Convenient Colorimetric Detection of Cholesterol Using Multi-Enzyme Co-Incorporated Organic-Inorganic Hybrid Nanoflowers.

We have developed a microscale well-plate colorimetric assay for the multiplexed detection of cholesterol in clinical human blood samples. This system utilizes a novel multi-enzyme incorporated organic-inorganic hybrid nanoflower which entrap both cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) as the organic components with copper phosphate as the inorganic component to detect cholesterol levels in blood samples. The hybrid nanoflowers, synthesized via an extremely simple but rapid sonication-mediated method within 5 min at room temperature, enable an efficient one-pot two-enzyme cascade reaction. The ChOx in the nanoflowers catalyze the generation of H2O2 only in the presence of cholesterol in the sample. This subsequently activates the HRP co-entrapped in the nanoflowers, thereby leading to the conversion of the employed chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), into a blue-colored product. This strategy can be used to detect target cholesterol concentrations as low as 8 µM, with a linear range from 10 to 70 µM, which is suitable to diagnose high levels of cholesterol (hypercholesterolemia) with excellent stability over three weeks at room temperature. The biosensor also exhibited an excellent selectivity to detect target cholesterol even in the presence of common interfering biomolecules in human blood and showed a high degree of precision when employing human blood serum samples. Therefore, this hybrid nanoflower-based assay can be used in clinical practice for the multiplexed and reliable quantification of cholesterol, and readily extended to other enzymes to prepare multi-step cascade enzymatic reactions for various biotechnological applications.

N, S, and P-Co-doped Carbon Quantum Dots: Intrinsic Peroxidase Activity in a Wide pH Range and Its Antibacterial Applications.

Nanozymes have drawn significant scientific interest due to their high practical importance in terms of overcoming the instability, complicated synthesis, and high cost of protein enzymes. However, their activity is generally limited to particular pHs, especially acidic ones. Herein, we report that luminescent N, S, and P-co-doped carbon quantum dots (NSP-CQDs) act as attractive peroxidase mimetics in a wide pH range, even at neutral pH, for the peroxidase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the presence of H2O2. The synergistic effects of multiple heteroatoms doping in CQDs boost the catalytic activity in a wide pH range attributed to the presence of high density of active sites for enzymatic-like catalysis and accelerated electron transfer during the peroxidase-like reactions. A possible reaction mechanism for the peroxidase-like activity of CQDs is investigated based on the radical trapping experiments. Moreover, the multifunctional activity of NSP-CQDs was further utilized for antibacterial assays for both Gram-negative and Gram-positive model species, including Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. The growths of the employed E. coli and S. aureus were found to be significantly inhibited due to the peroxidase-mediated perturbation of cell walls. The present work signifies the current advance in the rational design of N, S, and P-co-doped CQDs as highly active peroxidase mimics for novel applications in diverse fields, including catalysis, medical diagnostics, environmental chemistry, and biotechnology.

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.

Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes.

The use of protein-inorganic hybrid nanoflowers for the immobilization of enzymes has received a significant degree of attention owing to their capability to retain high enzymatic activity and stability. However, the relative lack of reusability due to the weakness of the flower-like structure has limited their practical applications. Herein, we have developed a simple but efficient method to synthesize highly robust enzyme-inorganic hybrid nanoflowers, which relies on further crosslinking of the enzyme molecules entrapped in the hybrid nanoflowers by treatment with glutaraldehyde (GA). By employing lipase from Candida rugosa as a model enzyme with copper phosphate during 3days incubation followed by the additional GA treatment for only 1h, we could successfully synthesize GA-treated lipase nanoflowers having similar flower-like morphology and hydrolytic activity (ca. 95% compared with the free lipase) as conventionally synthesized lipase nanoflowers without GA treatment. Importantly, the conventional lipase nanoflowers seemed not to be reusable because they lost most of their activity (∼90%) after recycling 4 times, whereas GA-treated lipase nanoflowers exhibited higher retention of their initial activity (over 70%) after 4 reuses, which was also accompanied by an efficient maintenance of their flower-like morphology. Based on our results, we expect that this simple GA-mediated strategy to synthesize enzyme-inorganic hybrid nanoflowers can be readily extended to other enzymes for various biotechnological applications.

A simple and eco-friendly one-pot synthesis of nuclease-resistant DNA-inorganic hybrid nanoflowers.

A simple and eco-friendly method has been developed for the one-pot synthesis of DNA-copper nanoflowers that exhibit high loading efficiencies, low cytotoxicities, and strong resistance against nucleases.