Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Ultrahigh peroxidase-like catalytic performance of Cu-N4 and Cu-N4S active sites-containing reduced graphene oxide for sensitive electrochemical biosensing.

Carbon-based nanozymes possessing peroxidase-like activity have attracted significant interest because of their potential to replace native peroxidases in biotechnology. Although various carbon-based nanozymes have been developed, their relatively low catalytic efficiency needs to be overcome to realize their practical utilization. Here, inspired by the elemental uniqueness of Cu and the doped elements N and S, as well as the active site structure of Cu-centered oxidoreductases, we developed a new carbon-based peroxidase-mimicking nanozyme, single-atom Cu-centered N- and S-codoped reduced graphene oxide (Cu-NS-rGO), which preserved many Cu-N4 and Cu-N4S active sites and showed dramatically high peroxidase-like activity without any oxidase-like activity, yielding up to 2500-fold higher catalytic efficiency (kcat/Km) than that of pristine rGO. The high catalytic activity of Cu-NS-rGO might be attributed to the acceleration of electron transfer from Cu single atom as well as synergistic effects from both Cu-N4 and Cu-N4S active sites, which was theoretically confirmed by Gibbs free energy calculations using density functional theory. The prepared Cu-NS-rGO was then used to construct an electrochemical bioassay system for detecting choline and acetylcholine by coupling with the corresponding oxidases. Using this system, both target molecules were selectively determined with high sensitivity that was sufficient to clinically determine their levels in physiological fluids. Overall, this study will facilitate the development of nanocarbon-based nanozymes and their electrochemical biosensing applications, which can be extended to the development of miniaturized devices in point-of-care testing environments.

Graphene quantum dots on TiO2 nanotubes as a light-assisted peroxidase nanozyme.

Hybrid nanozyme graphene quantum dots (GQDs) deposited TiO2 nanotubes (NTs) on titanium foil (Ti/TiO2 NTs-GQDs) were manufactured by bestowing the hybrid with the advantageous porous morphology, surface valence states, high surface area, and copious active sites. The peroxidase-like activity was investigated through the catalytic oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, which can be visualized by the eyes. TiO2 NTs and GQDs comprising oxygen-containing functional groups can oxidize TMB in the presence of H2O2 by mimicking peroxidase enzymes. The peroxidase-mimicking activity of hybrid nanozyme was significantly escalated by introducing light illumination due to the photosensitive features of the hybrid material. The peroxidase-like activity of Ti/TiO2 NTs-GQDs enabled H2O2 determination over the linear range of 7 to 250 µM, with a LOD of 2.1 µM. The satisfying peroxidase activity is possibly due to the unimpeded access of H2O2 to the catalyst's active sites. The porous morphology provides the easy channeling of reactants and products. The periodic structure of the material also gave rise to acceptable reproducibility. Without material functionalization, the Ti/TiO2 NTs-GQDs can be a promising substitute for peroxidases for H2O2 detection.

A generalizable sensing platform based on molecularly imprinted polymer-aptamer double recognition and nanoenzyme assisted photoelectrochemical-colorimetric dual-mode detection.

Developing highly sensitive and selective methods that incorporate specific recognition elements is crucial for detecting small molecules because of the limited availability of small molecule antibodies and the challenges in obtaining sensitive signals. In this study, a generalizable photoelectrochemical-colorimetric dual-mode sensing platform was constructed based on the synergistic effects of a molecularly imprinted polymer (MIP)-aptamer sandwich structure and nanoenzymes. The MIP functionalized peroxidase-like Fe3O4 (Fe3O4@MIPs) and alkaline phosphatase mimic Zr-MOF labeled aptamer (Zr-mof@Apt) were used as the recognition elements. By selectively accumulating dibutyl phthalate (DBP), a small molecule target model, on Fe3O4@MIPs, the formation of Zr-MOF@Apt-DBP- Fe3O4@MIPs sandwich structure was triggered. Fe3O4@MIPs oxidized TMB to form blue-colored oxTMB. However, upon selective accumulation of DBP, the catalytic activity of Fe3O4@MIPs was inhibited, resulting in a lighter color that was detectable by the colorimetric method. Additionally, Zr-mof@Apt effectively catalyzed the hydrolysis of L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAPS), generating ascorbic acid (AA) that could neutralize the photogenerated holes to decrease the photocurrent signals for PEC sensing and reduce oxTMB for colorimetric testing. The dual-mode platform showed strong linearity for different concentrations of DBP from 1.0 pM to 10 µM (PEC) and 0.1 nM to 0.5 µM (colorimetry). The detection limits were 0.263 nM (PEC) and 30.1 nM (colorimetry) (S/N = 3), respectively. The integration of dual-signal measurement mode and sandwich recognition strategy provided a sensitive and accurate platform for the detection of small molecules.

Enhanced peroxidase-like activity of Cu-Cu2O composite film through PtPd immobilization for colorimetric glucose detection.

In this study, Cu-Cu2O/PtPd nanocomposites were synthesized and characterized for their peroxidase-like enzyme activity. X-ray diffraction and energy dispersive X-ray spectroscopy analyses confirmed the successful synthesis of the nanocomposites, which exhibited a flower-like morphology and a more uniform dispersion than Cu-Cu2O. The catalytic activity of Cu-Cu2O/PtPd was evaluated using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), finding that Cu-Cu2O/PtPd outperformed Cu-Cu2O. The optimal temperature and pH for the catalytic activity of Cu-Cu2O/PtPd were determined to be 40 °C and pH 4.0, respectively. A kinetic analysis revealed that Cu-Cu2O/PtPd followed Michaelis-Menten kinetics and exhibited a higher affinity toward TMB than the horseradish peroxidase enzyme. The catalytic mechanism of Cu-Cu2O/PtPd involved the generation of hydroxyl radicals, which facilitated the oxidation of TMB. Furthermore, the Cu-Cu2O/PtPd nanocomposite was successfully applied for the colorimetric detection of glucose, demonstrating a linear range of 8-90 µM, a detection limit of 2.389 µM, and high selectivity for glucose over other sugars.

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.

Bimetallic FeOx-TiO2@Carbon hybrid structure materials with notable peroxidase enzyme mimics applied to one-step colorimetric detection of glucose.

FeOx-TiO2@Carbon hybrid structure materials (FeOx-TiO2@CHs) with high peroxidase (POD)-like activity have been prepared by one-pot hydrothermal method. Based on the excellent POD activity of FeOx-TiO2@CHs, one pot colorimetric detection for glucose was constructed by using TMB as substrate with the synergistic reaction of glucose oxidase; the linear range and the limit of detection (LOD) are 25 ~ 1000 and 1.77 µM, respectively. Using this method, the glucose in serum real samples was detected with satisfactory results, and the results are consistent with that of the glucometer method in the hospital. The recovery in diabetic and artificial urine samples was 95.71 ~ 104.67% and 99.01 ~ 103.16%, respectively. The mechanism of the catalytic colorimetric reaction was also investigated by multiple measurements, and the results indicated that superoxide anions (O2•-) between FeOx-TiO2@CHs and substrate play a main role, but a small quantity of hydroxyl radical •OH and singlet oxygen 1O2 is also generated simultaneously. The one-pot reaction method is simple and fast; the detection process only requires a simple mixing, which is suitable for application in special environment.

Peroxidase-like activity and mechanism of gold nanoparticle-modified Ti3C2 MXenes for the construction of H2O2 and ampicillin colorimetric sensors.

Transition metal carbides modified by Au nanoparticles (Au/Ti3C2) were synthesized and developed as a colorimetric sensor for the determination of H2O2 and ampicillin. The surface electrical properties of Ti3C2 were changed, and Au nanoparticles (AuNPs) and gold growth solution were synthesized simultaneously. Au/Ti3C2 was obtained by seed growth method with AuNPs modified on the surface of transition metal carbides, nitrides or carbon-nitrides (Ti3C2 MXenes). The synthesized AuNPs and Ti3C2 had no peroxidase-like activity, but Au/Ti3C2 had. The peroxidase catalytic mechanism was due to electron transfer. The peroxidase activity of Au/Ti3C2 can be utilized for the determination of H2O2. The linear range of Au/Ti3C2 for H2O2 was 1-60 µM, and the detection limit was 0.12 µM (S/N = 3). A colometric sensor for ampicillin detection based on Au/Ti3C2 was further constructed since S in ampicillin formed an Au-S bond with Au/Ti3C2, leading to the weakening of its peroxidase-like property. The change of peroxidase-like property attenuated oxidation of TMB, and the ampicillin content was inversely proportional to the concentration of oxidized TMB, and the blue color of solution faded, which enabled the determination of ampicillin. The linear range for ampicillin was 0.005-0.5 µg mL- 1, and the detection limit was 1.1 ng mL- 1 (S/N = 3). The sensor was applied to the detection of ampicillin in milk and human serum.

Galvanic replacement synthesis of PtPdAu hollow nanorods as peroxidase mimic with high specific activity for colorimetric detection.

Noble metal nanomaterials have been widely demonstrated to possess intrinsic enzyme-like properties and have been increasingly applied in the fields of analysis and biomedicine. However, current exploration of high-activity noble metal nanozymes is still far from adequate. The construction of hollow structures and adjustment of their elemental composition are effective ways to improve the specific activity (SA) of nanozymes. In this study, trimetallic PtPdAu hollow nanorods (HNRs) were developed using a galvanic replacement reaction and Kirkendall effect. The catalytic experiment showed that the PtPdAu HNRs possessed outstanding peroxidase-like performance and their SA value was up to 563.71 U mg-1, which is remarkable among various previously reported nanozymes and higher than that of monometallic or bimetallic counterparts with similar structure and size prepared in this study. Electron paramagnetic resonance (EPR)measurements showed that the PtPdAu HNRs could contribute to the formation of hydroxyl radicals (˙OH) in catalyzing hydrogen peroxide. When using PtPdAu HNRs as a nanozyme in the colorimetric detection of H2O2 and ascorbic acid (AA), the limits of detection were as low as 1.8 µM and 0.068 µM, respectively. This study demonstrates that PtPdAu HNRs are high-activity nanozymes and have the potential to be applied in the field of analysis.

Portable Hydrogel Kits Made with Bimetallic Nanozymes for Point-of-Care Testing of Perfluorooctanesulfonate.

Perfluorooctanesulfonate (PFOS), an emerging organic contaminant, necessitates robust on-site detection strategies to safeguard human health and ecological balance. This study introduces a novel point-of-care testing (POCT) platform, combining a hydrogel kit with nanozymes and smartphone technology, for the highly sensitive detection of PFOS. The strategy utilizes copper-substituted cobalt-based Prussian blue analogue nanoboxes (CuCo-PBA NBs), which exhibit intricate hollow structures and remarkable peroxidase-like catalytic activity, efficiently catalyzing the oxidation of chromogenic substrates with hydrogen peroxide (H2O2). Density functional theory calculations elucidate the adsorption dynamics of H2O2 on CuCo-PBA NBs, identifying the factors that improve the catalytic efficiency. The colorimetric POCT platform, integrating the hydrogel kit with a smartphone interface, demonstrates practical utility and achieves a detection limit of 1.43 × 10-8 mol L-1 for PFOS. This research not only presents a new nanozyme design for PFOS detection in diverse matrices, such as lake water, whole blood, urine, and milk, but also paves the way for developing a portable and efficient POCT platform for a variety of emerging contaminants.

Scalable synthesis of Au@CeO2 nanozyme for development of colorimetric lateral flow immunochromatographic assay to sensitively detect heart-type fatty acid binding protein.

Nanozymes with core@shell nanostructure are considered promising biolabeling materials for their multifunctional properties. In this work, a simple one-pot strategy has been proposed for scalable synthesis of gold@cerium dioxide core@shell nanoparticles (Au@CeO2 NPs) with strong localized surface plasmon resonance (LSPR) absorption and high peroxidase-like catalytic activity by redox reactions of Ce3+ ions and AuCl4- ions in diluted ammonia solution under room temperature. A colorimetric lateral flow immunochromatographic assay (LFIA) has been successfully fabricated for sensitive detection of heart-type fatty acid binding protein (H-FABP, an early cardiac biomarker) by using the Au@CeO2 NPs as reporters. The as-developed LFIA with Au@CeO2 NP reporter (termed as Au@CeO2-LFIA) exhibits a dynamic range of nearly two orders of magnitude, and a limit of detection (LOD) as low as 0.35 ng mL-1 H-FABP with nanozyme-triggered 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric amplification. Furthermore, the practicality of Au@CeO2-LFIA has been demonstrated by profiling the concentrations of H-FABP in 156 blood samples of acute myocardial infarction (AMI) patients, and satisfactory results are obtained.

Carbon dot enhanced peroxidase-like activity of platinum nanozymes.

As one of the most intriguing nanozymes, the platinum (Pt) nanozyme has attracted tremendous research interest due to its various catalytic activities but its application is still limited by its poor colloidal stability and low affinity to substrates. Here, we design a highly stable Pt@carbon dot (Pt@CD) hybrid nanozyme with enhanced peroxidase (POD)-like activity (specific activity of 1877 U mg-1). The Pt@CDs catalyze the decomposition of hydrogen peroxide (H2O2) to produce singlet oxygen and hydroxyl radicals and exhibit high affinity to H2O2 and high specificity to 3,3',5,5'-tetramethyl-benzidine. We reveal that both the hydroxyl and carbonyl groups of CDs could coordinate with Pt2+ and then regulate the charge state of the Pt nanozyme, facilitating the formation of Pt@CDs and improving the POD-like activity of Pt@CDs. Colorimetric detection assays based on Pt@CDs for H2O2, dopamine, and glucose with a satisfactory detection performance are achieved. Moreover, the Pt@CDs show a H2O2-involving antibacterial effect by destroying the cell membrane. Our findings provide new opportunities for designing hybrid nanozymes with desirable stability and catalytic performance by using CDs as nucleating templates and stabilizers.

Dendritic-like MXene quantum dots@CuNi as an efficient peroxidase candidate for colorimetric determination of glyphosate.

Oxidized MXene quantum dots@CuNi bimetal (MQDs@CuNi) were firstly prepared through a simple hydrothermal method. Compared to the controlled samples, MQDs@CuNi1:1 showed the highest peroxidase-like activity. The catalytic mechanism of MQDs@CuNi1:1 was investigated using a steady-state fluorescence analysis, which showed that MQDs@CuNi1:1 efficiently decomposes H2O2 and produces highly reactive hydroxyl radicals (OH). Furthermore, theoretical calculations showed that the remarkable catalytic activity of MQDs@CuNi1:1 originates from the interaction between CuNi bimetal and MQDs to promote the activation and decomposition of H2O2, making it easier to combine with the hydrogen at the end of 3,3',5,5'-Tetramethylbenzidine (TMB). Accordingly, a sensitive colorimetric sensor is proposed to detect glyphosate (Glyp), displaying a low detection limit of 1.13 µM. The work will provide a new way for the development of high-performance nanozyme and demonstrate potential applicability for the determination of pesticide residues in environment.

Breaking through the pH Limitation of Fe1-xS Nanozymes Using Component-Modulated Coupled Nanoclay.

Overcoming the intrinsic low activity of most peroxidase mimics under neutral pH is crucial but still extremely challenging for the detection of disease markers in biological samples. Here, we chose nanoclay (i.e., montmorillonite K10, MK10) as a carrier to modulate the structure of Fe1-xS nanozyme components through an interfacial modulation strategy, aiming at breaking the neutral pH limitation of Fe1-xS. MK10 with abundant hydroxyl groups on its surface acts as a carrier to increase the ratio of Fe(II) and S(II-) content in surface Fe1-xS. We verify that Fe(II)-promoted surface hydroxyl radical generation and S(II-)-promoted regeneration of Fe(II) play key roles in endowing peroxidase-like activity to Fe1-xS at neutral pH. As expected, Fe1-xS/MK10 exhibited 11-fold higher Vmax and 52-fold higher catalytic efficiency than bare Fe1-xS. As a proof of concept, the sensor constructed based on Fe1-xS/MK10 achieved colorimetric detection of xanthine under neutral conditions with a linear range of 5-300 µM and a limit of detection of 2.49 µM. Finally, we achieved highly sensitive detection of xanthine in serum using the constructed biosensor. Our contribution is the novel use of a nanoclay-mediated interfacial modulation strategy for boosting the peroxidase-mimicking activity and breaking the pH limitation, which contributes to the in situ detection of disease markers by nanozymes under physiological conditions.

Plasmon-Stimulated Colorimetry Biosensor Array for the Identification of Multiple Metabolites.

Rationally designing highly catalytic and stable nanozymes for metabolite monitoring is of great importance because of their huge potential in early disease diagnosis. Herein, a novel nanozyme based on hierarchically structured CuS/ZnS with a highly efficient peroxidase (POD)-mimic capability was developed and synthesized for multiple metabolite determination and recognition via the plasmon-stimulated biosensor array strategy. The designed nanozyme can simultaneously harvest plasmon triggered hot electron-hole pairs and generate photothermal properties, leading to a sharply boosted POD-mimic capability under 808 nm laser irradiation. Interestingly, because of the interaction diversity of the metabolite with POD-like nanomaterials, the unique inhibitory effect of metabolites on the POD-mimic activity could be the signal response as the differentiation. Thus, utilizing TMB as a typical chromogenic substrate in the addition of H2O2, the designed colorimetric biosensor array can produce diverse fingerprints for the three vital metabolisms (cysteine (Cys), ascorbic acid (AA), and glutathione (GSH)), which can be precisely identified by principal component analysis (PCA). Notably, a distinct fingerprint of a single metabolite with different levels and metabolite mixtures is also achieved with a detection limit of 1 µM. Most importantly, cell lysis could be effectively discriminated by the biosensor assay, implying its great potential in clinical diagnosis.

A ratiomectic aptasensor with enhanced signals based on peroxidase-like enzymes and NH2-MIL-101@MoS2 for trace detection of deoxynivalenol in traditional Chinese herbs.

The accumulation of the deoxynivalenol (DON) in the human body poses a significant health risk that is often overlooked, and we urgently need an ultra-sensitive rapid detection platform. Due to the porosity of NH2-MIL-101@MoS2, an increased loading of toluidine blue (TB) serves to create a signal reference. Cobalt@carbon (CoC) derived from metal organic frameworks was combined with NH2-MIL-101(NH2-MIL-101@CoC) to form an enzyme-free Nanoprobe (Apt-pro) with significant catalytic properties. The ratio (IBQ /ITB) was changed by varying the electrochemical signal of benzoquinone (BQ) (IBQ) and the amount of TB deposition (ITB). This aptasensor was successfully applied to detect DON in malt and peach seed, which exhibited a great linear range from 1 fg/mL to 10 ng/mL and low detection limit of 0.31 fg/mL for DON.

Rational Biomimetic Construction of Lignin-based Carbon Nanozyme for Identification of Uric Acid in Human Urine.

Nanozymes have made remarkable progress in the field of sensing assays by replacing native enzyme functions. However, it is still a challenge to rationally design active centers from molecular structure to enhance the catalytic performance and develop low-cost nanozymes. In this work, guided by the catalytic site of horseradish peroxidase (HRP), iron source and histidine were coupled to the main chain of aminated sodium lignosulfonate (SL) through the self-assembly biomimetic strategy to construct His-SL-Fe with peroxidase activity. The inherent functional groups and basic framework of aminated SL provide a robust environment and promote the formation of active sites. His-SL-Fe shows excellent robustness over multiple test cycles and has a strong affinity for the substrate compared to HRP. His-SL-Fe had been effectively integrated in the sensing system for catalytic detection of uric acid (UA) to achieve accurate recognition of UA in the range of 0.5-100 µM with the limit of detection as low as 0.18 µM. The recovery of human urine samples is in the range of 96.8%-106.1 % and the error is within 4 %. This work not only provides a new approach for the directed design of high-performance nanozymes, but also demonstrates promising ideas for the refined application of biomass resources.

Immobilizing nanozymes on 3D-printed metal substrates for enhanced peroxidase-like activity and trace-level glucose detection.

The prevalence of 3D-printed portable biomedical sensing devices, which are fashioned mainly from plastic and polymer materials, introduces a pressing concern due to their limited reusability and consequential generation of substantial disposable waste. Considering this, herein, we pioneered a ground-breaking advancement, i.e., a 3D-printed metal substrate-based enzyme. Our inventive methodology involved the synthesis of a thermally degraded Fe-based metal-organic framework, DEG 500, followed by its deposition on a 3D-printed metal substrate composed of Ti-Al-V alloy. This novel composite exhibited remarkable peroxidase-like activity in a range of different temperatures and pH, coupled with the ability to detect glucose in real-world samples such as blood and fruit juices. The exceptional enzymatic behaviour was attributed to the diverse iron (Fe) oxidation states and the presence of oxygen vacancies, as evidenced through advanced characterization techniques. Fundamentally, we rigorously explored the mechanistic pathway through controlled studies and theoretical calculations, culminating in a transformative stride toward more sustainable and effective biomedical sensing practices.

Metal-phenolic networks derived CN-FeC hollow nanozyme with robust peroxidase-like activity for total antioxidant capacity detection.

A tannate-iron network-derived peroxidase-like catalyst loaded with Fe ions on carbon nitride (C3N4) was reported for detection of total antioxidant capacity (TAC) in food in this study. Metal-phenolic networks (MPNs) was employed to form a low coordination compound on C3N4, and calcined catalyst formed hollow structure with abundant and uniform Fe sites and surface folds. CN-FeC exhibited significant peroxidase-like activity and high substrate affinity. The homogeneous distribution of amorphous Fe elements on the C3N4 substrate provides more active sites, resulting in provided excellent catalytic activity to activate H2O2 to ·OH, 1O2 and O2·-. The established CN-FeC/TMB/H2O2 colorimetric system can detect AA in the concentration range of 5-40 µM, with the detection limits of 1.40 µM, respectively. It has good accuracy for the detection of vitamin C tablets, beverages. Taken together, this work shows that metal-phenolic networks can be an effective way to achieve efficient utilization of metal atoms and provides a promising idea for metal-phenolic networks in nanoparticle enzyme performance enhancement.

Nanohybrid nanozyme based colourimetric immunosensor for porcine gelatin.

Enzyme mimicking nanomaterials, nanozymes, have gained considerable interest in the scientific community because of their superior properties compared to natural enzymes, including their high stability at extreme conditions, cheaper availability, and ease of synthesis. Herein, we report novel colloidal gold nanoparticles - graphene nanoplatelets - chitosan (CS) with peroxidase mimicking properties used to carry out highly sensitive and selective immunoassay for porcine gelatin detection. The interaction between anti-gelatin antibody conjugated nanozyme with porcine gelatin proteins produced an ultrasensitive immunoassay response in the form of a colourimetric signal directly proportional to the porcine gelatin protein concentration. The nanozyme produced a colourimetric response in the presence of its substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), demonstrating its peroxidase mimicking properties. The results revealed that the nanozyme exhibited remarkable selectivity and sensitivity in the assay, detecting proteins at concentrations as low as 86.42 pg/mL. Additionally, the immunosensor demonstrated a broad linear detection range spanning from 200 pg/mL to 2 ng/mL.