When nanozymes meet deoxyribonucleic acid: Understanding their interactions and biomedical diagnosis applications

As emerging alternatives to natural enzymes, nanoscale materials featuring enzyme‐like catalytic behaviors (nanozymes) exhibit some attractive merits including robust activity, low cost, and easy‐to‐regulate performance. These merits have enabled them to be intensively used in the biomedical field in recent years. To remedy the lack of catalytic selectivity in most nanozymes, deoxyribonucleic acid (DNA) chains with specific recognition functions are utilized to integrate with nanozymes to produce various nanozyme–DNA combinations via adsorption/desorption. In the formed combinations, the DNA component provides the molecular/ionic recognition role, and the nanozyme part offers response with catalytically amplified signals, enabling them to detect analytes and biomarkers selectively and sensitively. To highlight this interesting topic, here we made a critical review of the interactions between nanozymes and DNA and their applications in biosensing and disease diagnosis. First, strategies for the conjugation of DNA chains onto nanozyme surface were introduced briefly. Then, the interactions between DNA and nanozymes were summarized in detail, where flexible modulations of nanozyme activity by DNA adsorption/desorption as well as various factors were analyzed, and potential impacts caused by nanozymes on the recognition characteristics of DNA chains were pointed out. After that, typical applications of DNA‐mediated nanozyme modulation in toxic ion sensing, health risk factor monitoring, and biomedical diagnosis were introduced. In the end, prospects of the combination of nanozymes and DNA chains were presented, and future challenges of the emerging field were also discussed, to attract more interest and effort to advance this promising area.


| INTRODUCTION
In the field of biology, enzymes are an extremely important class of biocatalysts.Most natural enzymes (NEs) are biological macromolecules (proteins, ribonucleic acid [RNA], or deoxyribonucleic acid [DNA]), which are high cost and easy to lose activity.Therefore, NEs are limited in practical applications and adverse to large-scale industrial production.Fortunately, the cross fusion of biology and nanotechnology has brought a revolution to the research on enzymes.In 2004, Manea et al. discovered that gold clusters capped by triazacyclonane-functionalized thiols possessed excellent catalytic performance in the cleavage of phosphate esters, and they proposed the "nanozyme" concept for the first time. 1In 2007, Yan's group found that magnetic Fe 3 O 4 nanoparticles (NPs) exhibited catalytic characteristics similar to horseradish peroxidase in triggering a series of color reactions. 2In 2013, Wei and Wang described the nanomaterials possessing enzymemimicking characteristics as nanozymes in their prestigious review. 3Since then, the research on nanozymes has entered to a rapid development era, and the concept is becoming more extensive and clearer. 4,5ccording to the definition, 3 it is not hard to deduce that nanozymes have the dual characters (Figure 1A): on the one hand, benefiting from the surface (interface) effect, small-size effect, quantum size effect, and macroscopic quantum tunneling effect, nanozymes present some nanoscale material properties that are different from that observed in bulk materials; on the other hand, they are artificial enzyme-mimicking catalysts, where the catalytic activity, specificity, and stability should be taken into consideration.At present, various materials, including noble metals, transitional non-precious metals and compounds, and carbon-based materials, have been explored as nanozymes, 6,7 to mainly mimic the catalytic functions of oxidoreductases (peroxidase, oxidase, catalase, superoxide dismutase, etc.) and hydrolases (nuclease, esterase, phosphatase, protease, etc.) (Figure 1B).It is widely recognized that nanozymes, compared to NEs, show several attractive superiorities, such as lower production cost, better robustness against harsh environments, and easier manipulation of catalytic performance.In consequence, they have found extensive applications in the fields of biochemical analysis, environmental control, chemical catalysis, and biomedicine. 8- 14Especially in biosensing and medical diagnosis, the catalytic nature of nanozymes provides amplified signals for sensitive detection. 15However, with the in-depth research and development of nanozymes, some shortcomings are exposed, one of which is their poor catalytic specificity.Unlike NEs that often have well-defined microstructures and channels for substrate access to their active centers, nanozymes without refined surfaces lack the selectivity for substrate recognition and conversion.][18] As one of the most important macromolecules in biological cells, DNA carries genetic information to guide the synthesis of RNA and proteins.It is an essential species in the development and functioning of organisms.Single-stranded DNA (ssDNA) is composed of a few to thousands of deoxynucleotides, and each deoxynucleotide consists of a base, a deoxyribose, and a phosphate group (Figure 1C).There are four types of bases, namely adenine (A), thymine (T), guanine (G), and cytosine (C).Two ssDNA chains can be coiled around a central axis, forming a double helix structure, where the deoxyribosephosphate chain is located on the outside of the helix structure, and the bases face the inside.The two ssDNA chains complement each other in reverse and are linked by A-T and G-C pairs via hydrogen bonds between bases, forming a stable double-stranded DNA (dsDNA).As typical oligonucleotide chains screened from a random library via the systematic evolution of ligands by exponential enrichment technique, aptamers possess welldefined deoxynucleotide sequences and can exhibit high specificity and affinity to targets of interest.Since the concept of aptamers was proposed in the 1990s, scientists have devoted intensive interest to the research of aptamers, and their appearance provides a new tool for efficient and rapid recognition in chemical biology and medicine. 191][22] Such DNA-encoded nanozymes not only present desired catalytic activity thanks to flexible DNAprogrammed tuning but also retain the recognition role partially, [23][24][25][26] endowing them with promising use in bioanalysis [27][28][29] ; on the other hand, nanozymes can be employed as signal tags conjugated on DNA chains (aptamers), and in such a configuration the nanozyme label produces catalytically amplified signals responsive to various recognition events. 30More interestingly, DNA chains (aptamers) can be used to deftly modulate the catalytic performance of nanozymes via adsorption and desorption, where the DNA component offers the molecular/ionic recognition function, and the nanozyme one provides amplified response, enabling them to detect analytes and biomarkers selectively and sensitively.In the last case, the two parts in the formed DNA-nanozyme combinations interact with each other in a complex mode: the conjugation of DNA chains (aptamers) on nanozymes can adjust the latter's catalytic activity; the recognition characteristics of DNA adsorbed on nanozymes may be different from that of free DNA chains in solution (Figure 1D).][33][34][35] To highlight such an interesting topic, here we try to make a critical review on understanding the interactions between nanozymes and DNA and their biomedical diagnosis applications.First, a brief introduction of different strategies used for the conjugation of DNA chains (aptamers) onto nanozyme surface was presented, followed by discussing the intricate interplay between the two components.In detail, various regulations of nanozyme activity by DNA adsorption/desorption were classified, and potential factors (DNA configuration, DNA base, DNA strand length, DNA concentration, nanozyme substrate, nanozyme type, reaction conditions, etc.) affecting the interactions were summarized and discussed.At the same time, potential impacts caused by nanozymes on the recognition function of DNA chains (aptamers) were pointed out.After that, this review introduced some application progress of DNA-mediated nanozyme modulation in toxic ion sensing, health risk factor monitoring, and biomedical diagnosis.Finally, we presented the opportunities brought by the marriage of DNA and nanozymes.Also, some challenges of the promising area were emphasized, hoping to attract more effort to advance this field.

NANOZYMES
Integrating nanozymes with DNA chains (aptamers) to fabricate functional materials and application platforms requires the conjugation of the two parts.Moreover, different coupling interactions between DNA and nanozymes can have different impacts on their features and functions.To expound the modulation of DNA on the catalytic performance of nanozymes, here we first summarized the main methods for the conjugation of the two components (Table 1).
First, DNA chains (aptamers) can adsorb onto the nanozyme surface via various physical forces (van der Waals force, electrostatic adsorption, π-π stacking, hydrogen bonding, hydrophobic interaction, etc.) when they are incubated together under normal conditions (Figure 2A).As mentioned above, ssDNA is formed by a F I G U R E 2 (A) DNA adsorption on nanozymes via various physical interactions.(B) Biochemical strategies for the binding of DNA chains to nanozyme surface.(C) Different DNA conjugation strategies had different impacts on nanozyme catalytic activity.Reproduced with permission. 51Copyright 2019, American Chemical Society.DNA, deoxyribonucleic acid.
T A B L E 1 Bioconjugation of nanozyme materials and deoxyribonucleic acid chains (aptamers) for bioanalytical applications.deoxyribose-phosphate backbone and four kinds of bases (A, T, G, and C).The physicochemical properties of the backbone and bases in DNA chains offer weak intermolecular forces (van der Waals interaction) for their coupling with nanozymes.As the backbone phosphate with a pK a value around 2 has rich negative charges and the bases are nearly non-charged in the pH 4-8 scope, 36 DNA is highly negatively charged in neutral environments.Thus, strong electrostatic adsorption occurs between DNA chains (aptamers) and positively charged nanozymes.In addition, when nanozymes containing multiple benzene ring structures or some carriers like graphene are applied to support enzyme-like active nanomaterials, DNA can interact with these materials via π-π stacking. 37Besides, hydrogen bonding and hydrophobic interaction are common forces triggering the physical combination of DNA chains and nanozymes.In fact, the conjugation characteristics of nanozymes and DNA chains (aptamers) often involve the co-existence and balance of multiple forces.Typically, the AuNPs synthesized by sodium citrate reduction are negatively charged due to the attachment of citrate, so DNA chains with the phosphate backbone are not likely to absorb onto AuNPs surface via electrostatic interaction.However, many studies have proved that the negatively charged AuNPs can adsorb ssDNA stably, because the nitrogen and oxygen atoms in the bases provide binding forces with the gold surface. 36Besides, van der Waals force exists between ssDNA and citrate stabilized AuNPs.

Material
It is worth mentioning that the conjugation behaviors of DNA chains (aptamers) highly depend on the nanozyme materials used.Liu's group proved that the adsorption forces of ssDNA on graphene oxide (GO) and MnO 2 rods were different.DNA was adsorbed on MnO 2 mainly through its phosphate backbone, 38 while π-π stacking and hydrogen bonding played important roles in the conjugation of DNA chains and GO. 37Attractively, the DNA-nanozyme complexes formed in response to the above interactions are usually reversible, meaning that DNA can desorb off nanozymes with sufficient external forces.Given that the activity of nanozymes often originates from their surface properties, DNA molecules conjugated on the surface of various nanozymes possess the capability of regulating the latter's activity reversibly through adsorption/desorption.Compared to the following chemical and biological coupling strategies, the combination of DNA chains (aptamers) and nanozymes via physical interactions is relatively unstable, but the behavior is reversible, conducive to the flexible modulation of nanozyme performance and its versatile use.Second, biochemical conjugation is an efficient way to make DNA chains (aptamers) and nanozymes bind together stably.It is based on several chemical bonding and/or biological affinity interactions (Figure 2B).Typically, the glutaraldehyde crosslinking method can bind amino-functionalized ssDNA onto nanozyme surface with rich amino groups. 41,42This process is driven by the nucleophilic addition reaction between the aldehyde groups in glutaraldehyde and the amino ones to form Schiff bases (a).The 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) co-reagents are often utilized to chemically conjugate DNA molecules with nanozymes. 43,44In the EDC/NHS method, the two parts are coupled by activating the amino and carboxyl groups to form a stable amide bond (b).In addition, the biotin-avidin bridging system is a common labeling method with high specificity and sensitivity (c), which has been utilized to bioconjugate DNA chains (aptamers) and nanozymes. 52][49][50] Apart from the above biochemical interactions, some special techniques like click chemistry are also feasible for the conjugation of DNA chains (aptamers) and nanozymes. 53Besides, many metal-based nanozymes can adsorb oligonucleotides via coordination. 54For instance, Liu and his co-worker demonstrated that Fe 3 O 4 NPs could adsorb DNA chains via the backbone phosphate, 39 where some coordination interactions existed between the Fe 3 O 4 surface and the phosphate groups in DNA.Similarly, strong adsorption of DNA on nanoceria was observed, which was driven by not only electrostatic interaction but also the phosphate backbone binding to cerium via Lewis acid-base interaction. 40In comparison with physical absorption, these chemical bonding and biological affinity interactions are much stronger.This benefits the stability of the formed DNA-nanozyme hybrids, but the strong interactions make it hard to desorb or unlink the DNA chains coupled on the nanozyme surface again.
It is worth mentioning that different conjugation methods can affect the characteristics of DNA chains (aptamers) and nanozymes differently.Typically, Tang's group compared the enzyme-like catalytic function of Fe 3 O 4 NPs conjugated by ssDNA via different strategies. 51They found that the DNA chain linked via either physical absorption or the streptavidin-biotin method could increase the POD mimicking the activity of Fe 3 O 4 NPs in catalyzing the 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) color reaction in the presence of H 2 O 2 (Figure 2C).Compared to simple absorption, the streptavidin-biotin mode performed better in promoting the POD activity, and the underlying reason for such a difference was not clear.
LIANG ET AL.

ACTIVITY BY DNA
As mentioned above, the combination of DNA chains (aptamers) and nanozymes via physical interactions can be used to affect the latter's catalytic performance.Thanks to the reversible adsorption/desorption behavior of DNA, the activity of enzyme mimics can be flexibly modulated, which has been widely employed to design and fabricate nanozyme-based biosensors. 31,35It is widely reported that ssDNA can promote the catalytic efficiency of nanozymes, while in some studies DNA chains (aptamers) play a role in inhibiting their performance.Although most researches declare the reasons why DNA can promote or inhibit, some controversies and even contradictory points on the modulation of nanozyme activity by DNA adsorption exist.1][62][63][64][65][66][67] As a matter of fact, the control of DNA on nanozyme activity is influenced by many factors, including DNA configuration, DNA base, DNA strand length, DNA concentration, nanozyme substrate, nanozyme type, and reaction conditions (Figure 3).To better understand the regulatory effect of DNA, in this section we summarize and discuss the flexible modulation of nanozyme activity by DNA adsorption.

| DNA configuration
As a long polymeric macromolecule composed of repeated nucleotide units, DNA has the primary structure, secondary structure, and higher configurations.The primary structure refers to the four nucleotide linkages and their order.Short ssDNA can be roughly considered as in a straight strand state.Long ssDNA folds easily to form specific configurations.By exquisite design, the bases at each end of a long ssDNA can be paired to form a stem-loop structure.Such a structure has been demonstrated as an elegant and practical design for molecular biology and biotechnology. 68As mentioned above, two matched ssDNA chains can be linked by A-T and G-C pairs to form a stable dsDNA.For ssDNA and dsDNA with the same length, they possess different negative charges, which can affect the adsorption binding through electrostatic interaction.The bases of ssDNA are exposed and those of dsDNA are masked inwards.Meanwhile, the bases of dsDNA are already paired by hydrogen bonds, making them less likely to interact with other substances unless they encounter a target with stronger binding forces.The different molecular forces existing between DNA and nanozymes during physical adsorption correspond to whether the nanozymes interact primarily with the phosphate skeleton of DNA or with the bases.Therefore, ssDNA and dsDNA interact differently with nanozymes and have different impacts on the latter's activity.Thanks to the varied configurations and characteristics of DNA, nanozymes can be constructed with designable interfaces to produce different regulatory effects on their catalytic activity. 51[58][59][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85] The activity enhancement is mainly attributed to the increased surface negativity of nanozymes after the adsorption of DNA, which can enhance the diffusion and adsorption of positively charged substrates like TMB, thus resulting in the enrichment of the substrates toward the nanozyme surface and the improvement of catalytic efficiency.Liu's group demonstrated that Fe 3 O 4 NPs showed certain POD-like activity.When ssDNA was adsorbed on the surface of the nanozyme with positive charges under acidic conditions, it could promote the catalytic activity in inducing the TMB þ H 2 O 2 chromogenic system (Figure 4A). 83They confirmed that the Fe 3 O 4 NPs-ssDNA conjugate increased the surface negativity of these particles, and more positively charged TMB molecules were accumulated on their surface through electrostatic interaction.At the same time, they employed polyacrylic acid, polystyrene sulfonate, phosphoric acid, and guanosine phosphate to replace the ssDNA and found that the DNA bases played a key role in its adsorption.Besides, π-π stacking (via the benzene ring structure of TMB) and hydrogen bonding (via the amino group of TMB) could accelerate the diffusion and adsorption of the substrate onto the surface of ssDNAconjugated Fe 3 O 4 NPs, also benefiting the enzyme-like activity enhancement.Tang et al. found that free kanamycin aptamer (Ky2) with rich G bases could adsorb onto the surface of layered WS 2 nanosheets (NSs) via van der Waals force, enhancing the affinity of WS 2 to the substrate TMB. 84As a result, obvious enhancement of the POD activity in the nanozyme was observed in converting colorless TMB to blue oxTMB.They declared that the activity enhancement was mainly due to the rich G bases in the aptamer.The 2D NSs had the strongest adsorption affinity to G compared to the other three bases.It was speculated that the G bases in DNA could interact with the aromatic ring and amino group of the substrate through π−π stacking, hydrogen bonding, and some other forces, so the aptamer could adsorb more TMB to improve the observed POD-like performance.Moreover, the adsorption of G would cause the aptamer phosphate backbone to approach WS 2 surface, promoting the electrostatic attraction between TMB molecules and the nanozyme, further leading to the enhanced POD catalytic activity.When kanamycin was introduced, the Ky2 aptamer was consumed because of the preferential binding to the target, no longer enhancing the catalytic performance of WS 2 NSs.According to this principle, a simple colorimetric system based on the reversible enzyme-like activity regulation of layered WS 2 by DNA chains (aptamers) was established for the detection of kanamycin (Figure 4B).Xing's group claimed that the POD-mimicking activity of Fe 3 O 4 NPs could be promoted by the adsorption of an aptamer of epithelial-celladhesion molecule expressed by most exosomes. 85The performance enhancement was confirmed by the increased affinity between the aptamer-adsorbed nanozyme and the substrate TMB.The addition of exosomes could desorb the aptamer from Fe 3 O 4 NP surface, reducing the observed catalytic performance.Thus, such a finding could be employed to detect exosomes conveniently (Figure 4C).
63][64][65][66][67][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] It is generally considered that the adsorption of DNA chains (aptamers) covers some surfaces of nanomaterials, and the shielding of active sites causes the decrease in their enzyme-like catalytic activity.At the very beginning, Bansal's group observed that AuNPs exhibited the PODlike activity originating from the interaction of their surface with the POD substrate TMB. 67The catalytic activity could be suppressed by shielding AuNPs active sites via the adsorption of target-specific ssDNA aptamer.With no acetamiprid, the catalytic performance of the aptamer-AuNPs conjugate remained masked; after the target was introduced, the aptamer underwent targetresponsive structural changes, followed by a desorption process from the AuNPs to allow aptamer-target binding.This made the AuNPs reverse to their original form and recovered the target-specific POD-like activity (Figure 5A).Liu's group demonstrated that nanoceria could adsorb citrate and phosphate tightly, causing a negatively charged surface, but it showed low affinity to nitrate, chloride, and acetate. 40In these cases, the oxidase (OXD)-like activity of nanoceria was retained to convert colorless TMB into blue oxTMB.The material could also adsorb DNA chains via the phosphate backbone.However, its catalytic activity was significantly suppressed upon DNA adsorption (Figure 5B).Control experiments indicated that capping the nanoceria surface with small anions did not suppress its nanozyme activity, and the DNA-induced inhibition observed was likely due to the steric hindrance of DNA to inhibit substrate accessibility.
It is worth noting that some studies have reported that ssDNA adsorption has an inhibitory effect on the enzyme The POD activity of gold nanoparticles was suppressed after S-18 aptamer adsorption for acetamiprid detection.Reproduced with permission. 67Copyright 2014, American Chemical Society.(B) CeO 2 particles could adsorb citrate and phosphate tightly, retaining the oxidase-mimicking activity in converting colorless TMB to blue oxTMB, but the adsorption of DNA on CeO 2 surface inhibited the catalytic activity.Reproduced with permission. 40Copyright 2013, American Chemical Society.(C) Different DNA configurations made different impacts on the enzyme-like performance of Fe 3 O 4 nanoparticles.Reproduced with permission. 104Copyright 2018, American Chemical Society.DNA, deoxyribonucleic acid; POD, peroxidase; TMB, 3,3 0 ,5,5 0 -tetramethylbenzidine.6][57][58][59] In fact, the AuNPs adopted by these studies can be divided into two types: tyrosinemodified AuNPs and citrate-capped AuNPs.Bansal's group synthesized tyrosine-capped AuNPs and joined them with different ssDNA chains to construct biochemical platforms based on the regulation of nanozyme activity for the determination of acetamiprid and kanamycin, respectively. 62,67In both the detection systems, ssDNA adsorption showed an inhibitory effect on the catalytic performance of tyrosine-modified AuNPs.However, due to the lack of in-depth comparative studies on enzymatic reactions and ssDNA adsorption, we are unable to make further discussion here.Moreover, many factors can affect the catalytic activity of AuNPs.Therefore, it is not surprising that ssDNA can either promote or inhibit the AuNPs enzyme activity under certain conditions.
In contrast to the above discussion on ssDNA, dsDNA is usually used as a reverse control of ssDNA in designing nanozyme-based biosensors.In other words, the configuration transition into dsDNA can be used to restore the original activity of nanozymes affected by ssDNA.4 The results showed that all kinds of DNA configurations promoted the catalytic activities of the two nanozymes.For Fe 3 O 4 NPs, the promoting trend followed the order of short dsDNA < ssDNA < H-DNA < HCR dsDNA (Figure 5C).For AuNPs, the trend was short dsDNA < ssDNA ≈ H-DNA < HCR dsDNA.The H-DNA had a single strand protruding from the stem, and the HCR dsDNA displayed a bare sticky end.The extended single strand of the two structures could be adsorbed on Fe 3 O 4 NPs and AuNPs, which might be one of the reasons for the significantly promoted catalytic effect of H-DNA and HCR products.

| DNA base
Nanozymes can interact with DNA bases, phosphate backbones, or both.Considering that different nanozymes display different binding strengths with DNA, the composition of DNA bases may affect the adsorption to nanozymes and consequently impact the regulation of enzyme-like activity.8][109][110][111][112][113][114] Tang et al. found that the G base could significantly enhance the POD activity of WS 2 NSs, while other bases (A, T and C) had little effect. 84The possible reason was that the adsorption of G on 2D materials was the strongest, which strengthened the electrostatic attraction between the WS 2 NSs-DNA conjugate and TMB.Zhao et al. found that the enhancement trend of the enzyme activity of MoS 2 NSs by ssDNA was in line with G20 ≈ T20 > A20 > C20. 113They claimed that the C base could be protonated in the pH 4.0 buffer, which might lead to lower affinity of the C20-MoS 2 NSs conjugate toward TMB and stronger electrostatic repulsion between C and TMB, thus resulting in the weakest catalytic performance after C20 adsorption.Xia et al. showed that the trend of DNA enhancing the enzyme activity of carboxyl-rich single-walled carbon nanotubes (SWCNTs) was C15 > G15 > A15 > T15. 70According to the charge feature of all the four bases (Figure 6A), 36 the C15 base was protonated at pH 4.0, leading to tighter adsorption of the C15 chain on SWCNTs with rich negative charges.As a result, more TMB molecules could be bound to the C15-SWCNT combination, presenting an accelerated color reaction.The A base has the strongest binding to AuNPs among the four bases.It can be expected that polyA may have the strongest effect on the enzymatic activity of AuNPs.Yang et al. and Hizir et al. have confirmed this, respectively. 107,108In Hizir's study, significant enzymatic enhancement of AuNPs was observed when polyA chains were adsorbed on the material surface. 108However, Yang and co-workers found that the G, C and T bases improved the catalytic activity of oxygen-functionalized polypyrrole quantum dots (Figure 6B), while the A base showed an inhibitory impact. 107They stated that the absence of carbonyl functional groups in A decreased the catalytic activity due to the blockage of original carbonyl active sites.
According to the above reports, it is concluded that different bases have different effects on different nanozymes.Generally, DNA molecules composed of bases that bind more strongly to nanozymes are more likely to be adsorbed on their surface.In terms of the promoting LIANG ET AL. effect, this phenomenon leads to the enrichment of more TMB molecules on the nanozyme surface, resulting in a higher enzymatic activity.In terms of inhibition, the phenomenon covers more active sites and thus inhibits the activity more.

| DNA strand length
Different compositions of DNA involve different types of bases as well as different lengths.In general, single nucleotides can interact with nanozymes, and linear macromolecules composed of multiple nucleotides may possess stronger binding.Zhao et al. 113 fixed the total nucleoside concentration of different sequences (T5, T10, T15, T20, T30, and T40) and found that the ability of longer ssDNA in enhancing the enzyme-mimetic activity of MoS 2 NSs was stronger.Song et al. 115 showed that short ssDNA less than 6 bases could not bind to MIL-53 (Fe) stably and had little effect on its enzyme activity.In contrast, ssDNA with more than 12 bases performed excellently in enhancing the intrinsic activity of the material.At the same dose of long ssDNA, the enzymemimetic catalytic activity increased along with the increase in ssDNA length (DNA36 > DNA24 > DNA12).Zhu et al. 109 and Fan et al. 111 demonstrated that the catalytic activities of g-C 3 N 4 NSs and 3DBC-C 3 N 4 were also enhanced with the increase in ssDNA chain length.Longer ssDNA may have higher affinity to nanozymes, suggesting that the enzyme-mimicking activity should originate from the biological interface effect after ssDNA binding to nanozymes.Thus, the catalytic efficiency of nanozymes can be tuned using different lengths of ssDNA.Interestingly, Ouyang et al. found that the oxidation rate of dopamine by a set of aptamer-modified nanozymes highly depended on the modes of the aptamer connected to Cu 2þ -functionalized carbon dots 71 : (1) the nanozyme consisting of the 5 0 -end-dopamine binding aptamer (dopamine binding aptamer [DBA]) and carbon dots (I) offered higher activity in comparison with the 3 0end-DBA modified one (II); (2) the activity of the 5 0 -end-DBA-modified nanozyme was influenced by the length of the spacer linking the aptamer to Cu 2þ -modified carbon dots.As the spacer units increased from (TGTA) to (TGTA) 2 , the catalytic kinetics increased compared to the material consisting of the DBA aptamer bound to the catalyst directly (I).In contrast, the nanozyme (V) composed of the longer spacer (TGTA) 3 linked to the 5 0end-amino DBA aptamer presented lower catalytic performance in comparison with (I), giving the nanozyme activity order of IV > III > I > V (Figure 7).

| DNA concentration
According to the principle of ssDNA improving the activity of nanozymes described previously, within a certain range, the greater the concentration of DNA, the more F I G U R E 6 (A) Structures of various DNA bases and their pK a values.Reproduced with permission. 36Copyright 2012, Royal Society of Chemistry.(B) G-rich and polyA DNA sequences had positive and negative impacts on the peroxidase activity of oxygen-functionalized polypyrrole quantum dots, respectively.Reproduced with permission. 107Copyright 2023, John Wiley and Sons.DNA, deoxyribonucleic acid.
DNA molecules are adsorbed on the surface of nanozymes.This means the greater the negative charge on the surface, which can enhance the binding to positively charged TMB and thus promote the enzymatic activity.In other words, the higher the DNA concentration, the stronger the catalytic activity of the nanozyme-DNA conjugates.Many studies have confirmed this assumption. 73,116Some of them further find that the ssDNA concentration beyond its optimal range can cause a decrease in enzymatic activity. 73,102,117However, current reports provide a little explanation for this phenomenon, and further research is needed to uncover the underlying reasons.In our opinion, excessive free ssDNA may adsorb a certain amount of TMB, reducing the binding of nanozyme-DNA conjugates to substrate molecules and thus decreasing the enzyme-like catalytic activity.

| Nanozyme substrate
In many studies, TMB is the most frequently used POD substrate, followed by 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and OPD. Figure 8A displays the molecular structures of the three chromogenic substrates.TMB and OPD are positively charged in their catalytic systems (acidic), whereas ABTS is negatively charged.According to the previous discussion, the charge properties play a key role in the regulation of nanozyme activity by DNA.Therefore, when discussing whether DNA promotes or inhibits the activity of nanozymes with TMB as a substrate, ABTS is also used for comparison.Several studies have shown that when TMB is used as a substrate of nanozyme-ssDNA conjugates, the enzymatic activity is enhanced, but switching to ABTS shows an F I G U R E 7 Aptamers with different linking ends and strand lengths affected the kinetics of Cu 2þ -functionalized carbon dots catalyzing dopamine differently.Reproduced with permission. 71Copyright 2021, American Chemical Society.
F I G U R E 8 (A) Molecular structures of substrates commonly used in nanozyme studies.(B) Impacts of deoxyribonucleic acid adsorption on the Zeta potential of MoS 2 nanosheets and their peroxidase catalytic performance toward different substrates.Reproduced with permission. 113Copyright 2020, Royal Society of Chemistry.opposite effect. 75,83,113,115,118These results indicate that the ssDNA-nanozyme system indeed enhances the enzymatic activity by increasing surface negativity and binding to more positively charged TMB molecules.Typically, Zhao et al. claimed that the van der Waals force offered the power for ssDNA adsorption by MoS 2 NSs. 113After the addition of ssDNA, the Zeta potential of MoS 2 NSs decreased to −36.0 mV from −19.2 mV, suggesting the adsorption of ssDNA onto MoS 2 NSs.Furthermore, both the MoS 2 NSs and DNA/MoS 2 NSs were less negatively charged after the addition of TMB, indicating TMB adsorption onto the surface of MoS 2 NSs and DNA/MoS 2 NSs.The DNA/MoS 2 NSs with more negative charges exhibited a stronger electrostatic attraction toward TMB than bare MoS 2 NSs.Consequently, the enzymatic activity of MoS 2 NSs was promoted by DNA adsorption.When the POD substrate TMB was substituted with the negatively charged substrate ABTS, the DNA/MoS 2 NSs catalysis was significantly inhibited due to reduced affinity (Figure 8B), which resulted from the electrostatic repulsion between negatively charged ABTS and ssDNA adsorbed on MoS 2 NS surface.Also, as a positively charged substrate, OPD possesses the same tendency as TMB. 118

| Nanozyme type
Recently, different types of nanozyme catalysis have been found to make a different response to the adsorption of DNA chains (aptamers). 113,119It is not difficult to understand that different enzyme types catalyze reactions with different pathways, mechanisms, and conditions, so ssDNA may have different impacts on nanozyme catalysis by affecting these factors.Qiu's group utilized 4,7-bis(4-formylphenyl)-2,1,3-benzothiadiazole (BT) and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin (Tph) as two blocks to design a covalent organic framework (COF), and they further tuned the two types of enzyme-mimetic activities of Tph-BT via ssDNA surface modification. 119In comparison with the BT and Tph monomers, the Tph-BT COF had a narrower energy band gap, wider absorption, and higher light harvest efficiency.Upon visible light irradiation, the Tph-BT COF exhibited poor POD and strong OXD activities.It was found that ssDNA adsorption could have a reversed regulation impact on the two activities (Figure 9).They claimed that the adsorption of ssDNA on Tph-BT surface suppressed the intersystem crossing and energy transfer procedures to inhibit the generation of singlet oxygen, which was generally considered as the power to drive the oxidaselike catalytic reaction, while the electrostatic interaction between TMB and ssDNA improved the Tph-BT's affinity to TMB to promote the electron transfer from TMB to hydroxyl radical, thus enhancing the POD activity observed.It is worth mentioning that most of the current nanozymes are related to the POD and OXD activities.In addition to the two catalytic activities, other activities like catalase also find promising applications in biosensing, [120][121][122] but few studies on the modulation of catalase-like nanozymes using DNA chains (aptamers) are available.

| Reaction conditions
The catalytic activity of nanozymes depends on a series of reaction conditions.The conditions, including solution 9 The POD and OXD activities of Tph-BT were reversely regulated by ssDNA.Reproduced with permission. 119Copyright 2023, John Wiley and Sons.DNA, deoxyribonucleic acid; OCD, oxidase; POD, peroxidase; ssDNA, single-stranded DNA.
pH, temperature, buffer type, ionic strength, and reaction time, can make significant impacts on the modulation of nanozyme activity by DNA chains (aptamers). 83,108,111,113,115For the TMB-H 2 O 2 chromogenic system, TMB with two amino groups will be positive in acidic environment.The regulatory mechanism of DNA on nanozyme activity is largely due to the charge effect.Therefore, the activity of nanozyme-DNA conjugates should be dependent on pH changes.Most studies using the TMB-H 2 O 2 chromogenic system show that nanozyme-DNA conjugates achieve optimal activity at pH ~4.0.Their POD catalytic activity decreases sharply and even disappears when the buffer pH exceeds 8.0.The results are consistent with the structural characteristics of TMB and the appropriate pH condition of the TMB-H 2 O 2 chromogenic system.As demonstrated by Zhao et al., 113 in acidic solutions the adsorption of ssDNA made an obvious influence on the enzyme-like activity of MoS 2 NSs, while the increase in solution pH from 4 to 8 gradually weakened the enhancement effect of DNA adsorption (Figure 10A).
In contrast to NEs highly affected by temperature, nanozymes are relatively stable.In addition to the direct effect of temperature on the catalytic activity of nanozymes, the conjugation between ssDNA and nanozymes may also be affected by temperature.Zhao et al. observed that reaction temperature impacted the enzymemimicking activity of both the bare MoS 2 NSs and DNA/MoS 2 NSs. 113In detail, the catalytic activity was improved with the increase in temperature in the range of 25-55°C, while at higher temperatures the catalytic activity gradually decreased, and the promotion effect of DNA adsorption disappeared (Figure 10A).This was because, on the one hand, the high temperature decreased the intrinsic catalytic activity of MoS 2 NSs, and on the other hand, at such high temperatures it became hard for ssDNA to adsorb onto the material surface.Interestingly, when pH 4.0 was selected to study the influence of the buffer used on the catalysis of MoS 2 NSs, the reaction system in NaAc/HAc was obviously higher than that in sodium citrate/citric acid or Na 2 HPO 4 /HAc (Figure 10A), indicating that the buffer type could affect the activity of nanozyme-DNA conjugates.
The importance of charge in regulating nanozyme activity by ssDNA is mentioned throughout the review.Therefore, ionic strength in the solution is an important factor that cannot be ignored.In many studies explaining the mechanism of DNA regulated catalytic activity of nanozyme-DNA conjugates, the increase in negative surface charge makes it easier for their binding to TMB.Typically, NaCl concentration was varied when using the ABTS-H 2 O 2 chromogenic system to investigate the effect of salt ion concentration. 115It was found that the nanozyme-DNA conjugate gradually enhanced the oxidation of ABTS from a low salt concentration to a high salt concentration (0-300 mM), indicating that the presence of Na þ played a role in shielding the electrostatic repulsion between DNA and ABTS (both negative charges).
Interestingly, Hizir et al. found that the positive or negative impacts of DNA adsorption could be reversed with reaction time. 108In the presence of 100 nM DNA, the POD activity of AuNPs was slowed down initially.In other words, the initial boost of the absorbance at 650 nm was suppressed by DNA adsorption on AuNPs.After 10 min, the signal intensified as the reaction proceeded, resulting in an almost 2.5-fold enhancement compared to bare AuNPs after 2 h reaction (Figure 10B).When the concentration of H 2 O 2 used decreased to 5 mM from 10 mM, the reverse time point was postponed to near 30 min.This offered a simple way to control the enhancement impact back and forward by changing some reaction conditions.

RECOGNITION
In turn, nanozymes may impact the recognition features of adsorbed biomolecules.For instance, AuNPs adsorb DNA too strong, thus disallowing the adsorbed DNA to hybridize with its complementary sequence.In the 1990s, Herne and Tarlov demonstrated that the binding configuration of HS-ssDNA on a gold electrode could be adjusted by the participation of 6-mercapto-1-hexanol (MCH). 123Before exposure to MCH, the HS-ssDNA molecules interacted with the electrode surface through both the thiol group and the nitrogen-containing nucleotide bases.After exposure to MCH, the HS-ssDNA species was stably modified onto the electrode surface through only the thiol group, and the nucleotide bases did not interact with the surface.Unlike free DNA in solution, the adsorbed DNA chains (aptamers) exhibit different recognition behaviors toward targets of interest.Moreover, the immobilization configurations make a non-negligible impact on the binding ability of targets.Typically, Liu's group compared the adsorption and desorption behaviors of DNA chains (aptamers) on AuNPs and GO, respectively. 116They used KCN to dissolve the AuNPs to calculate the percentage of the desorbed DNA toward complementary DNA or aptamer target.It was found that the DNA desorbed from AuNPs was less than 5% for all the targets, suggesting quite strong DNA adsorption affinity.They concluded that AuNPs were unlikely to provide a good interface for developing biosensors solely depending on the desorption of DNA chains (aptamers).Currently, very little information about the DNA recognition changes caused by adsorption is available.It is very important to pay attention on revealing the impact of nanozymes on DNA recognition for better design and use of related biosensors.

| BIOSENSING APPLICATIONS
Based on the tuning of DNA on the catalytic performance of nanozymes, various biochemical systems have been constructed for biosensing and disease diagnosis.One can fabricate nanozymes with different biological interfaces using DNA.By changing the conformation of DNA chains, the binding between DNA and nanozymes and their enzymatic activity are flexibly modulated.Thus, highly sensitive and specific sensing systems can be constructed and applied in various fields, such as toxic ion detection, health risk factor monitoring, and biomedical diagnosis.It should be noted is that the same nanozymes can also be bio-conjugated in aptamers as catalytic labels to develop aptasensors.In this review, we focus on the regulation of DNA on the activity of nanozymes for fabricating biosensors.For the nanozymelabeled aptasensors, one can refer to other publications. 30,124-128

| Detection of toxic metal ions
With the development of industries and the continuous rise of human demand, various environmental pollutants emerge at a growing rate.They not only have a great impact on the environment but also gradually accumulate through biological chains, posing significant hazards to human health.As a typical class of environmental pollutants, toxic metal ions can be detected using the DNA-or aptamer-mediated nanozyme catalytic systems. 57,61,78,102,129,130For instance, Taghdisi et al. designed a DNA hairpin with a stem and a Pb 2þ -specific aptamer-based loop. 61The loop was able to recognize Pb 2þ and open the stem to release a short intermediate ssDNA (STP) from the triple-helix structure.The process caused a change in the colorimetric signal through the inhibitory effect of STP on AuNPs enzymatic activity.Consequently, the fabricated turn-off colorimetric aptasensor could be applied to detect toxic Pb 2þ selectively and sensitively, providing a limit of detection (LOD) of 602 pM.Qi et al. simply used the principle of Hg 2þ -specific aptamer adsorbed on AuNPs to promote the enzymatic activity to construct a fast colorimetric Hg 2þ aptasensor. 57With the sensor, the detection could be completed in 10 min, and the analytical results in tap water and lake water samples verified its good precision and accuracy.Interestingly, Li et al. combined  78 As illustrated in Figure 11A, adsorption of ssDNA could promote the enzymatic activity of P-CeO 2 NR but decreased the intrinsic fluorescence response of the carboxyfluorescein-labeled aptamer (FAM-apt).The fluorescence signal could be restored by aptamer shedding from P-CeO 2 NR through conformational transition after binding to the target.At the same time, the nanozyme recovered its original weak activity in catalyzing the TMB color reaction.With this principle, the two signals could be employed to construct a dual-mode platform for Hg 2þ sensing.Attractively, the bimodal outputs could complement each other to meet different detection requirements.All the above examples indicate that nanozymes with various characteristics can combine with structurally variable DNA chains (aptamers) to construct powerful biochemical sensing systems.
Very recently, Qiu's group found that aptamers could reversely modulate the oxidase-and POD-mimetic activities of some materials. 119,129In their study, 119 they synthesized a photosensitive COF (Tph-BT) as a nanozyme with strong OXD activity and weak POD activity.It was found that the UO 2þ 2 -specific DNA aptamer could obviously suppress the OXD-like activity but promote the POD catalytic activity.The introduction of UO 2þ 2 interacted with the DNA aptamer and detached the latter from the Tph-BT surface, thereby restoring the nanozyme activities again.According to the finding, both the OXD and POD channels provided a reversive response for the sensing of UO 2þ 2 (Figure 11B).

| Monitoring of health risk factors
It is undeniable that humans are exposed to various health risk factors all the time from the environment via water, food, and air.Numerous studies have confirmed the close relationship between emerging diseases and poor living surroundings.The environment becomes one of the most important factors leading to various diseases.Seriously, the rapid expansion and development of modern society are releasing increasing risk factors.Identifying these risks and taking corresponding preventive measures is a major demand for protecting human health.]133 The emergence and application of antibiotics is a major revolution in the history of human medicine.However, with the widespread use of antibiotics, including medical antibiotics and antibiotics for animals, new problems that seriously threaten human health have soon emerged.5][136] Therefore, strict control of the antibiotic residue level is essential to ensure human health and safety.To this end, Zhang et al.

F I G U R E 1 1 (A)
Illustration of the dual-mode sensing of Hg 2þ based on the modulation of enzyme-like P-CeO 2 NR by FAM-labeled aptamer.Reproduced with permission. 78Copyright 2022, Elsevier.(B) Colorimetric detection of UO 2þ 2 based on aptamer modulating the oxidase and peroxidase activities of Tph-BT reversely.Reproduced with permission. 129Copyright 2023, American Chemical Society.
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synthesized gold nanoclusters with POD activity and achieved the specific visual detection of tetracycline antibiotics (TCs) in drugs and milk samples based on the promoting effect of the TC aptamer on nanozyme activity. 59Zhu et al. 76 and Tang et al. 84 utilized kanamycinspecific aptamers to enhance the POD activity of boron nitride-anchored CeO 2 NR (BNQDs/CeO 2 NR) and WS 2 NSs, respectively, to construct sensitive, simple and stable colorimetric aptasensors for the detection of kanamycin in agricultural and animal products.Both the methods took only 10 min to complete the detection process, and the detection limits were 4.6 pM and 0.06 μM, respectively.The simple strategy of directly conjugating aptamers with nanozymes can greatly reduce detection time, but the detection limit highly depends on the intrinsic properties of nanozymes.Therefore, exploring nanozymes with strong activity is one of the key points for the construction of high-performance biochemical sensors.Zhu et al. constructed a bimetallic nanozyme (NiCo@C HCs) with a unique hollow structure, 91 which could catalyze nearly 5 times faster than other reported nanozymes.By combining the aptamer-mediated decrease of NiCo@C HC enzymatic activity with the specificity and strong affinity of the aptamer, an ultrasensitive colorimetric method was developed for the detection of enrofloxacin.Furthermore, they developed a colorimetric detection device and an APP to join the method with a smartphone for the integration of data reading and signal processing for on-site, quantitative monitoring.Different from common optical detection, Zhou's group achieved the direct electrochemical detection of kanamycin based on the aptamer-mediated POD activity modulation of AuNPs. 65The AuNPs synthesized by employing tyrosine as a reducing and capping agent presented certain POD-like activity.With the presence of kanamycin-specific aptamer, ssDNA adsorption on the AuNP surface blocked the active sites of the latter, thus suppressing the catalytic activity.When the target kanamycin was further introduced, it bound with the adsorbed aptamer on AuNPs with higher affinity, thus exposing the active surface of AuNPs and recovering the POD-mimetic activity again.They employed the AuNPs to catalyze the reaction between H 2 O 2 and reduced thionine to generate oxidized thionine (Figure 12A).The latter provided a notable reduction signal on the gold electrode, which could be employed to quantify the content of kanamycin.Under optimal conditions, the established electrochemical assay presented high sensitivity for kanamycin detection, with a LOD of 0.06 nM and a linear detection range of 0.1-60 nM.
Biotoxins are a class of toxic substances produced by various organisms.Food poisoning caused by biotoxins often occurs, which seriously threatens human health.
Currently, some studies have realized the monitoring of biotoxins using the DNA or aptamer-mediated nanozyme activity modulation principle. 56,69,80,100,103,131,132Typically, Li et al. designed a terminal-fixed anti-saxitoxin (STX) aptamer with a loop recognition structure, 56 and the extended two-terminal sequences could hybridize to form a stem-loop structure.It was found that the terminal-fixed aptamer could significantly increase its affinity to the target.By designing a capture DNA (cDNA) complementary to the loop, STX could compete with the cDNA for aptamer binding (Figure 12B), thus releasing the cDNA for promoting the enzymatic activity of AuNPs.In principle, STX as low as 142.3 pM could be detected, and the detection time was only 15 min.
5]133 Among these, Bansal's group established a colorimetric and electrochemical dual-signal aptasensor for Pseudomonas aeruginosa. 63In comparison with immunosensing, fluorescence hybridization-based detection, and quantitative PCR, the method possessed better results within a short time (10 min) and could be used for rapid, on-site analysis.Zhu et al. reported a label-free, ultrasensitive colorimetric aptasensor for the monitoring of food-borne Staphylococcus aureus (S. aureus) using the oligonucleotide-mediated OXD-mimetic activity of Mn 3 O 4 NPs. 94Their study demonstrated that oligonucleotides like SA31 aptamer could adsorb onto the surface of Mn 3 O 4 NPs and seriously suppress their enzymemimetic activity by blocking the electron transfer to TMB (Figure 12C).In the presence of S. aureus, the aptamer would bind to bacterial cells preferentially, leading to the recovery of the OXD activity.Naked eyes were able to recognize the color changes from green to yellow within the increasing level of S. aureus from 10 to 2 � 10 5 CFU/mL.
Also, several studies have demonstrated the feasibility of the aptamer-mediated nanozyme modulation principle in monitoring pesticide residues. 90,97Weerathunge et al. 90 utilized the dynamic interactions between PODmimicking AgNPs and chlorpyrifos-specific aptamer to realize pesticide sensing. 90As illustrated in Figure 12D, the aptamer adsorption on AgNPs blocked the latter's active sites and surfaces for the reaction, while the further introduction of the target chlorpyrifos could despoil the adsorbed aptamer off the nanozyme, thus recovering the catalytic activity of AgNPs again.As a result, the catalytic performance observed was highly dependent on the amount of aptamer adsorbed on the nanozyme surface.With the help of the catalyzed TMB color reaction, rapid and sensitive monitoring of chlorpyrifos was obtained.

| Biomedical diagnosis
According to the DNA regulation of nanozyme activity, it is easy to establish simple, convenient, and sensitive methods for biomedical analysis and provide technical support for clinical diagnosis.At present, many studies have reported a variety of nanozymes for the establishment of biomedical analytical methods.These nanozymes can be enhanced by the adsorption of ssDNA in terms of their catalytic activity.The flexible modulation of nanozyme activity by the adsorption and desorption procedures of aptamers has been successfully used in the analysis of DNA/RNA, 60,101,105,137 enzyme activity, 72,73,87,115 proteins, 79,118,138,139 and disease markers. 70,77,86,99,113or instance, considering that bladder cancer becomes one of the most common tumors in the urinary system and threatens human health seriously, Xu's group developed a method based on HCR-mediated Fe 2 MoO 4 nanozyme for the risk prediction of bladder cancer. 105gure 13A illustrates the colorimetric aptasensor for the detection of PSCA rs2294008 (C > T) with Fe 2 MoO 4 NPs as a sensing probe and HCR DNA as a signal amplifier.In their study, bladder cancer related genes were designed as an initiator, and the H1 and H2 sequences were designed to form a stable hairpin configuration.They could not interact spontaneously without the presence of the target.When the initiator existed, the HCR process was triggered, producing long duplex DNA nanowires connecting to Fe 2 MoO 4 NPs.The sticky end was attached to the nanozyme by van der Waals force between the DNA bases and Fe 2 MoO 4 , making the nanozyme surface covered by DNA, thus preventing the redox reaction with TMB.The long double-strained part having rich negative charges could compete with Fe 2 MoO 4 NPs toward positively charged TMB.Meanwhile, the part could repel the nanozyme.Besides, the length of the formed HCR products could reach 40 nm, extending the diffusion distance of the generated hydroxyl radicals.Thus, the nanozyme activity modified by the HCR products would F I G U R E 1 2 (A) Electrochemical detection of kanamycin based on the modulation of peroxidase-like AuNPs by aptamer.Reproduced with permission. 65Copyright 2016, Elsevier.(B) Label-free colorimetric sensing platform for saxitoxin detection using a terminal-fixed aptamer to mediate the enzyme-like activity of AuNPs.Reproduced with permission. 56Copyright 2021, Elsevier.(C) Colorimetric detection of Staphylococcus aureus based on nanozyme catalysis, aptamer recognition, and magnetic separation.Reproduced with permission. 94opyright 2021, Elsevier.(D) High-specificity determination of chlorpyrifos based on the interactions between silver nanozyme and targetspecific aptamer.Reproduced with permission. 90Copyright 2021, Elsevier.
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of 23 be suppressed.This phenomenon could be used to detect the PSCA rs2294008 (C > T) gene, which was further employed for bladder cancer risk prediction.
In addition to DNA, RNA is also considered as a potential target for disease diagnosis and treatment. 140,141In a representative study, Wu et al. developed a soft template-guided wet-chemical approach for the controllable preparation of 2D MnO 2 nanoflakes with dual enzyme-like activities and applied them to construct a homogeneous electrochemical biosensing system for microRNA. 101The 2D MnO 2 NSs were found to possess OXD-and POD-like activities and exhibited highly catalytic oxidation in converting dissolved O 2 into reactive oxygen species and reduced the peak current of methylene blue.Through van der Waals interaction, P let-7a was adsorbed on the surface of 2D MnO 2 NSs to form P let-7a /MnO 2 , which further enhanced the catalytic activity of the nanozyme.Interestingly, when let-7a was present, P let-7a hybridized with let-7a and subsequently left the 2D MnO 2 NSs, clearly reducing the enzyme-like activity of MnO 2 .With this principle, uniform electrochemical detection of let-7a was demonstrated as a proof-of-concept assay.
Very recently, Su's group constructed a dual-signal method based on the DNA promoted POD-activity of FeCo oxide NSs for sensing thrombin. 73In their work, one ssDNA (S1) as the first target aptamer was employed to promote the catalytic performance of FeCo oxide NSs, and another ssDNA (S2) as the second target aptamer was modified onto the surface of magnetic beads (MBs) to form MBs-S2 for magnetic separation.When thrombin was introduced, both MBs-S2 and S1 combined with thrombin quickly, and the level of S1 in the solution decreased after magnetic separation.The POD activity enhancement of S1/FeCo oxide NSs was reduced, leading to the suppression of the catalytic TMB oxidation reaction.Both the formation of blue oxTMB and the fluorescence quenching of MoS 2 QDs were affected (Figure 13B).Based on this strategy, quantitative analysis of thrombin was achieved through recording the UV-vis signal of oxTMB or the fluorescence signal of MoS 2 QDs.
Exosomes are extracellular vesicles with the size in the scope of 30-100 nm.They are secreted from many cell types and carry cellular cargoes including nucleic acids and proteins.Given that they exist in the circulation system and play important roles in the communications F I G U R E 1 3 (A) Hybridization chain reaction-mediated Fe 2 MoO 4 nanozyme sensor for analyzing the PSCA rs2294008 (C > T) fragment as a risk prediction factor of bladder cancer.Reproduced with permission. 105Copyright 2022, Elsevier.(B) Dual-signal platform based on the deoxyribonucleic acid promoted POD activity of FeCo oxide nanosheets for detecting thrombin.Reproduced with permission. 73Copyright 2023, Elsevier.(C) CD63 aptamer accelerated POD activity of g-C 3 N 4 nanosheets for the detection of exosomes.Reproduced with permission. 77Copyright 2017, American Chemical Society.(D) Nanozyme sensor array integrating with a solventmediated signal amplification strategy for the ratiometric fluorescence analysis of exosomal proteins and cancer identification.Reproduced with permission. 79Copyright 2021, American Chemical Society.POD, peroxidase.
between cells and disease development, they are widely considered as excellent noninvasive biomarkers for disease diagnosis. 142To this end, Wang et al. designed a simple system based on the regulation of the intrinsic POD-mimicking activity of g-C 3 N 4 NSs by aptamers for the analysis of exosomes. 77Initially, the ssDNA aptamer for CD63 was adsorbed on g-C 3 N 4 NSs and enhanced the latter's POD activity, thus accelerating the oxidation of TMB by H 2 O 2 and generating an intense blue product, oxTMB (Figure 13C).When exosomes carrying the surface protein of CD63 were introduced, the ssDNA aptamer preferably bound to the exosomes in a folded structure, which had lower affinity to g-C 3 N 4 NSs, and such that the nanozyme activity was no longer improved.This method could be used to quantify exosomes based on the level of CD63.Moreover, the fabricated sensor array could recognize different expressions of CD63 in the exosomes from a breast cancer cell line (MCF-7) and a control cell line (MCF-10A).Also, a similar trend was obtained in circulating exosomes from breast cancer patients and healthy controls.
As mentioned above, exosomes carry molecular information on their parent cells.The difference in protein distribution on exosomes brings some challenges to sensitive, specific, and accurate cancer diagnosis based on a single marker.The combination detection of multiple markers is supposed to enhance the accuracy of cancer diagnosis.The levels of exosomal proteins are usually associated with disease status and employed to evaluate the therapeutic response of precision medicine.With this consideration, Liu and co-workers developed a nanozyme sensor array combining with a solvent-mediated signal amplification strategy to realize the ratiometric fluorescence determination and discrimination of different exosomal proteins. 79The adsorption of aptamers promoted the catalytic efficiency of C 3 N 4 NSs as a nanozymes in oxidizing OPD to 2,3-diaminophenazine (DAP) with a strong fluorescence emission.When the target exosomes existed, the strong affinity between aptamer and exosome resulted in the disintegration of aptamer/ C 3 N 4 NSs, leading to a decrease in the catalytic performance, thus suppressing the generation of DAP.The ratiometric fluorescence signals based on photoinduced electron transfer between C 3 N 4 NSs and DAP highly relied on the amount of DAP produced, making it possible to realize the facile and robust detection of exosomal proteins (Figure 13D).Interestingly, the addition of 1,4-dioxane was found to sensitize the luminescence response of DAP with no notable impact on the fluorescence signal of C 3 N 4 NSs, thus obtaining the amplification of the exosome-aptamer recognition events.The developed method could be used to detect exosomes with a low LOD.Moreover, accurate discrimination of cancers could be achieved by machine learning to assess the difference in exosomal proteins collected from different patients.

| SUMMARY AND PROSPECT
Since the discovery of common Fe 3 O 4 NPs exhibiting intrinsic enzymatic activity, 2 nanozymes have gained increasing importance in scientific research.The rapid development of nanozymes has injected a new force into the fields of chemistry, materials science, biology, and even medicine.Undoubtedly, nanozymes show outstanding advantages including simple preparation, low cost, good stability, easy modification, and flexible combination with biomolecules.They can be used to solve some drawbacks caused by NEs, so they are widely considered as powerful alternatives to NEs.More excitingly, DNA can affect the catalytic behaviors of nanozymes.Particularly, the introduction of aptamers largely solves the poor selectivity problem in most nanozymes developed currently.Moreover, the designability and programmability of DNA chains as well as the specificity and diversity of aptamers provide strong theoretical and technical support for the development of biochemical sensors for various targets.Due to the advantages of combining nanozymes with DNA, their interactions have been intensively utilized in biosensing and disease diagnosis.It is believed that, after fully understanding the interactions between DNA and nanozymes, the DNAnanozyme combinations will find a more extensive use in the biomedical field.What should be noted is that the combination of nanozymes and DNA/RNA not only benefits the clinical diagnosis of various diseases, as demonstrated by the above examples but also brings a new avenue for targeted drug delivery and precise therapy.For drug delivery, DNA/RNA can be used to guide the delivery of drugs to particular disease areas, and the combined nanozymes can act as catalytic stimuli to trigger the release of the loaded drugs.For direct disease therapy, the DNA-nanozyme combinations can be guided to particular disease areas and play a "nanocatalytic medicine" role.
Although great progress has been made in exploring various materials as enzyme mimics in the past decade, the catalytic mechanisms of nanozymes are still not fully understood.In fact, limited reports on the in-depth analysis of nanozyme catalytic mechanisms are available.In addition, except a few single-atom nanozymes, the key structural information of the catalytic sites of most nanozymes remains poorly understood.The surface structure of nanomaterials is complex, and it is not clear whether the catalytic activity of nanozymes is dominated by some particular nanostructures.One of the future research directions of nanozymes is likely to be from the perspective of customized nanozymes.According to different needs, nanozymes are rationally designed to maximize their performance for desired use.At the same time, the mechanism studies can be integrated with emerging techniques like artificial intelligence to enrich the types of nanozymes through theoretical predictions and calculations, further improving their enzymatic activity, stability and selectivity and adding more brilliant properties to nanozymes for versatile applications.
For the modulation of DNA on the catalytic performance of enzyme mimics, in most reports the reasons for the promoting or inhibitory effects observed have been given.However, the modulation effect of DNA on nanozyme activity needs further effort.For instance, in some studies the reasons for the regulatory effect of DNA on AuNPs can be totally opposite.At present, studies are limited to the effects of ssDNA or simple dsDNA on the catalytic efficiency of nanozymes.In fact, DNA is a biological molecule that can be precisely programmed through base pairing, and it is able to be used to precisely prepare various DNA assemblies with different structures.Modulation of nanozyme surface by DNA assemblies holds promise for the precise regulation of nanozyme properties.
In addition, novel nanozymes are being developed at any time, and most attention is focused on developing nanozymes with higher activity.However, nanozymes with a single catalytic property will inevitably reduce the designability and limit their use to some extent.For the biomedical applications, it is expected to design multifunctional nanozymes and employ them to improve the diagnostic performance. 143Also, much effort is still needed to promote these biomedical sensing applications to clinical diagnosis in hospital, community and personal healthcare.
Fu et al. fixed one end of hybridization chain reaction (HCR) products on the surface of Fe 2 MoO 4 NPs. 105Due to the long extension of HCR, a part of positively charged TMB molecules adsorbed by the HCR products were far away from the surface of Fe 2 MoO 4 NPs, reducing the catalytic efficiency of the nanozyme.Park et al. amplified the target sequence by polymerase chain reaction (PCR), and the PCR products obtained had relatively dense coverage on Fe 3 O 4 NPs.The excess PCR products were stably adsorbed with the positive substrate o-phenylenediamine (OPD), thus decreasing the catalytic efficiency observed. 106Interestingly, Zeng et al. studied the effects of different DNA configurations (ssDNA, dsDNA, hairpin DNA [H-DNA], and HCR dsDNA) on the enzymemimicking activities of Fe 3 O 4 NPs and AuNPs.

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ET mesoporous ceria nanorods (P-CeO 2 NR) featuring both POD activity and fluorescence quenching property with fluorophore-modified aptamer to construct a colorimetric and fluorescent bimodal sensor for the specific detection of Hg 2þ .