Effective Cryopreservation of a Bioluminescent Auxotrophic Escherichia coli-Based Amino Acid Array to Enable Long-Term Ready-to-Use Applications.

Amino acid arrays comprising bioluminescent amino acid auxotrophic Escherichia coli are effective systems to quantitatively determine multiple amino acids. However, there is a need to develop a method for convenient long-term preservation of the array to enable its practical applications. Here, we reported a potential strategy to efficiently maintain cell viability within the portable array. The method involves immobilization of cells within agarose gel supplemented with an appropriate cryoprotectant in individual wells of a 96-well plate, followed by storage under freezing conditions. Six cryoprotectants, namely dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol, sucrose, and trehalose, were tested in the methionine (Met) auxotroph-based array. Carbohydrate-type cryoprotectants (glycerol, sucrose, and trehalose) efficiently preserved the linearity of determination of Met concentration. In particular, the array with 5% trehalose exhibited the best performance. The Met array with 5% trehalose could determine Met concentration with high linearity (R2 value = approximately 0.99) even after storage at -20 °C for up to 3 months. The clinical utilities of the Met and Leu array, preserved at -20 °C for 3 months, were also verified by successfully quantifying Met and Leu in spiked blood serum samples for the diagnosis of the corresponding metabolic diseases. This long-term preservation protocol enables the development of a ready-to-use bioluminescent E. coli-based amino acid array to quantify multiple amino acids and can replace the currently used laborious analytical methods.

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.

Reagent-Free Colorimetric Assay for Galactose Using Agarose Gel Entrapping Nanoceria and Galactose Oxidase.

A reagent-free colorimetric method for galactose quantification using a composite of cerium oxide nanoparticles (nanoceria) and galactose oxidase (Gal Ox) entrapped in an agarose gel was developed. In the presence of galactose, the Gal Ox entrapped within the agarose gel catalyzed the oxidation of galactose to generate H2O2, which induced a color change from white to intense yellow. This reaction occurred without any chromogenic substrate. This color transition is presumed to be due to the H2O2-mediated alteration of the oxidation state of cerium ions present on the surface of the nanoceria. The intensity of color change was quantified by acquiring an image with a conventional smartphone, converting the image to cyan-magenta-yellow-black (CMYK) mode, and subsequently analyzing the image using the ImageJ software. Using this strategy, galactose concentration was specifically determined with excellent sensitivity of as low as 0.05 mM. The analytical utility of the assay was successfully verified by correctly determining diverse levels of galactose in human serum, which is enough to diagnose galactosemia, a genetic disorder characterized by the malfunctioning of enzymes responsible for galactose metabolism. The assay employing a hydrogel composite with entrapped nanoceria and Gal Ox, is a simple, cost-effective, and rapid colorimetric assay for galactose quantification, without using any chromogenic reagent. This cost-effective method has great potential for the diagnosis of galactosemia and is highly promising in comparison to the laborious instrumentation-based methods currently in use.

Self color-changing ordered mesoporous ceria for reagent-free colorimetric biosensing.

A reagent-free colorimetric detection method using mesoporous cerium oxide with a large pore size trapping an oxidative enzyme has been developed and glucose is sensitively detected with a limit of detection of 10 µM by supporting glucose oxidase on mesoporous cerium oxide.