Nanozymes in Point‐of‐Care Testing: Classification, Biosensing Strategies, and Integrated Applications

Abstract

Nanozymes, a class of artificial enzyme mimics, combine the catalytic properties of natural enzymes with the high stability inherent to nanomaterials. This unique combination of features has established them as core functional materials in advanced fields such as biosensing and disease therapy. This review systematically examines recent advances in point‐of‐care testing (POCT) biosensors based on nanozymes, focusing on three fundamental aspects: the first is the classification of nanozymes; the second is the mechanism of action of nanozymes in biomolecular recognition, signal conversion and output, and signal amplification processes; and the third is the innovative applications of nanozyme‐based POCT technology in biomarker detection. By in‐depth analyzing the multifunctional roles of nanozymes as recognition units, signal converters, and signal amplifiers, this article outlines the design strategies for constructing nanozyme‐integrated POCT biosensors, with particular emphasis on the functional positioning of nanozymes within these systems. Furthermore, the application potential of nanozyme‐based POCT in detecting diverse biomolecules is discussed, providing a reference for newcomers and researchers to quickly grasp current trends in the field. Finally, key challenges are highlighted, and future research directions are proposed.

This review presents a three‐dimensional framework linking nanozyme classification, biosensing roles, and point‐of‐care testing (POCT) applications to bridge material innovation with practical application.

1. Introduction

In the field of health monitoring and disease diagnosis, rapid and accurate detection of biomarkers is key to achieving early diagnosis and treatment. Traditional laboratory detection methods offer high accuracy but rely on large‐scale instruments and professionals, making them unsuitable for on‐site needs in resource‐limited areas or emergency scenarios. This limitation has driven the development of point‐of‐care testing (POCT) technology, whose core is to deliver reliable results instantly at the sample collection site via portable devices [1]. Since 1995, when the National Committee for Clinical Laboratory Standards (NCCLS) of U.S. released the “Guidelines for Rapid Diagnostic Testing” and first proposed the concept of POCT, its connotation has evolved into a technical system meeting the “ASSURED” standards (Affordable, Sensitive, Specific, User‐friendly, Rapid and robust, Equipment‐free, Deliverable) [2]. With the innovation of POCT technology, new detection platforms have emerged continuously. The design of detection strategies directly determines a platform's efficiency, sensitivity, and accuracy, so developing new methods is the core foundation for POCT device research and development.

The design of conventional POCT methods covers two aspects: material foundation and organizational framework. For materials, functional substances such as fluorescent quantum dots [3, 4, 5], metal organic frameworks (MOFs) [6, 7, 8], noble metal nanoparticles [9, 10, 11], and enzyme‐mimetic materials [12, 13, 14] are used for signal output, enabling the construction of colorimetric [15], fluorescent [16], photothermal [17], and SERS‐based sensors [18]. For the framework, it includes three units: recognition units (antibodies, aptamers) for capturing targets, signal conversion units (fluorescent probes, electroactive materials) for signal transformation, and signal amplification units for enhancing trace detection capability.

Material selection is fundamental to POCT platform construction, yet traditional materials have obvious limitations: colloidal gold and organic fluorescent dyes suffer from insufficient sensitivity [19, 20] and poor stability [21, 22]; antigens and antibodies are prone to degradation or denaturation, while conventional reaction systems are often constrained in both specificity and sensitivity. The emergence of inorganic/organic materials with enzyme‐mimetic activity provides a new solution to these bottlenecks. In 2007, the Yan team first reported the peroxidase (POD)‐like activity of Fe₃O₄ nanoparticles and proposed the concept of “nanozymes” [23]. These materials integrate the catalytic activity characteristic of natural enzymes with the intrinsic stability of nanomaterials. Through precise regulation of composition and structure, they can mimic a wide range of enzymatic functions. Furthermore, advantages such as tunable catalytic activity, capacity for multifunctional integration, robust environmental stability, and ease of surface modification establish them as ideal candidates for constructing next‐generation POCT platforms.

This review focuses on the latest applications of nanozymes in POCT biosensors: it first sorts out the classification of nanozymes, then elaborates on their role in POCT design from the aspects of target recognition, signal conversion and amplification, subsequently summarizes the application cases of nanozyme‐based POCT in the detection of biomarkers such as small biological molecules, nucleic acid molecules, proteins, cells, and pathogens, and finally looks forward to the development trend of POCT as well as the challenges and prospects faced by nanozymes (Figure 1).

FIGURE 1.

Schematic illustrating the roles of nanozymes with different activities in point‐of‐care testing (POCT) platform construction and their applications in the detection of biomarker.

2. Classification of Nanozymes

The choice of materials plays a critical role in determining the sensitivity and portability of POCT platforms characterized by stable catalytic performance, tunable activity, and ease of integration, nanozymes effectively address the limitations associated with conventional biological enzymes [24] and have thus emerged as a core material in POCT development. Currently, nanozymes with diverse compositions have been reported, including metal and metal oxide nanoenzymes [25], carbon nanoenzymes [26], MOF nanozymes [27], as well as covalent organic framework (COF) nanozymes [28], and single‐atom nanozymes [29], all of which play pivotal roles in the construction of advanced POCT systems [13]. Nanozymes combine environmental tolerance (resistance to high temperatures and extreme pH) with multifunctionality (simultaneous targeted recognition and signal output). Moreover, their activity can be precisely tuned via size, morphology, and surface modification [30], offering diverse options for high‐sensitivity and portable POCT design. Classified by catalytic reaction types, nanozymes fall into hydrolase‐like and oxidoreductase‐like categories, each with specific application scenarios in POCT but also certain limitations.

2.1. Redox Nanozymes

Nanozymes with oxidoreductase‐mimetic activity are among the most intensively studied and widely applied nanozyme categories at present. These nanozymes function by participating in redox reactions within the reaction system, and can exhibit multiple enzyme‐mimetic activities, such as POD‐like, catalase (CAT)‐like, and oxidase (OXD)‐like activities. Among them, the catalytic activity of POD‐like nanozymes has reached or even exceeded that of their corresponding natural PODs [30].

2.1.1. Peroxidase‐Like Nanozymes

POD‐like activity is the earliest discovered enzyme‐mimetic function in metals and metal oxides nanomaterials. Similar to natural PODs in terms of mechanism, these nanozymes use hydrogen peroxide (H₂O₂) as a substrate: while catalyzing the decomposition of H₂O₂, they can oxidize multiple chromogenic substrates such as 3,3′,5,5′‐tetramethylbenzidine (TMB), 3,3′‐diaminobenzidine (DAB), and o‐phenylenediamine (OPD), thereby generating detectable signals. Their catalytic mechanism is mainly based on Fenton‐like reactions or electron transfer processes [31]. During the reaction, nanozymes activate H₂O₂ through their own metal active sites (e.g., Fe²⁺, Cu²⁺), producing highly reactive hydroxyl radicals (·OH); these hydroxyl radicals further oxidize chromogenic substrates, causing obvious color changes. This process is similar to the catalytic cycle of natural horseradish peroxidase (HRP); however, nanozymes typically exhibit a broader operational range across varying pH and temperature conditions [32]. Such enhanced environmental adaptability underscores their considerable potential for biosensing applications.

Due to the high substrate affinity for H₂O₂ and efficient catalytic properties, POD‐like nanozymes are widely used in constructing biosensors with high sensitivity and selectivity. Since Yan et al. [23] discovered that Fe₃O₄ magnetic nanoparticles (MNPs) exhibit intrinsic enzyme‐mimetic activity similar to that found in natural PODs, the Wei's group [31] has utilized the POD‐like activity of Fe₃O₄ to catalyze the oxidation of 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS) into a colored product in the presence of H₂O₂, enabling the colorimetric detection of H₂O₂ based on color changes. In addition, by combining Fe₃O₄ with glucose oxidase (GOx), they achieved the quantitative analysis of glucose. Based on similar sensing strategies, a series of nanozymes with POD‐like activity have been synthesized, which have successfully realized the rapid detection of substances such as uric acid [33] and cholesterol [34]. Furthermore, based on the enzyme activity regulation strategy of POD‐like nanozymes, which refers to the enhancement or inhibition of nanozyme activity by target substances and reaction intermediates, researchers have also achieved the detection of various biomarkers, including nucleic acids [35] and proteins [36, 37].

In addition to metal and metal oxide nanomaterials, studies have reported that carbon‐based materials, MOFs, and other materials also exhibit POD‐like activity. Zheng's group [38] used citric acid, ethylenediamine, copper salts, and hemin as raw materials to prepare copper‐doped carbon dots (Cu‐CDs) and hemin‐adsorbed carbon dots (Hemin@CDs) with POD‐like activity via microwave‐assisted one‐pot synthesis and stepwise in‐situ composite synthesis, respectively. Through copper ion doping and hemin molecule adsorption, not only the POD‐like activity of CDs was effectively enhanced, but their excellent biocompatibility, optical properties, and ease of synthesis were also retained. By leveraging the differences in interaction forces between biothiols and metal ions, they successfully achieved the identification of three types of biothiols and the authentication of different biological samples.

MOFs with enzyme‐mimetic activity offer distinct advantages over other types of nanozymes, such as well‐defined molecular structures and catalytic sites, high porosity, and large specific surface areas. Formed through the coordination‐driven self‐assembly of metal ions and organic ligands, MOFs can stabilize nanozyme structures, prevent aggregation or deactivation, and enhance catalytic efficiency and reusability. Interestingly, a large part of MOFs exhibit enzyme‐like characteristics [39, 40]. Current research on POD‐like nanozymes focuses not only on expanding their main application fields but also on improving their catalytic conditions and performance. Natural OXDs are often used in cascade catalytic reactions with POD‐like nanozymes, however, the pH mismatch between natural enzymes and nanozymes leads to low cascade reaction efficiency. Fortunately, based on the structural characteristics of MOFs, precise engineering and highly customized microenvironments of MOF‐based nanozymes (MOFzymes) can be achieved through controlled defect generation, regulation of metal‐support interfaces, heterometallic coordination, atomic‐level doping, and hybrid component design [41, 42, 43]. For example, Wei et al. [44] used MOF as a nanozyme model, adopted low‐molecular‐weight polyacrylic acid (PAA) as a Brønsted acid, and confined it within the pores of MOF‐based POD‐like nanozymes. With protons provided by PAA, an acidic microenvironment was formed inside the MOF pores, enabling the MOF‐based POD‐like nanozyme to exert optimal catalytic activity even under neutral conditions.

In addition, some sulfide nanozymes also exhibit broad pH stability. Yang et al. [45] reported a type II FeS₂/Co₃S₄ heterostructured nanozyme, which showed excellent POD‐like activity under near‐physiological conditions, successfully overcoming the pH range limitations of traditional nanozymes. Based on this nanozyme, the research group constructed a colorimetric biosensor for rapid detection of H₂O₂; by combining it with xanthine oxidase (XOD), they further achieved rapid and accurate detection of xanthine. These functional materials with excellent POD‐like activity have become core modules connecting biological recognition and signal output in biosensing, laying a key foundation for high‐sensitivity and high‐specificity detection.

2.1.2. Catalase‐Like Nanozymes

CAT‐like nanozymes efficiently catalyze the decomposition of H₂O₂ into H₂O and O₂. These nanozymes play a pivotal role in biotheranostics, contributing to regulating reactive oxygen species (ROS) homeostasis, protecting cells from oxidative stress, and alleviating hypoxia. Moreover, they also serve as a critical link between biorecognition events and signal output in biosensing systems. The mechanisms of H₂O₂ decomposition catalyzed by these nanozymes are mainly divided into two categories: heterolytic cleavage pathway and homolytic catalytic pathway, which depend on the preferential cleavage mode of chemical bonds (H—O bond or O—O bond) in H₂O₂, and are regulated by the composition, surface state of nanomaterials and reaction environment (e.g., pH) [46]. A typical feature of the heterolytic cleavage pathway is the accompaniment of proton transfer and metal ion valence cycling during the reaction. Taking CeO₂ nanoparticles as an example, the redox cycle of Ce³⁺/Ce⁴⁺ on their surface is the catalytic core: after H₂O₂ is adsorbed on the surface of CeO₂, the H—O bond breaks and releases protons, and Ce⁴⁺ is reduced to Ce³⁺; subsequently, another H₂O₂ molecule combines with protons to form H₂O, and Ce³⁺ is reoxidized to Ce⁴⁺, completing the catalytic cycle [47]. The homolytic catalytic pathway is characterized by the preferential cleavage of the O—O bond in H₂O₂ to generate hydroxyl radical (·OH) intermediates, which then form H₂O and O₂ through subsequent reactions [48].

Regardless of the decomposition pathway adopted, macroscopically, the core function of such nanozymes is to catalyze the decomposition of H₂O₂ to produce O₂. Their activity is commonly found in metal‐based nanoparticles, metal oxide nanoparticles, carbon nanomaterials, and sulfide nanoparticles. Combined with the diversity of material structures and morphologies and O₂‐producing properties, CAT‐like nanozymes are often used in biosensing systems for quantifying target analytes based on oxygen production or dissolved oxygen levels. In our previous work, Au@Pt NPs with CAT‐like activity were synthesized, which were layer‐by‐layer stacked on the inner wall of EP tubes through base complementary pairing, and target‐triggered release aptamer hairpin probes (AHP) were modified on the EP tube caps. Based on target‐triggered Ag⁺ release, DNAzyme activation, and incremental release of nanozymes, the O₂ signal catalyzed by Au@Pt NPs was converted into a total dissolved solids (TDS) signal, ultimately achieving the visual detection of prostate‐specific antigen (PSA) [49]. Liu et al. [50] synthesized hollow CeO₂‐encapsulated Pt/Au core–shell hybrid materials (CeO₂@Pt/Au), using the O₂ pressure generated by the reaction between CeO₂@Pt/Au and H₂O₂ as a signal converter. A POCT immunoassay has been successfully constructed by combining a coil device for visual detection and a pressure sensor.

In summary, with the efficient ability to decompose H₂O₂ and tunable catalytic properties, CAT‐like nanozymes have emerged as crucial modules bridging biological signals and detectable physicochemical signals. They provide highly specific and stable detection tools for POCT applications, such as the diagnosis of ROS‐related diseases and environmental monitoring.

2.1.3. Oxidase‐Like Nanozymes

OXD‐like nanozymes offer distinct advantages and hold considerable application potential in biosensing systems. Their core catalytic feature involves the direct utilization of dissolved oxygen as an electron acceptor to oxidize substrates, eliminating the need for exogenous H₂O₂. Unlike PODs and CATs, which require H₂O₂ as a cosubstrate, OXD‐like nanozymes directly catalyze the oxidation of electron‐donor substrates using molecular oxygen. This characteristic eliminates interference arising from the volatility and instability of H₂O₂, substantially simplifies assay procedures, and enhances suitability for complex biochemical analysis scenarios. Their catalytic mechanisms primarily involve two core pathways: ROS‐mediated pathway and electron transfer pathway. In the ROS‐mediated pathway, nanozymes adsorb and activate a large amount of dissolved oxygen via their large specific surface area, generating ROS intermediates such as ·O₂ ⁻ to oxidize substrates [51, 52]. For enzyme‐mimetic materials including single‐atom catalysts (SACs) [53] and metal oxide nanoparticles [54], the generation of ROS can be further enhanced by regulating the metal doping content and utilizing photoinduced electron–hole pairs, which effectively improves the OXD‐like activity of the materials. In the electron transfer pathway, nanozymes act as electron acceptors and transfer media. The catalytic process proceeds through sequential steps involving electron transfer from substrates to nanozymes, oxygen adsorption with formation of OOH adducts, and multiple proton‐coupled electron transfer events. Their catalytic activity can be further modulated via substituent groups [55]. It is noteworthy that the catalytic efficiency of OXD‐like nanozymes is influenced by the type of nanomaterials (e.g., Rh > Ru > Au in noble metal nanozymes) [56], substrate species, and environmental conditions. Systematic investigation into the variation rules of their catalytic mechanisms and the influencing factors is of great significance for the rational design and development of biosensors.

2.2. Hydrolase‐Like Nanozymes

Hydrolase‐like nanozymes refer to a class of nanomaterials with the ability to catalyze hydrolysis reactions [57]. Since Scrimin et al. [58] proposed the concept of hydrolase‐like nanozymes, a wealth of relevant research has been reported. The substrates that can be hydrolyzed by these nanozymes include phosphates [59], carboxylates [60], as well as amides [61], glycosidic bonds [62], etc. That materials include noble metal nanozymes, polymer nanozymes, peptide assemblies, metal and metal oxide nanoparticles, MOFs, and so on [57]. According to the type of catalytic substrates, hydrolase‐like nanozymes can be classified into phosphatase‐like, protease‐like, esterase‐like, and other categories.

Current research on nanozymes mainly focuses on oxidoreductase‐like nanozymes (accounting for 92.9%, statistical analysis covering literature through August 2018), while studies on hydrolase‐like nanozymes are relatively limited (only 7.1%) [63]. Moreover, existing research on hydrolase‐like nanozymes mostly focuses on enhancing the hydrolysis efficiency of ester bonds through approaches such as surface functionalization, element doping, and coordination microenvironment modification [64]. However, due to the complex active sites and coordination microenvironment of natural enzymes, the catalytic efficiency and selectivity of current hydrolase‐like nanozymes are still unable to match those of natural enzymes [65]. Although numerous studies on hydrolase‐like nanozymes have been reported in the field of biosensing, parameters such as their sensing performance are still limited by their own enzyme‐mimetic activity and selectivity. Therefore, improving the activity and selectivity of hydrolase‐like nanozymes through various approaches is a necessary and crucial research direction in this field.

Phosphorylation and dephosphorylation are the core reversible regulatory mechanisms in intracellular signal transduction. Natural phosphatases catalyze the hydrolysis of phosphomonoesters into free phosphate ions, enabling the precise removal of phosphate groups from substrates. As a core subclass of hydrolase‐like nanozymes, phosphatase‐like nanozymes can efficiently mimic this process; relying on their specific hydrolytic capacity toward phosphate ester substrates, they play a key role in biosensing signal conversion, phosphorylated biomarker detection, and disease diagnosis. The catalytic activity of these nanozymes mainly depends on the unique active site structure and action mechanism of the materials, and exhibits diverse characteristics due to different material compositions. For Ce‐based phosphatase‐like nanozymes, their catalytic core lies in the redox cycle of Ce³⁺/Ce⁴⁺ and surface oxygen vacancies—Ce³⁺ activates the phosphate groups of phosphate ester substrates through electron transfer, while oxygen vacancies provide stable sites for substrate adsorption, and Kuchma et al. reported that cerium nanoparticles can efficiently hydrolyze ester bonds in p‐nitrophenyl phosphate (pNPP), adenosine triphosphate (ATP), O‐phospho‐L‐tyrosine, and DNA, confirming their phosphatase‐like activity [66]. In the case of Zr‐based phosphatase‐like nanozymes, they rely on the strong Lewis acid property of Zr⁴⁺ and surface hydroxyl groups (—OH): Zr⁴⁺ weakens the P—O bond through coordination with phosphate ester groups, and meanwhile, surface —OH provides proton transfer channels, thereby accelerating substrate hydrolysis [67, 68]. Meanwhile, MOF‐based phosphatase‐like nanozymes improve substrate diffusion efficiency via porous structures; the metal clusters (e.g., Zr—O clusters, Ce—O clusters) in their frameworks serve as active centers, and combined with the ligand‐regulated microenvironment, they achieve highly selective catalysis of phosphate ester substrates [69, 70].

In terms of material systems, phosphatase‐like nanozymes cover diverse types, including Ce‐based nanoparticles, Zr‐based nanoparticles, MOFs, metal oxides, and single‐atom nanozymes. Each type can efficiently achieve phosphate ester hydrolysis by leveraging its unique catalytic mechanism. Their core application in biosensing therefore lies in two key steps. First, catalyzing the hydrolysis of phosphate ester substrates such as disodium 4‐nitrophenyl phosphate (p‐NPP) [71], 4‐methylumbelliferyl phosphate (4‐MUP) [72, 73], 3‐(2′‐spiroadamantyl)‐4‐methoxy‐4‐(3′‐phosphoryloxyphenyl)‐1,2‐dioxetane (AMPPD) [74], and 2‐phospho‐L‐ascorbic acid (AAP) [75]; Second, converting target recognition events into quantifiable colorimetric, fluorescent, electrochemical, or chemiluminescent signals. Together, these steps enable the accurate detection of various biological targets.

In recent years, various nanozyme materials with excellent performance have been continuously developed, providing important support for the development of biosensing and diagnostic fields. However, to further translate the superior catalytic performance and biosensing capabilities of nanozymes into practical diagnostic applications, the rational design and controllable construction of nanozyme‐based biosensing systems are particularly critical. Therefore, systematic exploration of the construction strategies of nanozyme‐based POCT is of great significance for connecting high‐performance nanozyme catalysis with on‐site rapid detection and promoting the practical application of diagnostic technologies.

3. Construction Strategy of Nanozymes‐based POCT

Traditional biosensors rely on the synergy of target recognition units, signal conversion components, and signal output components to achieve analyte detection. POCT biosensors are based on the construction strategy of traditional biosensors and need to possess the “ASSURED” attributes; thus, the construction of POCT biosensors also requires matching functional components with efficient synergy.

For heterogeneous targets such as small molecules, nucleic acids, protein macromolecules, cells, and pathogens, nanozymes, which act as multifunctional components integrating diverse enzymatic catalytic activities and nanomaterial properties, play key roles in target recognition, signal output, and signal amplification in POCT biosensors. Their diverse material categories enable flexible adaptation to varying component requirements. These materials overcome key limitations associated with natural enzymes, such as susceptibility to denaturation and high production cost, while rapidly generating detectable signals through catalytic reactions. This combination of properties provides fundamental support for the practical implementation of POCT biosensors.

3.1. Nanozymes‐Based Target Recognition

In nanozyme‐based POCT biosensors, nanozymes can serve as recognition units to bind target analytes through specific interactions. Their recognition mechanisms are mainly achieved through two pathways: direct recognition and recognition via specific site modification. Direct recognition relies on the intrinsic structural characteristics of nanozymes and the inherent properties of target analytes to form specific binding. For recognition via specific site modification, the recognition specificity for target analytes is enhanced by modifying the surface of nanozymes with targeting molecules such as aptamers, antibodies, or nucleic acid probes.

3.1.1. Direct Recognition via Enzymatic Activity Modulation

There are abundant active sites on the surface of nanozymes, however, these active sites are often affected by pH, temperature, and redox substances. In addition, relying on their own stable ligands and enzyme‐like activity, nanozymes can directly interact with target to be detected, which in turn leads to changes in nanozyme activity or the generation of new compounds with specific physicochemical properties. Based on these characteristics, the target recognition can be realized by using the inhibition/promotion effect of related substances on the activity of nanozymes and the optical and electrochemical properties of the products (Figure 2A). Shu et al. [77] developed a colorimetric/fluorescent probe based on carbon quantum dots (O‐CDs) and cytochrome C (Cyt C) for sensitive and specific trypsin detection and trypsin inhibitor screening. The O‐CDs prepared in this work exhibit stable fluorescence, providing a reliable signal readout basis. In the presence of trypsin, Cyt C is hydrolyzed to release heme with OXD‐like activity; this heme can oxidize colorless TMB to form blue TMB oxidation (oxTMB). The generated oxTMB then quenches the fluorescence of O‐CDs through the inner filter effect, a process that enables the accurate quantification of trypsin. Dashtian's group [76] synthesized Cu‐ and Ni‐doped Ce/Fe fumarate bio‐MOF (Ce/Fe‐Fum Bio‐MOF) via a hydrothermal method, and further loaded Ag nanoparticles on its surface by reducing AgNO₃ with NaBH₄, finally obtaining a nanozyme with multi‐enzyme activity. This nanozyme exhibits diverse colorimetric responses in the presence of substrates such as TMB, 2,4‐dichlorophenol (2,4‐DCP), 4‐aminoantipyrine (4‐AAP), catechol, OPD, and ABTS. When diabetic biomarkers, including uric acid, L‐tryptophan, acetone, and glucose, are present, their antioxidant properties inhibit the color reaction, inducing observable changes in the system's color. This color variation enables quantitative detection of multiple biomarkers via smartphones (Figure 2B). Deng's group [78] synthesized high‐performance prussian blue nanozymes with POD‐like activity via an ethylene glycol‐based mixed solvent approach, and integrated them with biological enzymes such as GOx. Leveraging H₂O₂ produced by enzymatic reactions, they developed a vertical flow assay strip platform. Within this platform, Prussian blue nanozymes, specific biological enzymes, and chromogenic reagents are incorporated into distinct functional layers, thereby enabling multiplex POCT detection of low‐molecular‐weight metabolites including glucose, cholesterol, uric acid, and triglycerides.

FIGURE 2.

(A) Schematic of direct recognition by nanozymes via target molecule‐mediated activation or inhibition of their activity and colorimetric signal output via the POD‐like activity. (B) The mechanism of oxidation of TMB, OPD, CAT, 4‐AAP, 2,4‐DCP, and ABTS by Cu:Ce/Fe‐Fum Bio‐MOF nanozyme with different mimic enzyme activities and detection of uric acid, L‐tryptophan, acetone, and glucose. Reproduced with permission [76]. Copyright 2025, Elsevier.

3.1.2. Indirect Recognition

By conjugating specific recognition elements such as antibodies, aptamers, or peptides to the surface of nanozymes, the resulting nanozymes acquire the capacity to target nonenzymatic substrates or directly interact with stimuli‐related targets. This strategy addresses the inherent poor selectivity of nanozymes while meeting the requirements for specific recognition and detection of targets in complex samples (e.g., serum and urine) for POCT applications. In POCT systems, antibody‐based immunoassays represent the most widely used approach, with broad target coverage, high specificity, and well‐established standardized detection protocols such as enzyme‐linked immunosorbent assay (ELISA). Traditional ELISA‐based detection strategies, including ELISA kits and lateral flow assay (LFA) strips, achieve target recognition and signal output via detection antibodies modified with gold nanoparticles (AuNPs) or HRP, but they possess inherent limitations in terms of detection sensitivity and stability. The substitution of conventional AuNPs and HRP with nanozymes represent an effective technical approach to enhance the detection performance of POCT systems. Zeng et al. [79] prepared nitrogen and sulfur codoped carbon dot‐supported nitrogen‐rich porous carbon nanozymes (N/S‐CDs@NC), which were subsequently conjugated with carcinoembryonic antigen (CEA) detection antibodies (Ab2). Meanwhile, Fe₃O₄ modified with CEA capture antibodies (Ab1) served as the target recognition and capture unit. After Fe₃O₄ magnetic beads–Ab1 (Fe₃O₄ MB–Ab1) specifically captured the target from the sample, they selectively bound to N/S‐CDs@NC‐polyethyleneimine (PEI)‐luminol‐Au NPs–Ab2, forming Fe₃O₄ MB–antibody–antigen–antibody–luminol nanocomposite conjugates, and then the electrochemiluminescence (ECL) signals, positively correlated with CEA concentration, were generated. This system not only significantly improved the detection sensitivity of CEA but also markedly enhanced its stability (Figure 3A). Based on a similar strategy, they further synthesized europium (Eu) MOFs and loaded isoluminol‐Au NPs on their surface. Integrating antigen–antibody specific recognition and magnetic separation technology, a sandwich‐type immunosensor was constructed for the simultaneous detection of ovarian cancer‐related biomarkers CA125 and HE4. The as‐constructed ECL immunosensor exhibited excellent detection performance, with a linear response range of 0.005–500 ng/mL, and the limits of detection (LOD) for CA125 and HE4 were as low as 0.37 and 1.58 pg/mL, respectively [82]. However, critical challenges remain to be addressed in this field, such as the inherent instability of antibodies and the incompatibility of small‐molecule targets with ELISA detection systems due to their extremely low surface area [83].

FIGURE 3.

(A) Preparation of N/S‐CDs@NC nanoenzyme, antibody modification and electrochemical recognition of CEA. Reproduced with permission [79]. Copyright 2022, ACS Publishing. (B) Schematic of colorimetric detection of Alzheimer's disease based on aptamer recognition and carbon quantum dots (CQDs) nanoenzyme release. Reproduced with permission [80]. Copyright 2025, ACS Publishing. (C) Schematic illustration for integrating recognition peptide into RGPT‐PdNP@rGO nanozyme to mimic the binding pocket of uricase for uric acid detection. Reproduced with permission [81]. Copyright 2025, Elsevier.

Aptamers are single‐stranded DNA or RNA molecules engineered via in vitro selection, and have garnered significant attention due to their unique advantages, including potential unlimited target recognition capability, enhanced binding efficacy and stability, ease of chemical modification, and low immunogenicity [84]. More importantly, aptamers exhibit distinct superiority in recognizing small‐molecule targets, thus being widely regarded as alternatives to antibodies. In recent years, they have been extensively investigated as targeted separation and recognition units in nanozyme‐based biomarker detection systems. Omer et al. [85] synthesized Cu‐MOFs nanozymes with POD‐like activity, and the thrombin aptamers (ssDNA) modified on their surface can effectively inhibit the catalytic activity of the nanozymes and weaken the fluorescence. When the COVID‐19 biomarker thrombin binds to ssDNA, it induces the recovery of the nanozymes’ catalytic activity and fluorescent signals, thereby enabling the detection of COVID‐19 biomarkers. Chen's group [80] developed a universal and modular probe for artificial urine biomarkers. Through an innovative “aptamer‐DNAzyme‐nanozyme” three‐module design, ultrasensitive detection was achieved for Aβ oligomers (AβO). The probe employs silica nanoparticles (SiO₂ NPs) as the carrier, with AβO aptamers, DNAzymes, and CQDs with POD‐like activity covalently conjugated to its surface. Upon specific recognition of AβO in blood by the aptamers, a conformational change is triggered to activate DNAzymes, which then cleave substrates to release a large quantity of CQDs. These CQDs can catalyze H₂O₂ to oxidize colorless TMB into a blue product, thus realizing the visual detection of AβO (Figure 3B). This strategy allows for early diagnosis solely via urine testing, with a sensitivity 67‐fold higher than that of the traditional ELISA method.

Some studies have confirmed that certain peptides can target specific molecules. By modifying nanozymes with peptides, they can be endowed with the ability to recognize relevant receptors and protein domains, thereby enabling POCT detection of various targets. Liu's group [81] integrated a recognition peptide (RGPT) into nanozymes to mimic the binding pocket structure of natural enzymes, achieving specific recognition of uric acid with a linear range of 0.2–110 μM and a detection limit of 120 nM (Figure 3C). In addition, research based on chemical affinity recognition has also been reported. Yu et al. [86] synthesized folic acid (FA)‐functionalized MnO₂ nanosheet/graphene oxide (FA‐MnO₂/GO) composites with OXD‐like activity. Leveraging the targeted binding ability of FA to folate receptors (FRs) overexpressed on the surface of cancer cells, combined with the signal conversion function of MnO₂ in catalyzing the oxidation of chromogenic substrate TMB, they achieved highly robust and sensitive colorimetric detection of cancer cells. This effectively addresses issues such as the instability of HRP, H₂O₂, and antibodies in traditional immunosensing platforms. For biological targets containing cis‐diol structures, strategies based on boronate affinity reactions are commonly used in POCT.

3.2. Nanozymes‐Based Signal Amplification Strategies

In traditional biosensors, the qualitative and quantitative analysis of targets with a certain abundance can be achieved by molecular modification of simple colored materials (such as AuNPs) and fluorescent dyes. However, for trace biomarkers such as circulating tumor DNA (ctDNA) and early infection markers, the signal output from traditional signal molecules is not enough to fully meet the detection requirements. Early‐stage colloidal gold test strips serve as a representative example of this limitation. To address this challenge, integrating nanozyme catalysis offers a viable solution. Both direct signal amplification using nanozymes and indirect strategies incorporating nucleic acid amplification, nanozyme‐cascade catalysis, or physical encapsulation‐controlled release systems enable effective qualitative and quantitative analysis of trace biological targets.

3.2.1. Nanozymes‐Based Direct Signal Amplification Strategies

Relying on the intrinsic catalytic activity of nanozymes, direct signal amplification enhances target‐induced signal output by modulating active site exposure, redox cycling, or substrate utilization, eliminating the need for additional amplification steps. The core mechanism involves the direct or indirect activation or inhibition of nanozyme catalytic activity by the target, thereby altering the generation of signal molecules. Consequently, the essence of this strategy lies in improving the catalytic efficiency of nanozymes to enhance the sensitivity of POCT platforms. Noble metal nanoparticles such as Pt, Pd, Ru, and Ir, along with related bimetallic nanocomposites, exhibit outstanding CAT‐like and POD‐like activities, making them widely applicable in nanozyme‐based POCT systems. Wang et al. [12] synthesized a CuTe@PtPd composite nanozyme with excellent catalytic performance for the ultrasensitive lateral flow immunoassay (LFIA) detection of FA in serum. In this work, the synergistic catalytic effect of Pt and Pd endowed the CuTe@PtPd nanozyme with superior POD‐like activity, characterized by a higher V _max (3.23 μM·s⁻¹) and a lower K _m (0.17 mM) compared to HRP. This system achieved a detection limit as low as 0.10 ng/mL for FA, representing a 9.50‐fold improvement in sensitivity over conventional colloidal gold‐based LFIA (Figure 4A). In another study, Peng et al. [87] prepared a Fe₃O₄@MOF@PtPd nanozyme with high catalytic activity. After modification with vancomycin, the nanozyme effectively captured and isolated Staphylococcus aureus (S. aureus). Furthermore, an aptamer combined with the nanozyme bound to the bacteria forms a sandwich structure. Based on this mechanism, an LFIA strip was fabricated that could detect S. aureus at concentrations as low as 2 CFU/mL within 30 min, achieving a 43‐fold enhancement in sensitivity compared to traditional LFIA (Figure 4B). Notably, noble metals are considered ideal materials for synthesizing highly efficient nanozymes due to the abundant free electrons and stable chemical states. Several strategies have been developed to enhance the enzyme‐like catalytic activity of noble metal nanozymes, primarily including modulation of electronic structure, surface ligand engineering, and excitation of plasmonic effects. These approaches have significantly broadened the application of noble metal nanozymes in direct signal amplification for POCT.

FIGURE 4.

(A) Illustration of the CuTe@PtPd‐LFIA for the ultrasensitive detection of FA. Reproduced with permission [12]. Copyright 2025, Elsevier. (B) Schematic diagram of bacteria detection, (a) the assembly of HCR‐multi‐Apt and (b) bacterial detection based on MA‐MN LFIA and its detection advantages. Reproduced with permission [87]. Copyright 2024, Elsevier. (C) Fe‐NC SAC based hydrogel sensor for the colorimetric detection of trypsin. Reproduced with permission [90]. Copyright 2023, Elsevier. (D) Schematic diagram of electrochemical sensing based on CuFe‐MOF nanozyme, (a) synthesis of CuFe‐MOF nanozyme and (b) fabrication of label‐free electrochemical immunosensor and immunoassay procedure for IgG. Reproduced with permission [91]. Copyright 2024, Elsevier.

Beyond noble metals, non‐noble metal nanozymes have also been found to have rich activity enhancement strategies [88, 89]. Hu et al. [90] synthesized a Fe‐NC SAC, which has excellent POD‐like activity because of its well‐defined atomic active centers and maximum utilization rate of active metal atoms. As a signal amplification element, Fe‐NC SAC was integrated into trypsin‐responsive gelatin hydrogel, and a new hydrogel sensor was constructed. In the presence of trypsin, the gelatin was specifically hydrolyzed, releasing the pre‐encapsulated Fe–N–C SAC and generating a colorimetric signal with a detection limit of 1 ng/mL (Figure 4C). Additionally, the preparation of bimetallic nanomaterials by compounding two or more metals is an effective strategy to enhance the enzyme‐like activity. Yang's group [91] developed a spindle‐shaped copper–iron bimetallic MOF (CuFe‐MOF) nanozyme with enhanced POD‐like activity, and constructed a label‐free electrochemical immunosensor based on catalytic amplification induced by the MOF nanoenzyme. The CuFe‐MOF can effectively oxidize electroactive probes with strong electrochemical signals, thus outputting enhanced differential pulse voltammetry (DPV) signals and enabling ultrasensitive detection of immunoglobulin G with a detection limit of 0.45 pg/mL (Figure 4D).

3.2.2. Nanozymes‐Based Indirect Signal Amplification Strategies

In the direct signal amplification strategy based on nanozyme, nanozyme acts like biological enzyme, and continuously catalyzes the substrate to generate various signals such as optical, photothermal and electrochemical signals, so as to achieve the purpose of signal amplification. However, in certain scenarios involving ultralow target concentration or complex sample matrices, direct signal amplification may prove insufficient. Therefore, on the basis of direct signal amplification, researchers have developed indirect signal amplification strategies using enzymes. Common methodologies include nucleic acid amplification‐based nanozyme catalysis, nanozyme‐cascade catalysis, and encapsulation‐controlled release nanozyme catalysis.

Common nucleic acid‐based signal amplification techniques include rolling circle amplification (RCA) [92], strand displacement amplification (SDA) [93], hybridization chain reaction (HCR) [94], catalytic hairpin assembly (CHA) [95], and entropy‐driven strand displacement reaction (ESDR) [96]. The integration of these nucleic acid amplification methods with nanozymes represents a powerful strategy for achieving ultrasensitive POCT of low‐concentration biomarkers. In a recent work of Wei et al. [97], a “dual‐functional” sensing system was constructed by employing Au nano pumpkin arrays (Au NPAs) as a high‐performance plasmonic substrate and Au@Pt nanorods (Au@Pt NRs) as nanozyme probes, together with a target‐triggered CHA amplification strategy. Upon target recognition, the CHA process induced the assembly of Au@Pt NRs on the Au NPA surface, enabling the nanozymes to catalyze TMB oxidation into oxTMB with strong SERS activity. The CHA cascade further amplified the SERS signal, achieving ultrasensitive detection of the gastric precancerous lesion biomarkers miR‐196b and miR‐221 at attomolar levels (Figure 5A). Similarly, Lu et al. [98] reported a novel photoelectrochemical (PEC) biosensor based on a dual‐amplification strategy combining ESDR and HCR. Target miRNA‐21 triggered the formation of long double‐stranded DNA structures on the electrode surface. Manganese porphyrin (MnPP) intercalated within the duplex functioned as a HRP mimic, catalyzing the generation of a measurable PEC signal. This approach enabled miRNA‐21 detection with a limit as low as 33 aM (Figure 5B).

FIGURE 5.

(A) Functionalization of Au NPAs, CHA signal amplification, and detection of miRNA. Reproduced with permission [97]. Copyright 2025, Elsevier. (B) Schematic diagram of ESDR amplification, HCR amplification, and PEC biosensor preparation. Reproduced with permission [98]. Copyright 2022, Springer Nature. (C) Illustration of (a) the synthesis LaNiCo/Pd nanozymes, (b) Exo‐DNAzyme cascade program, and (c) the PEC/VCL sensor based on LaNiCo/Pd nanozymes and Exo‐DNAzyme cascade program for CTCs assay. Reproduced with permission [99]. Copyright 2025, Elsevier. (D) Schematic illustration of the Au/Pt@ZIF‐90 nanozyme capsule‐based “explosive” signal amplifier for POCT. Reproduced with permission [100]. Copyright 2024, ACS Publishing.

Cascade catalytic amplification strategies integrating DNAzymes, bio‐enzymes and nanozymes have been widely employed in POCT of biomarkers. In Yang's work [99], La‐doped NiCo₂O₄ sea‐urchin structures with palladium modification (LaNiCo/Pd SUs) nanozymes were synthesized. By combining the LaNiCo/Pd nanozymes with an Exo‐DNAzyme cascade strategy, ultrasensitive detection of CTCs was achieved (Figure 5C). In another work by Zhou et al., [101], a MOF‐based nanozyme MIL‐53(Fe) served as a carrier for covalent immobilization of G4‐DNAzyme, forming a MIL‐53(Fe)/G4‐hemin composite with exceptional POD‐like activity. Target alkaline phosphatase (ALP) hydrolyzed AAP to generate ascorbic acid (AA), which subsequently inhibited the ABTS oxidation catalyzed by MIL‐53(Fe)/G4‐hemin. This method enabled ultrasensitive ALP detection through monitoring the colorimetric signal of ABTS oxidation.

In contrast to signal amplification techniques such as nucleic acid amplification and nanozyme cascade catalysis, encapsulation‐release strategies represent a more straightforward “one‐to‐many” signal amplification approach. Essentially, these strategies involve pre‐encapsulating nanozymes within a carrier structure. Upon target recognition by surface‐conjugated recognition elements, the carrier structure undergoes disruption, leading to massive release of nanozymes and consequent signal amplification. In our previous work (Figure 5D), Au/Pt NPs with exceptional POD‐like activity were synthesized and encapsulated within ZIF‐90 frameworks, forming size‐controllable nanozyme capsules capable of “explosive” release of Au/Pt NPs. Utilizing these nanozyme capsules as signal output modules and aptamers as recognition elements, an ultrasensitive detection platform for adenosine and cortisol was established through this one‐to‐many amplification strategy [100]. Similarly, Ji's group [102] developed a colorimetric immunosensing system for recombinant SARS‐CoV‐2 nucleocapsid antigen (CoVN1) by loading ultrasmall manganese dioxide quantum dots into liposomes (MnO₂ QDs@Lip). Based on the excellent catalytic activity of MnO₂ QDs and the high encapsulation efficiency of liposomes, the system can detect COVID‐19 virus in 20 min with a detection limit of 65 fg/mL.

It is worth noting that the related indirect signal amplification often requires extra steps, such as specific solution environments or the introduction of exogenous primers. These additional requirements tend to complicate the analytical process. Therefore, achieving a balance between high sensitivity and operational simplicity remains an essential direction for future development, with enhancing user‐friendliness representing a key consideration.

3.3. Nanozymes‐Based Signal Conversion and Output

Of particular note, nanozymes serve as the core component in constructing POCT platforms. Their another unique appeal lies in the ability to transform target concentrations into multimodally readable signals through catalytic reactions. In contrast to conventional signal output methods, the innovative advantage of nanozymes is their tunable and programmable signal output types and intensities. This programmability effectively overcomes the reliance on large‐scale instrumentation typically required for traditional biosensing. Current common signal outputs derived from target recognition include optical, photothermal [103, 104], chemiluminescence [105], and electrochemical signals, as well as signals detectable by miniaturized handheld devices, such as glucose levels, pressure, and distance [106].

3.3.1. Optical Signal

Optical signals are the most prevalent output modality in nanozyme‐mediated POCT biosensors, owing to their straightforward readout and compatibility with portable devices. By mimicking enzymatic activities such as POD and OXD, nanozymes catalyze specific substrates to generate colorimetric, fluorescent, or Raman signals. These signals can be directly coupled with POCT platforms such as LFA strips, smartphones, and portable detectors, enabling rapid on‐site target detection.

Within optical signaling systems, colorimetric signals have emerged as the most readily deployable format in POCT scenarios due to their “naked‐eye readability” and minimal dependence on instrumentation. Leveraging the POD‐like or OXD‐like activities of nanozymes, a “catalysis‐color development” signaling pathway can be established. In such biosensors, nanozymes catalyze chromogenic substrates (e.g., TMB, ABTS, OPD) in the presence of electron acceptors to produce visually detectable signals, with TMB being the most widely employed substrate [76, 107, 108, 109, 110]. LFA strips represent the most common nanozyme‐based colorimetric POCT platform, allowing for qualitative or semiquantitative readout of targets simply by applying a sample. The global COVID‐19 pandemic greatly accelerated the development of LFIA‐based rapid diagnostics. Their speed, affordability, and ease of use have not only gained public acceptance but also demonstrated advantages over conventional medical diagnostic systems in numerous settings. Zhang et al. [111] developed a label‐free LFIA platform using PtMnIr nanozymes for Salmonella typhimurium detection. The synthesized PtMnIr nanozymes which possesses excellent POD‐like activity can directly adhere to the pathogen surface and catalyze the color change of oxidized TMB. The LOD reached 10⁴ CFU/mL in colorimetric mode and was further improved to 10³ CFU/mL in catalytic mode (Figure 6A). For highly sensitive detection of S. aureus, Liu et al. [114], designed and prepared Pd/Pt nanoparticles exhibiting outstanding POD‐like activity. By leveraging the color change and the strong photothermal effect of oxTMB, a colorimetric‐photothermal dual‐mode LFIA platform was constructed for S. aureus analysis.

FIGURE 6.

(A) Schematic diagram of PtMnIr nanozyme based LFIAs and its advantages. Reproduced with permission [111]. Copyright 2025, Elsevier. (B) Schematic representation of early diagnosis of prostate cancer based on PtCd nanozyme and smartphone signal reading. Reproduced with permission [112]. Copyright 2024, Elsevier. (C) Illustration of the CoPt₃@HA nanozyme based microfluidic chip for colorimetric detection of colorectal cancer. Reproduced with permission [113]. Copyright 2024, ACS Publishing.

As consumer demands for POCT, there is a growing need not only for qualitative analysis but also for rapid quantitative detection of biomarkers. While LFA strips allow qualitative detection of various targets, their capacity for accurate quantification remains limited. A promising approach to achieve rapid, low‐cost, and quantitative detection lies in combining colorimetric signal outputs with the growing ubiquity of smartphones, which enable fast signal acquisition and subsequent data processing. Several reported systems have utilized POD‐like nanozymes in smartphone‐based colorimetric biosensors for detecting targets such as H₂O₂ and glucose [115, 116, 117, 118, 119]. The high catalytic efficiency of nanozymes, together with their specificity toward various targets, coupled with the ability of smartphones to analyze multiple types of signals, positions such platforms as powerful tools for multianalyte detection. Zhang et al. [112] developed a smartphone‐integrated immunosensor employing platinum–cadmium nanoclusters (PtCd NCs) with POD‐like activity as the key sensing element for detecting PSA in blood samples. A sandwich immunoassay constructed in a microtiter plate enabled specific capture of PSA via antibodies. The PtCd NCs then catalyzed the conversion of H₂O₂ into ·OH, which oxidized TMB to generate a blue‐colored oxTMB. Quantification of PSA was accomplished using the smartphone's built‐in red‐green‐blue (RGB) color analysis system. Notably, to further simplify operational procedures, minimize sample consumption, and advance POCT capabilities, microfluidic devices supporting visual detection of biomarkers have attracted considerable interest (Figure 6B). Li's group [113] developed a lateral displacement patterned, pump‐free microfluidic chip (LP‐chip) based on CoPt₃ nanozymes for detecting colorectal cancer stem cells (CCSCs) in peripheral blood and stool samples. By employing HA‐functionalized CoPt₃ nanoparticles that specifically bind to the CD44 marker on CCSCs, the chip enables magnetic separation of the target cells. Under an external magnetic field, the isolated CCSCs complexes are guided into the chip and captured at an HA‐modified detection line. Subsequently, the CoPt₃ nanozymes catalyze the reduction of dopamine to polydopamine in the presence of H₂O₂, generating a colorimetric signal that is quantified using a smartphone. This integrated approach achieves automated and portable CCSCs detection (Figure 6C).

3.3.2. Photothermal Signal

Photothermal signals utilize the synergistic combination of nanozymes’ photothermal conversion capability and catalytic activity to transform target concentration into quantifiable temperature readings, making them particularly suitable for accurate detection in complex sample matrices encountered in POCT environments. Compared to optical signals that are vulnerable to interference from sample turbidity and background absorption, photothermal signals rely on temperature as a parameter, effectively avoiding optical noise. Moreover, temperature detection devices such as portable thermometers and infrared thermal imagers are cost‐effective and easily miniaturized, establishing photothermal signaling as a valuable complementary approach in nanozyme‐based POCT systems [17].

In photothermal sensing platforms, nanozymes function through two main mechanisms. The first mechanism involves photothermal agent–catalyst synergy, where the nanozyme possesses both photothermal conversion ability and enzymatic activity, with targets indirectly modulating the photothermal effect by regulating enzyme activity. The second mechanism relies on catalytic product‐mediated photothermal conversion, where nanozymes catalyze substrates to generate products with photothermal properties, and the resulting photothermal signal intensity correlates with the target concentration.

The photothermal agent–catalyst synergy mechanism centers on the interconnected regulation of nanozymes’ inherent photothermal properties and enzymatic activities. For instance, Su et al. [120] synthesized MnO₂/Cu‐Mn nanosheets possessing exceptional laccase‐like catalytic activity and photothermal properties. They discovered that glutathione S‐transferase (GST) could restore both the laccase activity and the photothermal performance of the Cu–Mn nanozyme, which had been attenuated by glutathione (GSH). Based on this mechanism, they developed a GST‐modulated colorimetric/photothermal dual‐mode sensing method. This approach enables quantitative measurement of GST levels in human serum and has been successfully applied in preliminary assessment of hepatitis patients (Figure 7A).

FIGURE 7.

(A) Synthesis of Cu–Mn nanozymes and its photothermal sensing of GST. Reproduced with permission [120]. Copyright 2024, Elsevier. (B) Illustration of the dual‐mode microfluidic device for electrochemical detection of PCC‐CTC. Reproduced with permission [129]. Copyright 2022, Elsevier. (C) Schematic illustration of commercial glucometer‐based label‐free read‐out of PCR amplification using GOx‐like activity of CeO₂ NP. Reproduced with permission [137]. (D) Schematic diagram of sensing principle based on portable pressure meter. Reproduced with permission [138]. Copyright 2015, Wiley‐VCH. (E) Schematic diagram of a volumetric bar chart chip based on nanozymes with CAT‐like activity for the detection of CTCs, (a) sample preparation and aptamer conjugation, (b) sample loading in the sample chambers, (c) addition of H₂O₂ and ink, (d) adjust the signal reading channel, and (e) nanozyme catalyzed O₂ generation and signal readout. Reproduced with permission [145]. Copyright 2019, Wiley‐VCH.

In contrast, the catalytic product‐mediated photothermal mechanism relies on the photothermal activity of specific products generated through nanozyme catalysis. A representative example involves the substrate TMB, which exhibits negligible photothermal effect in its native state. However, upon nanozyme‐catalyzed oxidation, the reaction product oxTMB demonstrates strong absorption in the near‐infrared (NIR) region and exhibits excellent photothermal conversion efficiency [121]. In this mechanism, the target analyte influences the catalytic efficiency of the nanozyme toward TMB, thereby regulating the production amount of oxTMB and consequently modifying the system's photothermal signal. Xue's group [122] developed an enhanced photothermal sensor for cholesterol (CHOL) detection by integrating the photothermal effect and POD‐like activity of an inorganic/organic nanocomposite. In their design, CHOL selectively binds to the PB@PDA@β‐CD nanoprobe, effectively suppressing both its photothermal response and POD‐like activity. This suppression significantly reduced the photothermal response from nanozyme‐catalyzed oxTMB formation, enabling the development of a dual‐channel signal‐amplified photothermal detection method successfully applied to CHOL quantification.

Photothermal signals demonstrate notable advantages in POCT applications involving complex samples, with strong anti‐interference capacity and compatibility with inexpensive readout devices, including conventional thermometers, making them particularly suitable for resource‐limited settings. However, limitations remain, including the requirement for external NIR light sources, which increases equipment costs and imposes relatively strict environmental requirements. Consequently, addressing challenges related to batch‐to‐batch stability and reproducibility represents a crucial issue requiring resolution in photothermal signal‐based POCT development.

3.3.3. Electrochemical Signal

Electrochemical signals offer significant advantages for POCT of trace targets, owing to their high sensitivity, miniaturization capability, and quantitative nature. By leveraging the catalytic activity or interfacial electron transfer modulation of nanozymes, these systems convert target concentrations into measurable current, potential, or impedance signals. The operational mechanisms of nanozyme‐based electrochemical POCT platforms can be primarily categorized into two distinct types. The first is electrocatalytic activity modulation, where target molecules quantitatively regulate detection by either activating or inhibiting the nanozyme's catalytic activity, thereby altering the oxidation/reduction current of electroactive species in the system. This class of nanozyme‐based electrochemical sensors has been extensively utilized for detecting various metabolites including H₂O₂ [123, 124, 125], glucose [126, 127], and uric acid [128]. The second mechanism employs a signal label amplification strategy, where nanozymes function as “signal tags” that, through specific binding interactions such as immunorecognition or nucleic acid affinity, indirectly influence the activity or loading amount of electrode‐immobilized nanozymes. This results in significant changes in electrochemical signals, enabling sensitive detection of nonelectroactive targets including proteins and nucleic acids. Li et al. [129] developed an integrated dual‐mode microfluidic device for rapid, sensitive detection of pheochromocytoma‐derived circulating tumor cells (PCC‐CTCs). First, target CTCs are captured and enriched from blood samples using antibody‐functionalized Fe₃O₄@SiO₂ magnetic particles; then, a platinum‐based COF (COF@Pt) functionalized with meta‐iodobenzylguanidine (MIBG) and octreotide‐2,2′,2′′,2′′′‐(1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrayl)tetraacetic acid (DOTA) labels is employed as a POD‐like nanozyme, which can catalyzes the reduction of H₂O₂ for electrochemical detection while simultaneously generating an amplified colorimetric signal. The microfluidic chip automates the entire process, enabling dual‐mode quantitative assessment of PCC‐CTCs with high sensitivity through integrated electrochemical detection and smartphone‐based colorimetric analysis (Figure 7B). This approach mirrors broader applications where antibodies or aptamers enable ultrasensitive detection of proteins [130, 131], nucleic acids [132, 133], and cells [134]. Owing to structural simplicity and ease of integration, nanozyme‐based electrochemical biosensors hold significant potential for POCT applications, particularly in the development of portable or wearable devices [135].

3.3.4. Other Signals

Recent technological advances have spurred the widespread development of low‐cost, simple, and robust portable devices. This progress enables researchers to utilize nanozymes for converting target recognition events in POCT into signals such as glucose concentration, pressure, or distance. Relying on portable instruments like glucometers and pressure meters, these strategies substantially broaden the signal output dimensions for POCT.

The miniaturization of healthcare devices benefits from continuous progress and integration of multidisciplinary technologies. The glucometer, being one of the earliest such devices introduced for home use, is widely employed for blood glucose monitoring due to its operational simplicity and efficiency. Expanding this miniaturized platform to detect nonglucose targets is highly meaningful. Essential to this approach is the transduction of a target‐specific signal into a measurable glucose signal. Nanozymes exhibiting GOx‐like activity can directly or indirectly catalyze glucose production or consumption [136]. Based on this strategy, Park et al. [137] synthesized cerium oxide nanoparticles (CeO₂ NPs) exhibiting GOx‐like activity and developed a POC method for on‐site readout of polymerase chain reaction (PCR) results using a glucometer. In the presence of target DNA, PCR amplicons bind to CeO₂ NPs via electrostatic interactions, inducing nanoparticle aggregation and consequently decreasing the catalytic efficiency in glucose oxidation. This process establishes a correlation between DNA amplicon concentration and glucose level, permitting ultrarapid analysis of PCR results using only a commercial glucometer as the detection component (Figure 7C).

Pressure signals often closely relate to the CAT‐like activity of nanozymes. Conventional strategies involve the direct or indirect modulation of CAT‐like nanozyme activity by biological targets, or the triggering of binding and release of CAT‐like nanozymes via biological targets, thereby inducing changes in internal pressure within the system. The target can be visually read by using a handheld pressure meter. Yang's group [138] pioneered the use of a handheld pressure meter for highly sensitive bioanalysis. In this design, CAT or Pt NPs served as catalysts in a sandwich ELISA. Target presence induced rapid decomposition of H₂O₂ into O₂, generating a detectable pressure change within a sealed container. This allowed target concentration to be rapidly determined using a pressure meter (Figure 7D). Then, numerous pressure meter‐based biosensors have since been developed for various targets, including small molecules [139], nucleic acids [140, 141], proteins [142, 143], and pathogens [9].

To further eliminate instrumental requirements, subsequent research has focused on converting pressure signals into other readable outputs. Yang [144] reported a volumetric bar‐chart chip, termed V‐Chip. This chip was preloaded with ink and a target‐responsive aptamer‐crosslinked hydrogel. Target presence induced hydrogel decomposition, releasing embedded Au@Pt NPs. These nanoparticles then catalyzed H₂O₂ decomposition to produce O₂, which propelled the movement of an ink bar, thereby generating a target‐dependent, equipment‐free distance signal readable by the naked eye. Furthermore, the V‐Chip platform was adapted for detection of CTCs. As shown in (Figure 7E), aptamer‐conjugated nanoparticles were used to label target CTCs. Subsequent analysis in the V‐Chip quantified cells based on the ACNP‐catalyzed decomposition of H₂O₂ and production of O₂. The resulting distance of the ink bar showed a positive correlation with target cell concentration, enabling visual quantification of CTCs with a sensitivity sufficient to detect a single cell [145].

Beyond CAT‐like nanozymes‐based platforms, nanozymes with POD‐like or OXD‐like activities also facilitate distance‐based signal transduction. Khachornsakkul [146] developed a distance‐based paper analytical device (dPAD) for monitoring sarcosine levels in human samples. This dPAD incorporated a sample zone predeposited with sarcosine oxidase (SOx) and CDs with POD‐like activity, together with a detection zone containing TMB. Introducing a sarcosine‐containing solution to the sample zone generated H₂O₂ via SOx oxidation. The CDs then catalyzed H₂O₂ to produce hydroxyl radicals, which migrated to the detection zone via capillary action and oxidized TMB, causing a color change from colorless to blue. Measuring the length of the colored zone enabled visual quantification of sarcosine. By using a similar strategy, the group also achieved distance‐based detection of uric acid [147].

4. Integrated Application of Nanozymes‐Based POCT

Based on the role and advantages of nanozyme in target recognition, signal conversion and output, and signal amplification, it is an important research trend and direction of POCT to integrate them with related simple and portable devices/equipment to realize rapid, sensitive, and low‐cost detection of biomarkers such as small molecules, nucleic acid molecules, protein, cells, and viruses. Here, the application of nanozyme‐mediated POCT in the determination of heterogeneous biological targets is discussed comprehensively (Table 1).

TABLE 1.

List examples of POCT platform based on nanozyme for detection of small molecules, nucleic acids, proteins, cells, and pathogens.

Classification	Target	Label	Enzymes‐like activities	Signal	Detection methods	LOD	References
Small molecules	H2O2	Fe3O4@Chitosan	POD	Colorimetric	Microfluidic paper‐based analytical device (µPAD) (Smartphone)	69 nM	[148]
	Adenosine	Au/Pt@ZIF‐90	POD	Colorimetric	Smartphone	0.71 nM	[100]
	Uric acid	AuNS	OXD	Colorimetric	Smartphone	6.2 μM	[149]
	Adenosine triphosphate triphosphate	Pt NCs@ZIF‐90	OXD	Colorimetric	Spectrophotometer	0.062 μM	[7]
	Dopamine	FeCuAgCeGd–HEAzyme	POD	Colorimetric	Spectrophotometer	0.77 µM	[150]
	Glucose	Fe‐MIL‐101	POD	Colorimetric	μPAD (Digital camera)	2.5 μM	[151]
	Vitamin C	CuFe‐MOF	POD	Colorimetric	Hydrogel patch (smartphone)	1.707 μM	[152]
	Cholesterol/Glucose	V‐porous Co3O4	POD	Colorimetric	Mpad (Smartphone)	0.0275 mM, 0.0281 mM	[153]
	Glutathione	MnO2 nanosheets	OXD	Colorimetric	Spectrophotometer	300 nM	[154]
	AA	MOF‐808	POD	Colorimetric	Spectrophotometer	15 μM	[155]
	H2O2	Au@PtNP/GO	POD	Electrochemical	Film‐based microfluidic chips, (Electrochemical analyzer)	1.62 μM	[156]
Nucleic acid	Nucleic acid of SARS‐CoV‐2	FeS2	POD	Colorimetric	Test strips (Immunoquantitative analyzer)	200 copies/mL	[157]
	ctDNA	CoB NS	POD/CAT	Colorimetric/barometric	Smartphone/pressure meter	95.81 pM/79.88 pM	[141]
	ctDNA	MnB	OXD	Colorimetric	Smartphone	52.18 pM	[158]
	mircoRNA	Co‐MOF	POD	Electrochemical	Electrochemical work station	0.12 pM	[133]
	miRNA‐21	Pt@Au	POD	Colorimetric	Spectrophotometer	0.5 pM	[159]
	miRNA‐107	Au nanospheres	POD	Raman	Portable IM‐52 Raman microscope	0.7 fM	[160]
	HIV‐DNA	Pt/Ti3C2Tx	POD	Colorimetric	LFIA strips (Smartphone)	0.089 nM	[161]
	HIV‐DNA	NiFe2O4@UiO‐66	POD	Colorimetric/Fluorescence	Spectrophotometer	1 fM	[162]
	miRNA‐141	Au@CD NP, CeO2	GOx, POD	Photoelectrochemical	Paper‐based photoelectrochemical sensor (Electrochemical work station)	0.6 fM	[163]
Proteins	CEA	PtNPs	CAT	Resistance	Microfluidic plate	167 pg/mL	[164]
	PSA	Au/Pt NPs	POD/CAT	Colorimetric/TDS	Spectrophotometer/Portable water quality testing pen	0.15 ng/L, 0.57 ng/L	[49]
	Human gonadotropin (hCG)	MPt/CS NPs	POD	Colorimetric	LFIA strips (Smartphone)	0.025 ng/mL	[165]
	Urease	Cu@Zr	Phosphatase/Laccase	Colorimetric/Fluorescence	Portable swab (Smartphone)	3.56 U/L, 1.83 U/L	[166]
	Thrombin	CeO2 NRs	Phosphatase	pH	pH meter	1.17 nM	[167]
	PSA	Fe3O4 NPs	POD	Colorimetric/ Photothermal	Spectrophotometer/Thermometer	1.0 ng·mL–1	[168]
	Thrombin	PtNPs	CAT	Weight	Electronic balance	2.8 nM	[169]
	CEA	PtNPs	CAT	Temperature	Thermometer	0.6 pg/mL	[170]
	Amyloids	AuNPs	GOx	Fluorescence	Spectrophotometer (machine learning algorithms)	∼	[36]
	Alkaline Phosphatase	Fe–N–C	OXD	Coulometric/ Colorimetric	Spectrophotometer (Smartphone)	3.38 U L−1, 2.12 U L−1	[171]
Cells and viruses	S. aureus	BCNNTs	POD	Colorimetric	Spectrophotometer	1.05 CFU/ mL	[172]
	S. Enteritidis, E. coli O157:H7	Pd@Pt NPs	POD	Colorimetric	LFIA strips (Smartphone)	≈20 CFU/mL, ≈34 CFU/mL	[161]
	CTCs	FA‐PtCPs NRs	OXD	Colorimetric/ Photothermal	Spectrophotometer/Thermometer	2 cells/mL	[173]
	Cancer Cells	FA‐PEG‐Au NFs	POD/CAT	Pressure/ Photothermal	Pressure meter/Digital thermometer	6 cells/mL, 5 cells/mL	[174]
	Escherichia coli O157:H7	MPnS	POD	Colorimetric	LFIA strips (Smartphone)	≈103 CFUs	[175]
	CTCs	MNPs	POD	Colorimetric	Spectrophotometer	13 cells/mL	[176]
	CTCs	Au@CuMOF	GOx/POD	Glucose	Microfluidic device (portable glucose meter)	3 cells/mL	[177]
	CTCs	AuIrPt	SOD/POD/CAT	Electrochemical	Electrochemical workstation	2 cells/mL	[178]
	CTCs	CoPt3	POD	Colorimetric	LP‐chip (Smartphone)	3 cells mL−1	[113]

4.1. POCT of Small Molecules

As an important ROS, H₂O₂ plays critical roles in biological signal transduction and oxidative stress processes. Its detection is particularly valuable in disease diagnosis, as it assists clinicians and researchers in studying disease progression and mechanisms, such as in the early diagnosis of vascular diseases [179]. As a standard substrate for both CAT and POD, researchers have developed various nanozymes with POD‐like and CAT‐like activities, enabling simple, rapid, and highly sensitive POCT of H₂O₂. Huo et al. [180] prepared porous Fe single atoms/Co‐doped carbon nanomaterials (Fe SAs/Co CNs) with a high specific surface area using FeCo porous carbon nanosheets (FeCo PCNs) and graphitic carbon nitride (g‐C3N4) as raw materials. Based on the excellent POD‐like activity, the Fe SAs/Co CNs was integrated with a portable electrochemical sensing chip equipped with microfluidic channels, achieving sensitive detection of H₂O₂ in serum with a detection limit as low as 0.36 μM. Furthermore, TMB was preloaded into a CuBDC MOF by Wang et al. [181], followed by the synthesis of CuBDC‐TMB nanoparticles (CuBDC‐TMB‐NPs). Leveraging the excellent POD‐like activity of CuBDC‐TMB‐NPs, test strips and a smartphone were jointly applied, and the rapid visual detection of H₂O₂ was finally realized.

Notably, as a downstream product of small molecules such as glucose, uric acid, purine, and cholesterol, H₂O₂ can be in‐situ generated via the catalytic reaction of relevant OXDs; based on this characteristic, the indirect POCT detection of upstream biomarkers can be achieved through the detection of H₂O₂ content. Glucose is the most typical biomarker in this kind of detection, and its concentration changes are closely associated with diseases like diabetes. In Zhang's work [182], Au NPs with GOx‐like activity were encapsulated into the tip layer of microneedles (MNs), and a colorimetric MN patch was thereby designed. This MN patch can rapidly absorb a large amount of skin interstitial fluid, and the rapid detection of glucose was realized by combining a smartphone with Image J software (Figure 8A). Du et al. [183] synthesized PDAFeNi foam with excellent POD‐like activity by growing polydopamine nanoparticles (PDNPs) on activated FeNi foam, immobilized GOX on DAFeNi foam to realize cascade enzyme catalysis, and developed a sensing device including low‐light photographic equipment and digital image application, which can detect glucose in urine within 6 min by using smartphone (Figure 8B).

FIGURE 8.

(A) Schematic diagram of glucose detection based on MN patch. Reproduced with permission [151]. Copyright 2024, Elsevier. (B) Smartphone‐based rapid visual detection of urine glucose with PDAFeNi foam. Reproduced with permission [152]. Copyright 2024, Elsevier. (C) The synthesis of Fe‐CQDs nanozymes and their application in uric acid detection. Reproduced with permission [153]. Copyright 2025, Elsevier. (D) Schematic diagram of uric acid detection based on MPB nanozyme. Reproduced with permission [154]. Copyright 2020, Elsevier. (E) Schematic diagram of cortisol photothermal and colorimetric detection based on Au/Cu₂S/MoS₂ nanozyme. Reproduced with permission [156]. Copyright 2024, Elsevier.

As another typical upstream molecule of H₂O₂, elevated levels of uric acid in the blood often indicate the occurrence of gout, hyperuricemia, and kidney diseases. Gong et al. [184] synthesized Fe‐functionalized carbon quantum dots (Fe‐CQDs) with POD‐like activity; by constructing a cascade reaction system between these nanozymes and uricase, and integrating smartphone‐assisted visible light imaging, the visual detection of uric acid was finally achieved (Figure 8C). Based on the reducibility of uric acid, Yang's group [185] developed a uric acid POCT platform, which comprises a lateral flow pad (LFP), mesoporous Prussian blue nanoparticles (MPBs) nanozyme, and automatic calculation software. The main mechanism is that TMB can be oxidized to blue oxTMB in the presence of nanozyme, however, when uric acid is present, oxTMB is reduced, resulting in a distinct color change of the system. By capturing and automatically analyzing the color signals with a smartphone and custom software, the rapid detection of uric acid in whole blood can be realized (Figure 8D).

Beyond POCT detection of conventional small‐molecule disease biomarkers such as glucose, POCT strategies for small‐molecule biomarkers based on aptamer recognition, in which nanozymes undertake signal conversion, output, and amplification, have also been extensively developed. To enhance the catalytic activity and selectivity of nanozymes, Willner [186] proposed the concept of “Aptananozymes": by utilizing the high‐affinity binding between nucleic acid aptamers and substrates, an anchoring system for substrate accumulation at the solid‐phase catalytic interface was established; meanwhile, through adjusting the length of the spacer unit between the aptamer and catalyst, precise regulation of the catalytic activity of “Aptananozymes” was achieved. This work verifies that through strategic design, the specificity of nanozymes can be effectively improved, while their catalytic performance in biosensing systems can also be significantly boosted. Stress is ubiquitous in modern society, and excessive stress can trigger a variety of psychological and physical health issues. As a core biomarker for stress detection, cortisol can be used to monitor stress levels in different populations, providing accurate and reliable evidence for the diagnosis and treatment of related diseases. In Liu's work [187], maple leaf‐shaped Au/Cu₂S/MoS₂ (ACM) nanomaterials with photothermal conversion capability and excellent POD‐like activity were successfully synthesized. Via DNA modification and mixed incubation, “Y”shaped signal probes were prepared and immobilized on the surface of cortisol aptamer‐functionalized magnetic beads (MB‐Aptcortisol). When cortisol is present, the Y‐shaped signal probe was released to oxidize TMB. Finally, the signal transduction, signal amplification and smartphone‐based visual reading are realized by cascading enzyme reactions (Figure 8E).

4.2. POCT of Nucleic Acid

Nucleic acid diagnostic technologies provide a powerful technical means for acquiring disease‐related key information at the molecular level. This information is of great significance for timely diagnosis of patients’ diseases, making treatment plans and blocking the spread of harmful pathogens [188]. Circulating nucleic acids (i.e., DNA and RNA fragments) are widely present in body fluids and cancer cells. The content changes usually indicate the occurrence and development of related diseases. Therefore, conducting rapid quantitative analysis of nucleic acid fragments is of great value for the early diagnosis of diseases [189].

Over the past few decades, researchers have developed numerous nucleic acid detection strategies based on principles such as nucleic acid amplification, nanozyme‐mediated signal conversion, and signal amplification. MicroRNAs (miRNAs) are a class of small‐molecule RNAs that can precisely regulate gene expression, and their expression levels are closely associated with the occurrence and development of various diseases including cancer. Therefore, the detection of specific miRNAs serves as a highly valuable disease diagnostic approach, providing important evidence for early disease detection and condition monitoring [190]. Yu et al. [191] developed an ultrasensitive catalytically amplified electrochemical biosensor based on microfluidic paper‐based analytical devices (μPADs) for the detection of microRNA‐21. In this study, CeO₂‐Au@GOx with both POD‐like activity and GOx‐like activity was synthesized and used as the electrochemical probe. Meanwhile, μPADs modified with gold nanorods (Au NRs) (Au‐μPADs) were employed to immobilize hairpin probe DNA (HP). When the target miRNA is present, it binds to HP and unfolds the hairpin structure of HP; subsequently, HP further hybridizes with capture DNA (capDNA) labeled by the electrochemical probe. Finally, dual outputs of colorimetric and electrochemical signals were achieved on Au‐μPADs and providing a rapid and sensitive method for microRNA‐21 detection. Hu et al. [192] from West China Hospital of Sichuan University developed an on‐site visual detection technology for tumor‐associated miRNAs based on Ru@TiO₂ nanozymes. In this study, the strand displacement reaction triggered by miRNAs was used to initiate the RCA process and continuously release pyrophosphate (PPi). PPi can coordinate with Ru@TiO₂ nanoenzyme with POD‐like activity, block its active site and inhibit H₂O₂ decomposition, thereby realizing the regulation of miRNA‐related colorimetric signals and rapid quantitative analysis (Figure 9A).

FIGURE 9.

(A) Schematic illustration of colorimetric miRNAs detection using the POD‐like activity of Ru@TiO₂ nanozyme. Reproduced with permission [192]. Copyright 2024, ACS Publishing. (B) Synthesis of CoB NS nanozyme and detection of ctDNA based on colorimetric/pressure signal. Reproduced with permission [141]. Copyright 2025, ACS Publishing. (C) Illustration of Pt/Ti₃C₂Tx‐amplified dual‐readout CLFB for HIV‐DNA detection. Reproduced with permission [193]. Copyright 2023, Elsevier. (D) Schematic illustration of CL detection of HIV‐DNA by a handheld luminometer. Reproduced with permission [194]. Copyright 2024, Elsevier. (E) SARS‐CoV‐2 nucleic acid detection based on IMSN with POD‐like activity. Reproduced with permission [35]. Copyright 2023, Wiley‐VCH.

ctDNA is a free DNA fragment with tumor mutation, which contains tumor‐specific gene characteristics and epigenetic characteristics. Its half‐life is short and its content is extremely low, which can reflect tumor dynamics in real time. Therefore, the rapid detection of ctDNA can realize early screening, curative effect evaluation and early recurrence warning of tumor. Yang's group from Chongqing University [158] was the first to apply MnB₂ nanozymes with OXD‐like activity to ctDNA detection. Through experiments and density functional theory (DFT) calculations, they investigated the electronic structure and catalytic reaction pathway of MnB₂ nanozymes, and revealed their catalytic active sites and reaction mechanisms. By combining MnB₂ nanozymes with μPADs and based on a developed android‐based smartphone application (which can analyze the reaction color of μPADs via image processing), portable and efficient detection of ctDNA was achieved, providing a new POCT tool for early cancer diagnosis. Moreover, they also proposed a new POCT platform for ctDNA, as shown in Figure 9B, cobalt boride nanosheets (CoB NS) with double enzyme‐like activities of POD‐like and CAT‐like was synthesized and designed a card‐like paper‐based chip with a three‐layer structure of top shunting layer, middle detection layer and bottom collection layer. Combined with the dual‐mode reading system of smartphone and pressure meter, the convenient and efficient POCT of lung cancer ctDNA was realized [141].

In addition to endogenous pathogenic nucleic acid molecules, rapid detection of pathogenic bacteria and viral nucleic acid molecules is also crucial for disease prevention and diagnosis. In recent years, nanozymes have been widely applied in POCT detection of nucleic acids from pathogenic bacteria and viruses such as HIV and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Due to the large specific surface area and abundant binding sites of Ti₃C₂T _x nanosheets, Wen et al. [193] synthesized Pt/Ti₃C₂T _x with excellent POD‐like activity by loading multiple Pt NPs onto Ti₃C₂T _x nanosheets, and used it as a signal marker to construct a colorimetric lateral flow biosensor (CLFB) and realize the dual‐signal POCT of HIV‐DNA (Figure 9C). Jin et al. [194] constructed a chemiluminescence POCT platform for HIV‐DNA detection, enabling quantitative reading of HIV‐DNA using a handheld photometer. Its core lies in that it can induce signal amplification reactions such as SDR and assemble in situ G‐triplex hemin DNAzyme (G3‐hemin DNAzyme) with POD‐like activity in the presence of HIV‐DNA, and then output ECL signals (Figure 9D). The global pandemic of coronavirus disease 2019 (COVID‐19) has seriously threatened human life safety, making sensitive and rapid detection of severe acute respiratory SARS‐CoV‐2 of great significance. Gao's group [157] developed a platform combining recombinase polymerase amplification (RPA) with an FeS₂ nanozyme test strip. By leveraging the exceptional POD‐like activity of FeS₂ nanozymes, this method enables simplified nucleic acid amplification with subsequent colorimetric signal enhancement, achieving a detection limit of 200 copies/mL for SARS‐CoV‐2 RNA, thus demonstrating sensitivity comparable to that of RT‐PCR. Liu et al. [35] designed a nanozyme‐based naked‐eye colorimetric method for detecting SARS‐CoV‐2 nucleic acids by utilizing the inhibitory effect of PPi‐massively produced during nucleic acid amplification‐on the POD‐like activity of nanozymes. In their work, iron manganese silicate nanozyme (IMSN) with POD‐like activity was synthesized; reverse transcription and PCR amplification of the samples were performed, and based on the inhibitory effect of PPi generated during reverse transcription and amplification on the activity of IMSN, naked‐eye visual detection of the ORF1ab and N genes in COVID‐19 clinical samples was finally achieved (Figure 9E).

4.3. POCT of Biomacromolecules

As core functional executors of life activities in organisms, proteins are closely associated with the occurrence and development of diseases due to abnormalities in their expression levels, alterations in post‐translational modification states, and dysregulation of their interactions. Such molecules constitute one of the most important categories of biomarkers in the clinical diagnosis of various diseases, including tumors, cardiovascular diseases, neurodegenerative diseases, and infectious diseases. Therefore, achieving POCT detection of relevant protein biomarkers is of great significance for disease prevention, early diagnosis, and prognostic evaluation.

Early screening of tumor marker proteins is crucial for early diagnosis and treatment of cancer. In the detection of tumor marker proteins, the signal conversion and amplification effect of nanozyme significantly improves the detection performance. Capitalizing on the unique advantages of nanozymes in signal transduction and output, Yu et al. [195] ingeniously converted target recognition events into colorimetric, thermal, and distance‐based signals, enabling visual detection of glucose and CEA. Based on the OXD‐like activity of MnO₂ nanoflowers (MnO₂ NFs), specific antigen–antibody interactions, and the gel–sol transition behavior of gelatin, the team successfully transformed CEA concentrations into visually interpretable signals. The platform ultimately facilitated target detection through integrated readouts using smartphones, portable thermal imagers, and pH test strips (Figure 10A). In addition, Ju et al. [196] achieved dual‐signal amplification for tumor biomarker detection by enhancing antibody utilization efficiency and employing Au@Pt NPs with POD‐like activity. In their work, an enhanced LFIA (DEOAN‐LFIA) platform was designed, integrating targeted antibody anchoring with nanozyme‐catalyzed signal amplification. The platform utilized phenylboronic acid‐functionalized Au@Pt NPs to selectively capture and orient antibodies, thereby improving antibody utilization efficiency and achieving the first stage of signal amplification. Subsequently, the intrinsic POD‐like activity of Au@Pt NPs catalyzed chromogenic substrate oxidation, leading to a second amplification step. The DEOAN‐LFIA has realized the rapid detection of alpha‐fetoprotein (AFP) and CEA at picogram‐level within 25 min (Figure 10B).

FIGURE 10.

(A) Illustration of the biosensors for the detection of glucose (GOx and HRP) and CEA (MnO₂ NFs nanozyme). Reproduced with permission [195]. Copyright 2024, Elsevier. (B) Schematic of DEOAN‐LFIA with Au@Pt NPs antibody orientation by boronic acid and nanozyme catalytic amplification for ultrasensitive AFP and CEA detection. Reproduced with permission [196]. Copyright 2025, ACS Publishing. (C) Schematic of the immunosyringe sensor's working principle and its integration with bar‐chart chips for PSA detection. Reproduced with permission [197]. Copyright 2024, ACS Publishing. (D) Schematic of the construction of LFIA sensor based on Pd@Ir NPs nanozyme for PGI and PGII. Reproduced with permission [198]. Copyright 2024, ACS Publishing. (E) Schematic of LFIA sensing platform construction based on FeWO₄ nanozyme for CRP detection. Reproduced with permission [199]. Copyright 2025, ACS Publishing.

As a protein secreted by prostate epithelial cells, PSA is widely utilized in screening for prostate diseases and diagnosing prostate cancer. Fu et al. [197] developed an integrated system combining an immunosyringe sensors with a bar‐chart chip for PSA POCT. In their design, a sandwich immunoassay was constructed on the inner wall of a disposable syringe. The detection process involved immobilizing a capture antibody, followed by binding of the target antigen and subsequent recognition by a detection antibody conjugated with Pt NPs exhibiting CAT‐like activity. The catalytically active Pt NPs decomposed H₂O₂ within the sealed syringe to generate O₂, increasing internal pressure and autonomously driving fluid into the bar‐chart chip. This fluid then dissolved a solid dye in the chip's storage zone, propelling the dye through a microchannel. This mechanism transduced the immunorecognition event into a visually interpretable distance signal, enabling rapid PSA quantification via a simple “prick‐and‐read” operation (Figure 10C). Pepsinogen I (PG I) and pepsinogen II (PG II), as specific biomarkers of gastric cancer, hold significant diagnostic value for early gastric cancer detection. Ju et al. [198] prepared a bimetallic nanozyme palladium@iridium core–shell nanoparticles (Pd@Ir NPs) with excellent POD‐like activity, then systematically investigated the catalytic performance of Pd@Ir NPs with varying iridium shell thicknesses. Under optimized conditions, Pd@Ir NPs served as labeled probes that not only recognized targets but also catalyzed the in‐situ deposition of chromogenic substrates, thereby amplifying colorimetric signals. Based on this mechanism, the constructed LFIA realizes the ultrasensitive detection of PG I and PG II (Figure 10D).

Beyond tumor biomarker proteins, nanozyme‐based POCT systems for detecting inflammatory and cardiovascular disease markers such as C‐reactive protein (CRP), troponin, and acute myocardial infarction indicators have also been extensively reported. Wang et al. [200] proposed a straightforward, cost‐effective, and portable POCT platform for CRP detection using a pressure‐based readout system. By utilizing the CAT‐like activity of Pt NPs, the target recognition signal based on antibody‐antigen interaction was converted into a measurable pressure change, thereby enabling quantitative CRP detection with a handheld pressure meter. Yang et al. [199] synthesized PAA function FeWO₄ nanomaterials with exceptional POD‐like activity, and conjugated them with anti‐CRP detection antibodies to form a detection probe. This probe was subsequently integrated into a LFIA and developed a FeWO₄‐based LFIA sensing platform that enabled visual detection of CRP with a detection limit as low as 19.38 ng/mL (Figure 10E). Song et al. [201] developed a novel type of nanoparticles termed assembling allochroic nanoparticles (SANs). By encapsulating enzyme substrates within SANs during synthesis, this design eliminates the need for exogenous substrate addition during the analytical procedure, thereby enhancing detection sensitivity and significantly reducing assay time. The encapsulated substrates undergo catalytic oxidation in the presence of Cu²⁺ to generate colorimetric signals. Based on this mechanism, they constructed a nanozyme‐based LFIA platform and achieved sensitive detection of cardiac troponin I‐troponin C complex (cTnI‐TnC) and myoglobin (Myo). Heart‐type fatty acid‐binding protein (h‐FABP) serves as a crucial biomarker for early diagnosis of acute myocardial infarction. Qin et al. [202] developed a dual‐channel microfluidic PEC–photothermal immunosensor utilizing Au@Pt nanozyme‐mediated signal amplification for efficient h‐FABP detection. In this work, secondary antibodies were anchored alongside Au@Pt NPs with ascorbate OXD‐like activity, which catalyzed AA oxidation to quench the PEC signal. Concurrently, NIR light‐induced surface plasmon resonance effectively enhanced the enzyme‐mimicking activity of Au@Pt NPs. Capitalizing on this dual‐mechanism, the designed microfluidic PEC sensor incorporating dual working electrodes demonstrated significant advantages for rapid h‐FABP detection.

4.4. POCT of Cells and Pathogens

Cells (such as CTCs) and pathogenic microorganisms (including Escherichia coli O157:H7, Salmonella, and SARS‐CoV‐2) represent critical “solid” biomarkers in clinical diagnostics. Their detection, however, must overcome three major bottlenecks: challenges in efficient separation and enrichment, difficulties in trace‐level identification, and limitations in on‐site operation. Consequently, achieving rapid and accurate detection of such cells and pathogens within complex matrices holds considerable importance for early tumor screening as well as for the prevention and control of infectious diseases.

CTCs are malignant cells that detach from primary or metastatic lesions of solid tumors and enter the peripheral blood circulation. Evidence confirms that CTCs are present even during early stages of invasive tumor development [203], highlighting their significant potential for early cancer detection. Nevertheless, the detection of CTCs requires highly sensitive analytical technologies due to their exceptionally low concentration in blood. Li et al. [177] developed an integrated microfluidic system for CTCs identification, separation, and detection by combining smartphone technology with a commercial glucometer. The system employed Au@Cu MOF nanozymes functionalized with DOTA and norepinephrine (NE) as signal probes. The combination of functionalized Au@Cu MOF and CTCs endows cell complexes with the activity of GOx‐like and POD‐like, the cascade catalytic system of cell complexes subsequently enabled glucose oxidation followed by chromogenic reaction, generating simultaneously measurable colorimetric signals and glucose consumption readings. With this dual‐mode detection strategy employing smartphone imaging and portable glucometer, the platform achieved highly sensitive detection of PCC cells at a limit of 3 cells mL⁻¹ (Figure 11A). Moreover, based on the signal conversion capability of nanozymes, the system that utilizes portable devices such as pressure meters and thermometers for signal readout can effectively reduce reliance on large precision instruments and liberate the detection process from laboratory constraints. Wang et al. [174] developed a pressure and temperature dual‐mode sensing strategy for CTCs POCT by utilizing the photothermal properties and CAT‐like activity of NIR II‐responsive Au nanoframes (AuNFs). Through FA functionalization, the AuNFs specifically targeted cancer cells overexpressing FRs. This specific molecular recognition between FA and FRs was subsequently converted into amplified pressure and temperature signals, which could be readily quantified using portable pressure meter and thermometer respectively (Figure 11B).

FIGURE 11.

(A) Schematic of CTC capture based on a microfluidic device and nanozyme‐based colorimetric and commercial glucometer signal output. Reproduced with permission [177]. Copyright 2024, Elsevier. (B) Schematic of cancer cell detection, (a) synthesis of Au‐NFs nanozyme and (b) construction of a pressure‐ and temperature‐based detection platform. Reproduced with permission [174]. Copyright 2021, ACS Publishing. (C) Schematic of Escherichia coli O157:H7 detection based on Cu_2−xSe@MPBA nanozyme, (a) synthesis of Cu_2−xSe@MPBA, (b) preparation of Ab1‐MNPs, and (c) Cu_2−xSe@MPBA‐based Escherichia coli O157:H7 detection procedures. Reproduced with permission [204]. Copyright 2025, Elsevier. (D) Pressure‐based signal output and detection of Escherichia coli O157:H7 with a pressure meter based on Au@AuPt NPs nanozyme. Reproduced with permission [9]. Copyright 2024, ACS Publishing. (E) Principle of the MDN nanozyme based “All‐on‐a‐Tube” POCT platform for screening of Salmonella. Reproduced with permission [205]. Copyright 2024, Elsevier.

Despite significant advances in nanozyme‐based CTC detection platforms over the past decade, current technologies face substantial challenges. The ultralow abundance of CTCs in peripheral blood, combined with difficulties in efficient enrichment and the inability to specifically capture heterogeneous CTC subpopulations, limits practical application of existing POCT platforms. Furthermore, current systems cannot achieve precise individualized analysis. Therefore, further improvements in detection sensitivity and thorough investigation of clinical feasibility remain imperative for future development.

Escherichia coli O157:H7 is a major foodborne pathogen worldwide, posing a serious threat to public health. Its rapid detection is crucial for ensuring food safety and controlling epidemic outbreaks. Zhao et al. [204] utilized the glycolipid interaction between 4‐mercaptophenylboronic acid‐modified Cu₂‐ _x Se nanoparticles and bacterial cells to construct a multifunctional integrated platform for immunomagnetic separation coupled with a smartphone. Based on the excellent POD‐like activity of Cu₂‐ _x Se nanoparticles, this platform enabled the development of an unlabeled colorimetric biosensor for Escherichia coli O157:H7 (Figure 11C). In another study, Yang et al. [9] synthesized Au–Pt alloy‐coated AuNPs (Au@AuPt NPs) with enhanced CAT‐like activity and excellent stability. First, the Au@AuPt NPs nanozymes were modified with H1 or H2 hairpin DNA. Subsequently, the H1/H2 hairpin DNA‐modified Au@AuPt NPs were incubated with MBs immobilized with the initiator–aptamer complex. Target binding triggered the release of the initiator strand, which subsequently activated a HCR between the H1 and H2 hairpin DNA on the Au@AuPt NPs surface. This resulted in extensive aggregation of Au@AuPt NPs on the MBs, which significantly catalyze the decomposition of H₂O₂ to generate O₂, inducing a pressure change in the system. Finally, ultrasensitive quantitative detection of Escherichia coli O157:H7 was achieved by measuring the air pressure signal using a portable pressure meter (Figure 11D).

Salmonella represents another high‐incidence foodborne pathogen of global concern, underscoring the importance of developing rapid detection technologies for public health protection. Lin [206] developed a colorimetric biosensor for Salmonella detection by integrating automated immunosandwich reactions in a microfluidic chip with nanozyme‐catalyzed signal amplification. In a specially designed chip, the sample was efficiently mixed with immunoMBs and Au@PtPd nanozyme to form “MB–SalmonellaNnanozyme” immunocomplexes. This complex was subsequently separated and enriched via the chip's built‐in sliding magnetic field. Leveraging the exceptional POD‐like activity of Au@PtPd, a colorimetric signal was output via mixing with TMB/H₂O₂. Finally, quantitative detection of Salmonella was achieved using a smartphone. Most notably, the entire process is precisely actuated by air pressure generated from finger‐pressing an air chamber, requiring no external power source and thereby achieving truly rapid and highly sensitive POCT. Based on a similar strategy, their group, the same group developed an integrated “All‐on‐a‐Tube” POCT platform by incorporating immune recognition, magnetic separation, and nanozyme‐catalyzed signal amplification within a modified centrifuge tube (Figure 11E). The detection process involved three sequential steps: first, efficient mixing of large‐volume samples with immunoMNPs and manganese dioxide nanoflowers (MDNs) through mechanical stirring to form MB–bacteria–nanozyme complexes; second, target enrichment via magnetic separation; and ultimately, colorimetric signal generation through the POD‐like activity of MDNs upon catalytic substrate addition, enabling smartphone‐based quantification of Salmonella [205].

5. Conclusion

Owing to their excellent stability, tunable catalytic activity, signal conversion and amplification capabilities, and cost‐effectiveness, nanozymes have garnered extensive attention in the field of biosensors—particularly in POCT. This review summarizes the roles of nanozymes in POCT and the recent advances in nanozyme‐based POCT applications. By leveraging their superior catalytic and signal amplification properties, the integration of nanozymes into POCT platforms significantly enhances analytical sensitivity and diversifies signal output modalities. Additionally, the superior stability of nanozymes endows nanozyme‐integrated POCT platforms with unique advantages in diverse and complex environments. However, despite substantial progress achieved in recent years, the research and practical implementation of nanozyme‐based POCT platforms remain at a nascent stage; thus, the field continues to present numerous opportunities alongside persistent challenges for future research.

To address the demands of practical applications, research focused on enhancing the analytical performance of nanozyme‐based POCT will remain the mainstream direction. While the advantages of nanozymes facilitate the development of diverse POCT platforms, achieving high specificity and sensitivity for complex biological samples and ultralow‐concentration targets remains a significant challenge. First, the catalytic activity of most nanozymes remains lower than that of natural enzymes and is susceptible to environmental factors. To overcome this, optimizing material design, incorporating single‐atom catalysis, and constructing cascade catalytic systems represent effective strategies to enhance the catalytic activity of nanozymes. Furthermore, surface functionalization of nanozymes, the design of stimuli‐responsive nanozymes, and machine learning‐based prediction of structure–activity relationships for nanozyme architecture may serve as effective approaches to improve their environmental stability. Second, owing to the absence of substrate‐binding pockets, nanozymes suffer from poor specificity and false positives caused by redox‐active interferents. Beyond conventional modification, recent advances include aptamer–nanozyme conjugates via click chemistry for precise target recognition, bioinspired construction of artificial binding pockets using amino acid residues or peptide chains, and single‐atom nanozymes with tunable coordination environments that exhibit excellent substrate specificity. Finally, nanozymes currently employed in POCT are predominantly redox‐active types, with hydrolytic nanozymes being extremely limited. This restricts applications such as phosphorylated protein or lipid metabolite detection. Future efforts should focus on developing novel hydrolytic nanozymes, for example, transition metals (Zr⁴+, Ti⁴+) are doped into MOF framework to realize the regulation of hydrolase activity.

While traditional POCT standards represented by the “ASSURD” criteria primarily address fundamental detection requirements, evolving societal demands have prompted the emergence of the “SMART” standard (Specificity, Sensitivity, Multiplexity, Applicability, Rapidity, and Throughput), which places greater emphasis on versatility and practical applicability. Among the core requirements of the SMART standard, multiplexing (multiplex detection) is particularly critical for meeting the increasingly complex clinical diagnostic needs, as it enables simultaneous detection of multiple analytes in a single sample, greatly improving diagnostic efficiency, reducing sample consumption and detection cost, and providing comprehensive and accurate diagnostic information for disease screening, differential diagnosis and prognosis evaluation. However, the commercialization of nanozyme‐based POCT platforms faces substantial challenges due to variations in synthesis methods and material properties, resulting in inconsistent catalytic performance across production batches. The absence of unified manufacturing standards and evaluation criterion further impedes quality control and reliability assurance. Consequently, widespread adoption of the SMART standard requires broader industry consensus. Establishing standardized synthesis methodologies and developing comprehensive nanozyme material databases would significantly facilitate the transition of nanozyme‐based POCT platforms from laboratory research to commercial applications.

In summary, nanozyme research and nanozyme‐based POCT platforms hold enormous potential for personalized medicine and home health monitoring. While their commercial implementation faces numerous challenges, the future advancement of portable, integrated, and automated biosensing technologies is emerging as a promising direction that is set to significantly optimize and streamline medical service processes, including disease marker detection and pathogen analysis. It will also provide powerful diagnostic tools for patients in resource‐constrained settings. This not only enhances the accessibility and efficiency of healthcare services but also contributes to the universalization of high‐quality medical support.

Author Contributions

Wei Liu and Yi Wang: writing – original draft, writing – review and editing. Zhongwei Jiang, Xue Gong, and Ruili Li: investigation. Yi Wang: supervision and validation. Yi Wang, Wei Liu, Zhongwei Jiang, and Ruili Li: funding acquisition.

Funding

This study was supported by Chongqing Normal University (24XLB031, 25XLB019); Chongqing Municipal Education Commission (KJQN202500541, KJZD‐M2025005); and National Natural Science Foundation of China (22204011).

Conflicts of Interest

The authors declare no conflicts of interest.

Biographies

Wei Liu received his Ph.D. in chemistry from Central South University in 2024 under the supervision of Prof. Xiaoqing Chen. He then joined the Chongqing Normal University as a researcher. Liu has been dedicated to the study of nanozyme for POCT application of biomarker.

Zhongwei Jiang is currently an associate professor in College of Chemistry and Materials Science, Chongqing Normal University. He received his Ph.D. in College of Chemistry and Chemical Engineering, Southwest University. His research interests focus on chemical sensors as well as on porous material.

Xue Gong is currently an associate professor in College of Chemistry and Materials Science, Chongqing Normal University. She received her Ph.D. in College of Chemistry and Molecular Sciences, Wuhan University. Her research interests focus on functional DNA circuits for theranostics.

Ruili Li is currently affiliated with the College of Chemistry and Materials Science at Chongqing Normal University. She received her Ph.D. from the School of Chemistry and Chemical Engineering, Central South University. Her research focuses on the development of surface‐enhanced Raman spectroscopy (SERS)‐based detection strategies integrated with machine learning algorithms for early disease diagnosis and prediction, rapid detection of emerging pollutants, and investigation of their associated biological toxicity.

Yi Wang is a professor in College of Chemistry and Materials Science, Chongqing Normal University. He received his B.S. from Southwest University in 2007, and received his Ph.D. at Southwest University in 2014. As a visiting Ph.D. student, he joined Prof. Younan Xia's group at Washington University and Georgia Institute of Technology during 2011–2013. His current research interests focus on the controlled synthesis of nanomaterials and their applications in biosensing and biomedicine.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.