Co3O4/Au Hybrid Nanostructures as Efficient Peroxidase Mimics for Colorimetric Biosensing.

Nanomaterials with enzyme-like characteristics (nanozymes) have emerged as potential replacements for natural enzymes due to their potential to overcome several critical limitations of natural enzymes, including low stability as well as high costs in preparation and purification. Herein, we have developed hybrid nanostructures that incorporate cobalt oxide nanoparticles (Co3O4 NPs) and gold nanoclusters (AuNCs) through electrostatic attraction induced by simple incubation in an aqueous buffer for 2 hours. Owing to the synergistic effect of Co3O4 NPs and AuNCs, the constructed Co3O4/Au hybrid nanostructures yielded highly enhanced peroxidase-like activity and enabled rapid catalytic oxidation of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), producing a blue colored solution depending on the amount of H2O2. Moreover, we observed catalytic activity of the Co3O4/Au hybrid over a broad pH range, especially at physiologically relevant pH in the range of 5.0-7.4, which is advantageous for applications in biological systems. Using the hybrid as peroxidase mimic, we successfully determined the level of target H2O2 or glucose by coupling with glucose oxidase with excellent specificity and sensitivity. Based on this study, we expect that Co3O4/Au hybrid nanostructures can serve as potent peroxidase mimics for the detection of clinically important target molecules.

Colorimetric Detection of MPT64 Antibody Based on an Aptamer Adsorbed Magnetic Nanoparticles for Diagnosis of Tuberculosis.

We have developed a colorimetric biosensing system for the detection of antibody against MPT64, a protein secreted by Mycobacterium tuberculosis, using aptamer DNA adsorbed Fe3O4 magnetic nanoparticles (MNPs) for diagnosis of tuberculosis (TB). In this system, MNPs were first incubated with single stranded (ss) DNA-type aptamer having a high affinity toward target antibody against MPT64 (anti-MPT64), resulting in quick inhibition of the peroxidase-like activity of MNPs via the adsorption of aptamer on the surface of MNPs. By the addition of sample solutions containing anti-MPT64, aptamer bound on the surface of MNPs would strongly interact with free anti-MPT64 and be detached from the MNPs, thereby increasing the available surface area of the MNPs and consequently yielding enhanced peroxidase activity. Using this strategy, target anti-MPT64 was successfully detected by displaying increased colorimetric intensities from the higher oxidation of employed peroxidase substrate, 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS). Based on these results, we anticipate that aptamer adsorbed MNPs can serve as a potent probe system for the detection of clinically important target molecules.

Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles.

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

Glucose oxidase-copper hybrid nanoflowers embedded with magnetic nanoparticles as an effective antibacterial agent.

Bacterial contamination causes various problems ranging from bacterial infection to biofouling. As an effective and non-toxic agent for bacterial de-contamination, glucose oxidase (GOx)-copper hybrid nanoflowers embedded with amine-functionalized magnetic nanoparticles (NH2-MNPs), called 'MNP-GOx NFs', are developed. Positively-charged NH2-MNPs and negatively-charged GOx molecules are first interacted via electrostatic attraction which can be controlled by changing the buffer pH, and the follow-up addition of copper(II) sulfate leads to blooming of nanoflowers (MNP-GOx NFs) after incubation at room temperature for 3 days. MNP-GOx NFs show effective antibacterial activity by generating H2O2 from GOx-catalyzed glucose oxidation. For example, 99.9% killings of Staphylococcus aureus and Escherichia coli are achieved after 3 h treatment of 106/mL cells with 0.2 and 3.0 mg/mL MNP-GOx NFs, respectively, revealing that Gram-positive S. aureus with mono-layer membrane system is more vulnerable to the treatment of MNP-GOx NFs than Gram-negative E. coli with two-layer membrane system. MNP-GOx NFs can maintain 97% of bactericidal activity even after recycled uses by magnetic separation for eight times iterative bacterial killings. Finally, MNP-GOx NFs are employed for the fabrication of antibacterial gauzes. MNP-GOx NFs have also opened up a great potential for their applications in biosensors, biofuel cells and bioconversion as well as bacterial de-contamination.

Magnetic Nanoparticles-Embedded Enzyme-Inorganic Hybrid Nanoflowers with Enhanced Peroxidase-Like Activity and Substrate Channeling for Glucose Biosensing.

It is reported that glucose oxidase (GOx)-copper hybrid nanoflowers embedded with Fe3 O4 magnetic nanoparticles (MNPs) exhibit superior peroxidase-mimicking activity as well as substrate channeling for glucose detection. This is due to the synergistic integration of GOx, crystalline copper phosphates and MNPs being in close proximity within the nanoflowers. The preparation of MNP-embedded GOx-copper hybrid nanoflowers (MNPs-GOx NFs) begins with the facile conjugation of amine-functionalized MNPs with GOx molecules via electrostatic attraction, followed by the addition of copper sulfate that leads to full blooming of the hybrid nanoflowers. In the presence of glucose, the catalytic action of GOx entrapped in the nanoflowers generates H2 O2 , which is subsequently used by peroxidase-mimicking MNPs and copper phosphate crystals, located close to GOx molecules, to convert Amplex UltraRed substrate into a highly fluorescent product. Using this strategy, the target glucose is successfully determined with excellent selectivity, stability, and magnetic reusability. This biosensor based on hybrid nanoflowers also exhibits a high degree of precision and reproducibility when applied to real human blood samples. Such novel MNP-embedded enzyme-inorganic hybrid nanoflowers have a great potential to be expanded to any oxidases, which will be highly beneficial for the detection of various other clinically important target molecules.