Emerging single‐atom catalysts in electrochemical biosensing

Single‐atom catalysts (SACs) have attracted extensive interest owing to their maximized atomic utilization, low cost as well as outstanding catalytic activity, selectivity, and stability for diverse applications. Due to their excellent performance in electrocatalysis, SACs can be applied to electrochemical sensors, which have been a predominant tool employed in biosensing. In very recent studies, SAC‐based electrochemical biosensors have demonstrated enhanced sensing performances in biomarker detection and in vivo analysis. However, a comprehensive review of SAC‐based electrochemical biosensors has not been reported yet. Herein, we present a summary of the synthesis methods of SACs with their application in electrochemical sensor establishment and electrochemical characterization methods in electrochemical sensing. Biomedical applications utilizing SAC‐based electrochemical biosensors are introduced. Finally, the existing challenges and future prospects of SACs in the field of electrochemical biosensing are discussed.


INTRODUCTION
The catalytic performance of metal nanocatalysts is significantly affected by their structural features, in which the size of the metal particles plays the most critical role in catalysis. 1 Noble metals can generally demonstrate superior catalytic performance compared to non-noble metals or metal oxides. 2 However, conventional metal nanocatalysts loaded on sensors are primarily nanoparti-cles (NPs), featuring limited exposure of active sites. The downsizing of metals from NPs to isolated single atoms is a direct and effective method to achieve higher specific activity and lower cost simultaneously. Single-atom catalysts (SACs) are emerging heterogeneous materials with catalytically active and isolated metal atoms dispersed on solid supports that have attracted considerable interest due to their maximized metal atom efficiency as well as outstanding catalytic activity, selectivity and stability for VIEW. 2023;4:20220058.
wileyonlinelibrary.com/journal/view 1 of 11 https://doi.org/10.1002/VIW.20220058 diverse applications. The main benefit of SACs is their high specific surface area, enabling sufficient active sites to be exposed on the catalyst surface. Unlike traditional NPs, these isolated metal atoms are surrounded by a specific number of coordination atoms, and thus, a more uniform distribution of active sites can be achieved across the surface that are easily accessible to reactants, leading to increased reactivity and selectivity. Besides, active atoms are embedded in the supporting substrate through either covalent or coordination bonds, and thus, catalysts are less susceptible to migration and agglomeration that often occur with NP electrocatalysts, exhibiting high stability. 3 The low-coordination environment of the metal atoms, the high atom utilization and the enhanced strong metal-support interactions result in their excellent catalytic performances. 4 Since the first demonstration of SACs by Zhang et al., 5 significant advancements have been achieved in this field. Nowadays, SACs have been widely studied in electrochemical reactions, water-gas shift reactions, and hydrogenation reactions. 6 Various sensing strategies have been applied in biosensing, such as electrochemical, electrical, and optical methods, featuring fast response and high flexibility in employing different recognition elements. 7 Optical sensors benefit from their high sensitivity and specificity. However, they require complex instrumentation with high cost. In comparison, electrochemical biosensors are simpler and more cost-effective in both manufacturing and operation. Their flexibility of easy surface modification and integration with various platforms further promotes wide application. Measuring electric signals induced by biological molecules bonding to the sensor surface, piezoelectric sensors bear high sensitivity in detecting small masses. However, they easily suffer from interference of substances in samples and generally require complicated signal recording instruments. In contrast, electrochemical biosensors offer a highly miniaturized platform for point-of-care applications with responsive and robust electrochemical signals. Moreover, electrochemical biosensing offers real-time monitoring capabilities. Currently, electrochemical sensing has been demonstrated to be a promising method in biomedical applications. Electrochemical sensing features excellent capability of in vivo analysis in terms of its miniaturization, flexibility, real-time response, and high sensitivity for trace detection. To further improve the electrochemical biosensing performance, single-atom electrocatalysts with unique catalytic properties and high stability have been attracting emerging interest in developing novel electrochemical biosensors. In addition to the large surface area and abundant active sites resulting in higher sensitivity and selectivity, single atoms are also highly stable and do not aggregate during the sensing process, which leads to a longer lifespan and lower cost than traditional NP-based sensors. As can be seen, the stud-ies concerning SAC-based electrochemical sensing to date have mainly focused on biological and biomedical applications, which will be comprehensively summarized for the first time in this review. The synthesis methods of SACs for the establishment of electrochemical sensors and electrochemical characterization methods are presented with insights into the mechanisms of single-atom catalysis. Finally, the existing challenges and future prospects of SACs in electrochemical biosensing are discussed.

SYNTHESIS METHODS
The electrocatalytic performance of SACs is significantly affected by the type of metal atom and support as well as the spatial distribution of the metal atoms in the support. The critical factor lies in the synthesis of SACs, which can be challenging because the metal particles tend to form clusters and aggregates with a dramatic increase in free energy at the metal surface as they approach the size of single atoms. Here, the synthesis methods of SACs applied in electrochemical sensing are summarized.

Impregnation
Impregnation is a common synthesis method of SACs. In this approach, liquids containing active substance precursors permeate various supports, followed by an ion exchange/reduction process to convert the adsorbed precursor molecules into stable, anchored single atoms on the support. The electrochemical stability and electrocatalytic activity of SACs mainly depend on the interaction between the metal precursors and the supports, which include carbon-based materials and metal and metallic compounds. The amount of metal loading and the dispersion of the metal anchored on the support surface can be significantly influenced by the interaction. 8 Impregnation exhibits excellent application prospects due to its high operability, but it can be challenging to achieve uniform dispersion of the metal atoms, especially for high-loading SACs. Zhou et al. 9 added tetraethyl orthosilicate to a mixture of ethanol, deionized water, and ammonia, followed by mixing ethanol and water containing dopamine (DA) monomer and Ni(acac) 2 . After centrifugation and drying, the product was heated to 900 • C and reacted for 3 h with the protection of argon. Then, the cooled samples were etched in sodium hydroxide solution for 24 h to obtain nickel single atoms anchored on N-doped hollow carbon spheres (Ni SACs/N-C). The transmission electron microscopy (TEM) images ( Figure 1A material as the support has the advantage of simple synthesis, but the weak interaction between carbon and metal atoms hinders the stable dispersion of single atoms, leading to insufficient electrochemical stability of the synthesized SACs. 10 Alternative supports such as metal and metal oxides/hydroxides/sulfides/nitrous are expected to achieve better performance. Sun et al. 11 dispersed the flower-like MoS 2 synthesized by the hydrothermal process, and then the prepared nickel complex was added into the solution. The homogeneous solution was then recrystallized, and the crystalline powder was heated to 900 • C and reacted for 2 h with the protection of argon ( Figure 1C). This synthesis method enables Ni single atoms to be axially anchored to the Mo atom in the MoS 2 basal plane with the Ni-S 3 structure. The characterization by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) ( Figure 1D) shows the microscopic Ni atomic arrangement, and the randomly and individually dispersed brighter spots refer to the Ni atoms. The catalytic effect may also be enhanced by synergistic interactions between metal atoms and metal compounds. Li et al. 12 injected K 2 PtCl 6 with a syringe pump into the ethanol solution in which MoS 2 nanosheets were dispersed to obtain Pt 1 /MoS 2 . Single platinum atoms and activated S atoms of Pt 1 /MoS 2 are active sites for synergistic adsorption. To facilitate sufficient single-atom loading, Liu et al. 13 reported a synthetic method using a hollow structure porphyrin zirconium-based metalorganic framework (MOF) HPCN-222 as the support to prepare the SAC PtHPCN-222. The synthesized hollow MOFs with increased porosity can enhance their performance by exposing more active sites and promoting the diffusion of target molecules through the porous structure. Scanning electron microscopy images of HPCN222 ( Figure 1F) and PtHPCN-222 ( Figure 1G) are almost identical in shape and size, indicating the insertion of a single Pt atom into the porphyrin ring without agglomeration.

Pyrolysis
Pyrolysis is a straightforward method to obtain SACs by pyrolyzing precursor materials of loaded metals and support at high temperatures, resulting in the formation of coordination between the metal atoms and defect sites on the support. 14 As an ideal substrate for dispersing and stabilizing single atoms, nitrogen-doped carbon materials are usually obtained by the pyrolysis of MOFs, 6 with metal-nitrogen-carbon (M-N-C) SACs as the most representative product. Shu et al. 15 mixed cobalt nitrate and zinc nitrate solutions together and added the mixture dropwise to 2-methylimidazole solution by magnetic stirring. The Co-ZIF-8 precursor obtained after centrifugation, washing, and drying was transferred to a tubular furnace under the protection of argon, heated to high temperature (700 • C-1000 • C), and reacted for 3 h. Atomically dispersed Co-N-C was obtained after cooling and treatment with HCl, whose electrochemically active surface could be optimized by adjusting the calcination temperature. Hydrogel is an ideal material for the flexible and convenient construction of metal-loaded precursors. Cong et al. 16 mixed a mixture containing acidified multi-walled carbon nanotubes (CNTs) and pyrrole (Py) monomer with a mixture containing PMo 12 , FeCl 3 powder, and 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin to form black hydrogel. The product was carbonized at 900 • C for 2 h to obtain Mo single atoms anchored on the N, P, O co-doped carbon support. By varying the amounts of PMo 12 , Mo nanoclusters, Mo 2 C nanodots, and Mo 2 C, NPs were formed ( Figure 1E) with different catalytic properties. Due to their large surface area, abundant mass transport channels and low cost, porous carbon aerogels have been widely used as SAC supports. 17

Coprecipitation
In the coprecipitation method, the mixing of precipitating agents causes the insoluble metal hydrate oxides or metal salts to precipitate. It is easy to prepare smallsize and uniformly distributed nanopowder materials by a chemical reaction in solution. Liang et al. 18 added 2-methylimidazole and heme to a methanol solution of Zn(NO 3 )⋅6H 2 O, and the precipitate was stirred for 24 h. The obtained precursor powder was carbonized and soaked in sulfuric acid solution to synthesize nitrogen-and carbon-doped Fe single atoms (FeSAs-N/C) ( Figure 1H).
Similarly, Wei et al. 19 injected FeCl 3 ⋅6H 2 O and ZnCl 2 into a dispersion of glucosamine and melamine. The resulting precipitate was further ground to a fine powder after sulfuric acid treatment to remove the nanocrystals. Despite the simplicity of the coprecipitation method, it is worth noting that the coprecipitation method has difficulty in achieving uniform distribution of metal atoms on the support and can be susceptible to reaction conditions. Besides the low loading of metal, active metal atoms can be buried in the support, leading to a decrease in reactivity of the obtained SACs.

ELECTROCHEMICAL CHARACTERIZATION
To confirm the existence of isolated single metal atoms with desired properties, a series of characterizations should be performed on synthesized SACs. Imaging characterization can provide information about the structural properties and composition of SACs, including TEM, HAADF-STEM, X-ray photoelectron spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, etc. 6 Compared to direct morphological characterization, electrochemical characterization emphasizes the demonstration of SACs' electrochemical performance through electrochemical reactions. It provides time-resolved measurements, enabling the study of dynamic processes and catalytic mechanisms of SACs. Besides, electrochemical characterization is more quantitative and easily crosscorroborated with theoretical calculations, allowing for precise determination of the electrochemical reaction kinetics. Here, an overview of electrochemical characterization methods for verifying electrochemical properties of SAC-based sensors is provided.

Cyclic voltammetry
Cyclic voltammetry (CV) is widely applied in electrochemical analysis as a preferred characterization method for new synthetic materials. Through the CV test, the electrode voltage range for the redox reaction can be easily determined, and the electrocatalytic activities of SACs toward the redox reaction of the target and the redox mechanism could be illustrated by the number and potentials of redox peaks in the cyclic voltammograms. In particular, linear sweep voltammetry facilitates the observation of current changes at a small potential increment. To reduce the activation overpotential which is essential to galvanic redox potentiometry (GRP) sensing in vivo, Pan et al. 20 compared the three redox peaks in the CV curves of H 2 S oxidation on a NiN 4 -SAC-modified glassy carbon electrode (GCE). The sole presence of peak 1 in the first segment was assigned to H 2 S oxidation to S 8 , while the emergence and elevation along with cycled scans of peaks 2 and 3 in the following segments indicated their direct association with S 8 . In the control scans, the poor electrocatalytic activity of the substrate led to the positive shift of peak 1 and the absence of peaks 2 and 3, which highlighted the significance of NiN 4 sites in lowering the oxidation potential of H 2 S. Long et al. 21 found that the single-atom Pt supported on Ni(OH) 2 nanoplates/nitrogen-doped graphene (Pt 1 /Ni(OH) 2 /NG) demonstrated much lower oxidation potential (0.480 V) to glucose than those of Pt 1 /Ni(OH) 2 (0.505 V), Ni(OH) 2 /NG (0.506 V), PtNPs/Ni(OH) 2 /NG (0.521 V), and Ni(OH) 2 (0.525 V), validating that singleatom Pt and NG could facilitate the electrochemical oxidation of Ni(II) to Ni III OOH. In addition to the redox peak potential, the peak currents also reflect the electrochemical properties. Liang et al. 18 proved that the FeSAs-N/C exhibited better electrical conductivity and better sensitivity to different concentrations of H 2 O 2 than its precursor hemin@zeolitic imidazolate framework-8 (hemin@ZIF-8) through the peak reduction current. The electroactive area was also analyzed according to the Randles−Sevcik equation. For a better understanding of the electrochemical reaction kinetics, theoretical calculations can be performed, such as the charge-transfer coefficient and the apparent electron transfer rate constant derived from the classical Laviron formula and the diffusion-controlled process. 22

Differential pulse voltammetry
Differential pulse voltammetry (DPV) can suppress the charging current effect caused by the non-faradaic current in potential sweep voltammetry, thus exhibiting ultra-low limit of detection (LOD) and higher sensitivity, making it ideal for trace detection. Taking the ultrasensitive DA sensor based on Ni-MoS 2 11 as an example, DPV technology was employed to characterize the sensing performance with an LOD of 1 pM. The same characterizations by DPV were performed on another Ru 3 /nanocluster (NC)-based sensor which achieved an LOD of 10 nM in uric acid (UA) detection. It can be applied for DA detection simultaneously with an LOD of 3.3 nM due to a large separation peak potential in DPV. 23

Chronoamperometry
To demonstrate the application for target-specific electrochemical sensing, SACs are usually modified onto the electrode and examined by chronoamperometric measure-ments. Through analysis of chronoamperometric curves, the linear response range with the corresponding calibration equation and the LOD can be obtained. The difference between the current response to the target and potential interference species illustrates the selectivity of SACs. Wu et al. 24 utilized the ratio of catalytic reduction currents of H 2 O 2 and O 2 at equal concentrations to quantitatively evaluate the electrocatalytic selectivity to hydrogen peroxide reduction reaction (HPRR) and oxygen reduction reaction (ORR). The ratios of Co-N 4 /C, Pt/C, and platinized GCE are 14%, 55%, and 57%, respectively, confirming the higher tolerance of Co-N 4 /C to H 2 O 2 than that of Pt in the catalysis of four-electron ORR. Chronoamperometry was also utilized to evaluate the repeatability, reproducibility, stability, and response time of SAC-based electrochemical assay.

Electrochemical impedance spectroscopy
Compared to voltammetric measurement, electrochemical impedance spectroscopy (EIS) is more sensitive for perceiving subtle changes on the electrode surface and the electrode-electrolyte interface. For SACs used as electrode modifications, electron transfer capability is essential, which can be illustrated by the charge-transfer resistance from EIS. According to Li et al., 25 EIS results revealed that the sensor modified by atomic Co-Nx moieties anchored on nitrogen-doped CNT arrays (Co-N/CNT) had lower charge-transfer resistance than that of the Co-N/C sensor. Therefore, CNT arrays served as a better substrate than regular nitrogen-doped carbon with rich active sites and substrate-connected structure. In the non-faradaic region of applied potential, the electrical double layer formed at the electrode-electrolyte interface can be described by the double-layer capacitance. 26 Shu et al. 15 employed the double-layer capacitance (C dl ) on the electrode surface to evaluate the amount of active sites of Co-N-C synthesized under different temperatures, by calculating the ratio of current density and corresponding scan rate.

APPLICATIONS IN ELECTROCHEMICAL BIOSENSING
Biosensing places high demands on the catalytic performance of electrochemical sensors, especially for in vivo and trace detection of biomarkers. SACs are applied in electrochemical biosensing mainly for modulating electron transfer kinetics and their ion transport behavior. 3 This section discusses the latest developments in electrochemical biosensors based on SACs by category of biochemical markers, as summarized in Table 1.

Gas molecules
Cellular metabolism can produce some gas molecules as important biochemical markers, transmitters or signaling molecules. The challenges of electrochemical sensors for in vivo gas detection are mainly in the following aspects: (1) high sensitivity to detect trace amount of gas release, (2) fast response and real-time monitoring, (3) high selectivity to detect the gas molecules of interest out of interfering species, and (4) good stability for long-term sensing against poisoning. Mao's team has been a pioneer in this emerging field whose researches involve nitric oxide (NO) sensor, hydrogen sulfide (H 2 S) sensor, and advanced oxygen (O 2 ) sensor. 9,20,24 To achieve high sensitivity and real-time recording of cellular levels of NO, Zhou et al. 9 employed nickel single atoms anchored on N-doped hollow carbon spheres (Ni SACs/N-C), which greatly reduced Gibbs free energy in activating NO oxidation with better electrocatalytic performance. The Ni SACs/N-C integrated flexible electrochemical sensor (Figure 2A)  also found to be conducive to overcoming the low stability and sensitivity loss of H 2 S sensors suffering from sulfur poisoning-caused electrode passivation. Combined with GRP, Ni-N 4 active sites on the electrode interface facilitate H 2 S oxidation at an extremely low potential by reducing the energy barrier of the first electron-transfer step (H 2 S* → SH* + H + ), determining the reaction rate and thus minimizing the accumulation of sulfur on the sensing surface under open-circuit conditions. The sensor ( Figure 2B) is highly selective to H 2 S against potential interferents with similar redox potentials and can accurately measure H 2 S release in the living mouse brain in a real-time manner. SACs can also be tuned to achieve high selectivity. For example, ORR and HPRR may occur simultaneously for most catalysts. To address this problem, Wu et al. 24 reported a single-atom Co-N 4 electrocatalyst for ORR, with high H 2 O 2 tolerance outperforming commercial Pt electrocatalysts. Due to the weak adsorption of H 2 O 2 on the porphyrin-like Co centers, Co-N 4 catalytic sites preferentially foster the direct four-electron pathway of ORR over the two sequential two-electron reduction pathways that involve H 2 O 2 as an intermediate. The adsorbate-metal interaction plays a key role in determining the electrocatalytic selectivity, which offers a new approach to designing electrocatalysts that can cater to various sensing needs in a complex environment.

H 2 O 2
H 2 O 2 has been widely recognized as a vital mediator in a range of physiological processes and a biomarker of aging and disease. For the detection of intracellular H 2 O 2 , electrochemical sensors are mainly based on horseradish peroxidase (HRP), which may be easily denatured and inactivated. As alternatives to HRP, a variety of nanomaterials have been reported with peroxidaselike catalysis activity, while most of them exhibit limited sensitivity. [27][28][29] Owing to the outstanding enzyme-like performances, single-atom nanozymes (SAzymes) have been deeply studied recently. 30 M-N-C SACs with homogeneous MNx active sites anchored on the carbon supports possess structures similar to those of natural heme enzymes. Among them, Fe-N-C SACs are the most representative choice because Fe-N x sites are similar to the active sites of HRP containing a single heme b cofactor with a proximal ligand. 31 Liang et al. 18 reported Fe SACs with distorted graphitic carbon inherited simply from the precursor hemin@zeolitic imidazolate framework-8 (hemin@ZIF-8). The FeSAs-N/C-modified GCE shows fast electron transfer rate and wide linear range in electrochemical sensing. Generally, heterogeneous catalysts with additional active sites have enhanced catalytic performance than homogeneous catalysts; thus, some metal NPs or nanoclusters are often coupled as co-catalysts in SACs to provide synergistic effects. [32][33][34] In the work of Wei et al., 19 the catalyst of singleatomic Fe sites coupled with carbon-encapsulated Fe 3 C crystals (Fe 3 C@C/Fe-N-C) ( Figure 2C) was synthesized through a one-step C 3 N 4 spatial confinement strategy. Fe 3 C@C/Fe-N-C demonstrated significantly better performance in H 2 O 2 detection than Fe-N-C with a larger specific activity, which is ascribed to the relatively lower adsorption barriers of H 2 O 2 molecules on single-atomic Fe sites due to the assistance of Fe 3 C nanocrystals in electron transfer. Moreover, Ding et al. 35 proposed a new form of Fe-N-C based on Fe-polypyrrole (PPy)-derived carbon nanowire (Fe-single-atomic site catalyst [SASC]/NW) ( Figure 2E). Synthesized with the assistance of zinc atoms, Fe-SASC/NW is composed of distorted graphitic carbon with large specific surface area and abundant nanopores, which can accommodate plentiful atomic Fe sites. This structure can significantly facilitate the loading of active sites and enhance the electrical conductivity, resulting in an impressive LOD of 46.35 × 10 −9 M toward H 2 O 2 .
In addition to single-atom Fe, single-atom Co-N-C materials have also been studied for electrochemical biosensing.
Shu et al. 15 increased the abundance of Co-Nx sites by regulating calcination temperature to the optimal 800 • C. The Co-N-C-800-modified electrode enables the simultaneous detection of H 2 O 2 (LOD = 0.13 μM) and DA at different potentials. Although effective in synthesizing M-N-C catalysts, high-temperature pyrolysis can cause loss of the unbonded nitrogen and discrete NP structure. To promote nitrogen content, Li et al. 25 introduced urea into the pyrolysis of ZIF-67 to obtain Co-N/CNT ( Figure 2F). While loading ZIF-67 particles, urea also acts as a carbon and nitrogen source, promoting the formation of N-doped carbon nanotubes (N/CNTs) and atomic cobalt-nitrogen (Co-Nx) sites within the catalyst structure. The resulting electrocatalyst offers more active sites, leading to improved conductivity and catalytic activity.
Corresponding to the ORR mentioned in Section 4.1, Mao's team proposed single-atom Cu on carbon nitride (Cu 1 /C 3 N 4 ) to improve the selectivity of HPRR. 36 The Cu-N 2 site and its adjacent carbon site synergistically affect the adsorption state of the *OH intermediate; thus, the electrochemical HPRR occurs preferentially over ORR with a lower energy barrier. The microsensor based on Cu 1 /C 3 N 4 shows a good and selective response to H 2 O 2 , demonstrating excellent sensing performance in the living rat brain.

Glucose
Commercial glucose sensors are generally based on enzymatic catalysis and electrochemical sensing. 37 To address enzyme-related limitations, various nanomaterials have been developed in establishing non-enzymatic glucose sensors; however, their sensing performance still needs to be improved. As a result, SACs have recently emerged as a promising nanomaterial in electrochemical glucose sensing.
The first SAC-based electrochemical nonenzymatic glucose sensor was constructed by Zeng et al. 21 Pt 1 /Ni(OH) 2 /NG were synthesized ( Figure 2D). From the electrochemical characterization results, Pt 1 /Ni(OH) 2 /NG has the highest electrocatalytic activity compared to Ni(OH) 2 /NG, Pt 1 /Ni(OH) 2 , Ni(OH) 2 , and PtNPs/Ni(OH) 2 /NG. The single-atom Pt active centers and their surrounding Ni atoms provide strong binding for glucose, along with the highly conductive NG promoting electron transfer. Subsequently, they further improved the SACs by replacing the support Ni(OH) 2 /NG with Ni 6 Co 1 layered double hydroxides/nitrogen-doped graphene (Ni 6 Co 1 LDHs/NG) ( Figure 2D). 38 The resulting Pt 1 /Ni 6 Co 1 LDHs/NG exhibits a lower oxidative potential of 0.440 V with higher sensitivity of 273.78 μA mM −1 cm −2 and faster response (1.8 s), exceeding the previous Pt 1 /Ni(OH) 2 /NG (0.48 V, 220.75 μA mM −1 cm −2 , 4.6 s, respectively). This may result from the doping of heteroatoms Co, which can create additional anchoring sites for the loading of Pt single atoms and improve the catalytic performance. Moreover, a variety of carbon derivatives has been utilized as supports, such as graphene and CNTs discussed above. Novelly, Chen's team applied hierarchical three-dimensional (3D) N-doped porous carbon aerogels (NCA) ( Figure 2G) derived pyrolytically from biomass hydrogels to serve as a support matrix for single atoms (Fe, 17 Co 39 ). The 3D porous skeletons of NCA with high surface area, microporous defects and nanowrinkles enable the dispersion of the single atoms and facilitate the adsorption and mass transfer during the electrochemical reaction.

Dopamine
DA acts as a critical neurotransmitter in the brain that transmits information and regulates emotions. 40 DA is redox-active and is readily electrochemically oxidized to DA orthoquinone catalyzed by SAzymes. By synthesizing a single Ni site catalyst (Ni-MoS 2 ) anchored on the flowerlike MoS 2 support, Sun et al. 11 designed an ultrasensitive DA sensor with a low LOD of 1 pM, resulting from the axial anchoring of Ni single atoms on the MoS 2 substrate in the Ni-S 3 structure. Ni has a high electronegativity with redox couple Ni(II)/Ni(III) and an abundant electron density in the region between Ni and DA, which will be more conducive to the adsorption and oxidation of DA molecules. In addition to carbon-based and metallic compound-based supports, MOF is also an ideal support for SACs because of its large specific surface area, stable porous structure, and tunable catalytic properties. The hollow-structure porphyrin zirconium-based MOF (HPCN-222) developed by Liu et al. 13 enables a stable dispersion of single Pt atoms through strong interactions and also modifies the electronic structure of Pt atoms, achieving the sensitive detection of levodopa (a metabolic precursor of DA) with an LOD of 3 nM. Moreover, the adsorption of levodopa is also facilitated by the interaction between the porphyrin ring of the SACs and the aromatic ring of levodopa through π-π and H bonds.

Uric acid
UA is a typical biomarker related to many diseases such as gout, kidney diseases and cardiovascular diseases. Hu et al. 41 established an electrochemical sensor for UA detection with an LOD of only 33.3 nM, which utilized a single-atom catalyst consisting of Co(II) atoms anchored on an N-doped graphene matrix (A-Co-NG). According to Wu et al., 23 more atomic sites can better enhance the catalytic performance of SACs. They designed an atomically dispersed Ru 3 site catalyst (Ru 3 /NC) to catalyze UA oxidation with an LOD of 10 nM. The adsorption energy of OH − on Ru 3 /NC (−1.14 eV) is almost three times greater than that of Ru 1 /NC (−0.43 eV) ( Figure 2H), leading to the more favorable adsorption of hydroxy anion groups with a stronger binding interaction between the active sites and the adsorbates. In addition to biomarker detection, SACs are involved in other applications in the field of electrochemical sensing, such as environmental protection and hazard detection. For example, Luo et al. 42 reported a template-sacrificed strategy for the synthesis of atomically dispersed Ir SACs, which was applied to detect organophosphorus pesticides (OPs) with an LOD of 0.17 ng mL −1 . Niu et al. 43 constructed Si-doped graphene nanosheets, which exhibited excellent electrochemical detection ability for nitroaromatic compounds. Li et al. 12 developed a catalyst of Pt single atoms anchored on MoS 2 (Pt 1 /MoS 2 ) for the determination of As(III) with a high sensitivity of 3.31 μA ppb −1 .

SUMMARY AND PERSPECTIVES
In this review, we present a summary of the development and application of SACs in electrochemical sensing over the last few years. Current study on SAC-based electrochemical sensors is still at early stage with limited applications. Its concentration in the biological field may be due to the unique properties of SACs with outstanding electrocatalytic activity and stability which can satisfy the high demand for sensor performance in biological detection. For SACs, the electrochemical properties of intermediates at each step of the synthesis process can be explored through electrochemical characterization, thus revealing the catalytic mechanism of SACs in a more comprehensive and thorough way. Moreover, electrochemical characterization can achieve theoretical calculations at a microscopic scale of active sites in SACs. Therefore, electrochemical characterization techniques are beneficial for revealing the mechanism of SAC-based electrochemical sensors and should be performed strategically.
SAC-based electrochemical sensors have increasing potential in biomedical applications, but there are still challenges to be resolved in future studies. Firstly, the high selectivity of SACs is mainly exhibited in a specific or controlled environment against a range of known interferents. However, in a real biological or physiological environment, SACs can be affected by a large amount of interferents that have similar electrochemical properties, resulting in reduced selectivity. This poses sophisticated demands on the design of active sites and the synthesis of SACs. Secondly, the catalytic efficiency of SACs is strongly influenced by the compositional structure of metal atoms and the support, which determines the dispersion and the loading amount of the single atoms, the specific surface available for adsorption and catalysis of the target, etc. The highest catalytic activity of SAzymes reported so far is still inferior to that of natural enzymes with delicate and complicated structures, suggesting that intensive investigation and innovation in structure would be a promising way to further improve the catalytic efficiency of SACs.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.