Recent Advances in Nanozyme-Mediated Strategies for Pathogen Detection and Control

Abstract

Pathogen detection and control have long presented formidable challenges in the domains of medicine and public health. This review paper underscores the potential of nanozymes as emerging bio-mimetic enzymes that hold promise in effectively tackling these challenges. The key features and advantages of nanozymes are introduced, encompassing their comparable catalytic activity to natural enzymes, enhanced stability and reliability, cost effectiveness, and straightforward preparation methods. Subsequently, the paper delves into the detailed utilization of nanozymes for pathogen detection. This includes their application as biosensors, facilitating rapid and sensitive identification of diverse pathogens, including bacteria, viruses, and plasmodium. Furthermore, the paper explores strategies employing nanozymes for pathogen control, such as the regulation of reactive oxygen species (ROS), HOBr/Cl regulation, and clearance of extracellular DNA to impede pathogen growth and transmission. The review underscores the vast potential of nanozymes in pathogen detection and control through numerous specific examples and case studies. The authors highlight the efficiency, rapidity, and specificity of pathogen detection achieved with nanozymes, employing various strategies. They also demonstrate the feasibility of nanozymes in hindering pathogen growth and transmission. These innovative approaches employing nanozymes are projected to provide novel options for early disease diagnoses, treatment, and prevention. Through a comprehensive discourse on the characteristics and advantages of nanozymes, as well as diverse application approaches, this paper serves as a crucial reference and guide for further research and development in nanozyme technology. The expectation is that such advancements will significantly contribute to enhancing disease control measures and improving public health outcomes.

1. Introduction

Bacterial infection remains a significant global security threat to human health [1,2,3]. Antibiotics derived from natural products or chemically modified natural compounds have become the most commonly used approach for treating bacterial infections [4,5]. Antibiotics have played a significant role in the prevention, containment, and treatment of infectious diseases in animals [6]. The extensive utilization of antibiotics has also contributed to the significant issue of bacterial resistance [7,8,9]. More significantly, the proliferation of bacterial resistance poses a grave menace to global public health [9]. However, conventional methods for pathogen detection, such as a cell culture [10,11,12], polymerase chain reaction (PCR) [13,14,15], enzyme-linked immunosorbent assay (ELISA) [16,17,18], and other techniques, are time-consuming, technically demanding, and require specialized equipment. Moreover, they cannot achieve rapid and efficient detection in non-laboratory settings, which hinders the prompt discovery and effective control of pathogens [19]. Hence, it poses a significant challenge to explore novel strategies for achieving efficient detection and control of pathogens.

To tackle this challenge, significant efforts have been directed toward the development of efficient, streamlined, and rapid pathogen detection methods as well as alternative broad-spectrum antibacterial agents or strategies. For instance, certain detection platforms and antibacterial materials that utilize nanomaterials, such as continuous cascade nanozyme biosensors [20], cascade methods based on a natural self-assembled nanozyme [21], a nanozyme-mediated CRISPR-Cas 12a system [22], photothermal therapy (PTT) [23], photodynamic therapy (PDT), and photocatalytic therapy (PCT) [24], are emerging [25]. Recently, nanozyme-based sensing platforms have demonstrated successful applications in the diagnosis of bacteria, viruses, and plasmodium parasites [22,26,27]. In addition, the natural peroxidase (POD) and oxidase (OXD) present in living organisms can catalyze a range of substrates to generate numerous reactive oxygen species (ROS), which are effective against bacterial invasion [28]. Therefore, nanozymes are anticipated to serve as a viable alternative to antibiotics owing to their exceptional catalytic activity, remarkable substrate selectivity, and gentle action conditions [23]. However, the utilization of natural enzymes in pathogen diagnoses and control is greatly limited due to their high production costs, complex conditions, low stability of catalytic activity, and difficulty in storage [29,30]. In 2004, Scrimin and his colleagues employed triazabicyclo-alkane-functionalized gold nanoparticles as a catalyst for the dephosphorylation reaction [31]. In 2007, Yan’s team initially reported that Fe₃O₄ magnetic nanoparticles could serve as a peroxidase mimic similar to horseradish peroxidase (HRP) [32]. Recently, nanozymes have emerged as a research hotspot due to their promising features of a low cost, a high stability, facile production, tunable catalytic activity, and convenient storage [33,34,35,36]. To date, nanozymes utilized for antibacterial and pathogen detection primarily consist of metal-based compounds, carbon-based nanomaterials, transition metal dichalcogenides/peroxides/oxides, single-atom nanozymes (SAzymes), and metal–organic frameworks (MOFs)-based compounds that have been extensively developed [31,37,38].

Previously, several comprehensive reviews have provided systematic overviews of the applications of nanozymes in pathogen detection and control. Nguyen et al. extensively discuss the sensing mechanisms, device structures, analytical characteristics, and relevant applications of nanomaterials in colorimetric paper-based biosensors for accurate pathogen detection [39]. Songca et al. investigate the enhancement of the mechanisms, design, and structural role of nanozyme nanoconjugates in the detection and identification of viral, bacterial, and fungal pathogens [40]. Additionally, Sun et al. present a comprehensive overview of various nanozymes and their applications in pathogen detection [31]. However, there is currently no exhaustive review or exposition on the antibacterial and detection mechanisms employed with nanozymes.

The aim of this review is to provide a comprehensive review on the recent advancements in various nanozymes for combating bacterial infections and detecting pathogens. Additionally, it aims to elucidate the physicochemical properties and mechanism of action of nanozymes in antibacterial detection and pathogenic bacteria identification. This study differs from previous research endeavors. By categorizing and summarizing the antibacterial and detection mechanisms employed with these nanozymes, we describe their scope, efficacy, as well as factors influencing their performance in controlling pathogenic microorganisms (Figure 1). Furthermore, we analyze the main challenges faced by nanozymes in pathogen detection and control, while also exploring their future potential. Our intention is that this review will inspire readers with novel insights and technologies for pathogen detection and management, fostering a more comprehensive understanding of antimicrobial strategies based on nanomaterials.

2. Nanozyme-Mediated Detection Mechanisms

Rapid and efficient pathogen detection is a crucial component of disease prevention and control. Currently, commonly used immunoassays for detection, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), gas chromatography/mass spectrometry (GC-MS), liquid chromatography/mass spectrometry (LC-MS), and ELISA, exhibit exceptional specificity and accuracy [41]. However, these methodologies often require complex pre-processing procedures, advanced instrumentation, and skilled technical personnel for implementation, resulting in extended detection timelines that present challenges in meeting the demands of real-time pathogen detection [42]. Enzyme-mimetic nanomaterials, also known as nanozymes, are highly promising tools for enhancing quality assessment and ensuring safety in the agri-food industry due to their ability to mimic enzymes and possess nanoscale features. Their robust detection capabilities and potential for future-oriented applications make them particularly attractive for utilization in this sector [43]. In contrast to their natural enzyme counterparts, nanozymes possess several advantageous features including cost effectiveness [44], facile scalability for mass production [45], resilience towards harsh environmental conditions [46], prolonged storage capability [47], exceptional stability, and the ability to customize their activities as per specific requirements [45]. Nanozymes have demonstrated tremendous potential in surpassing the limitations of conventional enzymes within the field of analyses. Consequently, it is of significant importance to categorize the underlying principles of nanozymes within the context of pathogen detection. The catalytic activity of a nanozyme relies on the surface electron transfer process [48]. Researchers employed electron spin resonance (ESR) [49], density functional theory (DFT) [50], X-ray photoelectron spectroscopy (XPS) [51], and ultraviolet-visible spectrophotometry (UV) [52] to investigate the catalytic reaction mechanism of a nanozyme while employing an enzyme steady-state kinetic analysis to examine its catalytic activity. Nanozymes can be classified based on their catalytic types, which are determined by their distinct catalytic mechanisms [53]. This discourse primarily covers five distinct categories of nanozymes, including peroxidase-mimicking, oxidase-mimicking, catalase-like, glucose-oxidase-mimicking, and clickase nanozymes. All of these demonstrate the ability to detect key analytes relevant to the agri-food industry.

2.1. Peroxidase Nanozyme

POD mimics, a widely utilized category of nanozymes in detection applications, play an indispensable role in facilitating the catalytic conversion of a substrate into its oxidized form. This enzymatic process generates a variety of signals, including colorimetric manifestations (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5,5′-tetramethylbenzidine (TMB), and o-phenylenediamine (OPD)), fluorescent manifestations such as Ampliflu Red, and chemiluminescent manifestations like luminol. These signals become distinguishable in the presence of hydrogen peroxide (H₂O₂) as a cosubstrate. The catalytic kinetics of POD-like nanozymes are known to exhibit variations in response to appropriate concentration ranges of standard substrates and H₂O₂ cosubstrates, resulting in the generation of divergent quantities of catalytic oxidation products. As a result, various analytical techniques have been developed for a food analysis utilizing POD nanozymes to measure the cosubstrate H₂O₂ [54,55,56,57]. Simultaneously, certain substances exhibit selective reactivity towards H₂O₂, enabling signal amplification facilitated by the catalytic activity of nanozymes. The concentration of the substrate can be determined by monitoring the consumption of H₂O₂, thus achieving quantitative detection objectives [57,58] (Figure 2). Furthermore, complex reactions can be achieved through the synergistic interplay of multiple nanozymes. In order to construct an artificial cascade detection system, the researchers integrated nanozymes with hydrogen peroxide generation ability and POD nanozymes [55,58]. Similarly, it is possible to devise detection systems that incorporate cascade reactions, in which two or more nanozymes are collaboratively utilized autonomously without the involvement of enzymes [43]. In summary, this description highlights the application of POD mimics as nanozymes in detection scenarios, demonstrating their versatility in generating diverse signals and potential for quantification in a food analysis. Furthermore, the synergistic actions of nanozymes present opportunities for intricate detection systems, offering innovative approaches for analytical purposes.

2.2. Oxidase Nanozyme

Furthermore, nanozyme-based detection platforms can be utilized through the consumption of catalytic oxidation products (ox-substrate), providing access not only to peroxidase-like nanozymes but also oxidase nanozymes. Zhou and colleagues demonstrated the application of MnO₂ nanosheets with oxidase-mimicking activity in catalyzing the oxidation of TMB to TMB²⁺. Subsequently, the gold nanorods (Au NRs) underwent rapid etching, resulting in a noticeable blue shift of their longitudinal local surface plasmon resonance peaks and polychromatic changes. In the presence of E. coli, a cascade reaction was initiated via β-galactosidase hydrolysis of p-aminophenol β-D-galactopyranoside (PAPG), resulting in the generation of p-aminophenol (PAP). Subsequently, PAP mediated the reduction of MnO₂ nanosheets, leading to the disruption of their oxidase-mimicking activity and subsequently affecting TMB²⁺ generation. Consequently, the sensing system is capable of easily detecting varying concentrations of E. coli through distinct color changes exhibited [60]. The integration of MnO₂ nanosheets and Au NRs not only facilitated the detection of E. coli but also enabled the observation of visual color changes, thereby providing a promising strategy for achieving sensitive and selective detection in the presence of the bacteria.

2.3. Catalase-like Nanozyme

Catalase (CAT) is a vital oxidoreductase in the human body, which plays a significant role in catalyzing the elimination of ROS, safeguarding cell membrane integrity, and inhibiting tumor cell growth [61]. CAT-like nanozymes are a class of nanomaterials that possess inherent catalase activity, enabling them to efficiently facilitate the decomposition of H₂O₂ into water (H₂O) and oxygen (O₂) molecules [62]. Nano-enzymes with CAT activity based on different nanomaterials include metals, metal oxides, metal–organic frameworks (MOFs), carbon-based nanomaterials, metal sulfides, and Prussian blue (PB) (Figure 3) [62]. Zhou’s research team successfully synthesized trimetallic AuPtCo nanopolyhedrons that exhibit peroxidase and catalase-like catalytic activities. In an alkaline environment, the presence of ABEI and H₂O₂ leads to their conversion into an ABEI anion (ABEI⁻) and HO²⁻, respectively. Furthermore, the H₂O₂ is decomposed by AuPtCo@Cys into HO^• and O₂^•− through catalysis. Subsequently, ABEI- reacts with the generated HO²⁻ and HO^• to form ABEI^•− and O₂^•−, respectively. Notably, O₂^•− continuously reacts with HO^• to produce a plentiful supply of O₂. Ultimately, ABEI^•− reacts with both O₂^•− and O₂, resulting in the formation of the excited-state oxidation product of ABEI (ABEI-ox*), which subsequently returns to its ground state accompanied by light emission [63]. The developed approach successfully achieved sensitive detection of H₂O₂ and lipoprotein-associated phospholipase A2 (Lp-PLA2) in actual samples, thereby demonstrating its potential to significantly broaden the application scope in the field of rapid pathogen detection. These findings offer promising prospects for the expansion of pathogen rapid detection applications, emphasizing the potential utility of the synthesized nanopolyhedrons in this specific field.

2.4. Clickase Nanozyme

Clickase has demonstrated its efficacy in streamlining reactions. Currently, a Cu(I)-based nanocatalyst has been developed for the detection and analysis of foodborne pathogens, which is rich in Cu(I) ions effectively stabilized by glutathione (GSH). Furthermore, the Cu(I)-containing nanomaterials-based clickase exhibits exceptional biocompatibility, which facilitates antibody modification due to the abundant presence of amino (-NH₂) and carboxyl (-COOH) groups on its surface (Figure 4) [64]. Moreover, a nanoflower-like artificial clickase, known as LCN clickase, has been introduced for the portable and sensitive detection of foodborne bacterial pathogens using a click SERS immunoassay. Benefiting from its remarkable catalytic activity that promotes the Cu(I)-catalyzed azide–alkyne cycloaddition reaction, LCN clickase has successfully enabled the development of a novel click SERS immunoassay. This innovative approach integrates the clickase-mediated SERS signal modulation in the Raman-silent region, thereby effectively eliminating interferences caused by Raman reporters and biological components while minimizing complex sample matrix interference. In comparison to conventional CuAAC-based immunoassays, the established method avoids the unnecessary requirement of a dissolution process for nanocatalysts and eliminates the necessity for a reducing agent in the detection process. As a result, the detection time is considerably shortened, and the detection reliability is enhanced [65]. Fundamentally, clickase and its derivatives, such as CCN and LCN clickase, have revolutionized the field of biochemistry, particularly in foodborne pathogen detection. These advancements have significantly streamlined reaction processes, improved the reliability and sensitivity of detection methods, and substantially reduced detection time.

Due to their distinctive advantages and comparable catalytic capabilities to enzymes, nanozymes have garnered increasing attention for their applications in detection. Specifically for pathogen detection, nanozymes offer a simplified preparation and purification process compared to natural enzymes, obviating the need for intricate equipment or instruments [45]. Motivated by extensive research on nanozymes and an increasingly profound understanding of nanoscale particles, contemporary preparations of nanozymes have exhibited reduced time requirements and demonstrated efficacy in meeting diverse analytical demands [66]. Nanomaterial-based platforms have demonstrated enhanced efficacy, accelerated detection speed, and broader pathogen coverage in contemporary applications for pathogen detection.

2.5. Application of Nanozymes in Bacteria Detection

Given the increasing prevalence of foodborne illnesses, foodborne bacteria have emerged as a significant threat to global public health in recent years [67]. The efficient identification of pathogenic bacteria is crucial for the successful implementation of control measures. In the field of foodborne pathogen detection, biosensors incorporating nanozymes have been widely used [38].

2.5.1. Escherichia coli

The successful implementation of effective control measures relies on the utilization of efficient, rapid, and highly sensitive detection methods specifically designed for targeting pathogenic bacteria [68]. Zhang et al. developed man-PB, a functional nanozyme, for rapid and label-free detection of E. coli O157:H7 using a multi-readout LFIA technique. The man-PB nanoparticles showed exceptional binding affinity towards the flagella of E. coli O157:H7 as a novel recognition agent, enabling efficient capture of the target pathogen [56]. Additionally, He et al. developed a detection strategy utilizing nanozymes in a cascade reaction. When exposed to an acidic solution, MnO₂ nanosheets exhibited catalytic activity and converted the colorless substrate TMB into TMB²⁺ ions. Acting as an oxidant, TMB²⁺ rapidly corroded Au NRs, resulting in noticeable color changes. In our sensing methodology, β-gal derived from E. coli initiated the cascade reaction. β-galactosidase catalyzed the hydrolysis of the PAPG substrate, resulting in the formation of a reducing agent called PAP. PAP was found to reduce MnO₂ nanosheets, inhibiting their oxidase activity and subsequently decreasing the production of TMB²⁺. Consequently, the etching process of Au NRs was impeded. The diverse colors resulting from the etching of Au NRs facilitated a semi-quantitative visual analysis of β-galactosidase in Escherichia coli. The synergistic implementation of enzyme–nanozyme cascade reactions and the unique optical properties of Au NRs significantly enhanced the detection sensitivity towards β-galactosidase in E. coli. Moreover, this approach offered a high-resolution and user-friendly visual method for determining the concentration of E. coli through direct observation with the naked eye [60]. These groundbreaking methodologies highlight the use of functional nanozymes and cascade reactions based on nanozyme technology as highly effective and sensitive means for detecting pathogenic bacteria.

2.5.2. Listeria monocytogenes

Listeria monocytogenes (L. monocytogenes) are Gram-positive foodborne pathogens that exhibit a significant presence across diverse food sources [69]. Shi et al. proposed an innovative approach for the detection of viable L. monocytogenes by integrating PMA-LAMP technology with a nanozyme strip. This integrated platform allows for instrument-free, highly sensitive, and visually perceptible identification of viable L. monocytogenes. By modifying primers with BIO and FITC moieties, the LAMP process can generate a modified hlyA gene that specifically targets L. monocytogenes. Subsequently, magnetic nanozymes functionalized with biotin antibodies are used to create a probe capable of specifically recognizing and isolating L. monocytogenes on the strip [70]. Wu et al. have developed a magnetic resonance spectroscopy (MRS) DNA sensor utilizing an Au@Pt nanozyme for the rapid and highly sensitive detection of L. monocytogenes in chicken samples. In the presence of L. monocytogenes’ DNA, Au@Pt-probe2 nanoparticles demonstrate specific binding affinity to the target DNA region. Subsequently, the formed Au@Pt-probe₂-target DNA conjugates are hybridized with probe 1-modified magnetic nanoparticles (MNP ₁₈₀-probe₁). Following magnetic separation, the Au@Pt nanozyme within the conjugates catalyzes an enzymatic reaction involving H₂O₂, thereby facilitating the conversion of paramagnetic ions from Mn(VII) to Mn(II). The conversion mentioned above induces a corresponding alteration in the signal of transverse relaxation time (ΔT₂), thereby facilitating the precise and sensitive detection of L. monocytogenes [71]. Both Shi et al.’s PMA-LAMP combined with a nanozyme strip and Wu et al.’s Au@Pt-nanozyme-mediated MRS DNA sensor represent innovative approaches for the detection of viable L. monocytogenes. The method proposed by Shi et al. offers an instrument-free and visually discernible qualitative detection mechanism through the integration of PMA-LAMP and a nanozyme strip. Conversely, the approach proposed by Wu et al. utilizes an Au@Pt-nanozyme-mediated MRS DNA sensor, which enables rapid and sensitive detection through monitoring changes in the transverse relaxation time (ΔT₂) signal. These significant advancements contribute to the field of food safety by providing efficient and reliable detection methods for L. monocytogenes across various food sources.

2.5.3. Salmonella

The World Health Organization (WHO) has made an estimation highlighting that approximately 10% of the global population experiences foodborne illnesses annually, leading to the loss of 33 million healthy life years on a yearly basis [72]. Salmonella is one of the four major global etiological factors contributing to the occurrence of diarrheal diseases [73,74]. Guo et al. have proposed a microfluidic immunosensor as an innovative approach for the detection of Salmonella. The immunosensor employs Fe-MOF/PtNPs as a signal amplifier, a smartphone-based thermal sensor as a signal detector, and a two-layer microfluidic chip as a research platform. In this system, magnetic nanoparticles (MNPs) coated with capture antibodies (CAbs, indicated in green), Salmonella, and detection antibodies (DAbs, marked in purple) modified with Fe-MOF/PtNPs are simultaneously introduced into the microfluidic chip. Efficient mixing is facilitated by a three-dimensional chaotic micromixer followed by adequate incubation in the dedicated channel to form sandwich complexes consisting of MNPs–Salmonella–Fe-MOF/PtNPs. Subsequently, a magnet is utilized to capture these complexes within a separation chamber. In order to initiate a reaction, H₂O₂ is injected and decomposed by the nanozyme present on the sandwich complexes, resulting in the production of O₂ within the sealed chamber. The rise in pressure within the separation chamber propels preloaded H₂O into the heating chamber containing calcium oxide (CaO) powder, thereby inducing heat generation. The quantity of Salmonella is ascertained by gauging the resultant thermal output using a smartphone-based thermal sensor [75]. Wang et al. have reported the synthesis of Fe₃O₄@Cu (His-Fe₃O₄@Cu) magnetic nanozymes, which demonstrate remarkable peroxidase-like activity and strong superparamagnetism. These nanozymes have been employed to establish a straightforward and highly sensitive colorimetric method for the detection of Salmonella typhimurium (S. typhimurium). In their study, an Apt-His-Fe₃O₄@Cu complex modified with aptamer 1 and effectively blocked non-specific binding sites using whey protein isolate (WPI) was developed as a capture agent for selective enrichment of S. typhimurium in food samples. Subsequently, biotin-aptamer 2 is immobilized onto a microplate via the specific binding between avidin and biotin, serving as the capture probe for selective recognition of S. typhimurium, resulting in the formation of microplate/S. typhimurium/His-Fe₃O₄@Cu “sandwich” complexes. After eliminating any unbound Apt-His-Fe₃O₄@Cu, a hydrochloric acid (HCl) etching solution is introduced to restore the peroxidase-like activity of His-Fe₃O₄@Cu. Ultimately, the detection signal produced in the presence of a TMB-H₂O₂ solution can be visually perceived by naked eyes or quantitatively measured using a SpectraMaxi3x Reader [76]. These methodologies free Salmonella detection from the requirement of bulky instrumentation, allowing for sensitive, rapid, accurate, and efficient detection. By utilizing microfluidic systems’ integration, signal amplification techniques, and nanozyme technology, these investigations have established sensitive, expeditious, and efficient approaches to discerning and quantifying Salmonella in food specimens. The aforementioned advancements thus make a significant contribution to the enhancement of food safety standards and the safeguarding of public health.

2.5.4. Staphylococcus aureus

Staphylococcus aureus (S. aureus) is acknowledged as a significant foodborne pathogen capable of producing enterotoxins, which are implicated in the etiology of various severe and multifaceted ailments. These encompass localized suppurative infections, pneumonia, pseudomembranous colitis, pericarditis, sepsis, and diverse systemic infections [77]. Based on available data, Staphylococcus aureus has been identified as one of the prominent pathogens contributing to acquired foodborne illnesses and ranks among the top five. In the United States alone, this bacterium is estimated to be responsible for approximately one million cases of such illnesses annually [78]. Consequently, the scientific community has placed significant emphasis on developing robust and highly sensitive detection methods for Staphylococcus aureus [79]. The conventional method for detecting Staphylococcus aureus involves a process of selective enrichment, followed by an enumeration of colonies based on morphological characteristics. Additional evaluation may involve staining techniques or enzymatic assays for identification purposes. Regrettably, these procedures typically require a time frame of 2–4 days to produce informative results, making them unsuitable for urgent situations such as public health emergencies or cases of foodborne illness [80]. Numerous highly sensitive detection methods for Staphylococcus aureus have been developed by researchers, utilizing the PCR technique [81]. Furthermore, the application of ELISA has been investigated as a viable alternative for detecting Staphylococcus aureus with favorable sensitivity levels [61,82]. Despite the inherent advantages of these detection strategies, such as their ability to achieve low detection limits and high specificity, their widespread implementation is hindered by certain challenges. Specifically, the need for expensive instrumentation and a demand for well-trained personnel act as barriers to their broader application [81].

Aptamers are functional single-stranded oligonucleotide sequences, possessing the unique ability to selectively bind to targets with remarkable affinity. These aptamers are obtained through an in vitro process known as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method [83]. Aptamers, akin to antibodies, demonstrate remarkable affinity and specificity toward a wide range of targets, including proteins, small molecules, ions, and even cells [84]. In a recent study conducted by Zhu et al., it was discovered that oligonucleotide sequences, specifically aptamers, possess the ability to adsorb onto the surface of octahedral Mn₃O₄ nanoparticles (NPs) and modulate their oxidase-mimicking activity. This intriguing phenomenon has facilitated the development of an advanced label-free and highly sensitive colorimetric aptasensor for Staphylococcus aureus detection [85]. The integration of oligonucleotides has facilitated the modulation of a Mn₃O₄ nanoparticle’s oxidase-mimicking activity, resulting in a cutting-edge colorimetric aptasensor for the highly sensitive and selective detection of Staphylococcus aureus in food samples [85]. The interaction between octahedral Mn₃O₄ NPs and the chromogenic substrate TMB results in a hindered binding effect. As a consequence, the oxidase-mimicking activity of the catalytic system is significantly impeded, leading to electron loss from a fraction of TMB molecules and subsequent formation of oxidized TMB (oxTMB), which is observed as blue or green products. In the presence of S. aureus, the SA31 aptamer sequences exhibit a preferential binding affinity towards their corresponding target bacteria, resulting in the formation of stable complexes. Consequently, this alleviates the inhibitory effect on the oxidase-mimicking activity of octahedral Mn₃O₄ NPs. The induction of a visible color change is observed in the developed colorimetric aptasensor, which exhibits exceptional sensitivity with a low detection limit of 3 colony-forming units (CFU) per milliliter (mL). Additionally, it demonstrates excellent selectivity for detecting S. aureus.

To overcome the limitations of conventional ELISA techniques, such as colorimetric reactions, substantial sample volume requirements (approximately 200 μL), extended processing time (1–2 h), and the need for bulky instrumentation to interpret test results [86], the utilization of paper-based enzyme-linked immunosorbent assay (P-ELISA) tests has emerged as a promising solution to address these challenges, particularly in point-of-care testing (POCT) settings [87]. P-ELISA tests offer additional advantages, such as cost effectiveness and ease of use. Moreover, the test results can be visually discerned without relying on specialized detection instruments. Currently, there is a convergence between the P-ELISA concept and Pd@Pt nanoparticles, leading to the development of a diagnostic test known as the “nanozyme-immobilized paper-based analytical device” (nPAD) [88]. To improve the stability of the P-ELISA system, the researchers developed a 3D-printed sample holder that facilitates secure alignment and optimal contact between the multi-layered paper components comprising the test platform. Subsequently, the integration led to the development of an nPAD that enables rapid and precise test results with a turnaround time of approximately 30 min. Additionally, the nPAD demonstrated exceptional sensitivity, achieving a low limit of detection (LOD) as minimal as 9.56 ng/mL when used for quantifying protein A, a specific biomarker utilized in Staphylococcus aureus detection.

Concurrently, the implementation of a nanozyme-based enzyme-linked immunosorbent assay (N-ELISA) demonstrates significant improvements in achieving lower LOD compared to conventional ELISA techniques [89,90]. Wang’s research team has developed a systematic and portable application of a Pd@Pt nanozyme for the detection of Staphylococcus aureus using N-ELISA. They conducted a comparative study investigating three different immunoassay methods to explore a more sensitive detection strategy. Wang’s research team has systematically and portably applied Pd@Pt nanozymes in detecting Staphylococcus aureus through N-ELISA. A comparative study was conducted on three immunoassay methods to explore a more sensitive detection strategy. Type I utilized the conventional ELISA method, while Type II employed a modification that replaced the HRP-conjugated goat anti-mouse IgG (HRP-IgG) with a Pd@Pt-nanozyme-labeled secondary antibody (Pd@Pt-IgG). Furthermore, Type III introduced an enhanced bioprobe (Pd@Pt-HRP-IgG) by coupling HRP-IgG with Pd@Pt nanozymes to leverage their surface properties and achieve improved catalytic performance. Through his strategic integration, the LOD was significantly improved from 1 × 10⁵ to 1 × 10³ colony-forming units per milliliter (CFU/mL). As a result, this innovative approach has the potential to meet more stringent detection regulations using traditionally simple methods such as ELISA [79].

Vancomycin demonstrates high affinity and binds to the D-Ala-D-Ala moiety present in the peptidoglycan structure of bacterial cell walls through robust hydrogen bonding interactions and other related mechanisms (Figure 5) [91]. The literature has documented the use of vancomycin-functionalized nanomaterials for selectively capturing pathogenic bacteria in complex sample solutions [92]. In this study, researchers utilized vancomycin-modified magnetic nanoparticles (Fe₃O₄@Au-Van MNPs) to efficiently capture and isolate bacteria in complex environmental conditions. Additionally, by synergistically utilizing the specific binding capabilities of aptamers and the oxidase-like activity of octahedral Mn₃O₄ NPs, a novel magnetic-assisted colorimetric biosensor was developed. This biosensor exhibited exceptional sensitivity and selectivity in detecting pathogens in complex samples, without the need for laborious pretreatment steps. Notably, the Fe₃O₄@Au-Van MNPs and Mn₃O₄ NPs utilized in the colorimetric biosensor maintained their high reactivity even after multiple cycles of use, presenting a reliable and cost-effective solution for industrial applications in pathogen detection. A comparative analysis with similar colorimetric approaches demonstrated several advantages of the constructed biosensor. These include a lower limit of detection (2 CFU·mL⁻¹), an extended linear range (1 × 10⁶ CFU·mL⁻¹), reduced interference from matrix effects, and enhanced specificity [93]. The advancement of immunoassay techniques, specifically P-ELISA and N-ELISA, in conjunction with the integration of nanomaterials like Pd@Pt nanoparticles, has brought about a paradigm shift in the field. These developments have resulted in significant enhancements in sensitivity, reduced sample volume requirements, shortened processing durations, cost effectiveness, and improved user friendliness. Consequently, these advancements offer significant potential to enhance diagnostic capabilities and facilitate more efficient and accessible testing across a range of applications, including disease detection and monitoring.

2.5.5. Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is an opportunistic, Gram-negative pathogen of significant clinical relevance due to its capacity to elicit a wide range of severe nosocomial bloodstream infections [94,95]. According to the guidelines set forth by WHO, PA is recognized as an indicator organism for evaluating drinking water quality. Additionally, due to its ubiquitous environmental presence, PA is among the most notorious pathogens responsible for water contamination in various regions worldwide, including Asia, Europe, and the United States [95,96]. The unique surface plasmon resonance and catalytic characteristics exhibited by gold nanoparticles (GNPs) have made them indispensable in the field of colorimetric detection. In this context, GNPs act as catalysts for the oxidation of TMB, leading to the formation of diamine, which subsequently undergoes conversion to diimine, an electroactive product of interest [97,98]. To exploit the electroactive properties of TMB for electrochemical sensing, researchers have developed an aptamer–nanozyme-based assay on commercially available disposable screen-printed electrodes. This integration enables the acquisition of an electrochemical readout that facilitates highly sensitive detection of PA. The integration of the nanozyme assay with an electrochemical platform presents significant enhancements in diagnostic sensitivity. The aptamer–nanozyme-based electrochemical approach demonstrates remarkable features, including rapidity (within 10 min), affordability, and exceptional sensitivity (detecting concentrations as low as ~60 CFUs/mL in water). Moreover, this platform exhibits potential as a versatile detection system for a diverse range of molecular and cellular analytes, thereby demonstrating its capacity for broader applications. Its profound advantages include a rapid analysis, cost effectiveness, and heightened sensitivity.

2.5.6. Yersinia pseudotuberculosis

Yersinia pseudotuberculosis, a food and waterborne bacterium, is primarily transmitted among humans through the fecal–oral route, resulting in the development of yersiniosis [99,100,101]. Reactive arthritis, Reiter’s syndrome, erythema nodosum, glomerulonephritis, myocarditis, and sepsis are among the notable complications that can arise from Yersinia pseudotuberculosis infection [102]. Yang and colleagues have developed a novel colorimetric system for the detection of Yersinia pseudotuberculosis, utilizing phage particles and Au@Pt nanozymes. Initially, they isolated a highly specific phage (vB_YepM_ZN18) that targets Y. pseudotuberculosis from hospital sludge. Subsequently, they synthesized and immobilized Au@Pt nanozymes with peroxidase-like activity onto the surface of the isolated phage. Concurrently, another set of phages was modified with magnetic beads (MB). Additionally, a mixture of phages@MB and AuPt@phages was introduced into the bacterial samples for co-incubation. In the presence of the targeted bacteria, the phages@MB and AuPt@phages selectively adhered to the bacterial cells through specific interactions with the phage. After magnetic separation, the AuPt@phages bound to bacterial cells catalyzed the reaction between TMB and H₂O₂, producing a chromogenic solution. The lack of precise recognition in the presence of non-target bacteria facilitates easy removal of AuPt@phages along with the supernatant. His phage-based detection approach demonstrates outstanding performance characteristics and holds immense potential for the rapid identification of pathogenic microorganisms in diverse settings, such as drinking water, food production, and hospital samples [103]. This innovative approach, which integrates phages and Au@Pt nanozymes, provides a reliable and highly efficient colorimetric system for the precise detection of Yersinia pseudotuberculosis. It demonstrates significant potential for diverse applications in various fields, including monitoring drinking water safety, ensuring food hygiene, and accurately diagnosing infections in hospital environments.

2.6. Application of Nanozymes in Virus Detection

Viruses possess remarkable resilience and exhibit high transmissibility. Furthermore, their capacity for rapid mutation and genetic recombination significantly increases the likelihood of a pandemic, particularly in the context of an intricately interconnected globalized society [104]. A plethora of viruses, including influenza viruses, human immunodeficiency virus (HIV), Ebola virus (EBOV), Zika virus (ZIKV), and coronaviruses, have had significant impacts on public health throughout history. These effects range from the historical smallpox epidemic in the Aztec Empire in 1520 to the ongoing global COVID-19 pandemic [105]. In view of the ongoing COVID-19 pandemic, the pressing demand for rapid and highly sensitive detection methodologies targeting pathogenic agents has been further emphasized [106].

2.6.1. HEV

Hepatitis E virus (HEV) infection has emerged as a significant public health concern, characterized by an increasing prevalence of Hepatitis E outbreaks in both developing and developed nations. The transmission of HEV primarily occurs through waterborne and zoonotic pathways, further emphasizing the seriousness of the situation [107]. Currently, significant progress has been achieved in the field of HEV detection platforms. These advancements mainly involve the application of RNA-targeting reverse transcription polymerase chain reaction (RT-PCR) techniques and the identification of specific antibodies (IgG, IgM, and IgA) generated as a result of HEV infection [108]. Due to the limited availability of diagnostic assays for the HEV, there is a pressing need for a reliable immunoassay targeting HEV antigens to effectively combat emerging outbreaks through early detection of the causative agent. In response to this demand, Khrois et al. have developed an innovative immunoassay platform utilizing nanozymes. The nanozymes are composed of anti-HEV IgG-antibody-conjugated gold nanoparticles (Ab-AuNPs) as the core, with an outer shell formed with in situ silver deposition on the surface of Ab-AuNPs. The virus is entrapped within these nanocomposites, while the silver shell can be decomposed into silver ions (Ag⁺) upon the introduction of tetramethyl benzidine (TMBZ) and H₂O₂. This decomposition process indirectly quantifies the concentration of the target virus. By incorporating the enhanced effect of the Ag shell on the AuNP-based nanozyme, the immobilization of advanced deposition has been verified to validate the proposed immunoassay’s signal amplification mechanism. This decomposition process indirectly measures the concentration of the target virus. The incorporation of an Ag shell in conjunction with an AuNP-based nanozyme enhances its effectiveness, and advanced deposition immobilization has been confirmed to validate the proposed immunoassay’s signal amplification mechanism. The implemented assay for Hepatitis E virus-like particles (HEV-LPs) demonstrated remarkable sensitivity, capable of detecting concentrations as low as 10 pg/mL within a linear range of 10 pg/mL to 10 ng/mL [109]. The proposed methodology possesses the potential to operate as a highly efficient surveillance system for virus detection. In conclusion, the advancement of this sophisticated immunoassay utilizing the nanozyme platform demonstrates significant promise in facilitating the identification and monitoring of HEV outbreaks, thereby making substantial contributions to enhancing public health outcomes.

2.6.2. Nov-LP

Norovirus (NoV) is a genetically diverse group of small, icosahedral, non-enveloped viruses with a positive-sense, single-stranded RNA (ssRNA) genome [110]. NoV demonstrates a significant level of contagion primarily attributed to its exceptionally low infective dose, with the estimated median infective dose (ID₅₀) ranging between 18 and 1015 genome copies [111]. Currently, the most reliable and sensitive approach for detecting and quantifying NoV is the reverse-transcription–quantitative-polymerase-chain-reaction (RT-qPCR) [112]. Detecting NoV in food poses significant challenges, requiring a virus concentration specific to the matrix and removal of inhibitory compounds for detection assays. As a result, the utilization of RT-qPCR faces obstacles in rapid in-field or point-of-care diagnostic applications. To address this issue, Weerathunge et al. propose a novel colorimetric nanozyme aptasensor strategy capable of enabling rapid (within 10 min) and highly sensitive detection of the infectious murine norovirus (MNV), with a calculated LOD of 3 viruses per assay (equivalent to 30 viruses/mL) and an experimentally demonstrated LOD of 20 viruses per assay (equivalent to 200 viruses/mL) [112]. The implementation of this novel assay format has significant implications, enabling rapid and highly sensitive norovirus detection within minutes. Additionally, it is user-friendly and eliminates the need for specialized laboratory facilities. The development of this colorimetric nanozyme aptasensor strategy represents a significant advancement in norovirus detection.

2.6.3. RV

The rubella virus (RV), a human pathogen, is responsible for the occurrence of German measles, an airborne childhood disease renowned for its high contagiousness and global prevalence. When rubella infection occurs during pregnancy, it leads to the development of congenital rubella syndrome, which encompasses the well-established triad of cataracts, cardiac abnormalities, and sensorineural deafness [113,114]. Due to the importance of detecting the rubella virus, it is essential to use highly sensitive and effective detection methods. Among conventional approaches, using serological testing for rubella immunoglobulin (Ig) M is a standardized method for confirming acute infections [115,116]. Peroxidases, such as HRP, are widely employed in enzyme-linked immunosorbent assays (ELISAs) for the detection and quantification of antigens, antibodies, viruses, or cells. However, due to its inherent instability, HRP may result in a significant number of false-negative outcomes [117]. Taking inspiration from mesoporous silica-coated nanocrystals, which preserve the functional properties of their core and offer favorable surface functionalization characteristics, Li’s research group has developed a novel nanozyme denoted as Au-core@Pt-shell@mesoporous silica (Au@Pt@SiO₂) for immunoassays. The synthesized Au@Pt@SiO₂ nanozyme demonstrates catalytic capabilities in chromogenic reactions within immunoassays, thereby presenting a potential substitute for natural enzymes in conventional ELISAs. Additionally, a novel conjugate utilizing the antigen-labeled Au@Pt@SiO₂ nanozyme has been designed as a nanoprobe for virus serodiagnoses. The antigen-labeled Au@Pt@SiO₂ nanozyme exhibits highly sensitive peroxidase-like activity and remarkable catalytic stability and robustness, making it suitable for use in biochemical assays and clinical diagnoses (Figure 6) [118]. The research conducted by Li and colleagues, which focuses on the development of the Au-core@Pt-shell@mesoporous silica nanozyme and its utilization as a nanoprobe for virus serodiagnoses, represents a significant advancement in the field of rubella virus detection. The enhanced stability and catalytic properties demonstrated by this nanozyme offer significant potential for enhancing biochemical assays and clinical diagnoses, thereby promoting advancements in the accuracy and reliability of rubella virus detection and diagnoses.

2.6.4. SARS-CoV-2

Since its emergence in late 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, resulting in the development of a worldwide pandemic known as COVID-19 [119]. SARS-CoV-2, classified as an enveloped positive-sense single-stranded RNA virus, exhibits a high degree of contagiousness primarily through two modes of transmission: direct person-to-person contact and respiratory droplets [120]. In the context of a global epidemic outbreak, the primary area of concern lies in achieving a precise and expeditious diagnosis that encompasses characteristics such as simplicity and cost effectiveness [91]. Bradbury’s research team has developed an innovative paper-based device that integrates multiple components, including a lateral flow assay (LFA) test strip, dehydrated signal enhancement reagents (nanozymes and associated chemicals), and a sealed chamber containing a liquid enhancement buffer. These components are enclosed within a 3D-printed casing. The implemented device has demonstrated its efficacy by enabling the detection of N-protein in undiluted serum within a timeframe of 40 min, even at concentrations as low as 0.1 ng/mL. The device’s all-in-one design simplifies the process by requiring a single step to activate signal enhancement after the initial LFA detection [121].

To meet the requirements of POCT, Liu’s research team has developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting SARS-CoV-2 NP antigens. Furthermore, the assay demonstrates exceptional sensitivity, enabling precise detection of NP antigens even at concentrations as low as 10 pg/mL. The simplicity, sensitivity, speediness, and cost effectiveness of this assay make it an excellent choice for detecting NP antigens in individuals suspected of SARS-CoV-2 infection [122].

Furthermore, Oh’s research team has developed a novel colorimetric assay called MagLISA, which is based on magnetic nanoparticles and nanozymes linked to immunosorbent assays. The aim of this technology is to achieve highly sensitive and precise detection of the influenza virus. This assay effectively combines newly synthesized superparamagnetic MagNBs and AuNZs (gold-nanoparticle-based nanozymes) to enhance peroxidase activity. The MagLISA integrates three essential components: MagNBs for biomarker enrichment, AuNZs as a peroxidase-like artificial catalyst, and an anti-hemagglutinin (HA) monoclonal antibody (mAb) for specific recognition facilitation. This innovative probe facilitates the identification, separation, and visualization of the influenza virus through a one-step colorimetric analysis. To evaluate the catalytic properties of the probe, molecular dynamics (MD) simulation is employed, considering their surface characteristics. The MagLISA demonstrates a significantly enhanced limit of detection (LOD) of up to 10–14 g·mL⁻¹, particularly when combined with conventional commercial microreader equipment [123].

Recently, CRISPR-Cas-based detection systems have emerged as a valuable approach for detecting various biological entities, including nucleic acids [124,125,126], proteins [127,128,129], and small molecules (Figure 7) [130,131,132]. The combination of fluorescence detection and the CRISPR-Cas system has become widely adopted as the primary methodology in this field. Wu’s research team has innovatively incorporated a colorimetric approach into their study by harnessing the catalytic properties of MnO₂ nanozymes within a CRISPR-Cas12a system. The synthesized MnO₂ nanorods demonstrate exceptional oxidase-like activity, facilitating an efficient catalytic reaction between TMB and the nanozymes that leads to a visible color transition from pale yellow to blue. These MnO₂ nanorods are utilized as nanozyme labels for magnetic beads (MBs) through attachment via single-stranded DNA (ssDNA). In the presence of SARS-CoV-2, the activated Cas12a enzyme cleaves the ssDNA linker, inducing a color change for colorimetric detection. Notably, the MnO₂-nanozyme-mediated CRISPR-Cas system provides a simplified alternative to traditional horseradish peroxidase (HRP)-catalyzed TMB-H₂O₂ systems by eliminating the need for hydrogen peroxide (H₂O₂) and exhibiting high robustness [22]. These studies present compelling evidence for the potential of colorimetric assays to achieve sensitive and specific virus detection. The incorporation of diverse nanomaterials, such as MagNBs and MnO₂ nanorods, as peroxidase-like catalysts highlights the versatility offered by colorimetric methods in the field of virus detection. These innovative approaches provide valuable tools for rapid and cost-effective virus screening, showing great potential in their application to diagnostics and surveillance efforts.

2.6.5. ZIKV

ZIKV, a member of the flavivirus (FLAV) genus, exhibits similarities with other members of this group, including dengue, chikungunya, and yellow fever viruses. As a result, distinguishing clinical symptoms associated with ZIKV infection from those related to these viral infections poses a challenge. ZIKV was first detected in Brazil in 2015 and rapidly spread globally, affecting over 50 countries across multiple regions including Central and South America as well as Southeast Asia [133,134]. The primary concern regarding ZIKV is centered on the increased risk of microcephaly and other neurological abnormalities observed in infants born to mothers who contracted the virus during pregnancy [135,136]. Hsu’s research team has pioneered the development of a point-of-care (POC) test for detecting ZIKV. This innovative test consists of an immunosensor vial integrated with platinum/gold core–shell nanoparticles (Pt@Au NPs), which are artificial nanozymes, and utilizes an iPhone 7 Plus smartphone. Remarkably, this POC test demonstrates a high level of specificity in detecting ZIKV in whole blood, exhibiting minimal cross-reactivity with other flaviviruses (such as dengue virus, DENV) during screening for Zika infection. The test procedure is designed to be user-friendly, allowing individuals to independently perform the Zika infection test without requiring any assistance from medical personnel. The procedure entails the straightforward application of a blood sample onto the vial immunosensor, which contains antibodies-modified Pt@Au NPs (Ab-Pt@Au NPs), followed by gentle agitation, capturing an image using the smartphone, and awaiting the automated assessment report generated by the computational algorithms embedded in the smartphone. This POC test is noteworthy for its ability to analyze whole blood without the need for filtration steps. Its user-friendly operation, combined with the quantification capabilities provided by a smartphone, make it highly suitable for deployment at ports of entry, airports, and other relevant locations frequented by travelers returning from endemic regions. Furthermore, this test shows potential for its application in surveillance efforts by enabling geographical tagging of infected individuals [137]. The innovative test has exceptional specificity in detecting ZIKV and is user-friendly, seamlessly integrating with smartphone technology. It serves as a valuable tool in fighting the spread of the ZIKV virus, especially in regions with limited access to conventional laboratories.

2.7. Plasmodium

Malaria is a prevalent infectious disease that has a significant global impact. According to data provided by the reputable WHO, there were an estimated 241 million reported cases of malaria in 2020 across 85 countries designated as malaria-endemic [138]. Timely diagnoses and prompt initiation of therapeutic interventions are crucial for effective disease management and minimizing malaria-associated mortality. Additionally, the development of precise detection methodologies is of paramount importance to reduce unnecessary administration of antimalarial medications and prevent the emergence of potential drug-resistant strains [139]. In a study conducted by Nicklen et al., Pt-coated Au nanozyme probes (PtGNPs) efficiently captured the target analyte, with the platinum coating exhibiting peroxidase-like enzymatic activity under low pH conditions. Notably, after biomarker preconcentration and signal enhancement, the LOD for pLDH was determined to be 0.01 ng/mL, representing a significant 1000-fold improvement compared to conventional LFA methodology [27]. It is crucial to emphasize that malaria remains a pressing global health issue, highlighting the urgency of expedited diagnoses and treatment. The LFA, due to its practicality, represents a feasible solution for effectively addressing challenges related to accessibility and time-to-result. The integration of platinum-coated gold nanozyme probes into LFAs has resulted in significant advancements in terms of enhancing sensitivity. This innovative approach not only enhances the accuracy of malaria detection but also shows promise for expanding the range of diagnostic applications toward identifying various biomarkers.

3. Nanozyme-Mediated Antibacterial Mechanisms

With the rapid development of nanozymes in the field of antibacterial mechanisms, this paper aims to provide a comprehensive overview and analysis of their physical and chemical properties, as well as their associated antibacterial mechanisms. The elucidation and synthesis of the antibacterial mechanisms facilitated by nanozymes have significant implications for the application and management of nanozymes in the fields of infectious disease control, combating biological fouling, and the development of related nanozyme-based biomedical technologies. Given the wide range of nanozymes available, their complex physical and chemical properties, as well as the presence of various interfering factors, it is crucial to systematically organize and summarize the universal antibacterial mechanisms. Drawing on recent research findings, we provide a comprehensive review of four key antibacterial mechanisms exerted by nanozymes: regulation of ROS, HOBr/Cl regulation, extracellular DNA clearance, and lysozyme and light-triggered carvacrol release.

3.1. ROS Regulation

ROS is a comprehensive designation employed to describe a group of exceptionally reactive radicals that originate from unpaired electrons associated with oxygen, namely the hydroxyl radical (•OH) and superoxide (•O²⁻) [140]. In vivo, the main source of ROS is located at the substrate end of the mitochondrial inner membrane, where the electron transport chain complex facilitates the transfer of electrons from mitochondria to O₂. As a result, a portion of O₂ undergoes reduction, leading to the generation of either superoxide (O²⁻) or H₂O₂ [141]. ROS are widely recognized as deleterious metabolites capable of causing damage to cellular proteins, lipids, and nucleic acids. Simultaneously, ROS also serves as an effective phagocytic armamentarium, enabling the host to counteract invasive pathogens [142]. Despite their benefits, conventional antioxidants face challenges such as reduced stability, increased toxicity, and limited bioavailability [143] (Figure 8). In recent years, the progress of nanoscience and technology has introduced innovative antioxidant methodologies based on nanozymes, which have gained significant attention and implementation in combating ROS and their associated antibacterial effects (

Table 1. Mechanisms of the antibacterial activities of nanozymes by ROS regulation.

Nanozymes	Enzyme Mimicked	Bacterial Types	Antibacterial Mechanism	References
CeO2 nanozyme	Peroxidase	E. coli	Generation of •OH	[144]
Fe-N-C single-atom nanozyme	Peroxidase	E. coli, S. aureus	Generation of •OH	[23]
hybrid Ag/CeO2 nanocomposite	Superoxide dismutase	S. aureus	Generation of •O2	[145]
graphdiyne-nanowalls-wrapped hollow copper sulfide nanocubes (CuS@GDY)	Photothermal activity	E. coli, methicillin-resistant S. aureus, S. aureus	Generation of •OH and •O2	[147]
zinc oxide nanorod@graphdiyne nanosheet (ZnO@GDY NR)	Peroxidase	methicillin-resistant S. aureus, P. aeruginosa	Generation of •OH and •O2	[153]
Au@CeO2 hybrid nanozyme	Peroxidase	E. coli, S. aureus	Generation of •OH	[148]
TA-chelated Ag (TA-Ag) nanozyme	Peroxidase	E. coli, S. epidermidis	Generation of •OH	[154]
Mo@ZIF-8 nanozyme	Peroxidase	E. coli, S. aureus	Generation of •OH	[151]
HMPB@Cur@PDA nanozyme	Superoxide dismutase	-	Generation of •OH and •O2	[155]
CoIITBPP(bpy) nanozyme	Peroxidase	P. aeruginosa, E. coli, B. amyloliquefaciens, and S. aureus	Generation of •OH	[150]

). The CeO₂ nanozymes [144,145] have been demonstrated to operate through a detection mechanism that relies on the crystal structure of CeO₂, which is characteristic of their catalytic activity. In the presence of bacterial suspensions, oxygen vacancies are created, resulting in the release of oxygen atoms accompanied by two valence electrons. These electrons can subsequently be captured by Ce⁴⁺ ions and suspended oxygen molecules, leading to the generation of Ce³⁺ ions and •O²⁻. The enhanced Ce³⁺/Ce⁴⁺ ratio promotes the manifestation of SOD-like activity through the following mechanism: •O²⁻ + Ce³⁺ + 2H⁺ →H₂O₂ + Ce⁴⁺ [146]. Furthermore, diverse strategies have been investigated to effectively enhance the production of ROS mediated by nanozymes, thereby augmenting their antibacterial efficacy. These approaches include promoting plasmon–exciton coupling [147], utilizing spherical mesoporous SAzyme materials [23], leveraging NIR-enhanced peroxidase activity [148], and incorporating enriched multi-porphyrin structures [30]. These methodologies collectively aim to enhance the antibacterial effect by boosting the generation of ROS through nanozyme-based interventions. Through manipulation of the initial substrate concentration (e.g., TMB or H₂O₂), a comprehensive set of enzyme kinetic parameters, including the Michaelis–Menten constant (K_m) and maximum initial velocity (V_max), can be determined. These parameters serve as critical indicators for evaluating the substrate binding affinity and catalytic efficiency of nanozymes [144,147,149,150,151]. Moreover, a diverse array of enzymes with enzyme-like activities, including REDOX nanozymes containing SOD, CAT, POD, laccase, hydrolytic nanozymes, cleavage nanozymes, and other component cascade catalysts, can be covalently linked to expand the spectrum of antibacterial properties and broaden the application domain. These integrated systems hold significant potential for combating biofilm formation and addressing inflammation associated with bacterial infections. In summary, ROS demonstrate exceptional reactivity, capable of causing damage to cellular components while simultaneously serving as an innate defense mechanism against pathogens. The conventional use of antioxidants encounters inherent limitations, thereby stimulating the pursuit of innovative antioxidant strategies founded upon nanozymes. Notably, CeO₂ nanozymes, characterized by their distinctive crystal structure, have demonstrated considerable potential in bolstering ROS-mediated antibacterial undertakings. Moreover, alternative approaches involving plasmon–exciton coupling, mesoporous SAzyme materials, and NIR-enhanced peroxidase have been successfully explored, further enhancing the antioxidant capabilities of nanozymes.

3.2. HOBr/Cl Generation

Biofouling, a significant environmental phenomenon, is commonly encountered in marine ecosystems, exerting adverse impacts on diverse forms of marine life [156]. Marine organisms constantly face the persistent risk of epibiont overgrowth, compelling them to develop intricate chemical or physical antifouling mechanisms [157]. Marine algae have developed sophisticated chemical mechanisms to combat microbial fouling by interfering with quorum sensing, a complex process in which bacteria assess cell density and regulate various phenomena, including biofilm formation (Figure 9) [158]. In modern times, the significance of seaweed and certain lichens has been elucidated in their pivotal role in combating biofouling. They achieve this by catalyzing the production of biocidal hypohalous acid HOX (with X denoting I⁻, Br⁻, or Cl⁻) through the oxidation of halide ions with H₂O₂ [159]. Halogenic acids possess the ability to effectively eliminate bacteria and biofilms, making them a crucial component in biological antifouling strategies. Currently, various nanozymes based on vanadium and cerium have demonstrated exceptional halogenated-peroxidase-like activity when combined with H₂O₂ [156,158,160,161] (

Table 2. Mechanisms of the antibacterial activities of nanozymes by HOBr/Cl generation.

Nanozymes	Enzyme Mimicked	Bacterial Types	Antibacterial Mechanism	References
Vanadium pentoxide nanoparticles	Haloperoxidase	E. coli, S. aureus	Generation of HOBr	[158]
Cobalt-doped MoS2 (Co−MoS2)	Haloperoxidase	E. coli, S. aureus	Generation of HOBr	[161]
CeO2−x NRs	Haloperoxidase	-	Generation of HOBr	[160]
CeO2−x	Haloperoxidase	-	Generation of HOBr	[156]

). The identification of vanadium- and cerium-based nanozymes exhibiting elevated halogenated-peroxidase-like activity represents a promising approach towards the development of effective antifouling solutions, capable of disrupting biofilm formation and eliminating bacterial populations in diverse environments.

3.3. Extracellular DNA Clearance

Bacterial biofilms are complex multicellular communities enclosed within an extracellular polymeric substance (EPS), exhibiting robust structural integrity and serving as a mechanically resilient protective barrier. The intricate physical and biological characteristics inherent in biofilms contribute to their heightened resistance against conventional therapeutic interventions, thereby posing challenges in effectively managing infections associated with biofilms [162]. Upon exposure to traditional antibiotics, bacteria have a tendency to develop resistance, which not only perpetuates existing resistance but also exacerbates the overall challenge associated with effectively combating bacterial infections [8]. Simultaneously, the bacterial membrane plays a crucial role in the innate defense mechanisms of bacteria. However, antibiotics face limitations in penetrating the biofilm barrier, which restricts their antibacterial efficacy [163,164]. Currently, the utilization of endogenous deoxyribonuclease (DNase) has emerged as a promising strategy for mitigating biofilm formation by targeting the extracellular matrix (ECM) for removal [165]. Despite its potential as an antibacterial agent against biofilms, the implementation of natural DNase is hindered by certain drawbacks such as elevated costs, fluctuating catalytic activity, and irreversibility. To address this challenge, DNase nanozymes have emerged as a promising approach to effectively target and remove DNA in combating biofilms (

Table 3. Mechanisms of the antibacterial activities of nanozymes by extracellular DNA clearance.

Nanozymes	Enzyme Mimicked	Bacterial Types	Antibacterial Mechanism	References
DNase-AuNCs	DNase	-	NIR-activated PTT and PDT	[165]
CS@Fe/CDs	DNase and peroxidase	E. coli, S. aureus	Generation of •OH and cleavage of extracellular DNA	[168]
Au-Pt nanoparticles	Oxidation	E. coli O157:H7	Absorb DNA, photothermal conversion under NIR irradiation	[167]
Ag-Fe3O4@MoS2 MNPs nanocomposite	DNase	S. mutans, E. faecalis	Sustained release of Vanc, magnetic force exposure augments the penetration of MNPs through the various layers of biofilm	[169]
CuxO-PDA	Peroxidase	E. coli, S. aureus	DNA degradation, lipid peroxidation, and biofilm eradication	[170]
MOF/Ce-based nanozymes	DNase and peroxidase	S. aureus	Generation of •OH, CeIV target and hydrolyze eDNA	[166]

). The nanozymes effectively overcome the limitations associated with expensive and unstable natural enzymes, demonstrating their ability to penetrate the biofilm matrix, strongly inhibit biofilm formation, and thus exert a rapid bactericidal effect [166] (Figure 10).

Furthermore, from a broader perspective, DNase exhibits remarkable capabilities in rapidly disrupting the structural integrity of the ECM and significantly enhancing its sterilization efficacy when compared to conventional antibiotics. This effective approach is particularly valuable in combating drug-resistant bacterial strains and has extensive potential for various applications. Present in both natural and nanozyme forms, DNase exhibits a broad spectrum of sterilization activity, surpassing that of traditional antibiotics. As a result, its application demonstrates significant potential and extensive utility in combating infections associated with biofilm formations.

3.4. Lysozyme and Light-Triggered Carvacrol Release

Nanozymes, renowned for their exceptional catalytic efficiency, have demonstrated the ability to synergize with other bactericidal agents, thereby exhibiting a more potent multifaceted bactericidal effect. Nong’s research team has reported the development of a metal–organic framework-based nanozyme hybrid that showcases coordinated actions of lysozyme and light-triggered carvacrol release in eradicating bacterial infections. This innovative nanozyme hybrid encompasses a range of functionalities, including magnetic aggregation, photothermal conversion, electrostatic attractions that facilitate bacterial capture, enzymatic hydrolysis, and light-controlled carvacrol release [171]. The synergistic aggregation of nanozymes and complementary bactericidal substances represents a highly promising approach for the development of a broad-spectrum bactericide, exhibiting remarkable potential in the domains of biomedicine, food safety, and environmental protection. This strategy holds significant promise for achieving comprehensive bactericidal properties. The applicability of nanozymes extends to various fields, such as biomedicine, food safety, and environmental protection. The integration of nanozymes with distinct bactericidal mechanisms provides new opportunities for enhancing bacterial eradication effectiveness and addressing the challenges associated with antibiotic resistance.

4. Applications of Nanozymes in Antibacterial Mechanisms

In recent years, the exploration of novel class substitutes possessing mimetic functions has become a prominent research area, inspired by the remarkable attributes exhibited by natural enzymes. In this regard, significant attention has been garnered by various nascent nanomaterials collectively referred to as nanozymes due to their unexpected and compelling enzyme mimetic activities [37]. The robust antibacterial efficacy demonstrated by nanozymes is particularly noteworthy, as it has sparked the exploration of diverse innovative antibacterial strategies [172]. Within this section, our objective is to present a comprehensive compilation of exemplary nanozymes and their associated antibacterial strategies, categorized based on their elemental composition.

4.1. Metal-Based Nanozymes

4.1.1. Iron-Based Nanozymes

The utilization of iron-based nanomaterials has attracted significant attention in the field of biomedical applications, primarily due to their distinct paramagnetic or superparamagnetic magnetization characteristics. A Fe₃O₄@MoS₂-Ag nanozyme with an abundant defect rough surface was prepared using a simple hydrothermal method and in situ photo deposition method, demonstrating a good bacteriostatic effect (~69.4%) against Escherichia coli. Additionally, the magnetic recovery of Fe₃O₄ provides power for efficient disinfection of the nanozyme [173]. Drums et al. developed a cost-effective and versatile approach by utilizing paramagnetic iron oxide particles (SPION) conjugated with fructose metabolites, which effectively enhanced the uptake and therapeutic efficacy of SPION against both Gram-positive MRSA and Gram-negative biofilms (e.g., E. coli and Pseudomonas aeruginosa) [174]. These materials have demonstrated immense potential in various biomedical domains, including bio-separation, targeted drug delivery, magnetic resonance imaging, biosensors, and hyperthermia therapy [173,175,176]. In 2007, Gao and Yan presented pioneering evidence that magnetite (Fe₃O₄) nanoparticles possess inherent peroxidase-like enzymatic activity due to the substantial presence of Fe²⁺ and Fe³⁺ ions on their surface [53]. This pioneering research has served as a catalyst for the exploration and subsequent implementation of iron-based nanozymes in diverse biological domains, encompassing bioassays, tumor therapy, and antibacterial interventions [177,178,179]. The research conducted by Sun’s group has involved developing an enzymatic antibacterial approach centered around thermogenic nanozymes, which aims to achieve synergistic bacterial inhibition by enhancing the activity of nanozymes while simultaneously reducing the activity of natural enzymes in bacteria [29]. Specifically, yolk-shell structured Fe₂C@Fe₃O₄ NPs with a uniform size of approximately 20 nm were synthesized as the thermogenic nanozymes, exhibiting exceptional magnetothermal efficiency and escalated peroxidase-like activities facilitated by thermal enhancements [29]. Furthermore, in order to achieve an elevated level of nanozyme activity, a Fe,N co-doped ultrathin hollow carbon framework (Fe,N-UHCF) was synthesized and characterized by the substantial presence of Fe-Nx bonding as well as its distinct morphology. The Fe,N-UHCF framework exhibits exceptional peroxidase-like activity, surpassing that of nearly all previously reported nanozymes [180]. Ali and his colleagues conducted a study on the fabrication of Fe-doped MoS₂ (Fe@MoS₂) nanomaterials, which exhibited an enhanced peroxidase-like activity through a co-catalytic mechanism. The remarkable improvement can be attributed to the synergistic effect resulting from the concurrent co-catalytic activities of Fe and MoS₂, as well as the inherent characteristics of MoS₂ layers [178]. Furthermore, Liao and colleagues have achieved a significant breakthrough by engineering FePO₄-hydrogel (FePO₄-HG) as an exceptionally effective antibacterial therapeutic approach. The FePO₄ nanoparticles (NPs) were synthesized using the hydrothermal growth method and subsequently modified with L-cysteine to produce FePO₄-Cys. These were then covalently linked to the hydrogel (HG) through an amidation reaction, resulting in the fabrication of FePO₄-HG. This novel construct exhibits notable trienzyme-like functionality, eradicating bacteria through peroxidase-like catalytic capabilities while safeguarding normal cells from exogenous H₂O₂-induced damage via synergistic antioxidant effects stemming from the superoxide dismutase- and catalase-like activities of FePO₄-HG [181]. A multifunctional GGFzyme was successfully synthesized using a one-pot method, effectively and promptly combining glucose oxidase (GOx), gallic acid (GA), and ferrous ions (Fe²⁺). This innovative GGFzyme utilizes endogenous glucose molecules in the local environment to generate a controlled cascade of ROS, acting as a cytotoxic agent against MRSA commonly found in wound surroundings with high mortality rates. Notably, this approach eliminates the need for exogenous H₂O₂ administration. By efficiently reducing glucose concentration, GGFzyme promotes simultaneous elimination of pathogenic bacteria and wound healing, offering a promising therapeutic strategy for hyperglycemic wound treatment (Figure 11) [182]. In summary, these investigations highlight the vast potential of iron-based nanozymes in various biomedical fields, offering innovative solutions for bioassays, tumor therapy, antibacterial interventions, and other emerging biomedical challenges.

4.1.2. Cu-Based Nanozymes

Copper (Cu), a crucial microelement in living organisms, plays a significant role in the redox processes of native enzymes, as demonstrated by Cu-Zn superoxide dismutase and tyrosinase [183,184]. In parallel, although Cu ions have demonstrated commendable antibacterial efficacy, their significant toxicity limits direct utilization within living organisms [37]. Currently, investigations have confirmed the more favorable applicability of Cu-based nanomaterials as nanozymes due to their broader catalytic activity pH range compared to their Fe-based counterparts [37]. Drawing inspiration from natural enzymes’ active site structure, Liang and colleagues used Cu²⁺ to functionalize melanin nanoparticles (NMPs) in cuttlefish-ink-derived ink. This approach produced a highly stable and active SOD-like nanozyme (Cu-NMPs) with strong free radical scavenging capabilities. Subsequently, the simulated enzyme was incorporated into carrageenan films for food packaging purposes. Results showed that at a concentration of 10 μg/mL, Cu-NMPs generated over 80% •O⁻₂ and exceeded natural SOD enzyme activity by reaching 60 U/mL [185]. The research group, led by He, developed a versatile nanozyme called polydopamine (PDA)-modified copper oxide (Cu_xO-PDA). Cu_xO-PDA demonstrated peroxidase-like behavior, which was further enhanced under near-infrared (NIR) irradiation. Under neutral or alkaline conditions, Cu_xO-PDA had a negative surface charge and showed minimal peroxidase activity. However, under acidic conditions, the surface charge of Cu_xO-PDA can become positive, allowing for specific targeting of negatively charged bacteria. Interestingly, well-dispersed Cu_xO-PDA quickly aggregates upon NIR irradiation, trapping bacteria and nanozymes in close proximity. The study revealed that a decrease in the distance between the nanozyme and bacteria led to an increase in antibacterial efficacy. The conducted experiments demonstrated that the Cu_xO-PDA nanozyme induced DNA degradation, lipid peroxidation, and eradication of biofilm formations [170]. Liu et al. developed and synthesized biodegradable Cu-doped phosphate glass (Cu-PBG) nanozymes, which exhibit potent antibacterial activity against both Gram-positive and Gram-negative bacteria. The antimicrobial mechanism of Cu-PBG involves the generation of ROS and copper release. In wounds, it functions akin to peroxidase by inducing lethal oxidative stress on bacteria by catalyzing the decomposition of H₂O₂ into •OH. Additionally, Cu-PBG possesses inherent degradability attributed to its phosphate glass properties (Figure 12) [186]. These studies emphasize the potential of Cu-based nanomaterials as nanozymes, providing a promising alternative for various applications such as food preservation and antibacterial interventions. Their unique characteristics, including a wide pH range and enhanced catalytic efficacy under specific circumstances, make them invaluable tools in both biomedical and environmental fields.

4.1.3. Noble Metal-Based Nanozymes

Due to their notable surface metal atom ratios, various noble metal-based nanozymes, including Au [167], silver [169], platinum [187], and palladium [176], exhibit substantial catalytic activity. As a consequence, these nanozymes have found widespread utilization in the realm of biomedical applications, specifically in biosensing ventures [188]. Previous studies have predominantly observed monometallic compositions at the branch ends of dendritic structures. It is well-established that the peroxidase-like activity exhibited by monometallic nanozymes consistently falls short compared to their multimetallic counterparts [189,190]. According to the Sabatier principle, it is hypothesized that maintaining an optimal level of catalyst–intermediate interaction is crucial for achieving a balanced and moderate interaction between the catalyst and intermediate species [191,192]. Alloying is a suitable strategy for finely tuning the interaction between the catalyst’s surface and intermediate species. Pd nanocrystals have been shown to exhibit facet-dependent oxidase and peroxidase-like activities, endowing them with exceptional antibacterial efficacy through the generation of reactive oxygen species (ROS) [193]. Sun’s research group successfully developed a nanoplatform, designated as Pd@Pt-T790, which was easily synthesized by conjugating enzyme-catalytic Pd@Pt nanoplates with the organic sonosensitizer meso-tetra(4-carboxyphenyl) porphine (T790). Notably, it was observed that the incorporation of T790 onto Pd@Pt resulted in a significant suppression of the catalase-like activity exhibited by Pd@Pt. Under ultrasonic irradiation, the nanozyme activity was effectively restored, facilitating the catalytic decomposition of endogenous H₂O₂ into O₂. This “blocking and activating” phenomenon is crucial in mitigating potential toxicity and adverse effects of nanozymes on normal tissues, while also offering significant potential for achieving active, controllable, and disease-targeted nanozyme catalysis [188]. The development of ultrasonic (US)-switchable nanozyme systems represents a highly promising avenue for enhancing the active, controlled, and precise eradication of deeply entrenched bacterial infections through sonodynamic modalities. This innovative strategy demonstrates remarkable potential for future prospects in the realms of biomedical research and clinical therapeutics, laying the foundation for transformative applications.

Simultaneously, Yang et al. employed Cu,I-doped carbon dots (Cu, i-CDs) as a reducing agent in conjunction with nanozymes to fabricate an Au-based nanozyme composite material (AuNPs/Cu,I). The AuNPs/Cu,I nanozymes not only emulate the intrinsic activities of superoxide dismutase, peroxidase, and catalase under diverse conditions but also serve as surface-enhanced Raman spectroscopy (SERS) enhancers. The integration of Cu, I-CDs, and AuNPs enhances electron transferability, leading to augmented peroxidase-like activity and superoxide-like activity. The multi-enzyme-like functionality of AuNPs/Cu,I nanozymes can be precisely modulated by altering the composition of Cu⁰/Cu⁺ and Au [194]. In addition, Prasad’s team synthesized bimetallic Cu-M nanoparticles (M = Au, Ag, Pt, or Pd) using an electrical substitution (GR) reaction. The catalytic activity of the bimetallic system was compared with that of Cu nanozymes, and the Cu-Pt nanozymes showed the highest catalytic efficiency [195]. The combination of both materials takes advantage of the two properties of the redox processes of native enzymes of Cu and the significant catalytic activity of novel metal for applications of nanozymes in antibacterial mechanisms.

4.1.4. Vanadium-Based Nanozymes

Apart from the previously mentioned nanozymes based on Fe, Cu, and noble metals, vanadium (V)- and cerium (Ce)-based nanomaterials, possessing intrinsic mimic enzyme catalytic properties, are commonly denoted as vanadium-based nanozymes [37]. The exceptional capacity of vanadium (V)-based nanomaterials to mimic multiple enzymatic activities has been well-documented, demonstrating their efficacy in combating bacterial infections. These nanomaterials exhibit remarkable potential for addressing such infections due to their superior multienzyme-mimicking properties [196]. Ma et al. successfully developed a highly efficient bienzyme system, which demonstrated potent synergistic antibacterial activity. Vanadium oxide nanodots (VO_xNDs) were synthesized using a streamlined ethanol–thermal strategy with vanadium (III) trichloride (VCl₃) as the precursor. The as-synthesized VO_xNDs exhibited remarkable peroxidase- and oxidase-like activities, mimicking natural enzymes’ functions. This antibacterial system showed exceptional efficacy against both non-resistant bacteria, including Gram-positive S. aureus and Gram-negative E. coli, and drug-resistant strains such as ESBL-producing E. coli, MRSA, and kanamycin-resistant E. coli. Importantly, due to their nanoscale dimensions, the VO_xNDs displayed favorable biosafety as confirmed with various assessments including MTT assays, examination of physiological indices, and H&E staining [196]. Hu’s research team successfully synthesized a targeted nanozyme, Co^IITBPP(bpy), using meticulous supramolecular self-assembly techniques. This unique nanozyme possesses peroxidase-like properties and can generate •OH radicals without light, showing exceptional production of ROS under irradiation. This causes bacterial membrane disruption and the release of intracellular contents, resulting in synergistic antibacterial effects. Co^IITBPP(bpy) also efficiently decomposes excess H₂O₂ into O₂, thanks to its outstanding catalase-like activity. The catalytic process improves PDT efficacy, and reduces tissue hypoxia and excessive H₂O₂ levels. The synthesized nanozyme shows good biocompatibility and achieves over 95% antibacterial efficiency in vitro. The supramolecular self-assembly of these multi-porphyrin structures offers great potential for use in antibacterial treatments and wound healing [150]. Furthermore, vanadium- and cerium-based nanomaterials have emerged as promising candidates for mimicking haloperoxidase activity to address the challenge of low stability in natural enzymes. These nanomaterials possess the capability to mitigate biofouling and enable the development of materials with prolonged stability even in demanding environments [197]. Hu’s research group developed free-standing nanofibrous mats using electrospinning PVA with CeO_2−x NRs. After cross-linking, the resulting PVA mats incorporated CeO_2−x NRs and exhibited haloperoxidase-like activity in water environments. The CeO_2−x activity within the PVA fibers was quantitatively assessed with bromination of phenol red, confirming that the nanozyme remained active even after being processed in the polymer matrix. These mechanically robust hybrid mats exhibited catalytic oxidation of Br⁻ and H₂O₂ to HOBr, similar to natural haloperoxidases. Leveraging the haloperoxidase-like activity of ceria NRs in PVA mats, along with their easy production, these PVA/CeO_2−x hybrid fibrous networks show promise for robust anti-biofouling coatings on surfaces exposed to humid and marine environments [160]. The present study elucidates the inherent potential exhibited by nanomaterials derived from vanadium and cerium as nanozymes, thereby offering a wide range of applications including antibacterial therapies, wound healing, and the fabrication of durable materials tailored for demanding conditions. The unique catalytic properties and excellent compatibility with biological systems make these nanomaterials highly promising candidates for future biomedical advancements and environmental initiatives.

4.2. Carbon-Based Nanozymes

Recently, carbon-based nanomaterials have emerged as highly promising entities in the field of biomedicine due to their exceptional physicochemical properties, commendable biocompatibility, cost effectiveness, and versatile mimicry of multiple enzyme functionalities [37]. Numerous efforts have been dedicated to exploring the antibacterial potential of carbon-based nanomaterials. Fang et al. successfully synthesized and modified a variety of oxygenated nanodiamonds (O-NDs) with different oxidation degrees using a simple yet efficient mixed acid-assisted reflux method, giving them remarkable peroxidase mimicking capabilities. A thorough spectroscopy analysis and chemical structure characterizations conclusively revealed the presence of carbonyl, carboxyl, and nominal hydroxyl groups in these O-NDs. Consequently, these nanomaterials exhibited enhanced enzymatic activity across a wide pH range. Importantly, spectroscopic observations and structural investigations suggested a significant increase in enzymatic activity during prolonged experimental durations. Furthermore, the addition of trace amounts of H₂O₂ resulted in exceptional antibacterial efficacy demonstrated by O-NDs, effectively eliminating periodontal bacteria and disrupting biofilms under physiologically relevant conditions both in vitro and in vivo [198]. Carbon-based nanomaterials, particularly oxygenated nanodiamonds, have shown great potential in the field of biomedicine due to their exceptional properties and surface modifications that enhance enzymatic activity and potent antibacterial effects. These significant advancements offer promising avenues for exploring novel antibacterial strategies and therapeutic interventions in biomedicine.

4.3. Transition Metal-Based Nanozymes

Transition metal nanomaterials have attracted significant attention due to their exceptional photo-thermal and electronic properties, as well as their commendable biosafety profile. These characteristics make them highly valuable and extensively utilized in various domains including sensing, imaging, catalysis, and biomedicine [199,200]. Bai’s research team developed a new heterostructure called ZnO@GDY NR, which consists of zinc oxide nanorods layered with graphdiyne nanosheets. This innovative configuration acts as a piezocatalytic nanozyme, where the graphdiyne layer provides electrical conductivity, adsorption, and catalytic nanozyme activity, while the zinc oxide layer is responsible for piezo catalysis and catalytic nanozyme activity. The resulting piezocatalytic nanozyme shows impressive antibacterial efficacy, with nearly 100% effectiveness against drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa in both in vitro and in vivo settings [153]. The present investigation not only demonstrates the exceptional antibacterial efficacy of the ZnO@GDY NR heterostructure as a nanozyme but also highlights its commendable biocompatibility characteristics. These valuable observations not only contribute essential knowledge but also serve as a pivotal reference for the conceptualization and advancement of forthcoming antibacterial nanozymes that prioritize enhanced biocompatibility, thereby fostering their potential in biomedical applications.

4.4. Single-Atom Nanozymes

Controlling metal species at the atomic level is a highly effective strategy for enhancing both the efficiency of metal utilization and the catalytic performance of nanomaterials [201]. In 2011, Qiao and his research group introduced the innovative concept of single-atom catalysts (SACs), which has since revolutionized the field of catalysis [202]. Following their creation, SACs have gained significant attention in the field of catalysis due to their advantages including efficient atom utilization, high catalytic activity, exceptional stability, and remarkable selectivity [34]. Recently, a group of SACs have emerged as single-atom nanozymes (SAzymes), exhibiting atomically dispersed active sites and coordination environments comparable to those observed in natural metalloenzymes. This novel development not only provides valuable insights into the catalytic mechanisms of nanozymes but also establishes a crucial connection between natural enzymes and nanozymes [37]. SACs exhibit strong catalytic ability and allow for efficient use of metals, which is particularly important in cancer and antibacterial treatments. This capability ensures effective therapeutic outcomes even at low metal concentrations (Figure 13) [203,204]. N-doped porous carbon (NPC) has gained widespread recognition as a specialized nanoplatform for constructing SACs, thanks to its commendable catalytic efficacy, outstanding biosafety profile, distinctive mesoporous structure, and substantial specific surface area. In this study, they present the synthesis of Cu single-atom sites/NPC (Cu SASs/NPC) via a PEAP strategy, which exhibits notable nanozyme properties for photothermal–catalytic antibacterial therapy. The Cu SASs/NPC composite demonstrates superior catalytic activity, glutathione-depleting capabilities, and photothermal effects compared to pure NPC without Cu doping. These Cu SASs/NPC specimens, being peroxidase-like nanozymes, efficiently catalyze the conversion of H₂O₂ into •OH, facilitating conspicuous antibacterial efficacy. Additionally, under laser irradiation, the photothermal effect of Cu SASs/NPC enhances the peroxidase-like catalytic function, driving increased production of ROS and consequently yielding enhanced in vitro antibacterial effects [205]. In addition, Wang’s research group successfully synthesized a Mo SA-N/C single-atom nanozyme with remarkable heterogeneous peroxalate (HPO)-mimicking activity and visible-light-responsive photothermal behavior, offering a promising approach to mitigate biofouling in seawater environments. As an HPO-like nanozyme, Mo SA-N/C effectively catalyzes the oxidation of bromide ions (Br⁻) to produce cytotoxic hypobromous acid (HOBr). Furthermore, the application of visible light irradiation significantly enhances the oxidation activity of Br⁻ on the surface of Mo SA-N/C through a pronounced photothermal effect. To better understand how Br- is oxidized, we conducted DFT calculations. Ultimately, Mo SA-N/C showed excellent antibacterial properties when exposed to both Br- and H₂O₂, as well as impressive performance in preventing biofouling. This accomplishment opens up possibilities for the advancement of biocompatible and photo-responsive synthetic enzymes, expanding our capacity to combat marine biofouling [206]. The application of Cu single-atom sites/N-doped porous carbon (Cu SASs/NPC) as nanozymes exhibits great potential in advancing photothermal–catalytic antibacterial therapy, thus emphasizing their promising role in future biomedical applications.

4.5. MOFs-Based Nanozymes

MOFs are crystalline materials composed of metal ions and organic bridging linkers, characterized by diverse porous structures and exhibiting attributes of exceptionally high porosity (up to 90% free volume) and a significant surface area within the organic framework [207]. Due to their porous structures, MOFs have gained significant attention as protective matrices for immobilizing enzymes through chemical grafting or physical adsorption, effectively preventing denaturation caused by external stimuli [208,209]. Liu et al. proposed a novel approach combining cerium complexes (DNase mimics) and MOFs with peroxidase-like activity to create an artificial nanozyme with dual enzyme-mimetic capabilities. This integrated nanozyme effectively disperses biofilms and eliminates bacteria without the adverse effects commonly associated with antibiotics. The cerium (IV) complexes acted as DNase mimics, effectively breaking down eDNA and disrupting mature biofilms. Meanwhile, the MOF with peroxidase-like activity killed bacteria in dispersed biofilms by using H₂O₂. The combination of these two types of nanozymes effectively prevented bacterial recolonization and biofilm reoccurrence, which is a rational strategy for combating biofilms considering the challenges associated with using single-modal antibacterial agents [166]. Hu et al. successfully synthesized ultrasmall gold nanoparticles (UsAuNPs) on ultrathin two-dimensional MOFs through an in situ reduction process. The resulting hybrid material, UsAuNPs/MOFs, combines the advantages of both UsAuNPs and ultrathin 2D MOFs. This hybrid material shows remarkable peroxidase-like activity, efficiently decomposing H₂O₂ into •OH. The antibacterial efficacy of the UsAuNPs/MOFs nanozyme was evaluated against both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), achieving excellent antibacterial properties with low concentrations of H₂O₂. Animal experiments showed that the hybrid material effectively promotes wound healing and is biocompatible, indicating the potential of hybrid nanozymes in antibacterial therapy for future clinical applications [210]. These innovative methodologies highlight the potential of MOFs and nanozymes in tackling the challenge of biofilm eradication. By leveraging the unique characteristics and synergistic interplay of these materials, researchers are striving to advance strategies that offer enhanced efficacy in combating biofilms. This is particularly crucial, as biofilms represent a persistent obstacle that is notoriously resistant to eradication using conventional monotherapy antibacterial agents.

Additionally, the integration of MOFs with other nanomaterials can manifest supplementary functional attributes. Through the amalgamation of MOFs and diverse materials, the resultant composite systems can exhibit synergistic effects, thereby augmenting antibacterial activity and efficacy [211]. In addition, by incorporating antibacterial agents into MOFs or utilizing MOFs as carriers for other materials, the controlled release of antimicrobial agents can be achieved, thereby ensuring prolonged and effective antibacterial activity [212]. Moreover, the porous structures of MOFs can function as protective matrices for immobilizing anti-bacterial agents, thereby preventing their degradation or premature release. This safeguarding mechanism contributes to the preservation of stability and efficacy of antibacterial agents, ultimately enhancing their durability and performance in antibacterial applications [213]. The combination of MOFs with other materials in antibacterial applications can provide synergistic antimicrobial properties, controlled release of antibacterial agents, and preservation of their efficacy.

5. Summary and Outlook

This review presents a comprehensive overview of recent advances in the utilization of nanozyme-based platforms for pathogen detection and control. Compared to their natural enzyme counterparts, nanozymes offer distinct advantages such as cost effectiveness, high stability, controllable size, ease of preparation, and superior catalytic activity. These features position nanozymes as highly promising agents for antibacterial applications and versatile detection media. Despite the remarkable performance of current nanozymes in detection and antibacterial activities, their application remains largely in the preliminary stage. The field of nanozymes for clinical antibacterial therapy and diagnoses has not made significant progress due to limited research efforts. Furthermore, the development of nanozymes still faces numerous challenges that must be addressed for further advancement and widespread implementation.

Primarily, it should be noted that the catalytic activity demonstrated by nanozymes is comparatively inferior to that of natural enzymes, thereby limiting their capacity to fully meet the demands of pathogen detection and control. Furthermore, recent investigations have confirmed that the catalytic performance of nanozymes is profoundly influenced by a multitude of intrinsic physicochemical properties such as shape, size, and surface modification, as well as extrinsic factors including temperature, pH, and substrate concentration. However, the understanding of how complex biological microenvironments impact the catalytic activity of nanozymes and their long-term effects remains largely unexplored.

Secondly, it is noteworthy that certain types of nanozymes exhibit limited substrate selectivity, which may lead to potential undesired effects in biological environments. Therefore, precise therapy based on nanozymes has emerged as a prominent research area requiring substantial efforts to enhance the selectivity of nanozymes for targeted therapeutic interventions. Building upon existing research, we envision that the achievement of substrate-specific identification and catalysis can be realized through the implementation of appropriate adaptations, such as incorporating aptamers, chiral molecules, or molecularly imprinted polymers onto the surface of nanozymes. Alternatively, the synergistic combination of nanozymes with natural enzymes possessing inherent substrate selectivity offers an alternative approach to overcome this limitation and advance targeted therapy applications.

Thirdly, it is worth noting that nanozymes are based on enzymatic principles; however, their properties significantly differ from those of natural enzymes. Therefore, conventional methods used to characterize enzyme performance face challenges when applied to nanozymes. The widely used Michaelis–Menten mechanism for analyzing natural enzymes is based on homogeneous catalysis in a uniform medium, while nanozymes operate through a heterogeneous mechanism that occurs on the surface of nanomaterials. Therefore, it is imperative to establish a consistent framework and standardized criteria for effectively evaluating and elucidating the performance attributes of nanozymes.

Fourthly, it is worth noting that current research endeavors pertaining to the detection of target analytes in food samples predominantly concentrate on liquid matrices, such as milk and juice beverages. Conversely, relatively inadequate attention has been directed towards the examination of solid food samples. Consequently, it is fundamental to allocate additional resources and conduct extensive investigations to further elucidate and explore this area of study.

Moreover, it is worth noting that a significant number of nanozymes designed for antibacterial applications exhibit notable inhibitory effects on bacterial growth in vitro. However, the translation of such findings into in vivo models and treatment strategies remains at an early stage. Considering the intricate nature of biological systems, intensifying in vivo investigations of nanozymes is advantageous for evaluating their therapeutic efficacy, biocompatibility, and biodegradability.

Lastly, despite the existence of several investigations proposing potential catalytic mechanisms of nanozymes, numerous challenges and obstacles persist in the comprehensive elucidation of their catalytic pathways. In forthcoming research endeavors, it is imperative to delve deeper into the catalytic reactions of nanozymes. The advancements achieved in the field of computer science, particularly in molecular dynamics’ simulations, offer promising opportunities for unraveling the intricate interplay between the structure, activity, and catalytic mechanisms exhibited by nanozymes. Accordingly, the utilization of collaborative computational simulations, theoretical calculations, and the integration of artificial intelligence techniques possesses significant potential in facilitating a comprehensive comprehension of the molecular-level catalytic mechanisms employed by nanozymes.

In conclusion, the field of nanozymes utilized in pathogen detection and control has witnessed significant advancements. However, it is crucial to acknowledge that current developments have not fully satisfied the requirements of practical applications thus far. Consequently, concerted efforts are imperative in addressing the unresolved challenges, which will contribute to substantial progress in forthcoming research endeavors. This comprehensive review is anticipated not only to enhance researchers’ engagement and enthusiasm in the field of nanozymes but also to provide invaluable information and insights that facilitate exploration of previously uncharted catalytic mechanisms inherent within nanozymes. Therefore, the aforementioned challenges represent the forefront of future nanozyme research, necessitating further exploration and investigation.