Nanoenzyme-Based Colorimetric Assay for Rapid Glutathione Determination Using Microwave-Synthesized Palladium Nanocrystals

Nanozyme, with enzyme-mimicking activity and excellent stability, has attracted extensive attention. However, some inherent disadvantages, including poor dispersion, low selectivity, and insufficient peroxidase-like activity, still limit its further development. Therefore, an innovative bioconjugation of ananozyme and natural enzyme was conducted. In the presence of graphene oxide (GO), histidine magnetic nanoparticles (H-Fe3O4) were first synthesized by asolvothermal method. The GO-supported H-Fe3O4 (GO@H-Fe3O4) exhibited superior dispersity and biocompatibility because GO was the carrier and possessed outstanding peroxidase-like activity because of the introduction of histidine. Furthermore, the mechanism of the peroxidase-like activity of GO@H-Fe3O4 was thegeneration of •OH. Uric acid oxidase (UAO) was selected as the model natural enzyme and covalently linked to GO@H-Fe3O4 with hydrophilic poly(ethylene glycol) as a linker. UAO could specifically catalyze the oxidation of uric acid (UA) to generate H2O2, and subsequently, the newly produced H2O2 oxidized the colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue ox-TMB under the catalysis of GO@H-Fe3O4. Based on the above cascade reaction, the GO@H-Fe3O4-linked UAO (GHFU) and GO@H-Fe3O4-linked ChOx (GHFC) were used for the detection of UA in serum samples and cholesterol (CS) in milk, respectively. The method based on GHFU exhibited a wide detection range (5-800μM) and a low detection limit (1.5μM) for UA, and the method based on GHFC exhibited a wide detection range (4-400μM) and a low detection limit (1.13μM) for CS. These results demonstrated that the proposed strategy had great potential in the field of clinical detection and food safety.

Appropriate addition of phosphate salt in food can improve the food quality and taste. However, extensive intake of phosphate salt may lead to some human diseases such as hyperphosphatemia and renal insufficiency. Thus, it is essential to establish a cost-effective, convenient, sensitive, and selective method for monitoring phosphate ion (Pi) to ensure food quality control. In this work, a Co-based metal-organic frameworks (Co-MOF) nanomaterial with dual functions (peroxidase-like activity and specific recognition) was designed for acting as a catalytic chromogenic platform for sensitive detection of Pi. The Co2+ nodes not only provide high enzyme-like activity to catalyze the 3,3′,5,5′--tetramethylbenzidine (TMB) substrate to blue oxTMB (652 nm) but also act as selective sites for Pi recognition. The use of cationic organic ligands (2-methylimidazole) and cationic metal ions (Co2+) endows the Co-MOF with a strong positive surface charge, which is beneficial to the capture of negative-charged Pi and the dramatically suppressed TMB oxidation. When Pi exists, it specifically adsorbs onto the Co-MOF through the Co-O-P bond and the strong electrostatic interaction, leading to the change of surface charge on Co-MOF. The peroxidase-like catalytic activity of Co-MOF is thus restrained, causing a different catalytic effect on TMB oxidation from that without Pi. Based on this principle, a colorimetric assay was established for rapid and sensitive detection of Pi. A good linear relationship was obtained between Pi concentration and the absorbance at 652 nm, with a linear range of 0.009–0.144 mg/L and a detection limit of 5.4 μg/L. The proposed assay was applied to the determination of Pi in actual food samples with recoveries of 92.2–108% and relative standard deviations (RSDs) of 2.7–7.3%, illustrating the promising practicality for actual samples analysis.

Bifunctional Cu0.4Co2.6O4 nanocube induced by Cu substitution with superior peroxidase-like activity: Application in hydroquinone multi-mode detection

In this work, AuAgPd trimetallic nanoparticles (AuAgPd TNPs) with intrinsic and broad-spectrum peroxidase-like activity were synthesized through a one-pot method by co-reduction of HAuCl4, AgNO3, and Na2PdCl4 with NaBH4. The morphology and composition of AuAgPd TNPs were characterized. The peroxidase-like activity of AuAgPd TNPs were highly dependent on the composition and nanostructure of AuAgPd TNPs. Rationally designed AuAgPd TNPs could catalyze the oxidation of various chromogenic substrates including 3,3'5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and o-phenylenediamine (OPD) by H2O2 to generate blue, green, and yellow products, respectively. Kinetic assays indicated that AuAgPd TNPs exhibited high affinity to H2O2. Then, sensitive colorimetric assays were developed for H2O2 detection by using ABTS, OPD, and TMB as chromogenic substrates, respectively. Lowest limit of detection (LOD) of 3.1 μM with wide linear range of 6-250 μM was obtained by using ABTS as substrate. Hydrogen sulfide ion (HS-) could effectively inhibit the peroxidase-like activity of AuAgPd TNPs. Thus, a selective colorimetric assay was further fabricated for HS- detection with LOD of 2.3 μM. This work provides an effective way for the synthesis of trimetallic nanozyme with peroxidase-like activity and also for tailoring their catalytic activity for desired use. Graphical abstract.

Nanozymes are nanomaterials that exhibit enzyme-like activity upon exposure to a substrate solution. The use of noble and platinum group metals enhances enzyme-like catalytic activity. However, noble metals are obtained at a high cost; therefore, their recovery after use is of high importance. Herein, we report the fabrication of indium tin oxide-silica nanoparticles decorated with palladium nanoparticles (ITO-SiO2-prS-PdNPs). The ITO-SiO2-prS-PdNPs were evaluated for peroxidase-like activity toward the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. A colour change from clear or colourless TMB to blue colour (oxidized TMB products) was observed confirming the peroxidase-like activity. A typical Michaelis-Menten enzyme-like behaviour is observed with Km values of 0.68 mM for H2O2 and 0.47 mM for TMB, which are better than the reported values for horse-radish peroxidase (HRP) for the same substrate. The peroxidase-like activity of ITO-SiO2-prS-PdNPs was found to proceed via the electron-transfer mechanism. The ITO-SiO2-prS-PdNPs were cleaned successfully after each use by rinsing with water and ethanol solution thus making the surface simple and easy to recover and reuse. A reusable and highly selective colorimetric assay for glucose detection based on the peroxidase-like activity of ITO-SiO2-prS-PdNPs gave excellent results. ITO-SiO2-prS-PdNPs exhibited a good linear range of 5.0-30 μM, a low limit of detection (LOD) of 1.84 μM and a limit of quantification (LOQ) of 6.14 μM. Finally, the nanozyme (ITO-SiO2-prS-PdNPs) was successfully used to detect glucose in a complex newborn calf serum (NCS), representing a real sample.

Ultra-fast colorimetric detection of glutathione by magnetic Fe NPs with peroxidase-like activity

Adenosine triphosphate (ATP) is produced mainly in the mitochondrion, and its primary task is to function as a ubiquitous energy currency to meet the cellular metabolic demands in biological system...

A dual-signal sensing platform based on single atomic iron-nitrogen-carbon nanozyme for the detection of neurodegenerative disease-related bioactive molecules acetylcholinesterase and dopamine

Introduction of Lewis acid sites to endow cobalt phosphide with peroxidase-like and oxidase-like activities and application in colorimetric sensing analysis

Ag nanoparticle-decorated Ti3C2 nanosheets (AgNPs@Ti3C2 NSs) were facilely synthesized via a self-reduction approach, in which Ti3C2 NSs acted as both reductant and supporter. The AgNPs@Ti3C2 NS nanocomposite exhibited excellent peroxidase-like activity with o-phenylenediamine (OPD) and H2O2 as substrates. The catalytic behavior followed the typical Michaelis-Menten kinetics; Michaelis constant (Km) and maximum initial velocity (Vmax) for OPD were 0.263mM and 43.2 × 10-8M-1s, indicating high affinity and high catalytic efficiency towards OPD. The catalytic mechanism was revealed to be an accelerated electron transfer process. Based on the inhibition effect on the peroxidase-like activity of AgNPs@Ti3C2 NSs, a simple, fast, and sensitive colorimetric method for detection of low-weight biothiols (cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)) was developed by measuring the absorbance at 425nm. The colorimetric method displayed wide linear range (50nM to 50μM for Cys, 10nM to 250μM for Hcy, 10nM to 50μM for GSH), low limit of detection (48.5nM for Cys, 5.5nM for Hcy, 7.0nM for GSH), and good selectivity and short assay time (3min). Moreover, the feasibility of this colorimetric sensor was demonstrated by accurately determining Cys in diluted human serum samples; good recovery (95.9-101.0%) and low relative standard deviations (2.8-4.9%) were obtained, showing great promise for point-of-care test in clinical samples.

Dual enzyme-mimicking bimetallic MOF for selective SERS detection of L-DOPA in human serum based on cascade catalytic reaction.

Objectives: To fabricate iron-doped WS2 (Fe-WS2) nanozymes with prominent peroxidase-like (POD-like) activity and explore their application in biomolecule detection. Methods: Fe-WS2 nanozymes were synthesized via a hydrothermal method, and their structural and chemical properties were characterized. The POD-like activity of the Fe-WS2 nanozymes was evaluated at different pH, temperatures, and substrate concentrations. Kinetic parameters were determined using the Michaelis–Menten equation. The detection capabilities of the Fe-WS2 nanozymes for H2O2 and glucose were assessed through colorimetric assays. Results: The synthesized Fe-WS2 nanozymes exhibited a uniform size distribution with an average diameter of approximately 300 nm. The successful loading of iron ions onto the WS2 nanosheets was confirmed by mapping and energy-dispersive X-ray spectroscopy analyses. The Fe-WS2 nanozymes demonstrated enhanced POD-like activity compared to unloaded WS2 nanozymes, as evidenced by the increased oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The optimal conditions for POD-like activity were found to be at pH 4 and 55 °C. Kinetic analysis revealed that the Fe-WS2 nanozymes had a high affinity for both H2O2 and TMB, with low Michaelis constants (Km). The Fe-WS2 nanozymes-based colorimetric assay exhibited a wide linear range (0.2 to 1 mM) for H2O2 detection, with a low detection limit of 7.27 × 10−7 M. For glucose detection, the assay showed good selectivity and sensitivity, attributed to the specificity of glucose oxidase in catalyzing glucose oxidation. Conclusions: This study successfully developed Fe-WS2 nanozymes with remarkable POD-like activity, which were utilized to construct a sensitive and specific colorimetric biosensing system for the detection of H2O2 and glucose. The results indicate that the Fe-WS2 nanozymes have great potential for applications in biomedical diagnostics due to their high catalytic efficiency, stability, and excellent biocompatibility. This work not only provides a novel inorganic nanozyme biosensor but also opens up new avenues for the application of other nanozyme biosensors in the biomedical field.

In recent years, nanozymes have attracted particular interest and attention as catalysts because of their high catalytic efficiency and stability compared with natural enzymes, whereas how to use simple methods to further improve the catalytic activity of nanozymes is still challenging. In this work, we report a trimetallic metal-organic framework (MOF) based on Fe, Co and Ni, which was prepared by replacing partial original Fe nodes of the Fe-MOF with Co and Ni nodes. The obtained FeCoNi-MOF shows both oxidase-like activity and peroxidase-like activity. FeCoNi-MOF can not only oxidize the chromogenic substrate 3,3,5,5-tetramethylbenzidine (TMB) to its blue oxidation product oxTMB directly, but also catalyze the activation of H2O2 to oxidize the TMB. Compared with corresponding monometallic/bimetallic MOFs, the FeCoNi-MOF with equimolar metals hereby prepared exhibited higher peroxidase-like activity, faster colorimetric reaction speed (1.26-2.57 folds), shorter reaction time (20 min) and stronger affinity with TMB (2.50-5.89 folds) and H2O2 (1.73-3.94 folds), owing to the splendid synergistic electron transfer effect between Fe, Co and Ni. Considering its outstanding advantages, a promising FeCoNi-MOF-based sensing platform has been designated for the colorimetric detection of the biomarker H2O2 and environmental pollutant TP, and lower limits of detection (LODs) (1.75 μM for H2O2 and 0.045 μM for TP) and wider linear ranges (6-800 μM for H2O2 and 0.5-80 μM for TP) were obtained. In addition, the newly constructed colorimetric platform for TP has been applied successfully for the determination of TP in real water samples with average recoveries ranging from 94.6% to 112.1%. Finally, the colorimetric sensing platform based on FeCoNi-MOF is converted to a cost-effective paper strip sensor, which renders the detection of TP more rapid and convenient.

The Au-Hg amalgam anchored on the surface of reduced graphene oxide nanosheets (Au-Hg/rGO) has been synthesized successfully and characterized by various techniques such as transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The Au-Hg/rGO nanocomposites were found to possess excellent peroxidase-like catalytic activity and can quickly catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxTMB in the presence of H2O2. The obvious color change offered accurate determination of the H2O2 concentration by recording the absorbance at 652 nm using a UV-vis spectrophotometer. The linear response range for H2O2 was from 5 μM to 100 μM and the detection limit was 3.25 μM (S/N = 3). Furthermore, a kinetic study indicated that the catalytic behavior of Au-Hg/rGO nanocomposites followed the typical Michaelis-Menten theory and Au-Hg/rGO nanocomposites showed good affinity for H2O2. We envision that the simple and sensitive colorimetric detection system holds great promising applications in clinical diagnostics and food and environment monitoring.

Instant synthesis of bimetallic CuCo PBA nanozyme for efficient colorimetric immunoassay of carcinoembryonic antigen.