From a biological perspective, the active site of a natural enzyme is defined as the region where substrate molecules undergo specific chemical reactions with a significant reduction in activation energy. It serves as the critical component responsible for enzyme function. Consequently, the most effective approach to achieving comparable catalytic performance to natural enzymes is to mimic their active sites. To date, there has been a growing utilization of diverse nanomaterials in the construction of electrochemical nanozymes for the detection of reactive oxygen species (ROS). This can be attributed to the distinctive physicochemical characteristics exhibited by nanomaterials, including their diminutive size, expansive surface area, and remarkable reactivity. Prominent examples of such nanomaterials encompass noble-metal-based materials, transition-metal-based materials, carbon-based materials, metal–organic framework-based materials, and various other emerging nanomaterials. These materials have garnered significant attention in the field of electrochemical nanozymes due to their unique properties and potential applications in ROS detection.

Nano-carbon materials, including carbon nanotubes, graphene, and other related structures, exhibit exceptional characteristics such as high strength, outstanding electrical conductivity, efficient charge transfer capabilities at high current densities, low resistance, substantial surface area, and remarkable chemical stability. These inherent properties make them highly desirable for various applications, particularly in the field of electrochemical sensing and nanozyme-based systems.

Detection of O₂^•−. The most commonly used carbon material is graphene. A. Olean-Oliveira et al. recently reported on the development of a nonenzymatic chemiresistor sensor for the detection of superoxide radicals, utilizing an azo-polymer in conjunction with reduced graphene oxide (rGO) employed as the resistive platform for the sensor application ^[1][12]. The sensor platform was fabricated by sequentially depositing poly (azo-Bismarck Brown Y) and reduced graphene oxide films using a layer-by-layer assembly technique . The resulting nanocomposite film demonstrated intriguing synergistic properties, combining the redox properties of the azo-polymer with the excellent electronic conductivity and stability of graphene. Real-time impedance measurements (chrono-impedance) using the poly(azo-BBY)/rGO sensor exhibited a linear relationship between the real impedance and the concentration of superoxide anions (ranging from 0.12 to 2.6 mM), with a detection limit of 81.0 μM. Xuan Cai et al. further introduced a novel approach for fabricating enzyme-mimicking metal-free catalysts, specifically for the electrochemical detection of O₂^•−, incorporating phosphate groups into a graphene-based foam ^[2][13]. This was achieved through a template-free hydrothermal process, involving the treatment of graphene oxide (GO) with varying amounts of phytic acid (PA) to obtain a three-dimensional porous graphene-based foam (PAGF). The characterization results confirmed the successful fabrication of the sensors, which were effectively employed for the determination of O₂^•− released by cells, showcasing exceptional performance in the dynamic monitoring of cellular O₂^•− levels.

Mesoporous carbon materials possess a significant number of edge-plane-like defective sites, which effectively facilitate electron transfer to analytes and enhance the electrochemical activity at the electrode interfaces. The presence of mesoporous channels within the carbon shells of Hollow Mesoporous Carbon Spheres (HMCSs) offers advantageous mass transport and/or charge transfer properties between the sensors and analytes. Li Liu et al. successfully developed an enzyme- and metal-free electrochemical method with remarkable sensitivity for detecting O₂^•−. This method utilized a screen-printed carbon electrode (SPCE) that was modified with nitrogen-doped hollow mesoporous carbon spheres (N-HMCSs) ^[3][14]. The electrochemical reduction of O₂^•− that takes place on the surface of the modified electrodes is represented by the following equation:

Detection of H₂O₂. The generation of H₂O₂ was first discovered in 1966, and since then, a great deal of work has been performed to investigate the production of hydrogen peroxide and its important role in the body. According to some works, H₂O₂, an essential and potent oxidant, plays a pivotal role in various biological processes, encompassing intercellular signaling, immune cell recruitment, and modulation of cellular morphology and differentiation. Its significance lies in its simplicity, importance, and remarkable oxidative capabilities, which contribute to the intricate mechanisms underlying fundamental biological functions. Despite its lower oxidant power compared to O₂^•−, H₂O₂ is recognized as an extremely potent cytotoxic agent. However, in a groundbreaking study conducted by J. Q. Tian et al. in 2013, it was demonstrated for the first time that ultrathin graphitic carbon nitride (g-C₃N₄) nanosheets possess exceptional electrocatalytic properties, making them a cost-effective, environmentally friendly, and highly efficient catalyst for the reduction of hydrogen peroxide ^[4][15]. Another noteworthy contribution in this field was made by the research group led by J. Bai ^[5][16] who developed H₂O₂ sensors utilizing carbon dots (CDs) and multi-walled carbon nanotubes (MWCNTs). Notably, the CDs/MWCNTs/GCE sensor exhibited a significant synergistic effect, leading to enhanced performance, including an improved LOD of 0.25 μM.

H₂O₂ detection using carbon-based nanomaterials often relies on the electrochemical sensing principle, where H₂O₂ undergoes a redox reaction at the surface of the carbon nanomaterial, leading to measurable electrical signals. As represented by the following equation:

Noble metal nanomaterials, such as gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs), have captured significant interest within the scientific community due to their remarkable stability, high conductivity, facile preparation, expansive specific surface area, and outstanding biocompatibility. Notably, emerging research has highlighted the crucial influence of nanoparticle size, shape, and distribution on their electrocatalytic activity. These findings underscore the significance of tailoring these parameters to optimize the electrochemical performance of noble metal nanomaterials.

Detection of O₂^•−. W. Z. Fan. et al. successfully obtained uniformly dispersed silver nanoparticles (AgNPs) by pyrolyzing a novel silver-based metal–organic framework, as reported in their study ^[6][17]. Specifically, the AgNPs@C nanocomposites were synthesized through thermal treatment of the Ag-based metal–organic frameworks, utilizing silver as the metal center and benzimidazole as the organic ligand. An electrochemical response towards the reduction of O₂^•− was observed in the obtained AgNPs@C nanocomposites, exhibiting an ultra-wide linear range (3.032 × 10⁻¹³ to 5.719 × 10⁻⁵ M) and an exceptionally low detection limit (1.011 × 10⁻¹³ M). Furthermore, the fabricated sensor was successfully employed for real-time detection of O₂^•− released from HeLa cells under both normal and oxidative stress conditions. Based on the experimental results, the authors presented the reduction mechanism of O₂^•− on the AgNPs@C/GCE as:

As shown in Equation (3), O₂^•− rapidly decomposes into H₂O₂ and O₂ in aqueous solution due to its inherent instability. It is well-known that Ag nanoparticles possess catalytic activity towards the decomposition of H₂O₂ ^[7][18]. Consequently, it was observed that AgNPs exhibit enhanced catalytic efficacy in the decomposition of H₂O₂ as depicted in Equation (4). Subsequently, the liberated oxygen originating from Equations (3) and (4) undergoes diffusion in the vicinity of the electrode, allowing for its detection through reduction on the modified electrode, as illustrated in Equation (5). Other common methods involve the combination of nanoparticles and carbon materials. For instance, T. D. Wu. et al. fabricated a nanocomposite (NCF-Ag) comprising nitrogen-doped cotton carbon fiber and silver nanoparticles. The fabricated sensor exhibited remarkable electrochemical performance in the identification of O₂^•−, demonstrating notable attributes such as a low limit of detection (LOD) of 2.53 × 10⁻¹⁴ M, an extensive linear detection range spanning from 7.59 × 10⁻¹⁴ to 7.22 × 10⁻⁵ M, and exceptional selectivity ^[8][19]. In a separate investigation, a nanocomposite comprising silver nanoparticles and multi-walled carbon nanotubes (AgNPs/MWNTs) was employed as an efficient electrode material, enabling sensitive detection of superoxide anions.

Detection of H₂O₂. D. Z. Zhu ^[9][20] drew inspiration from self-assembling peptide nanofibers (PNFs) and successfully designed and synthesized a novel hybrid material called PtNWs-PNFs/GO, which consists of biomimetic graphene-supported ultrafine platinum nanowires (PtNWs) integrated with PNFs. The controllable self-assembly process allowed the PNFs to act as a bridge between the GO nanosheets and PtNWs. The presence of PtNWs, known for their high catalytic activity, imparts remarkable electrochemical properties to the PtNWs-PNFs/GO hybrid-based sensor. The sensor demonstrates an extended linear detection range of 0.05 mM to 15 mM and a low detection limit of 0.0206 mM. Euna Ko et al. ^[10][21] immobilized bimetallic Au and Pt nanoparticles on the surface of agarose microbeads through chemical means, resulting in a hybrid nanostructure termed Au@PtNP/GO. The synergistic effect between the bimetallic nanoparticles and GO conferred strong peroxidase-like catalytic activity to the hybrid nanostructure, particularly towards the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of H₂O₂. This hybrid nanostructure enables dual applications of colorimetric and electrochemical detection. Upon the introduction of the TMB substrate solution containing H₂O₂, the catalytic oxidation of TMB takes place. Consequently, on the electrode surfaces, the oxidized TMB is subsequently subjected to electrochemical reduction, thereby leading to the achievement of an expanded detection range for H₂O₂ spanning from 1 μM to 3 mM, along with a reduced lower limit of detection quantified as 1.62 μM. Furthermore, the developed point-of-care (POC) devices exhibited accurate determination of H₂O₂, demonstrating strong repeatability and reproducibility in real sample testing using artificial urine.

Detection of •OH. As is commonly known, among all the free radicals, hydroxyl radicals (•OH) exhibit exceptional reactivity and pose significant dangers due to their nanosecond-scale lifetime. Consequently, the electrochemical detection of hydroxyl radicals presents a formidable challenge, necessitating rapid response times, ultra-high sensitivity, and low detection limits. Regrettably, the current literature offers limited insight into the electrochemical detection of hydroxyl radicals, and the majority of developed sensors have not been employed for the analysis of biological samples. In this regard, two groups of modifier materials have emerged as potential candidates for •OH detection: thiol self-assembled monolayers (SAM) and CeOx nanoclusters.

X.W. Xu et al. ^[11][22] have successfully developed a novel electrochemical sensor utilizing a self-assembled nanoporous gold layer (NPGL) modified with 6-(Ferrocenyl) hexanethiol (6-FcHT) on a glassy carbon electrode (6-FcHT/NPGL/GE). This sensor demonstrates remarkable sensitivity and selectivity in detecting the release of •OH from living cells. The enhanced sensitivity can be attributed to the unique porous architecture of NPGL, which significantly increases the electrode’s surface area and facilitates rapid electron transport during electrochemical reactions. Moreover, NPGL offers abundant active binding sites for the efficient assembly of the •OH capture agent (6-FcHT), ensuring excellent selectivity.

In comparison, when 6-FcHT was solely immobilized on a glassy carbon electrode (6-FcHT/GE), the sensitivity for •OH detection was measured at 0.0305 mA nM⁻¹ with a detection limit of 0.133 nM within the linear range of 0.4 nM to 70 nM. After implementing the NPGL modification, the sensitivity of 6-FcHT/NPGL/GE towards •OH increased substantially to 0.1364 mA nM⁻¹, while the detection limit significantly decreased to 0.316 pM. Furthermore, the linear detection range was effectively extended from 1 pM to 100 nM. Importantly, the sensor exhibits additional merits in terms of reproducibility, repeatability, and stability, making it suitable for direct electrochemical detection of •OH in HepG2 cells.

Metal–organic frameworks (MOFs) have garnered significant attention due to their unique properties, such as a high specific surface area, tunable pore structure, and exposed activity. Comprising metal nodes and organic ligands, MOFs have emerged as versatile materials with diverse applications in the fields of electrochemistry, fluorescence, colorimetry, photo-electrochemistry, and electrochemiluminescence sensing. Their exceptional properties make them well-suited for these applications, enabling the development of advanced sensing platforms and devices. The high specific surface area of MOFs facilitates efficient analyte adsorption and interaction, while the tunable pore structure allows for the selective trapping and recognition of target molecules. Additionally, the exposed activity of MOFs contributes to their enhanced performance in various sensing modalities. As a result, MOFs have demonstrated great potential as promising materials for the development of next-generation sensing technologies.

Detection of O₂^•−. Y. H. Zhang et al. ^[12][38] conducted a study aimed at enhancing the sensing performance of O₂^•− by developing a straightforward one-step strategy for the morphology-controllable synthesis of a manganese–organic framework (Mn-MOF). Interestingly, they achieved the synthesis of Mn-MOF nanoparticles, asymmetric nano-lollipops, and nanorods with homogeneous components by carefully adjusting the solvent ratios and regulating the initial precursor concentrations. Subsequently, the authors found that the Mn-MOF nano-lollipops exhibited superior O₂^•−-sensing capabilities compared to other nanostructures due to their larger active surface areas, which can be attributed to the excellent dispersibility provided by the asymmetric structure, as well as the accelerated electron transfer rate facilitated by the stem structure. By utilizing the Mn-MOF nano-lollipops for O₂^•− detection, a high sensitivity of 105 μA cm⁻²·μM⁻¹ was achieved, enabling the successful real-time and in situ detection of O₂^•− released from living cells. This research not only provides valuable insights into the solvents engineered morphologies of other MOF nanomaterials but also advances the understanding and potential applications of Mn-MOF nanostructures in sensing technologies.

Detection H₂O₂. MOF-based electrochemical sensors have gained significant attention for the detection of H₂O₂. Gao et al. ^[13][39] synthesized a Pt-nanoparticle-modified metalloporphyrin MOF (Pt@PMOF(Fe)) with multienzyme activity, which was utilized to construct an electrochemical H₂O₂ sensor. Another study by Z. Q. Wei’s group ^[14][40] involved the synthesis of a 3D Co-based Zeolitic Imidazolate Framework (3D ZIF-67) for H₂O₂ detection. X. Liu’s group ^[15][41] reported the development of a nickel metal–organic framework nanosheet array on Ti-mesh (Ni-MOF/TM) as an enzyme-free electrochemical sensing platform for H₂O₂ determination. X. L. Yang et al. ^[16][42] selected MIL-47(V) as an electrocatalyst to explore the feasibility of electrochemical sensing of H₂O₂. Nevertheless, these conventional approaches were executed in the single-readout mode, which makes them vulnerable to false-positive or false-negative outcomes arising from external interferences, such as the intricacies of the biological milieu, non-standardized testing protocols, and discrepancies among operators or testing environments. These limitations pose substantial challenges to the accuracy of single-readout analytical methods and impose constraints on their practical applications in disease diagnosis.

To address this issue, K. Yu et al. ^[17][43] proposed a novel approach using a portable colorimetric and electrochemical dual-mode sensor for the detection of cell-secreted H₂O₂ and H₂S, based on the MOF-818 nanozyme. The trinuclear copper centers in MOF-818 catalyze the formation of •OH from H₂O₂, leading to the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) and the production of blue oxTMB. The authors developed a smartphone sensing system by capturing the Hue-Saturation Value (HSV) of the reaction solution using a “Color Identifier” App. Furthermore, MOF-818 exhibited outstanding electrocatalytic activity in H₂O₂ reduction, as evidenced by its reduction peak potential of 0.08 V vs. Ag/AgCl. This exceptional performance enabled the integration of a smartphone and a mini electrochemical analyzer, leading to the establishment of an ultra-sensitive sensing system for H₂O₂. The inherent advantage of a dual-modal assay lies in the fact that the two signals obtained can be utilized to calibrate the assay results and effectively mitigate false-positive/negative outcomes. Additionally, analytical methods featuring dual readout signals can readily adapt to different analytical conditions, catering to the diverse needs of assay tasks. This innovative dual-mode sensing strategy represents a significant advancement in MOF-based electrochemical sensors and holds great promise for enhancing their accuracy and practical applicability in disease diagnosis and beyond.

To sum up, within recent decades, numerous nanozymes were applied as electrochemical biosensors. However, researchers also can find that most of studies are focused on the detection of superoxide anions (Table 1) and H₂O₂ (Table 2). The elements commonly used to construct the active sites of nanozymes for detection of ROS are mainly Au, Ag, Pt, Ni, Co, Mn, Fe, Cu, Ti, and Ce. Specially, due to its special chemical properties, CeOx has an excellent antioxidant capacity and catalytic performance, so it has great application potential in detecting ROS. On the one hand, CeOx, as a potent antioxidant, is able to reversibly change its oxidation state in the presence of ROS through the redox cycle, thereby converting excess ROS into harmless substances and protecting cells from oxidative stress. On the other hand, CeOx nanozymes can enhance their selectivity and sensitivity to ROS through surface modifications or composite materials, so as to achieve accurate detection and monitoring of ROS concentration. With the in-depth study of the mechanism of ROS in vivo and the continuous optimization of the properties of nanozyme materials, it is believed that nanozymes will play an important role in the fields of life science, medicine, and environmental studies, providing new solutions for the prevention, diagnosis, and treatment of ROS-related diseases.

Electrode	Linear Range (μM)	LOD (nM)	Sensitivity (μA cm−2 mM−1)	Stability (Days)	Application Potential (V)	Ref.
AgNPs@C/GCE	7.422 × 10−4–0.5719	1.011 × 10−4	-	7	−0.7	[6][17]
Co3O4@CMWCNTs/GCE	5 × 10−9–10	1.6767 × 10−9	-	-	-	[18][23]
Mn-MPSA-HCS/SPCE	0–1257.4	1.25	224	-	0.75	[19][32]
Ni(PO4)NRs/C-MWCNTs/GCE	1–80	97	5.67 × 104	25	−0.3	[20][35]
2D-mNC@CeO2/SPCEs	8–536	179	401.4	20	−0.5	[21][44]
PAMAM-Au/GCE	3.69 × 10−5–37.2	0.0123	-	-	−0.7	[22][45]
AgNPs-MC/GCE	1.68 × 10−3–30.6	0.012	-	15	−0.5	[23][46]
Co-NPs–NG/GCE	1.67 × 10−3–0.575	1.67	628.86	-	0.9	[24][47]
Co3(PO4)2/I-rGO/GCE	2.4 × 10−3–2.195	2.4	177.14	30	0.6	[25][48]
Mn-MPSA-MWCNTs/SPCE	0–1817	127	77.47	30	0.7	[26][49]

Electrode	Linear Range (μM)	LOD (nM)	Sensitivity (μA cm−2 mM−1)	Stability (Days)	Application Potential (V)	Ref.
Fe SAs-N/C/GCE	764–9664	340	22.1	-	−0.05	[27][50]
Cu@Cu2O/GCE	2–860	460	1855.53	-	−0.5	[28][51]
rGO/Au-NPs/GCE	25–3000	6.55	0.0641	-	−0.8	[29][52]
Bi2S3/g-C3N4/GCE	0.5–950	78	1011	7	0.26	[30][53]
Pt-LEPG/GCE	0.01 × 10−3–0.029	0.65	575.75	14	0.5	[31][54]
CoHCF-NSp’s /GCE	2–1130	2.1	329	-	0.8	[32][55]
Ag/PNA/GCE	1–3000	0.972	1844.76	10	−0.42	[33][56]
RGO–Pt NPs/GCE	0.5–3475	0.2	459	14	−0.08	[34][57]
Ag-Au/RGO/TiO2/GCE	10–30,000	3	-	-	-	[35][58]
MOF-Au@Pt/GCE	0.8–3000	86	24.14	-	−0.12	[36][59]
LDH/PPy-Ag/GCE	30–800	280	257.64	30	−0.3	[37][60]
CuO-CeO2/MXene/GCE	5–100	1.67	84.44	-	−0.3	[38][61]
Ag-Cu nanoalloys/GCE	2000–961,000	152	-	-	−0.07	[39][62]
AgNPs/2D Zn-MOFs/GCE	5–70,000	1.67 × 103	358.7	6	−0.55	[40][63]
BiVO4/TiO2/GCE	5–400	5 × 103	3014	90	0.5	[41][64]
NiCo2S4/rGO/GCE	25–11,250	190	118.5	14	−0.45	[42][65]
Pt/C-CeO2/GCE	10–30,000	2 × 103	185.4	15	−0.4	[43][66]
Co3N NW/TM/GCE	2–28	1 × 103	139.9	30	−0.7	[44][67]
MnO2/Ta/GCE	1–2	60	1111.09	-	−1.21	[45][68]