Ficin-copper hybrid nanoflowers with enhanced peroxidase-like activity for colorimetric detection of biothiols.

The proteolytic enzyme ficin exhibits peroxidase-like activity but it is low and insufficient for real applications. Herein, we developed ficin-copper hybrid nanoflowers and demonstrated that they have significantly enhanced peroxidase-like activity of over 6-fold higher than that of free ficin, with one of the lowest Km and highest kcat values among all reported ficin-based peroxidase-like nanozymes. This was most likely caused by the synergistic catalysis of co-existing ficin and crystalline copper phosphate within nanoflower matrices having a large surface area. The nanoflowers were easily prepared by incubating ficin and copper sulfate at ambient temperature, causing coordination interactions between ficin's amine/amide moieties and copper ions, followed by concomitant anisotropic growth of petals composed of copper phosphate crystals with ficin. When compared to free ficin and natural horseradish peroxidase, the resulting nanoflowers' affinity toward H2O2 was greatly increased, yielding Km values of half and one-tenth, respectively, as well as noticeably improved stability. The nanoflowers were then applied to colorimetric determination of biological thiols (biothiols), such as cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), based on their inhibition of nanoflowers' peroxidase-like activity, producing reduced color intensities as the concentration of biothiols increased. This strategy achieved highly sensitive colorimetric determinations of Cys, GSH, and Hcy after only 25-min incubation. Additionally, using this technique, biothiols in human serum were successfully determined with excellent precision, suggesting the potential application of this technology in clinical settings, particularly in point-of-care testing environments.

Colorimetric determination of phenolic compounds using peroxidase mimics based on biomolecule-free hybrid nanoflowers consisting of graphitic carbon nitride and copper.

Hybrid nanoflowers consisting of graphitic carbon nitride (GCN) and copper were successfully constructed without the involvement of any biomolecule, by simply mixing them at room temperature to induce proper self-assembly to achieve a flower-like morphology. The resulting biomolecule-free GCN-copper hybrid nanoflowers (GCN-Cu NFs) exhibited an apparent peroxidase-mimicking activity, possibly owing to the synergistic effect from the coordination of GCN and copper, as well as their large surface area, which increased the number of catalytic reaction sites. The peroxidase-mimicking GCN-Cu NFs were then employed in the colorimetric determination of selected phenolic compounds hydroquinone (HQ), methylhydroquinone (MHQ), and catechol (CC). For samples without phenolic compounds, GCN-Cu NFs catalyzed the oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, producing an intense blue color signal. Conversely, in the presence of phenolic compounds, the oxidation of TMB was inhibited, resulting in a significant reduction of the color signal. Using this strategy, HQ, MHQ, and CC were selectively and sensitively determined in a linear range up to 100 µM with detection limits down to 0.82, 0.27, and 0.36 µM, respectively. The practical utility of this assay system was also validated by using it to detect phenolic compounds spiked in tap water, yielding a good recovery of 97.1-108.9% and coefficient of variation below 3.0%, demonstrating the excellent reliability and reproducibility of this strategy. Colorimetric determination of phenolic compounds using peroxidase mimics based on biomolecule-free hybrid nanoflowers consisting of graphitic carbon nitride and copper.

Highly Conductive Peroxidase-like Ce-MoS2 Nanoflowers for the Simultaneous Electrochemical Detection of Dopamine and Epinephrine.

The accurate and simultaneous detection of neurotransmitters, such as dopamine (DA) and epinephrine (EP), is of paramount importance in clinical diagnostic fields. Herein, we developed cerium-molybdenum disulfide nanoflowers (Ce-MoS2 NFs) using a simple one-pot hydrothermal method and demonstrated that they are highly conductive and exhibit significant peroxidase-mimicking activity, which was applied for the simultaneous electrochemical detection of DA and EP. Ce-MoS2 NFs showed a unique structure, comprising MoS2 NFs with divalent Ce ions. This structural design imparted a significantly enlarged surface area of 220.5 m2 g-1 with abundant active sites as well as enhanced redox properties, facilitating electron transfer and peroxidase-like catalytic action compared with bare MoS2 NFs without Ce incorporation. Based on these beneficial features, Ce-MoS2 NFs were incorporated onto a screen-printed electrode (Ce-MoS2 NFs/SPE), enabling the electrochemical detection of H2O2 based on their peroxidase-like activity. Ce-MoS2 NFs/SPE biosensors also showed distinct electrocatalytic oxidation characteristics for DA and EP, consequently yielding the highly selective, sensitive, and simultaneous detection of target DA and EP. Dynamic linear ranges for both DA and EP were determined to be 0.05~100 µM, with detection limits (S/N = 3) of 28 nM and 44 nM, respectively. This study shows the potential of hierarchically structured Ce-incorporated MoS2 NFs to enhance the detection performances of electrochemical biosensors, thus enabling extensive applications in healthcare, diagnostics, and environmental monitoring.

Comparison of Optical and Electrical Sensor Characteristics for Efficient Analysis of Attachment and Detachment of Aptamer.

Nucleic acid aptamer-based research has focused on achieving the highest performance for bioassays. However, there are limitations in evaluating the affinity for the target analytes in these nucleic acid aptamer-based bioassays. In this study, we mainly propose graphene oxide (GO)-based electrical and optical analyses to efficiently evaluate the affinity between an aptamer and its target. We found that an aptamer-coupled GO-based chip with an electrical resistance induced by a field-effect transistor, with aptamers as low as 100 pM, can detect the target, thrombin, at yields as low as 250 pM within five minutes. In the optical approach, the fluorescent dye-linked aptamer, as low as 100 nM, was efficiently used with GO, enabling the sensitive detection of thrombin at yields as low as 5 nM. The cantilever type of mechanical analysis also demonstrated the intuitive aptamer-thrombin reaction in the signal using dBm units. Finally, a comparison of electrical and optical sensors' characteristics was introduced in the attachment and detachment of aptamer to propose an efficient analysis that can be utilized for various aptamer-based research fields.

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.