Yang et al. synthesized iron and nitrogen co-doped CDs (Fe-N-CDs), which with peroxidase activity. 3,3′,5,5′-tetramethylbenzidine (TMB) was catalyzed to blue in the presence of hydrogen peroxide. On this basis, Fe-N-CDs conjugated antibody was applied to detect carcinoembryonic antigen (CEA) by immunosorbent assay. The detection limit was as low as 0.1 p g/mL within 5 min (Yang et al., 2017). Based on the similar principle of enzyme-linked immunosorbent assay, iron and cobalt co-doped CDs with high peroxidase-like activity and palladium-iridium nanocubes with CDs as reference fluorophores can detect histamine and cardiac troponin I, respectively (Tan et al., 2019; Li et al., 2021). Even more striking, Guo et al. used Fe₃(PO₄)₂·8H₂O-CDs-FA hybrid nanoflower realized the naked eye immunoassay of as few as 25 HeLa cells (Guo et al., 2019).

Based on the above TMB discoloration principle, Fe-N/C single-atom nanozyme was used to screen alkaline phosphatase activity in the range of 0.05–100 U/L, with a detection limit of 0.02 U/L (Chen Q. et al., 2020). The cascade colorimetric biosensor combined with cholesterol oxidase demonstrated excellent selectivity and high sensitivity to the target in the concentration range of 0.01–1.0 mM. The detection limit was as low as 7 mM (Zhao et al., 2019). Both V₂O₅-CDs nanocomposites and palladium/CDs composites (Pd-CDs) have also been proved to bind glucose oxidase and realize the colorimetric glucose sensing with a detection limit as low as 0.2 μM (Honarasa et al., 2019).

CDs have demonstrated significant application value in fluorescence detection due to their numerous unique physical and photochemical properties, and CDs-based nanozymes also exhibit fluorescence detection characteristics (Zhan et al., 2020).

Lu et al. synthesized Fe-doped CDs (Fe-CDs). Oxidative OPD (ox-OPD) can be generated when the oxidase substrate o-phenylenediamine (OPD) coexists with H₂O₂. Therefore, a dual fluorescence emission detection system can be established based on fluorescence characteristics of Fe-CDs and Ox-OPD. The results indicated that the limit of detection for cysteine was as low as 0.047 μM in the concentration range of 0.25–90 μM (Lu et al., 2020).

The advantages of electrochemical sensors include linear output, low power consumption, good resolution, repeatability, and accuracy (Chen et al., 2019; Teymourian et al., 2020; Wang Q. et al., 2021). Additionally, applying CDs-based nanozymes to electrochemical sensors is a hot topic.

The realization of electrochemical sensing based on CDs-based nanozymes is often the modification of electrodes. Wang et al. immobilized horseradish peroxidase on a glassy carbon electrode by simply mixing carbon nanodots and cobalt-iron layered double hydroxides (Wang et al., 2015). Qin et al. used hydroxyl-rich carbon dot-assisted gold nanoparticles (CDs @AuNP) as a marker of copper deposition reaction, and cooperated with chitosan to modify glassy carbon electrode (Qin et al., 2018). Hu et al. used coordination reaction and surface adsorption to prepare ferrous and ferrous ion modified CDs to regulate heterogeneous nucleation process of iron oxide, and its enzyme-like activity was more than 6 times higher than that of pure Fe₂O₃ nanomaterials (Hu et al., 2021) Fatemeh Honarasa et al. prepared Fe₃O₄/CeO₂/C-dot nanozyme with more complex structure, and its modified multi-walled carbon nanotube/ionic liquid paste (MWIL) electrode was used for electrocatalytic determination of H₂O₂, showing a linear range of 2.0 × 10⁻⁸ ∼ 1.0 × 10⁻⁶ M (Honarasa et al., 2021).

Compared with metal/metal oxide NPs or materials, CDs have the disadvantages of low product yield, difficulties in purification and precise size control, which significantly affect applying CDs-based nanozymes in biosensors.

Biomolecules such as H₂O₂, ascorbic acid, uric acid, and pyrophosphate have also been developed to detect the nanozyme complex CDs method.

Yang et al. synthesized carbon-based AuPd bimetallic nanocomposite (AuPd/C NC) with good catalytic activity and peroxidase activity. H₂O₂ can be detected in a wide linear concentration range of 5–500 µM and 500 µM–4 mM (Yang et al., 2016). Zhuo et al. demonstrated that manganese (II) doped CDs (Mn-CDs) have a similar catalytic ability to oxidase. They could be utilized for quantifying ascorbic acid in a concentration range of 50–2,500 nM based on the principle of “TMB discoloration reaction” (Zhuo et al., 2019). Shu et al. demonstrated that carbon quantum dots (CQDs) also exhibit peroxidase activity but with a narrower detection range and lower detection limit (Shu et al., 2020). Liang et al. synthesized carbon quantum dots co-doped with iron and nitrogen (Fe@NCDs). In the presence of H₂O₂, the response was linear in the uric acid concentration range of 2–150 μM (Liang et al., 2020). Chen et al. prepared nanozymes with complex CDs exhibiting peroxidase simulation properties, which could catalyze o-phenylenediamine oxidation in the presence of H₂O₂. The process was inhibited by pyrophosphate (PPI), and the degree to which it was inhibited could be monitored using the colorimetric method with generated yellow product 2,3-diaminophenazine (Chen Q. et al., 2020).

Although nanozyme-based colorimetry is a rapid method for detecting glutathione, it lacks the high efficiency and low toxicity of nanozyme. Luo et al. prepared Fe₃O₄/CNDs hybrid NPs with excellent peroxidase-like catalytic activity, and they could produce a rapid color reaction on glutathione (Luo et al., 2019). Similar studies have focused on peroxidase-like nanomaterials, which require H₂O₂ addition. Because H₂O₂ is extremely unstable, quickly decomposes, and even reacts with assay, applying this nanozyme mimicking peroxidase remains limited. Therefore, Jin et al. prepared titanium dioxide/carbon point oxidase nanozyme. The nanozyme possessed abundant thermodynamic metastable Ti atoms on MXene. The oxygen vacancy in TiO₂ on carbon matrix surface can facilitate O₂ adsorption in solution, generating ROS, thereby quickly oxidizing TMB to TMBox in the absence of H₂O₂ to detect glutathione (Jin et al., 2020).

Although colorimetric and fluorescence methods possess high selectivity, high sensitivity, low cost, and simplicity, such methods follow single-mode signal readout. It is easy to be disturbed by the environment and challenging to meet accurate bioassay requirements. In this case, colorimetric/fluorescence two-channel measurement provides a more reliable strategy for detecting H₂O₂ and related biomolecules.

Su et al. prepared for the first time a multifunctional hemin@CDs hybrid nanozymes (hemin@CDs) with peroxidase-like activity and fluorescence signal properties (Su et al., 2020). This is a two-channel fluorescent probe for H₂O₂ and H₂O₂-based biocatalytic systems. It catalyzes the oxidative coupling of 4-aminoantipyrine and phenol in the presence of H₂O₂, resulting in a pink quinone imine dye with a maximum absorbance at 505 nm. The probe can be deployed to detect glucose and xanthine due to the conversion of glucose/xanthine into H₂O₂ catalyzed by related oxidase.

Ren et al. synthesized active copper-containing CDs (Cu-CDs) with inherent laccase-like activity. Unlike Su et al.‘s work, this is a novel enzyme reaction that catalyzes phenylenediamine oxidation by laccase substrates, resulting in a typical color change from colorless to brown. Cu-CDs were further employed as a fluorescent probe for unlabeled hydroquinone (H₂Q) detection. The results indicate that a linear relationship is good in buffers with different pH values of 0.05–20 mM and 1–30 mM (Ren et al., 2015).

To further overcome the problem of obtaining fluorescence utterly dependent on a single signal output and a low signal background ratio in the method mentioned above, Yang et al. prepared CDs-doped CeO₂ (CeO₂-CDs) with peroxidase activity and fluorescent carbon dot. Fluorescent o-phenylenediamine (OPD), a peroxidase substrate, can be catalyzed by cerium oxide and cadmium sulfide to produce fluorescent o-phenylenediamine (palladium oxides). UV-Vis absorption of palladium oxides partially overlays the fluorescence emission of cadmium sulfide, reducing its intensity under the effect of an internal filter (Yang Z. et al., 2021). Based on this principle, a sensitive and selective fluorescence assay for the ratio of H₂O₂ to cholesterol was developed.

Gold and silver are typical SERS substrates. The SERS activity of precious metals/CDs nanocomposites was enhanced by improving probe molecule adsorption and amplifying electromagnetic fields.

Wang et al. prepared silver-CDs (Ag-CDs) nanocomposites with excellent peroxidase and SERS activity. The nanocomposite can be used to determine uric acid (UA) levels (Wang et al., 2019). In addition, the chain-like Au/CDs (GCDs) nanocomposite was simulated using finite-difference time-domain (FDTD) method to demonstrate how the aggregation of gold NPs enhanced the electromagnetic field, thereby increasing SERS signal based on diamond-like nanocomposite. The nanocomposite enables glucose detection at a concentration of 5 × 10⁻⁷ M (Gan et al., 2021). All these demonstrated that the synergistic method based on colorimetric reaction and SERS detection possessed the advantages of a low detection limit, a wide detection range, and high accuracy, which made the detection results more reliable and accurate.

Using a two-carbon point system as a peroxide-mimicking enzyme and a fluorescent probe, combining carbon point with catalytic activity or carbon point with fluorescence quenching effect greatly improves the sensitivity of the detection method.

Dhamodiran Mathivanan et al. synthesized double emission carbon spots of enzyme simulated N/Cl-CDs and N/Zn-CDs. N/Cl-CDs exhibited apparent intrinsic peroxidase-like activity, catalyzing OPD oxidation by H₂O₂ to form the yellow product 2, 3-diaminophenazine. N/Zn-CDs exhibited significant fluorescence properties, with a quantum yield of 27.52% (Mathivanan et al., 2020). Using similar construction, Wang et al. constructed a double-carbon point system with fluorescent CDs (N/Cl-CDs) and copper-doped CDs (N/Cu-CDs) that function as peroxide mimic and fluorescent probe and can fluoresce in hydroquinone determination. The fluorescence quantum yield of N/Cu-CDs was 37%. Compared with the study of Dhamodiran Mathivanan et al., the fluorescence quantum yield was significantly improved. N/Cl-CDs exhibits inherent peroxidase-like activity and catalyzes hydroquinone oxidation to p-benzoquinone and intermediates to determine H₂Q (Wang X. et al., 2020).

Although nanozymes with complex CDs have the advantages of rapid response, high sensitivity, and simplicity when applied to molecular detection, they possess some limitations and are unsuitable for in vivo and continuous analyses. However, they lay the foundation for enzyme-dependent biological research. In future studies, it is necessary to enhance the substrate specificity of CDs complex nanozymes by modifying their functional groups.

To clearly describe the application performance of CDs-based nanozymes in the field of detection, we summarized the existing reports in Table 1.