Nanozymes for Treating Ocular Diseases.

Nanozymes, characterized by their nanoscale size and enzyme-like catalytic activities, exhibit diverse therapeutic potentials, including anti-oxidative, anti-inflammatory, anti-microbial, and anti-angiogenic effects. These properties make them highly valuable in nanomedicine, particularly ocular therapy, bypassing the need for systemic delivery. Nanozymes show significant promise in tackling multi-factored ocular diseases, particularly those influenced by oxidation and inflammation, like dry eye disease, and age-related macular degeneration. Their small size, coupled with their ease of modification and integration into soft materials, facilitates the effective penetration of ocular barriers, thereby enabling targeted or prolonged therapy within the eye. This review is dedicated to exploring ocular diseases that are intricately linked to oxidation and inflammation, shedding light on the role of nanozymes in managing these conditions. Additionally, recent studies elucidating advanced applications of nanozymes in ocular therapeutics, along with their integration with soft materials for disease management, are discussed. Finally, this review outlines directions for future investigations aimed at bridging the gap between nanozyme research and clinical applications.

Wearable Aptalyzer Integrates Microneedle and Electrochemical Sensing for in Vivo Monitoring of Glucose and Lactate in live Animals.

Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel microneedle (HMN) arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of Type 1 Diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and addresses the need for reliable and multiplexed bioanalytical monitoring in vivo. This article is protected by copyright. All rights reserved.

Metal-Drug Coordination Nanoparticles and Hydrogels for Enhanced Delivery.

Drug delivery is a key component of nanomedicine, and conventional delivery relies on the adsorption or encapsulation of drug molecules to a nanomaterial. Many delivery vehicles contain metal ions, such as metal-organic frameworks, metal oxides, transition metal dichalcogenides, MXene, and noble metal nanoparticles. These materials have a high metal content and pose potential long-term toxicity concerns leading to difficulties for clinical approval. In this review, recent developments are summarized in the use of drug molecules as ligands for metal coordination forming various nanomaterials and soft materials. In these cases, the drug-to-metal ratio is much higher than conventional adsorption-based strategies. The drug molecules are divided into small-molecule drugs, nucleic acids, and proteins. The formed hybrid materials mainly include nanoparticles and hydrogels, upon which targeting ligands can be grafted to improve efficacy and further decrease toxicity. The application of these materials for addressing cancer, viral infection, bacterial infection inflammatory bowel disease, and bone diseases is reviewed. In the end, some future directions are discussed from fundamental research, materials science, and medicine.

Adsorption of DNA and Aptamers to Sodium Urate Crystals and Inhibition of Crystal Growth.

An elevated level of blood uric acid (UA) can cause the formation of kidney stones, gout, and other diseases. We recently isolated a few DNA aptamers that can selectively bind to UA. In this work, we investigated the adsorption of a UA aptamer and random sequence DNA onto sodium urate crystals. Both DNA strands adsorbed similarly to urate crystals. In addition, both the UA aptamer and random DNA can inhibit the growth of urate crystals, suggesting a nonspecific adsorption mechanism rather than specific aptamer binding. In the presence of 500 nM DNA, the growth of needle-like sodium urate crystals was inhibited, and the crystals appeared granular after 6 h. To understand the mechanism of DNA adsorption, a few chemicals were added to desorb DNA. DNA bases contributed more to the adsorption than the phosphate backbone. Surfactants induced significant DNA desorption. Finally, DNA could also be adsorbed onto real UA kidney stones. This study provides essential insights into the interactions between DNA oligonucleotides and urate crystals, including the inhibition of growth and interface effects of DNA on sodium urate crystals.

High-Density Au Anchored to Ti3C2-Based Colorimetric-Fluorescence Dual-Mode Lateral Flow Immunoassay for All-Domain-Enhanced Performance and Signal Intercalibration.

In this work, a novel MXene-Au nanoparticle (Ti3C2@Au) was synthesized with a high molar extinction coefficient, strong fluorescence quenching ability, ultrahigh antibody affinity, high stability, and good dispersibility, and it was used to develop a colorimetric-fluorescence dual-mode lateral flow immunoassay (LFIA). The detection limits of this method for the detection of dexamethasone in milk, beef, and pork were 0.0018, 0.12, and 0.084 µg/kg in the "turn-off" mode (colorimetric signal), and 0.0013, 0.080, and 0.070 µg/kg in the "turn-on" mode (fluorescent signal), respectively, which was up to 231-fold more sensitive compared with that of the reported LFIAs. The recovery rates ranged from 81.1-113.7%, and 89.2-115.4%, with the coefficients of variation ranging from 1.4-15.0%, and 1.9-14.8%, respectively. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.97). This work not only developed novel nanocarriers for antibody-based LFIA but also ensured high-performance detection.

Pt-Decorated Gold Nanoflares for High-Fidelity Phototheranostics: Reducing Side-Effects and Enhancing Cytotoxicity toward Target Cells.

Functionalized with the Au-S bond, gold nanoflares have emerged as promising candidates for theranostics. However, the presence of intracellular abundantly biothiols compromises the conventional Au-S bond, leading to the unintended release of cargoes and associated side-effects on non-target cells. Additionally, the hypoxic microenvironment in diseased regions limits treatment efficacy, especially in photodynamic therapy. To address these challenges, high-fidelity photodynamic nanoflares constructed on Pt-coated gold nanoparticles (Au@Pt PDNF) were communicated to avoid false-positive therapeutic signals and side-effects caused by biothiol perturbation. Compared with conventional photodynamic gold nanoflares (AuNP PDNF), the Au@Pt PDNF were selectively activated by cancer biomarkers and exhibited high-fidelity phototheranostics while reducing side-effects. Furthermore, the ultrathin Pt-shell catalysis was confirmed to generate oxygen which alleviated hypoxia-related photodynamic resistance and enhanced the antitumor effect. This design might open a new venue to advance theranostics performance and is adaptable to other theranostic nanomaterials by simply adding a Pt shell.

Characterization of nanozyme kinetics for highly sensitive detection.

Nanozymes have been widely used as enzyme substitutes. Based on a comprehensive literature survey of 261 publications, we report the significant differences in the Michaelis-Menten constants (Km) between peroxidase-mimicking nanozymes and horseradish peroxidase (HRP). Further, these differences were not considered in more than 60% of the publications for analytical developments. As a result, nanozymes' catalytic activity is limited, resulting in a potentially higher limit of detection (LOD). We used a peroxidase-mimicking Au@Pt nanozyme, which has Km for TMB comparable with HRP and three orders of magnitude higher Km for H2O2. Using the Au@Pt nanozyme as a label for immunoassays, non-optimized nanozyme substrate concentrations led to 30 times higher LOD compared to optimized conditions. The results confirm the necessity of measuring nanozymes' kinetic parameters and the corresponding adjustment of substrate concentrations for highly sensitive detection.

Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH.

Triplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex-forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low-entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNSds. Compared to the conventional clamp-like triplex switch, CTNSds increased the triplex-forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple-helix structure in FBS and cell lysate. CTNSds was also less sensitive to free-energy disturbances, such as lengthening linkers or mismatches in the triple-helix stem. The CTNSds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches-based switchable nanodevices, showing great promise in biosensing and biomedicine.

Characterization of the Binding Properties of Ten Aptamers Using the Intrinsic Fluorescence of Oxytetracycline.

Tetracyclines are a class of commonly used four-ringed antibiotics. A series of DNA aptamers were recently obtained using the capture-SELEX (systematic evolution of ligands by exponential enrichment) method to bind to oxytetracycline, and one of the aptamers can bind to a few other tetracycline antibiotics as well. Upon binding to the aptamers, the intrinsic fluorescence of tetracycline antibiotics can be enhanced. At least 10 different DNA aptamers were isolated from the previous selection experiment. In this work, a systematic characterization of these ten aptamers was performed. Each of these aptamers shows a different degree of fluorescence enhancement ranging from around 1-fold to over 20-fold. Fluorescence enhancement was boosted in the presence of Mg2+ . Isothermal titration calorimetry (ITC) studies were done and showed a great variety in dissociation constant (Kd ) from 62 nM to 1.6 µM. Steady-state fluorescence spectroscopy and fluorescence lifetime studies showed a correlation between fluorescence lifetime and degree of fluorescence enhancement. A few aptamers showed slow binding kinetics, although no correlation was found between the kinetics of fluorescence change and degree of fluorescence enhancement. This is the first study of ten different aptamers for the same target, providing fundamental insights into aptamer binding and bioanalytical applications.

Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays.

To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The Kd of apt2-OT was measured to be 2.86 µM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC50) of 3.26 µM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%-115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.

Affinity-Guided Coevolution of Aptamers for Guanine, Xanthine, Hypoxanthine, and Adenine.

The simultaneous evolution of multiple aptamers can drastically increase the speed of aptamer discovery. Most previous studies used the same concentration for different targets, leading to the dominance of the libraries by one or a few aptamers and a low success rate. To foster the best aptamers to grow independently in the sequence space, it is important to (1) use low target concentrations close to their dissociation constants and (2) stop at an early round before any sequence starts to dominate. In this study, we demonstrate this affinity-guided selection concept using the capture-SELEX method to isolate aptamers for four important purines: guanine (5 µM), xanthine (50 µM), hypoxanthine (10 µM), and adenine (10 µM). The round 9 library was split, and in round 10, the four targets were individually used to elute the binding sequences. Using thioflavin T fluorescence spectroscopy and isothermal titration calorimetry, we confirmed highly selective aptamers for xanthine, guanine, and adenine. These aptamers have Kd values below 1 µM and around 100-fold selectivity against most competing analytes, and they compare favorably with existing RNA aptamers and riboswitches. A separate selection was performed using hypoxanthine alone, and no selective aptamer was achieved, even with negative selection, explaining the lack of its aptamer in our mixed selection. This affinity-guided multiplex SELEX study offers fundamental insights into aptamer selection and provides high-quality aptamers for three important purines.

Intramolecular aptamer switches.

Aptamer switches as effective biosensing tools have become a focal point of research in engineered aptasensors. Intramolecular aptamer switches are more versatile, affordable, and simpler than classical "open-close" and strand displacement-based aptamer switches. Recently, many new aptamers with an overall hairpin structure have been reported. In this study, intramolecular aptamer switches were developed by adding new base pairs to the end of aptamers. The additional nucleotides can pair with the internal domains of the aptamer, causing a change in its conformation from the original secondary structure without a target. When a target binds to an aptamer, a marked change in the structure of the aptamer is expected. As models for testing this intramolecular aptamer switch idea, aptamers of oxytetracycline (OTC), 17ß-estradiol (E2), and adenosine were employed. When the additional base pairs are too long, binding the target to the aptamer becomes more challenging. This research offers valuable insights into the development of intramolecular aptamer switches and their potential applications in biosensor design.

Selection and Characterization of DNA Aptamers for Cytidine and Uridine.

Cytidine and uridine are two essential pyrimidine ribonucleotides, and accurate detection of these nucleosides holds significant biological importance. While many aptamers were reported to bind purines, little success was achieved for pyrimidine binding. This study employs the library-immobilization capture-SELEX technique to isolate aptamers capable of selectively binding to cytidine and uridine. First, a selection was performed using a mixture of cytidine and uridine as the target. This selection led to the isolation of a highly selective aptamer for cytidine with a dissociation constant (Kd ) of 0.9 µM as determined by isothermal titration calorimetry (ITC). In addition, a dual-recognition aptamer was also discovered, which exhibited selective binding to both cytidine and uridine. Subsequently, a separate selection was carried out using uridine as the sole target, and the resulting uridine aptamer displayed a Kd of 4 µM based on a thioflavin T fluorescence assay and a Kd of 102 µM based on ITC. These aptamers do not have a strict requirement of metal ions for binding, and they showed excellent selectivity since no binding was observed with their nucleobases or nucleotides. This study has resulted three aptamers for pyrimidines, which can be employed in biosensors and DNA switches.

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.

Surface Ligand Engineering Ruthenium Nanozyme Superior to Horseradish Peroxidase for Enhanced Immunoassay.

Nanozymes have great potential to be used as an alternative to natural enzymes in a variety of fields. However, low catalytic activity compared with natural enzymes limits their practical use. It is still challenging to design nanozymes comparable to their natural counterparts in terms of the specific activity. In this study, a surface engineering strategy is employed to improve the specific activity of Ru nanozymes using charge-transferrable ligands such as polystyrene sulfonate (PSS). PSS-modified Ru nanozyme exhibits a peroxidase-like specific activity of up to 2820 U mg-1 , which is twice that of horseradish peroxidase (1305 U mg-1 ). Mechanism studies suggest that PSS readily accepts negative charge from Ru, thus reducing the affinity between Ru and ·OH. Importantly, the modified Ru-peroxidase nanozyme is successfully used to develop an immunoassay for human alpha-fetoprotein and achieves a 140-fold increase in detection sensitivity compared with traditional horseradish-peroxidase-based enzyme-linked immunosorbent assay. Therefore, this work provides a feasible route to design nanozymes with high specific activity that meets the practical use as an alternative to natural enzymes.

Selective Hemin Binding by a Non-G-quadruplex Aptamer with Higher Affinity and Better Peroxidase-like Activity.

Previous aptamers for porphyrins and metalloporphyrins were all guanine-rich sequences that can fold in G-quadruplex structures. Due to stacking-based binding, these aptamers can hardly tell different porphyrins apart, and they can also bind other planar molecules, hindering their practical applications. In this work, we used the capture selection method to obtain aptamers for hemin and protoporphyrin IX (PPIX). The hemin aptamer (Hem1) features two highly conserved repeating binding loops, and it cannot form a G-quadruplex, which was supported by its Mg2+ -dependent but K+ -independent hemin binding and CD spectroscopy. Isothermal titration calorimetry revealed much higher enthalpy change for the new aptamer, and the best aptamer showed a Kd of 43 nM hemin. Hem1 can also enhance the peroxidase-like activity of hemin. This work demonstrates that aptamers have alternative ways to bind porphyrins allowing selective recognition of different porphyrins.

Aptamer-functionalized liposomes for drug delivery.

Among the various targeting ligands for drug delivery, aptamers have attracted much interest in recent years because of their smaller size compared to antibodies, ease of modification, and better batch-to-batch consistency. In addition, aptamers can be selected to target both known and even unknown cell surface biomarkers. For drug loading, liposomes are the most successful vehicle and many FDA-approved formulations are based on liposomes. In this paper, aptamer-functionalized liposomes for targeted drug delivery are reviewed. We begin with the description of related aptamers selection, followed by methods to conjugate aptamers to liposomes and the fate of such conjugates in vivo. Then a few examples of applications are reviewed. In addition to intravenous injection for systemic delivery and hoping to achieve accumulation at target sites, for certain applications, it is also possible to have aptamer/liposome conjugates applied directly at the target tissue such as intratumor injection and dropping on the surface of the eye by adhering to the cornea. While previous reviews have focused on cancer therapy, the current review mainly covers other applications in the last four years. Finally, this article discusses potential issues of aptamer targeting and some future research opportunities.

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.

Composite materials based on covalent organic frameworks for multiple advanced applications.

Covalent organic frameworks (COFs) stand for a class of emerging crystalline porous organic materials, which are ingeniously constructed with organic units through strong covalent bonds. Their excellent design capabilities, and uniform and tunable pore structure make them potential materials for various applications. With the continuous development of synthesis technique and nanoscience, COFs have been successfully combined with a variety of functional materials to form COFs-based composites with superior performance than individual components. This paper offers an overview of the development of different types of COFs-based composites reported so far, with particular focus on the applications of COFs-based composites. Moreover, the challenges and future development prospects of COFs-based composites are presented. We anticipate that the review will provide some inspiration for the further development of COFs-based composites.

TMB+-mediated etching of urchin-like gold nanostructures for colorimetric sensing.

The morphology-dependent localized surface plasmon resonance of gold nanostructures has been widely utilized for designing sensors. One method relies on the color change of gold nanoparticles upon etching. In previous work, TMB2+oxidized from 3,3',5,5'-tetramethylbenzidine (TMB) was found to etch gold nanorods (AuNRs), leading to a spectrum of different colors. However, the preparation of TMB2+needs the addition of a strong acid and other harsh conditions. Herein, a new colorimetric biosensing platform was developed using urchin-like gold nanoparticles (AuNUs). Compared with AuNRs, the etching of AuNUs can happen under mild conditions by TMB+at pH 6, protecting enzymes and proteins from denaturation. The role of CTAB surfactant was dissected, and its bromide ions were found to be involved in the etching process. Based on these observations, a one-step colorimetric detection of H2O2was realized by using horseradish peroxidase and H2O2to oxidize TMB. Within 30 min, this system achieved a detection limit of 80 nM H2O2. This work offered fundamental insights into the etching of anisotropic gold nanostructures and optimized the etching conditions. These advancements hold promise for broader applications in biosensing and analytical chemistry.