In recent years, a large number of research reports have confirmed that many MOFs containing Fe redox nodes have peroxidase catalytic function, and their catalytic principle is usually Fenton-like mechanism, that is, hydrogen peroxide reacts with ferrous ions to produce hydroxyl radicals (· OH) with strong oxidizing ability^{[11,25⇓ ~27]}. In addition to iron-based MOF, some other transition-metal-containing MOF, including copper-based, ruthenium-based, nickel-based and zirconium-based, have also been found to have peroxidase catalytic ability^[10,28]. These nanoenzymes can oxidize colorless 3,3 ', 5,5' -tetramethylbenzidine (TMB) to blue 3,3 ', 5,5' -tetramethylbenzidine diimine oxide (TMBox) in the presence of hydrogen peroxide, and then turn yellow in the presence of acid^[29⇓~31]. Compared with the natural peroxidase, the MOF nanoenzyme has stronger peroxidase-like activity and higher stability. For example, the Michaelis constant K_m of Zr-based MOF nanocomposite (Zr-MOF-PVP) synthesized by Lu et al. For hydrogen peroxide and TMB is about 6 and 14 times lower than that of natural HRP enzyme, while the highest reaction rate is about 14 and 7 times higher than that of HRP enzyme^[12].

In addition to peroxidase activity, many cerium-based, cobalt-based, copper-based MOF nanozymes also show oxidase catalytic activity^[32⇓~34]. They can mimic the action of oxidase, activate oxygen in aqueous solution to produce reactive oxygen radicals (ROS), and then catalyze the redox reaction of substrates^[34]. At the same time, the MOF nanoenzyme can directly catalyze the oxidation of a substrate (such as TMB) by using dissolved oxygen in the reaction without the participation of hydrogen peroxide, and has the advantages of simple operation and rapid reaction^[10]. A series of studies have shown that its enzyme-like activity is much better than that of natural oxidase. For example, Mao et al. Simulated the active catalytic center of natural enzyme and synthesized a spherical porous copper-mimetic oxidase (Cu-MOF) with clear catalytic active center.It has good oxidase-like activity, the K_m value of Michaelis-Menten constant is as low as 1.09 mmol/L, and it shows good catalytic activity even at low concentration, and the enzyme activity can be maintained for about one month^[13]. At the same time, the nanoenzyme can also produce strong exogenous ROS.

MOF nanoenzymes synthesized from cerium-based, manganese-based and other materials are also often used to mimic catalase activity, as antioxidants to catalyze the decomposition of hydrogen peroxide into water and oxygen, and also have high affinity and catalytic effects on other ROS^{[35,36][17,19]}. The MOF nanoenzyme has higher catalytic activity and stability than natural catalase. In order to further improve the catalytic activity of MOF nanoenzyme, researchers introduced noble metal nanoparticles into the MOF substrate to form a composite MOF nanoenzyme with high enzymatic activity^[37,38]. He et al. Modified AuNPs on the surface of MOF in situ to form hydrogen peroxide nanoenzyme with high catalytic activity, which increased the decomposition ability of hydrogen peroxide by more than 6 times compared with the catalytic ability of AuNPs alone, and had good stability in buffer environment such as phosphate^[37].

At present, many bimetallic MOF nanoenzymes have been used by a large number of researchers to mimic superoxide dismutase^[39,40]. The function of this kind of nanoenzyme is similar to that of natural superoxide dismutase, which can catalyze the dismutation of ROS to oxygen and hydrogen peroxide, but it has higher stability and stronger catalytic activity^[30,31]. Qu et al. Prepared a Cu-TCPP MOF with superoxide dismutase-like activity, whose size and Cu active site coordination environment are similar to those of native superoxide dismutase, and whose superoxide dismutase activity is higher than that of other nanoenzymes (e.The activity of that MOF nanoenzyme is nearly 10 time higher than that of the CeO₂, and is 4 time higher than that of the MnO₂ nanoenzyme), the MOF nanoenzyme is closer to the natural superoxide dismutase, and the MOF Nanoenzyme can stably exist in high temperature, various organic solvents and a complex environment in a human body^[41].

In addition to oxidase activity, cerium-based and zinc-based MOF nanoenzymes have also been shown to have hydrolase-like activity, which can promote the hydrolysis of chemical bonds of the corresponding substrates^[42,43]. According to the different substrates, the hydrolase activity of MOF nanoenzyme is mainly divided into three types, namely, organophosphorus hydrolase, protease and esterase^[22,44]. For example, Lin et al. Used ZIF-90 to simulate organophosphohydrolase (OPH) to realize the hydrolysis of organophosphorus methyl parathion (MP), the highest reaction rate of which is tens of thousands of times that of natural OPH, and has higher stability and recyclability^[45]; The MOF-808 nanoenzyme prepared by Ly et al. Showed high protease activity, especially the model dipeptide (glycine-glycine) hydrolysis effect was excellent, which could completely hydrolyze the peptide bond with a rate constant of 2.69×10^-4s^-1;Chen.It showed good enzymatic activity toward p-nitrophenylacetate with a Michaelis-Menten constant (K_m value) of 3.9 mmol/L and a maximum initial velocity of 317 nm/s, and was easily separated from the system for recycling^[24][46].

The above five common MOF nanoenzymes have their own advantages and disadvantages: ① Many peroxidase-like MOF nanoenzymes have higher activity than natural enzymes, but most of them need moderate acidic conditions to exert higher activity; (2) The catalytic activity of MOF nanoenzyme with oxidase-like activity is also high, but the substrate selectivity is insufficient in complex samples; (3) catalase-like MOF has good stability and biocompatibility, but its catalytic activity is greatly affected by pH; (4) The stability of superoxide dismutase and hydrolase MOF nanoenzymes is better than that of natural enzymes, but the former is more cytotoxic and difficult to be used for in vivo analysis, while the biological activity of the latter is easily affected by pH. In addition, many MOF nanozymes can possess two or more enzymatic activities at the same time, which can be used for the analysis of synergistic reactions of multiple enzymes, such as catalytic cascade reactions.

Nucleic acid, as a carrier of genetic material, is of great significance in the growth and development of organisms, as well as in the diagnosis, treatment and prognosis of diseases. The analysis of disease-related nucleic acid markers has become the basis of diagnosis and precise diagnosis and treatment of various diseases^[49]. At present, polymerase chain reaction (PCR) is the most widely used nucleic acid detection method in clinical practice, which requires professional operation and precise instruments and equipment. Isothermal amplification techniques (such as loop-mediated isothermal amplification, rolling circle amplification, etc.), as a newly developed detection technique, have high sensitivity, simple operation, and do not require professional analytical instruments^{[50⇓⇓~53]}. These methods can realize the sensitive analysis of nucleic acid, but their common shortcomings are that the experimental process is greatly affected by enzyme activity, and the sensitivity and specificity are insufficient.

As an enzyme-like active substance, the MOF nanoenzyme is less affected by the reaction environment, can realize the sensitive and specific detection of the target nucleic acid through specific functional modification, and has the advantages of simple operation and rapid reaction^[14]. Based on this, Kong et al. Encapsulated glucose oxidase (GOx) in ZIF-8 as a nanocarrier, modified DNA strands on its surface to form a three-dimensional Walker, and achieved microRNA-21 (miRNA-21) sensitive electrochemical analysis on ZIF-8 nanocarrier through cascade catalytic reaction, with a minimum detection limit of 29 pmol/L.The implementation of this method provides a new idea for the clinical analysis of miRNA (Figure 1A); Li et al. Synthesized a loose and porous three-layer structure MOF @ Pt @ MOF nanoenzyme, which has a large specific surface area and abundant porosity, and its efficient catalytic performance and cascade primer exchange reaction can achieve ultrasensitive detection of exosomal miRNA, with a detection limit as low as 0.29 fmol/L^[54][55]. Similarly, the researchers used MIL-101, a metal-organic framework with abundant surface area and catalytic sites, excellent hydrothermal and chemical stability, to effectively immobilize G-triad/heme DNase, which has dual peroxidase activity and achieves ultrasensitive detection of miRNA-721 with a detection limit as low as 0.25 fmol/L^[56].

In order to further improve the detection sensitivity and effectively realize the early monitoring of diseases, Zhang et al. Developed a dual-signal mode based on the luminescent metal-organic framework coupled cascade nucleic acid circuit by using the photophysical effect of luminescent MOF and its own multi-active site characteristics.Effectively normalizing the interference of environmental factors to achieve accurate and sensitive analysis of serum circulating miRNA solves the challenge of low abundance detection of circulating miRNA in peripheral blood (Figure 1B)^[57]. Meanwhile, Wang et al. Assembled palladium nanoparticles on the surface of the previously synthesized Fe-MIL-88NH₂MOF microcrystals to prepare a novel multifunctional MOF (PdNPs @ Fe-MOF). Compared with the previously synthesized MOF, the prepared MOF has a large specific surface area and multiple active sites, and the enzyme activity can be enhanced by using the synergistic effect between PdNPs and Fe-MOF, which can detect miRNA-122 at a concentration as low as 3×10^-3fmol/L in serum samples (Figure 1C)^[58].

Although a series of nucleic acid analysis technologies based on MOF nanoenzymes have improved the sensitivity of traditional analysis methods, the stability of MOF materials is insufficient. In order to solve this problem, Han et al. Prepared an ultrathin (only 1 nm) two-dimensional MOF nanoenzyme, which has a larger surface area and more accessible active sites than the traditional three-dimensional MOF nanoenzyme, and has a smaller diffusion barrier for substrate molecules, thus improving the stability of detection^[59]. At the same time, the MOF-modified electrode can be regenerated by flowing target molecules and phosphate buffer, which can achieve continuous monitoring of target molecules (Figure 1 D).

Nucleic acid detection based on MOF nanoenzyme is a great progress in nucleic acid molecular detection technology, which helps to achieve more sensitive, accurate and convenient nucleic acid detection, but the enzyme-like catalytic activity of MOF nanoenzyme limits its practical application. Enhancing the catalytic activity of MOF nanoenzymes by increasing the specific surface area, modifying the spatial structure or doping other metal materials is an important direction worthy of further study.

The change of protein level in organism is often used as an objective evaluation basis for disease development, and is an important reference object for disease prediction, diagnosis and treatment. At present, protein analysis is usually based on antigen-antibody reaction, mainly including enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence immunoassay, etc. These methods are cumbersome, time-consuming, dependent on the substrate of enzyme-catalyzed reaction, and lack of sensitivity and specificity^[60,61]. MOF nanoenzyme overcomes the limitations of natural enzymatic reaction to a great extent because of its high reaction rate and catalytic performance.

Wang et al. prepared peroxidase-like catalytic active Fe-MIL-88 A (ion-based MOF material) for TMB catalysis, and achieved rapid analysis of thrombin within 10 min, with a minimum detection limit of 0.8 nmol/L observed by naked eyes (Fig. 2A)^[62]; Furthermore, Jiang et al. Synthesized a highly catalytic catechol-oxidizing nanoenzyme MOF-818 on the surface of carbon cloth fiber in situ by hydrothermal method, and used the surface modified functional aptamer to achieve ultrasensitive analysis of thrombin, which was at least two orders of magnitude higher than the reported analysis strategy (detection limit of 6.4 pmol/L) (Fig. 2B)^[63]; At the same time, Jiang et al. Modified a platinum-coordinated titanium-based porphyrin metal-organic framework (Ti-MOF-Pt) on the surface of a glassy carbon electrode, and enhanced the analytical ability of thrombin (linear range of 4 pmol/L ~ 0.2 μmol/L, detection limit of 1.3 pmol/L) by taking advantage of the high specific surface area of MOF and the excellent electrochemical properties of platinum, which is superior to most electrochemical and fluorescence analytical techniques^[64].

In order to better demonstrate the excellent analytical performance of MOF nanoenzyme, the researchers also used it for the detection of some protein markers. Zeng et al. Used carcinoembryonic antigen (CEA) -specific aptamer-functionalized DNA/AuNPs/MOF complex (peroxidase mimetic activity) for highly sensitive detection of CEA, with a minimum detection limit as low as 0.742 pg/mL; Feng et al. Prepared peroxidase-like active Fe-MIL-88B-NH2 (Fe-MOF) as an immunoprobe.Combined with the electrochemical platform, the analytical sensitivity of prostate specific antigen was improved, and the detection limit was 0. 13 pg/mL; Wang et al used Zr-based MOF as a peroxidase mimic to quantify and identify phosphorylated protein, and the linear detection range was 0. 17-5. 0 mg/mL, and the detection limit was 0. 16 mg/mL^[65][66][67]. In addition, Wang et al. Developed a colorimetric method for quantifying ALP activity using 2D-MOF with peroxidase-like activity in combination with alkaline phosphatase (ALP) -catalyzed hydrolysis, and three different phosphates, PPi, ATP, and ADP, were used as inhibitors of peroxidase nanoenzymes.Three different linear detection ranges were achieved, namely, 2.5 – 20 U/L, 5 – 60 U/L, and 50 – 200 U/L. The analytical performance of the method was able to adapt to the detection of samples with different abundances (Fig. 2C)^[68]. The above research strategies have effectively utilized the efficient enzyme-like catalytic activity of MOF nanoenzymes, but the sensitivity of these techniques needs to be further improved to better apply to the analysis of clinical samples. Therefore, Mao et al. covalently linked MIL-53 (Fe) MOF with a G-quadruplex forming sequence (F3TC) to couple heme to produce peroxidase-mimetic activity, and achieved ALP detection through a cascade enzymatic reaction with a detection limit of 0.02 U/L, which was successfully used in the analysis of human plasma samples (Figure 2D)^[69]. In order to further meet the needs of clinical rapid detection, Zhou et al. Introduced the technology of smart phone to identify the color change. According to the color change caused by Fe/Zn MOF, the RGB value generated by the color reaction was obtained, and the minimum ALP of 0.03 U/L could be identified^[70].

MOF nanoenzyme has been widely used in the detection of various protein disease markers, which can not only be used to effectively enrich antigens, but also have stable and controllable properties, and its catalytic activity can be comparable to that of natural enzymes. Combined with specific recognition elements (such as aptamers and antibodies), it can well achieve specific, sensitive and stable detection of protein targets. Improving the enzymatic activity and stability of MOF nanoenzyme will help to enhance its practical application in clinical protein detection.

In clinical analysis, inorganic compounds, ions, short peptides and other small molecules play an important role in the homeostasis, metabolism, occurrence and development of diseases. The existing methods for the analysis of small molecules are generally detected by atomic absorption spectroscopy, atomic emission spectroscopy, cold vapor, gas chromatography, liquid chromatography, high performance liquid chromatography and the like, and the methods are often insufficient in specificity or complex in operation, long in detection period and the like^[71,72]. Based on this, MOF nanoenzyme can make up for the defects of existing detection methods through functional modification and high catalytic performance.

Blood glucose monitoring, as the main type of clinical small molecule detection, occupies an indispensable position in the diagnosis and treatment of diabetes. Although a large number of glucose sensing devices have been used in clinical practice, the key to diabetes diagnosis and treatment is to continue to explore sensitive, simple and low-cost glucose monitoring strategies. Based on the advantages of MOF nanoenzyme, such as high activity, high specific surface area and rich active sites, researchers have applied it to glucose monitoring^[73]. Xu et al. Synthesized a peroxidase-like active Fe-MIL-88B-NH₂(Fe based MOF) nanoenzyme to link GOx by amidation coupling reaction, and performed cascade catalytic colorimetric detection of glucose. The detection system (linear range of 1 – 500 μmol/L, detection limit of 0.487 μmol/L) had comparable analytical performance with a blood glucose meter and better repeatability (Fig. 3A)^[74]. However, due to the limitation of sensitivity, this kind of method is easy to miss the detection of people with impaired glucose tolerance, so the analysis performance of this kind of sensor needs to be improved. Based on this, Yuan et al. established an ultrasensitive colorimetric glucose biosensing platform using two-dimensional iron-based MOF nanosheets with peroxidase properties for the determination of glucose in people with impaired glucose tolerance or other biological matrices, with a linear range of 0.04 ~ 20 μmol/L and a minimum detection limit as low as 39 nmol/L^[27]. During the occurrence and development of diabetes mellitus, not only the increase of blood sugar, but also the increase of other substances in the process of glucose metabolism. Therefore, simultaneous monitoring of multiple target molecules is often required at the same time of blood glucose detection. Wang et al. Introduced H₂pydc and EuNO₃·6H₂O to synthesize peroxidase-active MOF Eu-pydc to establish a dual-target molecular sensor, which could not only detect glucose, but also analyze cysteine, with detection limits of 0.28 μmol/L and 6.9 μmol/L, respectively (Fig. 3B)^[75].

In addition to blood glucose monitoring, MOF nanozymes are also widely used in other clinically common small molecule assays. Cui et al. Constructed a cascade catalytic detector (MIL-88B(Fe)-NH₂@GLOX) to immobilize glutamate oxidase (GLOX) using Schiff base reaction for glutamate analysis (detection limit of 2.5 μmol/L, linear range of 1 – 100 μmol/L)^[76]. Zeng et al. Found that the biomineralized MOF of bovine serum albumin (BSA) could enhance the high catalytic activity of nanoenzyme, so the Bioinorganic hybrid nanoenzyme functionalized by cholesterol oxidase was used to achieve highly sensitive detection of cholesterol in serum samples, with a minimum detection limit of 4.85 μmol/L; Xu et al. Established a new indirect competitive MOF nanoenzyme linked immunosorbent assay.For highly sensitive detection of aflatoxin B1, this method uses functional MOF instead of natural enzyme to catalyze color development, and its detection limit is 0.009 ng/mL. Compared with traditional ELISA, the detection limit of this method is reduced by 20 times, and false positive and false negative results are effectively avoided during the detection process (Figure 3C)^[77][78]. While improving the catalytic performance, the specificity of MOF nanoenzyme needs to be enhanced urgently. Wu et al. Coated polydopamine on the surface of MIL-53 (Fe) by self-polymerization, which showed strong anti-interference ability to common ions, amino acids and other substances on the basis of enhancing peroxidase activity and target recognition ability^[79]. Wang et al. Combined the flexible structure of MOF materials to construct a catalytic and luminescent dual-function nanoenzyme for 17β-estradiol specificity analysis^[80]. In addition to the analysis of single target molecules, Zhu et al. Improved Hemin @ BSA @ ZIF-8 peroxidases and successfully used them for the sensitive detection of various small molecules such as hydrogen peroxide and bisphenol A, broadening the biological application scope of MOF nanozymes (Fig. 3D)^[81].

MOF nanoenzyme provides strong support for sensitive and high-throughput detection of glucose, hydrogen peroxide, aflatoxin B1 and other small molecules by its strong stability and uniformly dispersed active sites. Further improving the specific surface area of MOF nanoenzyme and modifying its spatial structure will help it react more fully with small molecules and improve the precision and accuracy of clinical analysis.

Increasing the stability of MOF nanozymes in aqueous environment is a major challenge for their clinical detection applications. Because most MOF nanoenzymes are synthesized in organic solvents, their skeleton structure is easily destroyed in the catalytic process in aqueous environment, resulting in great structural instability. In clinical detection, most of the analytical systems are aqueous environment, which is easy to destroy the structure of MOF nanoenzyme and reduce its catalytic function, thus affecting the clinical detection results. Therefore, improving the stability of MOF nanoenzyme in aqueous environment, such as reducing the size of MOF materials or doping metal nanoparticles such as platinum, is conducive to its reuse and application in clinical testing.

At present, the specificity of MOF nanozymes mainly depends on the interaction between nanozymes and analytes or their functionalized biological recognition elements (antibodies, aptamers, etc.). However, MOF nanoenzyme itself does not have the fine protein structure of natural enzyme, which is easy to cause the recognition element to be unable to exclude the influence of non-target substances to a certain extent, resulting in the limitation of analytical specificity. Strengthening the interaction between the target substance and MOF nanoenzyme is the key to improve the specificity. The construction of nanozymes targeting substrate specificity is an effective way to enhance their detection selectivity. In addition, changing the spatial structure of MOF nanozymes can also solve the problem of selective catalysis of substrates by MOF nanozymes to some extent.

In previous studies, MOF nanoenzymes were difficult to achieve ideal catalytic activity due to their morphological structure and active sites. Although some strategies have been used to improve the enzyme-like catalytic activity, such as the synthesis of 2D-MOF nanosheets to expand the contact area of the active site, the results still fail to meet the needs in clinical testing. On this basis, the emerging single-atom and complex metal (such as bimetallic) MOF nanoenzymes are expected to provide new ideas for the ultrasensitive detection of nucleic acids, proteins, and small molecules. However, how to adjust the composition ratio of the composite metal and the monoatomization of the active site while retaining its own excellent structure and performance remains a great challenge.

In view of the above key problems, researchers can take advantage of the easy functionalization of MOF nanomaterials to synthesize monatomic nanomaterials with natural enzyme structure in the follow-up research, which can not only ensure high catalytic activity, but also greatly improve the selectivity of target nucleic acids, proteins or small molecules. The catalytic process of MOF nanoenzyme and the interaction between the catalytic active center and the clinical target were theoretically analyzed by density functional theory calculation, and the relationship between the structure and activity of MOF nanoenzyme was explored to prepare high-performance nanoenzyme. Controlling different reaction conditions (precursor, temperature, etc.) To achieve high-density catalytic active sites, ideal dispersion, stability, etc. Of MOF materials, and improve its clinical applicability. With the progress of nanoscience and the further study of the properties and catalytic mechanism of MOF nano-mimetic enzymes, MOF nano-enzymes with better stability, catalytic activity and selectivity will be further applied.