The Nanozyme Revolution: Enhancing the Performance of Medical Biosensing Platforms

Nanozymes represent a class of nanosized materials that exhibit innate catalytic properties similar to biological enzymes. The unique features of these materials have positioned them as promising candidates for applications in clinical sensing devices, specifically those employed at the point‐of‐care. They notably have found use as a means to amplify signals in nanosensor‐based platforms and thereby improve sensor detection limits. Recent developments in the understanding of the fundamental chemistries underpinning these materials have enabled the development of highly effective nanozymes capable of sensing clinically relevant biomarkers at detection limits that compete with “gold‐standard” techniques. However, there remain considerable hurdles that need to be overcome before these nanozyme‐based sensors can be utilized in a platform ready for clinical use. An overview of the current understandings of nanozymes for disease diagnostics and biosensing applications and the unmet challenges that must be considered prior to their translation in clinical diagnostic tests is provided.


Introduction
Biological enzymes are known for their ability to accelerate the rate of specific reactions, principally in the context of physiological processes.As such, they exhibit extremely high catalytic activity and substrate specificity, which produce favorable reaction kinetics that can be exploited for the rapid formation of a measurable substrate.When enzymes are utilized in a system designed such that the reaction is initiated by the presence of a target of interest, the formation of product can be a rapid and potentially quantifiable indication of target presence.Whilst the most effective of these enzymes (e.g., horseradish peroxidase [1] and alkaline phosphatase [2] ) have found use as powerful tools in lab-centric DOI: 10.1002/adma.202300184diagnostics, their stability, [3] usability, [4] scalability [5] and biosynthetic manipulations [6] hinder their employment in clinical and, in particular, point-of-care diagnostics.Notably, since most natural enzymes are proteins, they can be readily denatured by environmental changes or digested by proteases and thus their catalytic performance is often detrimentally affected by harsh (and in some instances, physiological) conditions.
As such, considerable efforts have been made toward the development of nanozymes, a class of synthetic nanosized materials that mimic enzyme function (Figure 1).Akin to their biological counterparts, nanozymes are capable of drastically accelerating the rate of physiologically relevant reactions.Nanozymes have been synthesized in a plethora of different compositions, topologies and morphologies.For example, nanomaterials ranging from relatively simple noble metal-and metal oxide-based nanoparticles [7] to complex multiconstituent metalorganic frameworks [8] and/or covalent organic frameworks [9] have demonstrated enzyme-like catalytic activities.General advantages of nanozymes compared to natural enzymes include: a generally simple preparation process; a potential for significantly lower cost when produced at scale; and relative stability with regard to structural degradation, particularly in demanding conditions such as high temperatures and extreme pH. [10]Furthermore, the synthetic methodologies used to produce nanozymes offer precise control over structure-property effects, and this has been clearly demonstrated in the recent realization of "smartly designed" single atom nanozymes where nanomaterials with atomically interspersed active centers have shown superior activity, selectivity and stability. [11]wing to these benefits, there has been great interest in the incorporation of nanozymes in diagnostic assays where target detection combined with rapid signal amplification can result in greater sensor performance.At the forefront of research involving use of nanozymes in biosensors and bioassays is the realization of extremely effective peroxidase mimics.These materials effectively catalyze the disproportionation of H 2 O 2 to carry out the oxidation of organic and inorganic compounds.The most relevant of these are chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ophenylenediamine (OPD), which upon oxidation give rise to colored compounds with high extinction coefficients (Figure 2).High-performance peroxidase mimics are extremely pertinent as an alternative to horseradish peroxidase (HRP), which has been the lynchpin of biological enzyme use in laboratory-centric colorimetric immunoassays owing to its significant catalytic activity.It should also be noted that effective nanozymes have also been reported to emulate other biological enzyme types such as oxidase, [12][13][14] catalase [15,16] and superoxide dismutase; [17] though with regard to signal amplification, the ability of peroxidase and oxidase nanozymes to perform catalytic oxidation of chromogenic substrates is of particular importance.
Recent reports of nanozyme-assisted platforms for the effective sensing of clinically relevant biomarkers have shown detection limits and cross-reactivity profiles which compete with, and in some cases surpass, "gold-standard" techniques.However, there remains considerable boundaries that need to be addressed before nanozyme-based biosensors and bioassays can be widely utilized in a platform available for clinical diagnosis.Herein, we examine current progress of and future opportunities for nanozyme-based technologies in disease diagnostics and biosensing applications.In Section 2, we specifically describe how the introduction of nanozymes can enhance the performance of point-of-care diagnostic tests.In Section 3, we specifically examine promising nanozyme materials and platform methodologies involving nanozymes for in vitro diagnostic tests.In Section 4, we discuss the barriers to and potential for clinical translation of these tests for non-communicable and communicable diseases.Overall, this text seeks to inspire further development and adoption of nanozyme-based diagnostic and biosensing technologies by engineers, scientists, clinicians, and public health experts.

Nanozymes in Point-of-Care Devices
Point-of-care (PoC) tests are medical diagnostic tests that are administered at the point of need and have proven particularly useful as a decentralized healthcare technology for monitoring disease outbreak, enabling high patient throughput and improving patient access to healthcare.In 2003, the World Health Organi-zation (WHO) published a set of guidelines to describe the specifications of an ideal point-of-care test to be used in any resource setting worldwide.Although these criteria were developed with the diagnosis of communicable diseases in mind, it is broadly accepted to refer to these standard when developing PoC devices for both communicable (CDs) and non-communicable diseases (NCDs).Initially coined as the ASSURED criteria (affordability, sensitivity, specificity, user-friendliness, rapid and robust, equipment-free, and deliverable to the end user, respectively) [18] in 2019, Rosanna Peeling and her team proposed an amendment to the criteria to meet the demands of an increasingly technologically advanced society.These criteria, known as REASSURED, added the concepts of real-time connectivity (possibly through mobile health (mhealth), which allows for internet-connected real-time diagnostics using a smartphone as both an assay reader and a data analytics tool), [19] Ease of specimen collection, and Environmental friendliness to the specifications for PoC diagnostic platforms. [20]The emergence of nanozymes and their unique benefits in the context of PoC tests present an extremely promising opportunity for the realization of innovative REASSURED diagnostic platforms (Figure 3A) in accordance with these guidelines.Recent advancements in each of these areas are discussed below.
The cost of components used to produce PoCs is often a key driver in the development and uptake of new tests.The relative affordability of nanozymes can address this issue.In contrast to their natural enzyme counterparts, the synthesis of nanozymes is well-suited to large scale production, and thus economies of scale can be utilized to reduce material and chemical cost in overall test manufacture.Mimicking the widespread use of nanoparticle probes, nanozymes can be readily integrated with paper-based assays, which are highly cost-effective due to the wide availability of their low-cost materials. [21]Furthermore, to enable their employment in immunosorbent-based tests, nanozymes are often functionalized with biorecognition elements such as antibodies, aptamers or oligonucleotides. [22]This functionalization is typically achieved through either physisorption or covalent bonding strategies, as these techniques are widely accessible and utilize relatively inexpensive conjugation chemistries and are therefore not expected to incur significant cost not arising from the biorecognition binders themselves.
The potential for test signal amplification and the resulting improvement in assay sensitivity is arguably the biggest potential advantage of nanozymes in bioassays.The principal accepted disadvantage of current PoC technologies is the sacrifice of sensitivity for test portability; the most recognized example of this limitation is current lateral flow tests not meeting the detection limits possible with "gold standard" polymerase chain reaction (PCR) tests.Compared to gold nanoparticles (AuNPs) that are commonly utilized as an optical probe in many paper-based PoC tests, the intrinsic catalytic activity of nanozymes can be utilized to attain greater sensor performance through the auxiliary formation of a colored substrate, increasing the amount of material detectable by eye or absorbance based measurements.This improves the test sensitivity and identifying positive test results that would have been incorrectly classified as false negatives.Numerous approaches have been employed to design highly sensitive nanozymes; for instance, the properties of AuNPs have been synergistically enhanced to exhibit peroxidase activities by   A-C) Reproduced with permission. [27]Copyright 2021, American Chemical Society.D,E) Relative activity of AuNPs@MoS 2 -QDs at temperatures between 25 and 80 °C (D), and pH 1-12 (E).D,E) Reproduced with permission. [25]Copyright 2018, Elsevier.F) Response of reused ceria paper-based assay after 1 (left) and 10 (right) cycles, the top row is the sensing paper after reconditioning, the bottom row of dark yellow is the colorimetric response after H 2 O 2 exposure.Reproduced with permission. [21]Copyright 2011, American Chemical Society.
combining them with other metals. [23]As an example, Au-Nibimetallic NPs doped in graphite carbon nitride sheets have shown a higher substrate affinity toward TMB compared to that measured for their monometallic counterparts and HRP. [24]This was attributed to the synergistic electronic interaction between the Au and Ni atoms in the bimetallic nanostructure.Owing to their respective ionization potentials, an effective electron transfer from Ni atoms to Au atoms resulted in an increase in the electron charge density on Au and thus both Au and Ni atoms were able to act as active catalytic sites for the peroxidase-like activity in the nanocomposite.Utilization of this electron transfer effect to increase electron density and mobility on active catalytic sites appears to be common in the formation of effective peroxide mimics.For example, AuNPs decorated with molybdenum disulfide quantum dots (Au@MoS 2 ) where strong Au-S bonds facilitate an electron charge transfer from the sulfur to Au atoms have also been reported to show higher substrate affinity and catalytic rate than HRP, and a limit-of-detection (LOD) improvement when compared with the Au-Ni-bimetallic NPs for glucose detection. [25]Additionally, many nanozymes are designed by taking advantage of the peroxidase activities exhibited by platinumgroup metals. [26]This has been demonstrated to good effect with platinum nanoparticles, which show an LOD decrease when compared with HRP and can be further enhanced with the formation of bimetallic nanozymes.For instance, platinum nanoparticles fashioned with Ni-rich cores and Pt-rich shells have reported a LOD of 1.1 pg mL −1 for the cancer biomarker carcinoembryonic antigen (CEA) in a plate-based assay, displaying a k cat 10 4 times higher than natural peroxidases (Figure 3B). [27]he progressive enhancement in performance (HRP < PtNPs < Ni-Pt NPs) has been informed by DFT calculations, which show a decrease of free energy necessary to oxidate the substrate (Figure 3C).These examples demonstrate the tunability of nanomaterials and how size, shape, and composition can modulate the surface electron density to both exceed the catalytic rate of natural enzymes and engineer more sensitive biosensing devices.
The functionalization of nanozymes with biorecognition elements ensures test specificity, although this is known to reduce catalytic rates, and thus test sensitivity, by increasing steric blocking of active sites. [23]Hence, sensitivity and specificity are often inversely related.However, instances of lower specificity can be tolerated when the harm of overtreatment is much less significant than a missing diagnosis. [20]Nonetheless, a pioneering recent report successfully utilized nanozymes in a cross-reactive sensor array. [28]A signal was generated due to the differential competitive nonspecific interactions between nanozymes and analytes.An absorbance measurement was recorded, and the analytes were identified against each other using linear discrimination analysis.This technology was used to detect biothiols in serum and proteins in human urine.[31] Despite this progress, there is a shortage of validation studies with human samples that determine the sensitivity (false negatives) and specificity (false positives) of literaturereported nanozyme-PoC-based biosensors.This can be attributed to the difficulty of accessing human patient samples and lack of industrial partners that would be able to translate devices for clinical validation and possible commercialization.
The goal of the PoC field is to realize easy-to-perform tests with minimal user intervention that are equipment-free or operated with small portable devices.There are several examples of nanozymes employed in paper-based devices for qualitative and quantitative analyte detection.Paper-based assays are highly compliant with the ASSURED criteria due to their easy samplein-answer-out mechanism that requires very little user interaction.Colorimetric nanozyme-based lateral flow immunoassays (LFIAs) are desirable since they can generate a high signal intensity that is either visible to the naked eye (in cases of qualitative assays) or measured with a smartphone camera or portable scanner (in instances of quantitative assays).Where possible, nakedeye visualization for ultrasensitive tests is preferred as it excludes the requirement for and any costs associated with sophisticated transducers that would otherwise be necessary for assay readout.The deployment of nanozyme-assisted lateral flow devices in the field is dependent on the infrastructure needed to administer the chemicals required for the signal amplification step and the portability of the instrumentation needed to read the tests.The latter restrains the use of fluorescent and electrochemical nanozyme-based assays in PoC devices, despite their promise of more accurate analytical measurements, due to the involvement of sophisticated complex instrumentation.
PoC tests should be rapid, such that results can be obtained as soon as possible after sample collection.The high surface energy and area and increased electron density of nanozymes enable easier electron transfer, exhibit higher substrate affinity, and maximize reaction velocity.These features aid in the rate of development of measurable substrate and thus can support the rapidnature of PoC tests.Nanozymes should also be robust, meaning that they should have a long shelf-life and be able to withstand the circumstances of a supply chain without requiring additional storage conditions. [20]Fortunately, the robustness of nanozymes is widely acknowledged as they are known to be resistant to loss of catalytic activity under harsh conditions such as temperature and pH and have shown minimal loss in performance over prolonged periods of time. [23,30,31]This has been demonstrated for AuNPs@MoS 2 -QDs, which show minimal decrease in relative catalytic activity across a broad range of temperatures and pH values (Figure 3D,E) Compared to natural enzymes, nanozymes can also be environmentally friendly, due to the reduced need for refrigeration, possible reusability, and the previously mentioned comparative ease in manufacturing and compatibility with paper-based devices.For example, a CeO 2 nanozyme paper-based biosensor has been developed for the detection of H 2 O 2 and glucose, and this biosensor has been reported to be reusable up to 10 times without loss of performance (Figure 3F).In addition, long-term storage (79 days) at room temperature was shown to not cause any observable loss of analytical performance. [21]The sample collection process for PoC devices also has the potential to be improved through the use of nanozymes.In addition to their catalytic activity, nanozymes can be designed to exhibit additional physical properties such as magnetism.Magnetic nanozymes have been utilized to enable easy sample processing with regard to separation and enrichment of the target from a sample.Here, we allude to silica-shelled magnetic nanobeads and gold nanozymes (AuNZs) that were combined for the detection of influenza virus A. [32] AuNZs were tested with clinically isolated human serum samples and exhibited a LOD of 2.6 PFU mL −1 .Although this work was performed in a microplate assay, the underlying nanozyme technology has the potential to be translated to PoC and filter relevant biomarkers from complex matrices like whole blood.With sample enrichment techniques, it is possible that less invasive sample extraction techniques will lead to a smaller amount of sample required per assay.
It is common for studies employing nanozymes in PoC tests to have shown a level of clinical applicability by demonstrating performance utilizing complex samples (saliva, urine, plasma) spiked with the antigen of interest.[35][36] Notwithstanding, our group have shown the naked-eye detection of acute stage HIV in clinical plasma samples under 20 min through a nanozyme-amplified LFIA. [37]Here, platinum nanocatalysts (PtNCs) are employed for test line signal amplification as a means to increase the detection limit of the assay (discussed further vide infra), where assay validation against longitudinal clinical samples successfully followed HIV seroconversion by enabling naked-eye detection of p24 capsid protein.In another work, a PoC DNA-testing platform termed "POCKET" (point-of-care kit for the entire test) utilized a triple amplification methodology that employs an isothermal recombinase polymerase amplification with AuNP-catalyzed silver amplification and has been shown to detect nucleic acid mutations in blood, urine and buccal samples. [33]The clinical validation of this device compares patient samples against healthy cohorts, showcasing sensitivity, specificity, positive predictive values and negative predictive values close to 100% for most targets.In a different work, silica-coated magnetic Fe 3 O 4 with Pt nanoparticles decorated onto the outer surface (Fe 3 O 4 @SiO 2 @Pt) with controlled core-shell structures were designed for the visual detection of metabolic biomarkers. [34]This system was applied to the clinical diagnosis of pancreatic cancer patients with 84% sensitivity, 94% specificity, and area under the receiver operating characteristic (ROC) curve (AUC) of 0.958.Since nanozymes have the potential to revolutionize the field of PoC diagnostics, it will be exciting to see increasing clinical proof of their applicability.
Paper-based LFIAs are one of the most widely used examples of PoC platforms that have great potential to realize diagnostic assays heavily compliant with the REASSURED criteria, exhibiting characteristic low cost, ease-of-use, and rapid nature of paperbased tests.As such, LFIAs have seen extensive global use, for example, in pregnancy tests and notably in the recent COVID-19 pandemic.Since the introduction of this technology in the late 1950s, [38] a wide range of detection labels have been developed for LFIAs, which include colored latex beads, [39,40] colloidal gold nanoparticles, [41,42] magnetic nanoparticles, [43,44] quantum dots, [45,46] enzymes, [47] and fluorophores. [48]However, LFIAs generally suffer from substandard sensitivities when compared to "gold standard" techniques such as PCR.[51][52] We previously reported an ultrasensitive LFIA for the detection of p24, which is one of the earliest presenting HIV biomarkers, using a porous platinum coreshell structure (Figure 4A). [37]In this immunoassay, antibodyfunctionalized platinum nanocatalysts (PtNCs) displaying high internal surface area owing to an urchin-like morphology served as the detection probe.The high catalytic activity of the PtNCs enhanced the test signal by 100-fold, resulting in the naked-eye detection of p24 in the low femtomolar range (0.8 pg mL 1 ) in under 20 min. [37]Furthermore, owing to the dual sensitivity regime enabled by the inherent coloration of the PtNC and the ability to catalytically generate a colored product to enhance the test line, the technology exhibited an ultrabroad dynamic range spanning over 4 orders of magnitude (Figure 4B).This dynamic range is ca. 2 orders of magnitude broader than standard colorimetric enzymelinked immunosorbent assays (ELISAs).
More recently, the Xia group reported rough-surfaced Au-Ir core-shell structure nanoparticles as a state-of-the-art peroxidase mimic. [53]They demonstrated that the ca.40 nm Au-Ir NPs can enhance catalytic efficiency to a k cat as high as 10 7 s −1 , which is approximately an order of magnitude greater than those found in Au-Pt NPs of similar size.These nanoparticles were utilized in a LFIA format where, in a method analogous to our aforementioned PtNC, a rapid TMB substrate treatment process was applied at room temperature to amplify signal.When using the detection of carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) as model biomarkers, the LFIA based on Au-Ir NPs offered improved sensitivities when compared to alternative nanoparticle probes.Specifically, the LOD of Au-Ir NP-based LFIA was ≈200 and 3.3 times lower than AuNP-and Au−Pt-NP based benchmark LFIAs, respectively, and reached the pg mL −1 level.
Also of note, Liu et al. reported Co-Fe@hemin peroxidase mimicking nanoparticles for the detection of the SARS-CoV-2 spike protein in a LFIA format. [54]The iron-cobalt nanoparticles coated with hemin (Co-Fe@hemin nanocomposites) were utilized to generate a chemiluminescent signal through the catalysis of luminol under the presence of H 2 O 2 in alkaline conditions (Figure 4C).This resulted in the sensitive detection of recombinant spiked antigen with a LOD as low as 0.1 ng mL −1 in <16 min, with no noticeable cross-reactivity with other human coronaviruses.The assay also exhibited a linear range of 0.2-100 ng mL −1 , which is 32-fold wider than a traditional HRPbased ELISA. [54]The test was also verified using SARS-CoV-2 pseudovirus with a detection limit of 360 PFU mL −1 , rivaling the LOD attained with ELISA.
During the development of nanozyme-based LFIAs, unwanted interactions that can produce non-specific binding and/or high background should be viewed as particularly detrimental, as this effect will be amplified upon catalytic enhancement.This principle has been shown in numerous reports of nanozyme-assisted signal-amplified LFIAs, [55,56] where a test line can be observed in negative control samples following signal amplification.Therefore, development of paper-based assays should, in order to minimize unwanted interactions, include special consideration of: nanoparticle size to aid nanoconjugate flow; nanozyme surface coating to prevent aggregation; and nitrocellulose blocking and optimization of assay running buffers.The dual sensitivity regime employed by the PtNCs pre-and post-catalysis provides an ultrabroad dynamic range for biomarker detection.Reproduced with permission. [37]Copyright 2017, American Chemical Society.C) Schematic showing the nanozyme chemiluminescence paper test for SARS-CoV-2 S-RBD antigen.Recognition, separation and catalytic amplification by nanozyme probes is highlighted.Reproduced with permission. [54]Copyright 2020, Elsevier.

Nanozymes Utilized in Other In Vitro Biosensing Platforms
Emulating the use of natural enzymes in analyte detection assays, nanozymes have found application in the development of many in vitro biosensors and bioassays ranging from simple solutionbased colorimetric assays to more sophisticated aptamer-and antibody-based immunosorbent tests and even electrochemical sensors.Incorporation of nanozymes as a direct substitute for natural enzymes into these well-established platforms has not only enhanced the analytical sensitivity of the assays but has also made progress toward overcoming the shortcomings associated with the use of natural enzymes in such assays, such as their inhibition by sample components or digestion by proteases present in biological samples.In this section, incorporation of nanozymes as enzyme substitutes into different in vitro diagnostic platforms will be explored.

Nanozymes in Solution-Based Cascade Assays
Initially, nanozymes were not employed as direct replacements for natural enzymes in biosensing assays, but instead were utilized in tandem with natural enzymes in enzyme-nanozyme cascade assays.One of the preliminary works on detection of glucose through the use of nanozymes was reported by Wei and Wang in 2008. [57]Here, a biocatalytic cascade of peroxidasemimicking Fe 3 O 4 nanoparticles coupled with native glucose oxidase (GOx) enzyme was utilized for the oxidation of glucose to gluconic acid, and the subsequent production of H 2 O 2 was utilized for the conversion of ABTS to its green oxidized form.In a similar manner, an inverse approach to the cascade was reported by Zeng et al., where nanoconjugates were attained by immobilizing HRP enzyme onto the surface of 13 nm AuNPs. [58]ere, the gold-based nanozyme was responsible for the initial catalyzed conversion of glucose to gluconic acid and then the immobilized HRP enzyme utilized the released H 2 O 2 to oxidize ABTS.These studies provided proof-of-concept for inclusion of nanozymes in biosensing platforms.[61][62] In recent years, the application of nanozymes has been extended to more complex bioassays such as immunosorbent assays, electrochemical biosensors and even in vivo sensing platforms and each of these are discussed in turn below

Nanozymes in Immunosorbent Assays
Enzyme-linked immunosorbent assays (ELISAs) typically take place in a microtiter plate; take advantage of the extremely specific interaction between antibodies and their target antigens to detect proteins and molecules with exquisite specificity in the laboratory; and can achieve detection limits in the fg mL −1 range. [63]Traditional ELISA techniques typically rely on an enzyme label to produce a detectable substrate for signal development, where the use of HRP is commonplace due to its high catalytic activity, which provides excellent assay sensitivity.A breakthrough in the use of nanozymes was realized in 2007, when Gao et al. [64] reported immunoassays utilizing Fe 3 O 4 instead of HRP.[67][68][69] A relevant advantage for the use of nanozymes in immunosorbent assays is the forgoing of the need for chemoenzymatic labeling of detection or secondary antibodies, which is often a cumbersome process that requires sophisticated purification; [70] on the other hand, there are, as previously mentioned, a myriad of techniques for facile modification of nanozymes with biomolecules such as antibodies. [71]hmed et al. reported a size-controlled preparation procedure for the formation of peroxidase-like graphene-gold nanoparticle hybrids that have been utilized as a nanoprobe in immunoassays for the detection of norovirus-like particles in human serum. [72]or the nanozyme-assisted immunoassay, a final limit of detection of 92.7 pg mL −1 was observed, which was reported to be 112 times lower than that of a conventional ELISA and 41 times more sensitive than a commercial diagnostic kit.The method also exhibited a wide linear range from 100 pg mL −1 to 10 μg mL −1 and no notable cross-reactivity with H1N1 virus.
Wu and co-workers [73] reported an aptamer-based NLISA platform for detection of aflatoxin B1 (AFB1).In this competitive NLISA, AFB1 aptamers were conjugated onto magnetic Fe 3 O 4 nanoparticles to create the immunoassay capture component whilst mesoporous SiO 2 /Au-Pt nanozymes were functionalized by the complementary DNA (c-DNA) to AFB1 aptamers to create the detection probes.In the absence of the target toxin, the SiO 2 /Au-Pt carrying c-DNA attached to the capture aptamer, catalyzing the conversion of TMB.In the presence of AFB1 in the sample, however, the toxin would selectively bind onto the aptamers, hence blocking them from binding to the nanozymecarrying detection probe and thus eliminating the production of a colorimetric signal.The combination of both the aptamer and nanozyme into the immunoassay produced a LOD of 5 pg mL −1 for the detection of AFB1, which was found to be 600-fold greater compared to a traditional ELISA.
Recently, our research group reported the development of a nanozyme-catalyzed clustered regularly interspaced short palindromic repeats (CRISPR) assay, termed CrisprZyme, for the detection of long non-coding RNAs (lncRNA). [74]The assay consists of two principal steps: an initial Cas-based reaction and a subsequent nanozyme-linked immunosorbent assay, which utilizes porous platinum core-shell nanozymes for signal development (Figure 5A).Here, the sample was first incubated with a gRNA-Cas13 complex and reporter RNA modified with biotin at one end and a 6-carboxyfluorescein (FAM) at the other end.In the absence of target RNA, the Cas-13 enzyme was not activated and therefore the reporter RNA remained intact and was then detected through a NLISA.However, in the presence of the target RNA, the Cas enzyme was activated, cleaving the reporter RNA and thus eliminating signal in the NLISA (Figure 5B).This method achieved a LOD of 4.72 pm of target RNA (Figure 5C).The underlying method of the NLISA process was further demonstrated in a dipstick lateral flow format (alternatively known as half-strip format, in which the sample and conjugate pads of the lateral flow are omitted), where the platinum nanozymes were used as the detection probe.
Here, the absence of a test line was indicative of a positive indication of target RNA, and the performance of the platinum-based nanozymes was demonstrated where catalyzed oxidation of a colorimetric substrate resulted in a signal-amplified readout compared to the pre-developed optical visualization of the platinum nanozymes (Figure 5D).In this signal-amplified format, a LOD of 12.5 ng mL −1 of target RNA was achieved (Figure 5E).The successful translation of the NLISA to a paper-based format represents an important breakthrough in the field of PoC detection of nucleic acids and suggests that this assay has the potential to be translated toward a point-of-care test.Nucleic acids are an extremely significant class of disease biomarker that can detect both infectious and non-communicable diseases; therefore, portable, low-cost, disposable and highly sensitive biosensors for detection of nucleic acids are of great interest. [75]

Nanozymes in Electrochemical Biosensors
Nanozymes have also been incorporated into many novel electrochemical biosensors as an alternative to natural enzymes.81] With regard to the use of nanozymes as electrode modifiers, Li et al. [78] reported the fabrication of a label-free homogenous electrochemical sensor for detection of microRNA (Figure 6A).By introducing oxidase-mimicking MnO 2 nanoflakes as an electrode modifier, the group greatly enhanced the efficiency and specificity of the sensor for detection of microRNA compared to nanozyme-free electrode systems.To achieve this, they initially inhibited the catalytic activity of the nanozymes by blocking their catalytic active sites through adsorption of ssDNA (P let-7a ).Upon addition of a target microRNA (let-7a), the ssDNA was liberated from the nanozyme surface by the hybridization reaction,  A-E) Reproduced with permission. [74]Copyright 2022, The Authors, published by Springer Nature.Here, an increase in catalytic oxidation of methylene blue (MB) is observed in response to target miRNA as catalytic inhibiting ssDNA is liberated from nanozyme surface, resulting in removal of MB and subsequent reduction in observed current.B) A linear relationship between DPV peak current and let-7a dosage was observed between 0.4 and 100 nm of let-7a.A,B) Adapted with permission. [78]Copyright 2021, Elsevier.C) Schematic representation of the cytosensor and detection principle.D) Linear relationship observed between peak current and logarithmic MCF-7 concentration.C,D) Reproduced with permission. [80]Copyright 2018, Elsevier.
resulting in a restoration of the oxidase-mimicking activity of the MnO 2 nanozymes.This mechanism was exercised in an elegant assay utilizing methylene blue (MB) as the enzymatic substrate where in the instance of let-7a, the oxidation of MB was facilitated and a measurable change of the peak electrochemical current was observed.A linear relationship between differential pulse voltammetry (DPV) peak current and let-7a dosage was observed between 0.4 and 100 nm of let-7a (Figure 6B).In another study, Wang et al. [80] reported the use of CuO nanozymes as catalytic tags for current signal amplification in an electrochemical cytosensor for highly sensitive detection of MCF-7 circulating tumor cells (CTCs) (Figure 6C).Upon attachment onto the electrode through binding to the MCF-7 CTCs, the peroxidasemimicking CuO nanozymes amplified the electric signal through reduction of H 2 O 2 , which can be sensitively detected using CV (cyclic voltammetry) and DPV.Incorporation of CuO greatly enhanced the LOD of the cytosensor to as few as 27 cells mL −1 with a linear relationship observed between DPV peak current and MCF-7 CTC concentration between 10 2 and 10 4 cells mL −1 (Figure 6D).
To expand on the inherent advantages of nanozymes over natural enzymes, nanozymes can be engineered with additional functionalities that can be useful for electrochemical biosensors.
For instance, the outstanding electron conductivity of metal-and carbon-based nanozymes can greatly improve the electron transfer kinetics of nanosensors. [82]Nanozyme-modified electrodes have been reported for detection of glucose [83] and cancer cells via their released reactive oxygen species. [84,85]

Nanozymes for In Vivo Disease Sensing
Despite the substantial progress of nanozymes for the in vitro detection of important biotargets, efforts to develop nanozymes for biosensing in living systems have been limited to-date.This is to be expected, since nanozymes developed for in vivo administration must be suitable within a substantially more complex environment, where significant hurdles include biosafety and extraction of nanozymes ex vivo for detection.Further considerations include nanozyme stability in the circulation, accumulation in the liver, enhanced accumulation in tumors, enhanced penetration of cells, and ability to cross the blood-brain barrier.Ultrasmall (1-3 nm) nanoparticles are particularly wellsuited to in vivo biosensing: these particles provide the benefit of being renal clearable, allowing urine to be used as an appropriate sample matrix; [86] and since protein affinity to ultrasmall Figure 7. A) Schematic illustrating the AuNC-Nav (≈11 nm) complex.B) Protease-sensitive complex specifically disassembles in the presence of relevant dysregulated proteases.The liberated AuNCs (≈2 nm) are then filtered through kidneys and collected in the urine.C) The presence of AuNCs is detected by measuring their ability to catalyze the oxidation of TMB in the presence of hydrogen peroxide to generate a colored signal that can be read by the nakedeye.D) Catalytic activity assay on urine collected from healthy and LS174T tumor-bearing mice 1 h post-injection.The catalytic activity was measured by initial velocity analysis (A 652 min −1 ), and the dashed line represents the LOD.A-D) Adapted with permission. [87]Copyright 2019, The Authors, published by Springer Nature.
nanoparticles is considered to be generally weak, the particles show limited protein adsorption.As an example of the use of nanozymes for in vivo sensing, we would like to draw attention to a study from our research group where ultrasmall gold nanoclusters (AuNCs) were used as probes (Figure 7A). [87]These nanoclusters are an efficient peroxidase mimic so their presence can be detected in a TMB assay, and as mentioned, these particles precise nanometer-size allows for effective filtration through the kidney.Here, multifunctional protease nanosensors composed of AuNCs tethered to neutravidin through protease-cleavable peptides were fashioned.Upon interaction with the target protease in vivo, the peptides were cleaved and the AuNCs were thus liberated and became detectable in urine in <1 h (Figures 7B,C).In a mouse model of colorectal cancer, the catalytic activity of AuNCs in the collected urine was monitored and tumor-bearing mice exhibited a 13-fold increase in colorimetric signal compared to healthy mice (Figure 7D).Furthermore, the nanosensors were completely eliminated through hepatic and renal excretion within four weeks of administration with no evidence of toxicity.
Although nanozymes have been shown to be more stable and resilient than their natural enzyme counterparts, the complex in vivo environment remains a considerable challenge as the use of nanozymes may still result in unpredictable and undesirable side effects.Although iron oxide nanoparticle based reagents, such as Resovist, have already been approved for clinical use as a magnetic resonance imaging (MRI) contrast agent, [88] the biotoxicities and suitability of promising nanozymes still need to be extensively tested before clinical trials.

Translation and Commercialization
The use of nanozymes in medical diagnostics addresses an unmet clinical need for early diagnosis using rapid, low-cost, ul-trasensitive, and accurate diagnostic devices without the need for equipment.Existing early infection diagnosis is broadly conducted by biomolecular testing, such as PCR.In many middleand lower-income countries, limited access to PCR facilities and staff and/or disruption of reagent supply chains hinder use of biomolecular testing.In addition, the cost of widespread PCR testing is prohibitive even in high-income nations.Ultrasensitive rapid tests enabled by nanozyme technology can bridge the gap in the market from high-tech, high-cost, instrument-based testing to rapid, instrument-free, paper-based tests without compromising sensitivity.
However, the delivery of nanozyme-enabled diagnostic tests to the market is not without challenges.The integration of sophisticated labeling probes and reaction readout unfortunately contradicts a main advantage of paper-based assays-that of simplicity.This explains why, despite the many published developments, LFIAs still primarily use the traditional colorimetric readout with AuNP labels.The added complexity, cost, and development time needed to engineer and time the additional liquid handling steps needed to deliver the substrate to the test strip further restrict the deployment of nanozyme-based versus standard LFIAs.Technology readiness levels (TRLs), first described by NASA, [89] are a way to describe technology development from idea to product.Product development for point-of-care devices can be described within this framework as described in Figure 8.Most research councils fund academic research to early prototype level or TRL 3-4, device design to prototype validation are TRL level 4-5, leaving a gap in development that is not normally within the funding remit for academic institutions.This means that the academic labs that develop the nanozyme technology either must partner with an existing company or spin out from the university to bring the product to prototype stage for validation.Furthermore, the nanozymes used are often novel structures and compositions that are produced in the lab at a small scale.Careful consideration will need to go into the manufacture of the particles to ensure they retain their catalytic properties when produced at scale.
Lessons from the development of an antigen PoC test in response to the COVID-19 pandemic has spotlighted the need for more sensitive technologies that can be deployed at the scale.These assays should be developed to a level where they can be easily adapted to a new disease target and user experience should be carefully considered to ensure adoption.Continued research should focus on ensuring that patients can collect and process samples at the point-of-care.An important emerging platform with the potential to integrate nanozymes and reach the market is microfluidics, which can deliver liquid handling and mixing on-chip.Advances in microfluidics also mean that sample preparation can take place on chip, thus reducing user steps.

Considerations for Non-Communicable Diseases (NCDs)
NCDs-diseases not directly transmissible between individuals-are responsible for 74% of all deaths worldwide (41 million), with cardiovascular diseases and cancers being the major causes.Early detection and screening are the most important ways to prevent NCDs deaths.PoC devices have the advantage of being easily deployed to the community and via primary care, making screening affordable, fast, and easily available.
For the detection of NCDs, it is critical to quantitatively measure key biomarkers with high accuracy.This increases the challenge for PoC devices since a reader is required to accurately capture and analyze the results.92] As the biomarker discovery field advances, more is known, and biomarker panels are generated to accurately predict NCDs within clinical pathways.Standalone tests can be useful, for example, cardiac troponin I (cTnI) is a common target since it is released into the bloodstream within 90 min of the onset of acute myocardial infarction (AMI). [93]Detecting cTnI is challenging due to its low concentration in biological fluids, however, nanozymes have been employed successfully for its detection. [94]ultiplexed detection of several biomarkers would increase both the specificity and sensitivity of the diagnostic test.For example, multiplexing cTnI with myoglobin, CK-MB, and troponin T would help to better predict the risk of AMI after the onset of symptoms. [93]The same can be said about the detection of other classes of biomarkers like circulating nucleic acids (DNA, mRNA, and microRNA).These can give information about malignant and benign lesions, inflammatory diseases, and trauma.Circulating tumor DNA has genetic and epigenetic changes relevant to cancer development, progression, and responsiveness to treatment. [95]As mentioned previously (see Section 3.1), we have developed a test (CrisprZyme) where PtNCs were used for the amplification-free detection of nucleic acids. [74]The system was developed in a microplate format and transferred to an LFIA device where RNA targets were detected in the low picomolar range at room temperature.This platform was utilized to identify patients with AMI and monitor the cellular differentiation of prostate cancer patients in vitro and tissue biopsies.
Nanozymes have shown wide applicability for the diagnosis of NCDs. [96]Recent examples include: an electronic tongue with terephthalic-acid-modified graphene quantum dots with different transition metal ions to detect thiols, abnormal values of which have been associated with Alzheimer's, immune deficiency syndrome (AIDS), and cancers; [97] silica-coated magnetic particles to capture pheochromocytoma circulating tumor cells in samples and a platinum-based covalent organic framework to detect neuroendocrine, a rare type of tumor; [92] a ROSsensitive nanozyme-augmented photoacoustic nanoprobe with combined zinc phthalocyanine and ceria nanozyme as a theragnostic of acute liver failure; [98] and Au-decorated CoAl-layered double oxide nanozymes to detect aspartate transaminase, alanine transaminase, and alkaline phosphatase, which are liver damage biomarkers. [90]

Considerations for Communicable Diseases (CDs)
CDs or infectious diseases are caused by pathogens like bacteria, viruses, parasites, or fungi that spread directly or indirectly between individuals.In most low-income countries, CDs still account for >60% of total deaths. [99]Early detection and screening of the population are key to stopping the spread of pathogens.Although facilities to do so are scarce, it is of utmost importance to deploy PoC devices that enable the detection of these pathogens in community healthcare settings, at a price point that allows the test to reach the affected population.
The detection of CDs is usually performed by detecting the presence of pathogens, toxins, or antibodies in bodily fluids.Ultrasensitive detection is desired so that the infection can be diagnosed earlier, and the patient receive appropriate care.A quantitative measure is not usually necessary since the aim is to know if an infection is present through a binary response.Therefore, it is not necessary to develop costly readers and analyzers, although it might be of interest to capture the results with a smartphone camera connected to appropriate software for reporting purposes.
Nanozymes have been widely employed for the detection of CDs. [100]Recent examples include AuNP decorated 2D metalorganic frameworks (MOFs) for the electrochemical detection of pathogenic Staphylococcus aureus; [101] aqueous two-phase system to concentrate and purify samples in combination with Pt-NCs for the colorimetric detection of the malaria biomarker Plasmodium lactate dehydrogenase in human serum; [102] and PtNCs to perform a nanozyme linked immunochromatographic sensor for the detection of SARS-CoV-2 nucleocapsid protein in the blood. [103]lthough PoC devices were initially thought to be most advantageous for the detection of CDs, they have been widely applied to the detection of NCDs as well.In fact, most recent publications focus on the use of nanozymes to detect NCD-related biomarkers in PoC.This may be due to not only the remarkable performance of nanozymes, which is thought to be suitable for high degree multiplexing, but also due to a shift toward healthcare decentralization.

Final Words
Nanozymes and their advantages over natural enzymes are extensively documented and have been designed and developed as a relatively simple, cheap, and effective material to carry out sensitive catalytic methods for the detection of relevant biotargets.This Perspective promotes the use of many different enzymemimicking nanoparticles and their use in biosensing, with a special focus on PoC devices, which we consider to be the future of decentralized healthcare.Owing to their low cost, longer shelflife, high catalytic performance in aqueous environments, and easy immobilization into paper-based assays, there is an understandable drive toward nanozyme implementation in biotechnology and clinical diagnosis.However, nanozymes currently exhibit limitations such as poor bioavailability, reproducibility and multisubstrate activities, which hinder their clinical applications; additional device development to house the enzymatic substrate solution and address concerns about additional storage and potential hazard also contribute to limiting their translation to the PoC.
There is a vast wealth of reports for the generation of high activity nanozymes (peroxidases in particular), which is likely to continue, and whilst the motivation for even more sensitive tests is of course understandable, their implementation is unlikely to see further improvement unless logistical parameters that enable their use in clinical tests are addressed.We hope that this Perspective specifically highlights the unaddressed challenges of and stimulates further research activity in this field.

Figure 1 .
Figure 1.Nanozymes are nanomaterials that exhibit enzyme-like properties and thus encompass desirable properties of both nanoparticular and enzymatic moieties.

Figure 2 .
Figure 2. Schematic illustrating the enzymatic activities of various nanozymes and how peroxidase and oxidase nanozymes can be employed to catalyze the oxidation of chromogenic substrates.

Figure 3 .
Figure 3. A) Schematic illustrating how nanozyme-assisted PoC devices follow the REASSURED criteria.B) Calibration curves of ELISA-based antigen detection with Ni-Pt NPs, Pt NPs, and HRP.C) Free-energy diagrams for H 2 O 2 decomposition on Ni-Pt, Pt, and Ni surfaces and optimized adsorption configurations on Ni-Pt surface, based on DFT calculations.A-C) Reproduced with permission.[27]Copyright 2021, American Chemical Society.D,E) Relative activity of AuNPs@MoS 2 -QDs at temperatures between 25 and 80 °C (D), and pH 1-12 (E).D,E) Reproduced with permission.[25]Copyright 2018, Elsevier.F) Response of reused ceria paper-based assay after 1 (left) and 10 (right) cycles, the top row is the sensing paper after reconditioning, the bottom row of dark yellow is the colorimetric response after H 2 O 2 exposure.Reproduced with permission.[21]Copyright 2011, American Chemical Society.

Figure 4 .
Figure 4. A) Schematic illustrating the use of PtNCs as immunoassay tags for signal amplification.By catalyzing the oxidation of CN/DAB (4-chloro-1-naphthol/3,3′-diaminobenzidine, tetrahydrochloride) substrate in the presence of hydrogen peroxide, a 100-fold signal enhancement is achieved.B)The dual sensitivity regime employed by the PtNCs pre-and post-catalysis provides an ultrabroad dynamic range for biomarker detection.Reproduced with permission.[37]Copyright 2017, American Chemical Society.C) Schematic showing the nanozyme chemiluminescence paper test for SARS-CoV-2 S-RBD antigen.Recognition, separation and catalytic amplification by nanozyme probes is highlighted.Reproduced with permission.[54]Copyright 2020, Elsevier.

Figure 5 .
Figure 5. A) Schematic of the CrisprZyme Technology.B) Photograph of CrisprZyme results in a 384-well plate.Six replicates were performed for each concentration with low concentration of a lncRNA target (lnc-LIPCAR) to the left; high concentration to the right.C) Sigmoidal regression of the CrisprZyme of a lncRNA target.D) Detection of a serial dilution of lnc-LIPCAR with nanozyme-amplified LFIA.Photographs show the test bands of the lateral flow test strips after completion of the assay without (top) and with (bottom) the chromogenic substrate solution added for enhanced signal.E) Sigmoidal regression of the lnc-LIPCAR target for the nanozyme-amplified LFIA.Data represent the mean of test line pixel density normalized to the internal grid lines of the light box.A-E) Reproduced with permission.[74]Copyright 2022, The Authors, published by Springer Nature.

Figure 6 .
Figure 6.A) Schematic illustrating the electrode modifying principle underpinning the P let-7a /MnO 2 -based homogeneous electrochemical biosensor for miRNA.Here, an increase in catalytic oxidation of methylene blue (MB) is observed in response to target miRNA as catalytic inhibiting ssDNA is liberated from nanozyme surface, resulting in removal of MB and subsequent reduction in observed current.B) A linear relationship between DPV peak current and let-7a dosage was observed between 0.4 and 100 nm of let-7a.A,B) Adapted with permission.[78]Copyright 2021, Elsevier.C) Schematic representation of the cytosensor and detection principle.D) Linear relationship observed between peak current and logarithmic MCF-7 concentration.C,D) Reproduced with permission.[80]Copyright 2018, Elsevier.

Figure 8 .
Figure 8. Schematic illustrating a potential route for the translation of clinical diagnostic devices and a roadmap to benchmark technological developments.