.2 M, pH = 5.0, 2.95 mL). The UV-vis spectrum of TMB + MnO₂@G system was obtained after reaction at 30 °C for 30 minutes.

2.5 Steady-state kinetic study for oxidase-like

To determine the affinity between the MnO₂@G and substrate, steady-state kinetic experiments were implemented under the optimum reaction conditions by altering the TMB concentrations in the TMB + MnO₂@G system. Generally, a series of substrate concentrations of TMB (35 μL) were added into NaAc-HAc buffer solutions (2.95 mL, 0.2 M, pH = 5.0) containing MnO₂@G (15 μL, 1.0 mg mL⁻¹). On a UV-vis spectrophotometer, kinetic tests were conducted in time course mode to record the change in absorbance at 652 nm. According to the Michaelis–Menten equation,³⁷ ν = V_max[S]/(K_m + [S]), where is the beginning velocity, [S] is the substrate concentration, V_max is the maximum velocity, and K_m is the Michaelis constant, the kinetic constants were computed.

2.6 Catalytic mechanism for oxidase-like activity

To investigate the possible reason for the oxidase-like activity of MnO₂@G, TMB catalytic oxidization on O₂-dependent experiments were performed by the nitrogen injected the solution and keeping it for 30 minutes. In addition, in order to explore the reactive oxygen species (ROS) in TMB + MnO₂@G system, some radical scavengers were used. For example, iso-propyl alcohol (IPA, ˙OH scavenger), sodium azide (NaN₃, ¹O₂ scavenger) and p-benzoquinone (PBQ, O₂˙⁻ scavenger) were added respectively to the TMB + MnO₂@G system. The concentrations of IPA, NaN₃ and PBQ were 0.5 mM, 1.0 mM and 2.0 mM.

2.7 Dual-mode sensing platform for DA detection

For DA detection, 50 μL of DA with different concentration, 35 μL of TMB (10 mM), and 15 μL of MnO₂@G (1.0 mg mL⁻¹) were added to NaAc-HAc buffer solutions (2.9 mL, 2.0 M, pH = 5.0). After 30 minutes of incubation at 30 °C, the UV-vis spectrum of TMB + DA + MnO₂@G system at 652 nm was recorded. The linear relationship between the absorbance change value ΔA (ΔA = A₀ − A, A₀ is the absorbance of the blank solution, and A is the absorbance of DA sample solution) and the DA concentration was investigated.

The concentration of DA in a phosphate-buffered solution (PBS, 0.1 M, pH = 5.5) was determined by differential pulse voltammetry (DPV). All electrochemical detections were done in a typical three-electrode setup at room temperature to use a bare GCE or MnO₂@G/GCE as working electrodes, a platinum electrode as the counter electrode, and a calomel electrode as the reference electrode.

2.8 DA assay in human serum samples

The serum was collected from a group of volunteers from the Affiliated Hospital of Hubei Minzu University. The standard additive method was used to show the practical applicability. After diluting the serum with ultra-pure water to a final dilution of 100 times, the serum sample was added with DA standard solution at varying concentrations. Then, DA was detected with the same procedures described above.

3. Results and discussion

3.1 Characterizations of MnO₂@G

The morphologies of the MnO₂@G were examined through scanning electron microscope (SEM). In Fig. 1a shows the irregular sheets structure of MnO₂@G nanocomposite. It is noticed from SEM images with higher magnification (Fig. 1b) that MnO₂ nanoparticles of particle size of ∼10 μm was unevenness distributed on graphene sheets. This result showed that both MnO₂ nanoparticles and graphene lamellae did not show agglomeration, MnO₂ nanoparticles distribution were dis-uniformly deposited on the graphene surface. Additionally, the image from TEM depicts showed that the MnO₂ in the shape of nanosized fibrous morphology and some were intertwined nonuniformly on the graphene surface (Fig. 1c and d). The morphology implies that MnO₂ nanoparticles and graphene sheets should have favorable interaction, which would benefit their synergistic effect, and ensure the application of two-mold detection.

Fig. 1 SEM (a and b) and TEM (c and d) images of a MnO₂@G.

The chemical structure of the MnO₂@G was examined by XRD to determine the crystal form of MnO₂. The XRD patterns of the MnO₂@G contain two groups of feature diffraction peaks (Fig. 2): the broad feature diffraction peak at 26° is assigned to lattice plane of graphene (002), which implies that the graphene sheets are exfoliated. Furthermore, five sharp diffraction peaks appeared at 28.6°, 36°, 37.4°, 41.7°, 49.7° and 59.9° are the other group, corresponding to (301), (400), (211), (204), (411) and (512) of facts of K_xMnO₂ (JCPDS no. 44-1386), respectively, demonstrating the fine formation of the cryptomelane-type MnO₂ (K_xMnO₂, where x is non-stoichiometric, around 0.12) crystalline structure of MnO₂, which agrees the formation of K_xMnO₂ crystal observed in TEM. The XRD results proves the success deposition of K_xMnO₂ on graphene surface.

Fig. 2 XRD patterns of MnO₂@G.

Raman spectrum further confirm the chemical formation of MnO₂@G. Raman spectrum of the material in Fig. 3 clearly show two characteristic peaks of MnO₂@G, D peak (1355 cm^−l) and G peak (1592 cm^−l) attributing to the presence of defects and the graphite-derived sp² carbon structure, which proves the presence of graphene sheets.^30,35,38 Another Raman band at 639 cm⁻¹ is the characteristic band of K_xMnO₂.^36,39 According to the ref. 40, the sharp Raman bands at 639 cm⁻¹ is the indicative of a tetragonal structure with an interstitial space consisting of (2 × 2) tunnels, due to the symmetric Mn–O vibrations and assigned to the Ag spectroscopic species.

Fig. 3 Raman spectra of MnO₂@G.

To confirm the formation of MnO₂@G, XPS data of the material were recorded (Fig. 4a). As presented in Fig. 4a, the survey XPS spectrum exhibits the presence of Mn, K, C and O elements in the MnO₂@G with the atomic percentage of 9.4, 0.9, 60.9 and 28.8%, respectively. As shown in Fig. 4b, the peaks of O 1s at 529, 530.6 and 532.5 eV can be respectively attributed to Mn–O–Mn, C=O and C–OH.^41,42 In detail, the C 1s peak can be deconvoluted into three components, with binding energies at 284, 285.3 and 287.6 eV (Fig. 4c), which are ascribed to the graphitic carbons, C–O bonds and C=O bonds. Moreover, the two peaks at 291.1 eV and 293.8 eV were considered as the characteristic peak of K 2p.⁴³ The high-resolution Mn 2p spectra in Fig. 4d for all samples show the same composition of peaks, including two peaks at 652.9 eV and 641.4 eV, corresponding to the Mn 2p_1/2 and Mn 2p_3/2, respectively. The energy separation of these peaks was found to be 11.5 eV, which indicates the presence of Mn⁴⁺ in MnO₂.⁴⁴ The above result verified successfully in situ formation of MnO₂ on the graphene surface. In summary, in the nanocomposite of MnO₂ and graphene, the cryptomelane-type MnO₂ (K_xMnO₂) provides the metal active site and graphene enhances the electrochemical performance of the materials, which lays the foundation for the subsequent construction of the dual-mode colorimetric-electrochemical sensors.

Fig. 4 (a) XPS assay of MnO₂@G; the high-resolution XPS pattern of (b) O 1s, (c) C 1s + K 2p and (d) Mn 2p.

3.2 Electrochemical investigation of MnO₂@G

Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to examine the electrocatalytic characteristics of modified electrodes. The Nyquist curves of the GCE and MnO₂@G/GCE electrodes were shown in Fig. 5a. The diameter of the semicircle at high frequency relates to the electron transfer resistance, whereas the straight portion corresponds to the diffusion process.⁴⁵ As seen in Fig. 5a, a large diameter of the semicircle part was observed in the case of GCE. While on the MnO₂@G/GCE has a nearly straight line appeared with the electron transfer resistance value close to zero. This indicates that the electron transfer of Fe[(CN)₆]^3−/4− on the surface of MnO₂@G/GCE electrode is faster due to the good electron pathway provided by graphene, which improves the electron transfer efficiency to enhance the conductivity of bare GCE electrode. Besides, the slope of the MnO₂@G/GCE electrode in the low-frequency region is larger than that of the MnO₂@G/GCE electrode, which further indicates that the MnO₂@G can effectively reduce the interfacial resistance and can improve the conducting characteristics of the electrochemical sensor.⁴⁶

Fig. 5 EIS diagram (a) and CV curves (b) of the various electrodes in 0.1 M KCl solution containing 5 mM Fe[(CN)₆]^3−/4−.

As illustrated in Fig. 5b, GCE and MnO₂@G/GCE was characterized by CV methods. It can be seen from the figure that a pair of symmetric redox peaks were presented on each electrode, which show a quasi-reversible electrochemical process. The intensity of the redox peak corresponding to MnO₂@G/GCE was substantially greater than that of GCE. The anodic peak current (I_pa) on MnO₂@G/GCE were 1.07 times higher than that of CCE, and the cathodic peak current (I_pc) on MnO₂@G/GCE were 1.15 times higher than that of CCE. The results of CV indicated that MnO₂@G enhanced electro-catalytic activity and conductivity of the CCE electrode.

3.3 Kinetic analysis of MnO₂@G as an oxidase mimic

To determine the oxidase-like activity of MnO₂@G, the kinetic research was done under the following conditions: 5.0 μg mL⁻¹ of MnO₂@G, pH = 5.0 and 30 °C. As shown in Fig. 6a, the typical Michaelis–Menten curves was obtained by changing the concentration of substrate (TMB).¹² The double-reciprocal Lineweaver Burk plots (Fig. 6b) was used to obtain the kinetic parameters Michaelis–Menten constant (K_m) and maximum initial rate (V_max), and they are 0.026 mM and 2.52 × 10⁻⁸ M s⁻¹, respectively. The kinetic parameters reported in other references and this study were given in Table 1. The K_m represents the affinity between nanozymes and substrate, which the lower value of K_m means a stronger affinity.⁴⁷ The results indicated that the value of K_m of MnO₂@G toward TMB lower than that of other oxidase-like nanozymes, indicating a stronger affinity between MnO₂@G and substrates.

Fig. 6 Steady-state kinetics assays of MnO₂@G by varying concentrations of TMB (a) and corresponding Lineweaver–Burk plots (b).

Table 1 Comparison of kinetic constants of MnO₂@G with different nanozymes

Nanozymes	Km (mM)	Vm (10^−8 M s^−1)	Ref.
MnO2@G	0.026	2.52	This work
MnO2/PS	0.068	8.9	48
PtNPs@MnO2	0.095	26	49
Ag/His-GQD	0.6	22.5	50
V6O13-rGO + F^−	0.082	4.34	33
HPR	0.53	14.5	51
Laccase	0.19	4.5	52

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3.4 Investigation of the catalysis mechanism of MnO₂@G

The catalytic mechanism of the oxidase-mimicking activity may originate from the dissolved oxygen to convert reactive oxygen species (ROS), which the oxidize the substrate (TMB).³⁷ To reveal the role of dissolved oxygen in the oxidation of TMB, a comparative experiment was conducted under a N₂-rich atmosphere. It can be seen from Fig. 7a that the absorbance was decreased by 47.3% when N₂-rich coexist, which proved the important role of dissolved oxygen in the catalytic process and further verify the oxidase-like activity of MnO₂@G.

Fig. 7 (a) UV-vis spectrum of TMB + MnO₂@G system in N₂ saturated system. (b) The relative activity of MnO₂@G with different radical scavenger (PBQ, NaN₃ and IPA).

As shown in Fig. 7b different radical scavengers were used in order to further explore active species in TMB + MnO₂@G system. To determine the ROS coexist, some scavengers, such as p-benzo- quinone (PBQ), NaN₃ and iso-propyl alcohol (IPA) were added into the catalytic systems to capture O₂˙⁻, ¹O₂ and ˙OH reactive oxygen species. As can be seen from Fig. 7b, PBQ (O₂˙⁻ scavenger) and NaN₃ (¹O₂ scavenger) induces a significant decline in catalytic activity of MnO₂@G, which indicates the existence of O₂˙⁻ and ¹O₂ in this system. The IPA as scavenger of ˙OH radical has minor effect on the catalytic activity of MnO₂@G. It is indicated that the O₂˙⁻ and ¹O₂ play an important role in the catalytic oxidation of TMB by MnO₂@G.

3.5 Feasibility for colorimetry and electrochemical dual-mode sensing platform

The oxidase activity of MnO₂@G was investigated by conducting a catalyzed reaction with TMB, which resulted in a substantial color shift from colorless to blue. Fig. 8b reveals that MnO₂@G can effectively oxidize TMB to enhance absorbance, with the greatest absorption peak occurring at 652 nm in the MnO₂@G + TMB system. The findings confirmed that TMB oxidation was caused by MnO₂@G decomposing dissolved oxygen. Interestingly, when 10 μM DA was added to the TMB + MnO₂@G system, its blue immediately faded and its absorbance reduced considerably. This behavior may be explained by the inhibition of the catalytic oxidation of TMB by of DA. The above investigation results showed that MnO₂@G can be used as nanozyme for colorimetric analysis of DA.

Fig. 8 (a) UV-vis absorption spectra of in different systems in NaAc-HAc buffer (0.2 M pH = 5.0) at 30 °C for 30 min. (b) DPV curves of GCE and MnO₂@G/GCE in PBS (0.1 M pH = 5.5) containing 10 μM DA.

As shown in Fig. 8b, the DPV was used to study the electrochemical behavior of DA on GCE and MnO₂@G/GCE electrodes. The larger peak currents were obvious on the MnO₂@G/GCE than that of GCE. The peak currents on MnO₂@G/GCE were 7.13 times higher than that of CCE. It indicated that the capability of the MnO₂@G/GCE electrode to detect DA is greatly enhanced owing to the capacity of MnO₂@G to enhance the conductivity of the GCE, hence facilitating electron transfer. Therefore, the MnO₂@G modified electrode can be used as an electrochemical analysis platform for DA.

3.6 Condition optimization for the dual-mode sensing platform

For the purpose of more sensitive assay of the dual-mode sensing platform, several parameters in the detection system were optimized. Like natural oxidase, the oxidase-like catalytic activity of MnO₂@G is depends on the pH (Fig. 9a), temperature (Fig. 9b), concentrations of catalyst (Fig. 9c) and reaction time (Fig. 9d). As shown in Fig. 9a, the relative activity of MnO₂@G reached the maximum when the pH of the NaAc-HAc buffer solution was 5.0. Fig. 9b shows that the optimum catalytic temperature of MnO₂@G catalyst is 30 °C. As shown in Fig. 9c with the increase of MnO₂@G concentration, the relative activity of the catalyst gradually increased. However, the rate of increase of relative activity was slow when the concentration of MnO₂@G reached 5 μg mL⁻¹. As shown in Fig. 9d, the relative activity stabilized when the reaction time reached 30 minutes. In summary, the relative catalytic activity of MnO₂@G was most suitable at pH = 5.0, temperature of 30 °C, reaction time of 30 minutes and catalyst concentration of 5 μg mL⁻¹.

Fig. 9 Dependency of the MnO₂@G catalytic activity on (a) pH values, (b) temperature, (c) the catalyst concentration, and (d) interaction time. DPV curve (e), effect of pH on peak current (f) and linear fitting curve of peak potential and pH (f) of MnO₂@G/GCE in PBS containing 30 μM DA with different pH (pH = 4–8); (g) CV plots of DA with various scan rates from 40 to 200 mV s⁻¹ on MnO₂@G/GCE; (h) the linear relationship between peak current and scan rate.

To increase the sensitivity of the electrochemical sensor, DPV evaluated the effect of pH on the response current of the sensor. With 30 μM DA as the analyte, MnO₂@G/GCE as the working electrode, and 0.1 M PBS as the supporting electrolyte, the pH was optimized by the peak current response value of DPV. The detection outcome was depicted in Fig. 9e. The current value of DA reaches its maximum when the pH = 5.5. Consequently, the PBS with pH = 5.5 was utilized as the supporting electrolyte in subsequent analyses of the DA. As seen in Fig. 9f, as pH increased, the peak potentials of DA moved in a negative direction, showing that protons were involved in the electrochemical process.⁵³ Simultaneously, the correlation between peak potential (E_p) and pH was also investigated. According to Fig. 9f, the relationship between peak potential and pH is linear. The equation for linear regression was E_p (V) = −0.054 pH + 0.548 (R² = 0.997). The slope is −0.054, which is near to the Nernst value (−0.059), suggesting that the redox process at the MnO₂@G/GCE electrode involves an equal quantity of protons and electrons.^54,55 Using CV method, the impact of different scan rate on MnO₂@G/GCE was recorded. Fig. 9g demonstrates that when the scan rate increased, both the I_pa and I_pc of DA linearly grew. As shown in Fig. 9g, the two peak currents are proportional to square root of scan rate with a linear regression equation as I_pa = 0.0098ν^1/2 + 4.947 (R² = 0.993) and I_pc = −0.277ν^1/2−4.028 (R² = 0.993). It is indicated that the electrochemical reaction of DA on MnO₂@G/GCE is a diffusion control process.⁵⁶

3.7 Colorimetric and electrochemical detection of DA

Inspired by the oxidase-like activity of the MnO₂@G, we used TMB + DA + MnO₂@G system for sensitive colorimetric detection of DA. Under the optimal reaction conditions, the absorbance of the system was measured with DA concentration changing from 0.1 to 15 μM as shown in Fig. 10a. A linear curve of ΔA (Δ absorbance is the difference of the absorbance of the solution with and without DA) to DA concentration was shown in Fig. 10b. The results showed that the absorbance decreased with the DA concentration in the range 0.1–15 μM and the calibration equation was ΔA = 0.084[DA] + 0.08 (R² = 0.994) with the LOD of 0.14 μM (S/N = 3).

Fig. 10 (a) UV-vis absorption spectra of MnO₂@G combined with different DA concentrations (from top to down DA concentrations are: 0, 0.1, 0.2, 0.4, 0.6, 0.9, 1.2, 1.5, 2, 3, 4, 6, 8, 10, 13, 15 μM). The inset shows the corresponding images. (b) The plotted linear relationships between ΔA and the corresponding [DA]. (c) DPV curves of MnO₂@G/GCE in pH = 5.5 buffer containing different concentrations of DA (0, 0.4, 1, 2, 3, 4, 6, 10, 15, 20, 30, 40, 55, 70 μM). (d) Represent the calibration plot between peak current and DA concentration.

To farther broaden the application of MnO₂@G, the DPV method was used to analyze the relationship between concentration and the current response of DA under optimal conditions. The standard curves of the relationship between concentration and current response are shown in Fig. 10c and d. It can be seen from Fig. 10c, as the DA concentration increases from 0.4 to 70 μM, the peak current linear increases. The corresponding linear equation are I_pa = 0.072[DA] + 0.34 (R² = 0.993) (Fig. 10d) with the LOD of 1.16 μM (S/N = 3). Compared with these references as listed in Table 2, the proposed two method exhibits a relatively low LOD and wide detection range, confirming the superiority of the proposed complementary-dual-modal sensing platform for DA detection.

Table 2 Comparison of analytical performances of different DA assays

Methods	Materials	Linear range (μM)	Detection limit (μM)	Ref
UV-vis	MnO2@G	0.1–15	0.14	This work
Electrochemistry		0.4–70	1.16	This work
UV-vis	h-CuS NCs	2–150	1.67	57
UV-vis	NiCo2S4-rGO	0.5–100	0.43	32
UV-vis	Pt/hBNNSs	2–55	0.76	58
Electrochemistry	Pd-NCd/rGo	20–220	7.02	59
Electrochemistry	GO/Fe3O4	1–10	0.48	60
Electrochemistry	PtNP/rGO	1–10; 10–100	1.405	61

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3.8 Interferences, stability, and reproducibility

Selectivity is important for the colorimetric sensor for DA based on MnO₂@G. Under optimal conditions containing 10 μM DA, 50-fold inorganic ions such as Na⁺, Mg⁺, Ca²⁺, K⁺, SO₄²⁻, Cl⁻ and 5-fold biomolecules such as Gly, Leu, Lys, Arg, glucose and fructose as several potential interfering substances, signal changes below 5% in absorbance were observed in Fig. 10a. These results demonstrate that the present MnO₂@G based assay shows excellent specificity for DA. MnO₂@G has a stable oxidase-like activity and maintains 96.6% relative catalytic activity when stored at room temperature for 30 days.

The interference of coexisting species on experimental results was investigated. As shown in Fig. 10b, the results show that 100-fold of inorganic ions such as Ca²⁺, Na⁺, K⁺, Mg²⁺, SO₄²⁻, Cl⁻ and 10-fold biomolecules, such as Gly, Leu, Lys, Arg, glucose and fructose do not affect the test results (with signal change below 5% on the height of the peak current). It shows that the electrochemical sensor has sound interferences. Under the optimal experimental conditions, with MnO₂@G/GCE as the working electrode, 30 μM DA is tested in PBS buffer with pH = 5.5 for DPV. The current value changes were observed to verify the stability and reproducibility of the electrode. Using the same MnO₂@G/GCE electrode to scan 15 times under the above conditions, the relative standard deviation (RSD) was 2.4%. MnO₂@G/GCE electrode was placed at room temperature for 7 days. The response current of DA was 97.3% of the initial current, which proved that the electrode possessed good stability and reproducibility (Fig. 11).

Fig. 11 Selectivity of (a) the colorimetric sensor and (b) the electrochemical sensor.

3.9 Application of colorimetric and electrochemical sensor for real sample analysis

To explore the potential applications of the colorimetric and electrochemical sensor toward DA, standard addition method was applied for serum samples examination. The results were summarized in Table 3. The recoveries were obtained by the standard addition method, and the spiked recoveries were in the range of 96.5%–103.2%, with the relative standard deviations less than 5%. These data indicate that the constructed double-sensor can be successfully applied to simultaneously detecting the levels of DA in real biological samples.

Table 3 The determination results of DA (n = 3) in human serum

Sample	Added (μM)	Found (μM)	Recovery (%)	RSD (%)
Colorimetric
1	2	1.93	96.5	4.35
2	8	7.86	98.3	3.41
3	12	12.21	101.8	3.67
Electrochemical
1	5	5.16	103.2	3.46
2	35	35.73	102.1	2.15
3	65	64.56	99.3	1.78

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4. Conclusion

In this work, an in situ green synthesis method is proposed, and a novel composite produced by MnO₂ nanoparticles and graphene nanosheets (MnO₂@G) is prepared. The colorimetric and electrochemical analysis double-channel methods for the detection of DA are firstly designed based on the excellent oxidase-like activity and electrochemical performance of MnO₂@G. Through comparing the designed two strategy, the colorimetric sensing platform demonstrated lower detection limit with 0.14 μM and higher sensitivity. The electrochemical analysis methods exhibit the advantages of wider linear range with from 0.4 to70 μM and good selectivity. Furthermore, the MnO₂@G nanozyme-based double sensing platform with recoveries from 96.5% to 103.2%, and RSD values were less than 4.35% was used for the successful quantitative testing of DA in clinical serum samples. Therefore, the proposed detection platform provided complementary colorimetric and electrochemical modes to meet the requirements of different occasions. Furthermore, we believe the novel nano-composite MnO₂@G should also possess potential in other applications such as catalyst, absorption, etc., for its unique microstructure.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors acknowledge support from the financial support by the National Natural Science Foundation of China (No. 22066010); the Science and Technology Plan Research Project of Hubei Education Department (Q20211905); Incubation Project for High-Level Scientific Research Achievements of Hubei Minzu University (No. PY22004).

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