Nanozymes: Definition, Activity, and Mechanisms.

"Nanozyme" is used to describe various catalysts from immobilized inorganic metal complexes, immobilized enzymes to inorganic nanoparticles. Here, the history of nanozymes is dvescribed in detail, and they can be largely separated into two types. Type 1 nanozymes refer to immobilized catalysts or enzymes on nanomaterials, which were dominant in the first decade since 2004. Type 2 nanozymes, which rely on the surface catalytic properties of inorganic nanomaterials, are the dominating type in the past decade. The definition of nanozymes is evolving, and a definition based on the same substrates and products as enzymes are able to cover most currently claimed nanozymes, although they may have different mechanisms compared to their enzyme counterparts. A broader definition can inspire application-based research to replace enzymes with nanomaterials for analytical, environmental, and biomedical applications. Comparison with enzymes also requires a clear definition of a nanozyme unit. Four ways of defining a nanozyme unit are described, with iron oxide and horseradish peroxidase activity comparison as examples in each definition. Growing work is devoted to understanding the catalytic mechanism of nanozymes, which provides a basis for further rational engineering of active sites. Finally, future perspective of the nanozyme field is discussed.

Salt-Induced Adsorption and Rupture of Liposomes on Microplastics.

Microplastics have attracted considerable attention because of concerns regarding their environmental risks to living systems. The interaction between the lipid bilayer and microplastics is important for examining the potential harm to biological membranes in the presence of microplastics. In addition, membrane coatings may change the surface and colloidal properties of microplastics. Herein, phosphatidylcholine (PC) lipids, whose headgroup is most common in cell membranes, were used as model lipids. The adsorption and rupture of PC liposomes on microplastics were systematically studied. We found that divalent metal ions, such as Mg2+ and Ca2+, facilitate liposome adsorption onto microplastics and induce 40-55% liposome leakage at 2.5 mM. In contrast, to achieve a similar effect, 300 mM Na+ was required. Adsorption and rupture followed the same metal concentration requirements, suggesting that liposome adsorption was the rate-limiting step. After adsorption with liposomes, microplastics became more hydrophilic and were better dispersed in water. A similar behavior was observed for all five types of tested microplastics, including PP, PE, PVC, PET, and PS. Leakage also occurred in ocean water. This study provides fundamental insights into the interactions between liposomes and microplastics and has implications for the colloidal and transport properties of microplastics.

Metal-Mediated DNA Adsorption on Carboxylated, Hydroxylated, and Hydrogenated Nanodiamonds.

Nanodiamonds (NDs) have attracted considerable attention owing to their quantum properties and versatility in biological applications. In this study, we systematically investigated the adsorption of DNA oligonucleotides onto NDs with three types of surface groups: carboxylated (COOH-), hydroxylated (OH-), and hydrogenated (H-). Among them, only the H-NDs showed fluorescence quenching property that is useful for real-time DNA adsorption kinetic studies. The effect of common metal ions on DNA adsorption was studied. In the presence of Na+, the order of DNA adsorption efficiency was H- > OH- > COOH-, whereas all the NDs showed a similar DNA adsorption efficiency in the presence of divalent metal ions such as Ca2+ and Zn2+. Desorption studies revealed that hydrogen bonding and metal-mediated interactions were dominant for the adsorption of DNA, and the H-NDs exhibited extraordinarily tight DNA adsorption. Finally, a fluorescently labeled DNA was adsorbed on NDs for DNA detection, and the COOH-NDs had the highest target specificity, and a detection limit of 1.4 nM was achieved. This study indicates the feasibility of using metal ions to mediate the physical adsorption of DNA to NDs and compares various NDs with graphene oxide for fundamental understanding.

Cytosine-Rich DNA Binding Insulin Stronger than Guanine-Rich Aptamers: Effect of Aggregation of Insulin for Its Detection.

The detection of insulin is an important analytical task. Previously, guanine-rich DNA was believed to bind insulin, and an insulin aptamer was selected based on a few guanine-rich libraries. Insulin is a unique analyte, and it forms different aggregation states as a function of its concentration and buffer conditions, which may affect the detection of insulin. Herein, using fluorescence polarization assays, three insulin preparation methods were evaluated: direct dissolution, ethylenediaminetetraacetic acid (EDTA) treatment to remove Zn2+, and dissolution in acid followed by neutralization. All the insulin samples containing Zn2+ barely bind to the aptamer DNA, whereas monomers and dimers of insulin with Zn2+ removed were able to bind. Compared to the previously reported aptamer, C-rich DNA showed stronger binding affinities and faster binding kinetics. The sigmoidal binding curves and slow binding kinetics showed that multiple DNA strands and insulin molecules gradually bind, and it took approximately 1 h to reach saturation. This insulin binding was nonspecific, and other tested proteins also can bind to C-rich and G-rich DNA with even strong affinities. These results provide important information on the detection of insulin and further insights into the binding mechanisms between oligomeric insulin and DNA.

Comparison of the peroxidase activities of iron oxide nanozyme with DNAzyme and horseradish peroxidase.

Peroxidase-based assays are the most extensively used in bioanalytical sensors because of their simple colorimetric readout and high sensitivity owing to enzymatic signal amplification. To improve the stability, modification, and cost of protein-based enzymes, such as horseradish peroxidase (HRP), various enzyme mimics, such as DNAzymes and nanozymes, have emerged over the last few decades. In this study, we compared the peroxidase activities of HRP, a G-quadruplex (G4)-hemin DNAzyme, and Fe3O4 nanozymes in terms of activity and stability under different conditions. The reactions were much slower at pH 7 than at pH 4. At pH 4, the turnover rate of HRP (375 s-1) was faster than that of G4 DNAzyme (0.14 s-1) and Fe3O4 (6.1 × 10-4 s-1, calculated by surface Fe concentration). When normalized to mass concentrations, the trend was the same. Through observation of the reaction for a long time of 2 h, the changes in the color and UV-vis spectra were also different for these catalysts, indicating different reaction mechanisms among these catalysts. Moreover, different buffers and nanozyme sizes were found to influence the activity of the catalysts. Fe3O4 showed the highest stability compared to HRP and G4 DNAzyme after a catalytic reaction or incubation with H2O2 for a few hours. This study helps to understand the properties of catalysts and the development of novel catalysts with enzyme-mimicking activities for application in various fields.

Spherical DNA for Probing Wettability of Microplastics.

Wettability of microplastics may change due to chemical or physical transformations at their surface. In this work, we studied the adsorption of spherical nucleic acids (SNAs) with a gold nanoparticle core and linear DNA of the same sequence to probe the wettability of microplastics. Soaking microplastics in water at room temperature for 3 months resulted in the enhancement of SNA adsorption capacity and affinity, whereas linear DNA adsorption was the same on the fresh and soaked microplastics. Drying of the soaked microplastics followed by rehydration decreased the adsorption of the SNA, suggesting that the effect of soaking was reversible and related to physical changes instead of chemical changes of the microplastics. Raman spectroscopy data also revealed no chemical transformations of the soaked microplastics. Heating of microplastics over a short period induced a similar effect to long-term soaking. We propose that soaking or heating removes air entrapped in the nanosized pores at the water-plastic interface, increasing the contact surface area of the SNA to afford stronger adsorption. However, such wetted porosity would not change the adsorption of linear DNA because of its much smaller size.

A Label-Free, Mix-and-Detect ssDNA-Binding Assay Based on Cationic Conjugated Polymers.

The accurate, simple, and efficient measurement of the concentration of single-stranded DNA (ssDNA) is important for many analytical applications, such as DNA adsorption, biosensor design, and disease diagnosis, but it is still a challenge. Herein, we studied a cationic conjugated polymer (CCP)-based ssDNA assay taking advantage of the obvious fluorescence change of CCPs upon binding ssDNA. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) achieved an apparent dissociation constant (Kd) of 57 ± 4 nM for ssDNA, indicating a very high binding affinity between PMNT and ssDNA. This allowed us to develop a CCP-based ssDNA biosensor with a detection limit of 0.6 nM, similar to the fluorescence-dye-based method using SYBR Green I and SYBR Gold. Our CCP-based biosensor produced smaller differences among ssDNA samples with different base compositions. In addition, the existence of double-stranded DNA (dsDNA) at different concentrations did not interfere with the fluorescence of PMNT, indicating that our CCP-based biosensor was more suitable for the measurement of ssDNA. Compared with fluorescence-intensity-based quantification, our CCP system allowed ratiometric quantification, which made the calibration easier and more robust. We then applied our method to the quantification of ssDNA on AuNPs using both unmodified and thiolated ssDNA, and the accurate quantification of ssDNA was achieved without any fluorophore modification. This method provides an alternative approach for the measurement of ssDNA.

Gold Nanoparticles Synthesized Using Various Reducing Agents and the Effect of Aging for DNA Sensing.

Gold nanoparticles (AuNPs) are one of the most commonly used reagents in colloidal science and biosensor technology. In this work, we first compared AuNPs prepared using four different reducing agents including citrate, glucose, ascorbate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). At the same absorbance at the surface plasmon peak of 520-530 nm, citrate-AuNPs and glucose-AuNPs adsorbed more DNA and achieved higher affinity to the adsorbed DNA. In addition, citrate-AuNPs had better sensitivity than glucose-AuNPs for label-free DNA detection. Then, using citrate-AuNPs, the effect of aging was studied by incubation of the AuNPs at 22 °C (room temperature) and at 4 °C for up to 6 months. During aging, the colloidal stability and DNA adsorption efficiency gradually decreased. In addition, the DNA sensing sensitivity using a label-free method also dropped around 4-fold after 6 months. Heating at boiling temperature of the aged citrate-AuNPs could not rejuvenate the sensing performance. This study shows that while citrate-AuNPs are initially better than the other three AuNPs in their colloid properties and sensing properties, this edge in performance might gradually decrease due to constantly changing surface properties caused from the aging effect.

Removal and Degradation of Microplastics Using the Magnetic and Nanozyme Activities of Bare Iron Oxide Nanoaggregates.

Removal and degradation of microplastics are often carried out separately. In this work, hydrophilic bare Fe3 O4 nanoaggregates allowed efficient removal of the most common microplastics including high-density polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyethylene terephthalate. Full extraction was achieved using Fe3 O4 at 1 % of the mass of microplastics. Hydrogen bonding is the main force for the adsorption of Fe3 O4 . Unlike the more commonly used hydrophobically modified Fe3 O4 nanoparticles, the bare Fe3 O4 benefitted from the peroxidase-like activity of its exposed surface, enabling further catalytic degradation of microplastics with nearly 100 % efficiency and easy recovery of the Fe3 O4 .

Protection of DNA by metal ions at 95 °C: from lower critical solution temperature (LCST) behavior to coordination-driven self-assembly.

While polyvalent metal ions and heating can both degrade nucleic acids, we herein report that a combination of them leads to stabilization. After incubating 4 mM various metal ions and DNA oligonucleotides at 95 °C for 3 h at pH 6 or 8, metal ions were divided into four groups based on gel electrophoresis results. Mg2+ can stabilize DNA at pH 6 without forming stable nanoparticles at room temperature. Co2+, Cu2+, Cd2+, Mn2+ and Zn2+ all protected the DNA and formed nanoparticles, whereas the nanoparticles formed with Fe2+ and Ni2+ were so stable that they remained even in the presence of EDTA. At pH 8, Ce3+ and Pb2+ showed degraded DNA bands. For Mg2+, better protection was achieved with higher metal and DNA concentrations. By monitoring temperature-programmed fluorescence change, a sudden drop in fluorescence intensity attributable to the lower critical solution temperature (LCST) transition of DNA was found to be around 80 °C for Mg2+, while this transition temperature decreased with increasing Mn2+ concentration. The unexpected thermal stability of DNA enabled by metal ions is useful for extending the application of DNA at high temperatures, forming coordination-driven nanomaterials, and it might offer insights into the origin of life on the early Earth.

Surface Science of Nanozymes and Defining a Nanozyme Unit.

The field of nanozyme aims to use nanomaterials to replace protein-based enzymes. Nanozymes have attracted extensive interest because of their stability, cost-effectiveness, and versatility. While the focus of the nanozyme field has mainly been the discovery of new nanozyme materials and the exploration of their analytical, biomedical, and environmental applications, the number of fundamental studies is growing. Nanozymes are related to two important fields: enzymology and heterogeneous catalysis. Although fitting nanozyme kinetic data to the Michaelis-Menten kinetics is a very common practice, using the surface science methods of heterogeneous catalysis can provide insights about their catalytic mechanisms. The definition of a nanozyme unit is critical to understanding and comparing nanozyme activities. In this perspective, we articulate the use of a surface science approach to study nanozymes and discuss the various application scenarios of using different nanozyme units.

Adsorption of Linear and Spherical DNA Oligonucleotides onto Microplastics.

Microplastic pollution of water and food chains can endanger human health. It has been reported that environmental DNA can be carried by microplastics and spread into the ecosystem. To better comprehend the interactions between microplastics and DNA, we herein investigated the adsorption of DNA oligonucleotides on a few important microplastics. The microplastics were prepared using common plastic objects made of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), composite of PS/PVC, and polyethylene terephthalate (PET). The effect of environmentally abundant metal ions such as Na+, Mg2+, and Ca2+ on the adsorption was also studied. Among the microplastics, PET and PS had the highest efficiency for the adsorption of linear DNA, likely due to the interactions provided by their aromatic rings. The study of DNA desorption from PET revealed the important role of hydrogen bonding and metal-mediated adsorption, while van der Waals force and hydrophobic interactions were also involved in the adsorption mechanism. The adsorption of spherical DNA (SNA) made of a high density of DNA coated on gold nanoparticles (AuNPs) was also studied, where the adsorption affinity order was found to be PET > PS/PVC > PS. Moreover, a tighter DNA adsorption was achieved in the presence of Ca2+ and Mg2+ compared to Na+.

Modulation of DNAzyme Activity via Butanol Dehydration.

Understanding the activity of biomolecules in cosolvent systems is important for catalysis, separation and developing biosensors. The majority of previously studied solvents are either phase separated with water or miscible with water. Butanol was recently used to extract water for the conjugation of DNA to gold nanoparticles. In this work, the effect of butanol on the activity of a few RNA-cleaving DNAzymes was studied. A 130-fold improvement in sensitivity for the Na+ -specific EtNa DNAzyme was observed, and butanol also improved the activity of another Na+ -specific DNAzyme, NaA43T by a few folds. However, when divalent metal ions were used for both EtNa and 17E DNAzymes, the activity was inhibited. A main driven force for enhanced DNAzyme activity is the concentration effect due to butanol dehydration. This study provides insights into the interplay between DNA, metal ions and organic solvents, and such an understanding might be useful for developing sensitive biosensors.

Nanozyme Catalytic Turnover and Self-Limited Reactions.

Enzymes have catalytic turnovers. The field of nanozyme endeavors to engineer nanomaterials as enzyme mimics. However, a discrepancy in the definition of "nanozyme concentration" has led to an unrealistic calculation of nanozyme catalytic turnovers. To date, most of the reported works have considered either the atomic concentration or nanoparticle (NP) concentration as nanozyme concentration. These assumptions can lead to a significant under- or overestimation of the catalytic activity of nanozymes. In this article, we review some classic nanozymes including Fe3O4, CeO2, and gold nanoparticles (AuNPs) with a focus on the reported catalytic activities. We argue that only the surface atoms should be considered as nanozyme active sites, and then the turnover numbers and rates were recalculated based on the surface atoms. According to the calculations, the catalytic turnover of peroxidase Fe3O4 NPs is validated. AuNPs are self-limited when performing glucose-oxidase like activity, but they are also true catalysts. For CeO2 NPs, a self-limited behavior is observed for both oxidase- and phosphatase-like activities due to the adsorption of reaction products. Moreover, the catalytic activity of single-atom nanozymes is discussed. Finally, a few suggestions for future research are proposed.

Metal-Doped Polydopamine Nanoparticles for Highly Robust and Efficient DNA Adsorption and Sensing.

Controlling DNA adsorption on nanomaterials is crucial for a wide range of applications in analytical and biomedical sciences. Polydopamine (PDA) is a versatile material that can be coated on nearly any surface, and thus adsorbing DNA onto PDA can be a general method for indirect DNA functionalization of surfaces. Polyvalent metal ions were reported to promote DNA adsorption on PDA nanoparticles (NPs), but previous works added the metal ions after the formation of PDA. Herein, we compared the effect of polyvalent metal ions added during the synthesis of PDA NPs (called metal-doped) with the effect of polyvalent metal ions added after the synthesis (metal-adsorbed). A series of metal ions including Ca2+, Zn2+, Ni2+, Fe3+, and Gd3+ were tested, and Zn2+ was studied in detail due to its excellent ability for promoting DNA adsorption. With 100 µM Zn2+, metal-doped NPs were ∼30% more efficient than metal-adsorbed NPs for DNA adsorption in buffer attributable to a higher metal loading on the surface of the metal-doped NPs. Metal leaching was negligible from the metal-doped NPs, and they showed a remarkably higher robustness than the metal-adsorbed NPs, resulting in a 20-fold higher DNA extraction efficiency from serum. Based on the desorption studies, a higher adsorption affinity for the metal-doped NPs was confirmed. Finally, the Zn2+-doped PDA NPs were used for sensitive DNA detection with a limit of detection of 0.45 nM, and the sensor was highly resistant to nonspecific protein and phosphate displacement.

Spherical Nucleic Acid Mediated Functionalization of Polydopamine-Coated Nanoparticles for Selective DNA Extraction and Detection.

Magnetic nanoparticles have been widely used for the separation of biomolecules for biological applications due to the mild and efficient separation process. In previous studies, core-shell magnetic nanoparticles (NPs) were designed for DNA extraction without much sequence specificity. In this work, to achieve highly selective DNA extraction, we designed a core-shell magnetic structure by coating polydopamine (PDA) on Fe3O4 NPs. Without divalent metal ions, PDA does not adsorb DNA at neutral pH. The Fe3O4@PDA NPs were then functionalized with spherical nucleic acids (SNA) to provide a high density of probe DNA. Fe3O4@PDA@SNA was also compared with when a linear SH-DNA was covalently attached to the NPs surface, showing a higher density of the probe SNA than SH-DNA can be loaded on the NPs in a remarkably shorter time. Nonspecific DNA extraction was thoroughly inhibited by both probes. DNA extraction by the Fe3O4@PDA@SNA was more effective as well as 5-fold faster than by the Fe3O4@PDA@SH-DNA, probably due to the favorable standing conformation of DNA strands in SNA. Moreover, extraction by Fe3O4@PDA@SNA showed high robustness in fetal bovine serum, and the same design can be used for selective detection of DNA. Finally, the method was also demonstrated on silica NPs and WS2 nanosheets for coating with PDA and SNA. Altogether, our findings revealed an interesting and general surface modification strategy using PDA@SNA conjugates for sequence-specific DNA extraction.

Cooperative Metal Ion-Mediated Adsorption of Spherical Nucleic Acids with a Large Hysteresis.

Spherical nucleic acids (SNA) refer to nanoparticles attached with a high density of oligonuleotides. Linear and spherical nucleic acids have many differences such as hybridization affinity, melting transition, and cellular uptake. In this work, these two types of DNA of the same sequence were compared for adsorption on polydopamine (PDA) nanoparticles and graphene oxide (GO). We focused on the effect of metal ions including Na+, Ca2+, and Zn2+ since metal ions are indispensible for DNA adsorption on PDA and GO. Gold nanoparticles (AuNPs) of various sizes were used to prepare the SNAs. For both PDA and GO, a normal binding curve of one metal ion was obtained for adsorbing the linear DNA, while the spherical DNAs larger than 5 nm showed a sigmoidal binding curve requiring multiple metal ions. Urea and EDTA were used to probe DNA adsorption affinity, where the spherical DNA showed stronger adsorption in general. In the presence of 300 mM Na+, 4 M urea or 4 mM EDTA failed to desorb the 13 nm spherical DNA. The spherical DNA showed a very large hysteresis of metal-dependent adsorption. This study demonstrates another unique property of SNA compared to linear DNA, revealing interesting orientation and packing of DNA on AuNPs, which has deepened our understanding of DNA interface chemistry.

Transition Metal-Mediated DNA Adsorption on Polydopamine Nanoparticles.

Polydopamine (PDA) is a widely used universal coating for a broad range of materials. Interfacing PDA with various biomolecules, such as DNA, is critical for applications such as sensing, intracellular delivery, and material fabrication. Because of the negative surface charge of PDA at neutral pH, electrostatic repulsion exists between PDA and DNA. In previous studies, modified DNA or low pH was used to overcome this repulsion for DNA adsorption. More recently, divalent Ca2+ was found to bridge DNA and PDA. Herein, we studied four transition metals (Mn2+, Co2+, Zn2+, and Ni2+) and compared their efficiencies with Ca2+ for promoting DNA adsorption. These transition metals induced a more efficient and tighter DNA binding compared to Ca2+. In all these cases, the DNA phosphate backbone played a dominant role in adsorption, although DNA bases might also interact with strong binding metals such as Ni2+. Moreover, when the adsorption affinity was stronger, sensing was more selective to complementary DNA. Finally, aging of PDA appeared to be detrimental for DNA adsorption, which could be due to further oxidation of PDA. We showed that using Zn2+ or Ni2+ could considerably relieve the aging effect, while storing PDA at 4 °C could slow down aging.

Label-free and simple detection of endotoxins using a sensitive LSPR biosensor based on silver nanocolumns.

This paper describes the construction of a silver-based LSPR biosensor for endotoxin detection. We used GLAD method to procure reproducible silver nanocolumns. In this work, the silver nanostructures were considerably stabilized by a SAM of MPA, and the limit of detection of biosensor was measured to be 340 pg/ml for endotoxin E. coli. Considering endotoxin B. abortus as the second type of endotoxin contamination in our target samples (HBs-ag produced in Institute Pasteur, Iran), we investigated selectivity of the biosensor in various experiments. We showed that this biosensor can selectively detect both types of endotoxins compared to other biological species. Overall, this study proposes that LSPR biosensing can be considered as a sensitive, simple, and label-free method for endotoxin detection in the quality control laboratories.