Aihua Liu

Nanozymes have great potential as substitutes for natural enzymes. However, most nanozymes experience unsatisfactory catalytic activity and narrow optimal pH conditions, which greatly limit their wide applications. Herein, we report that copper nanoclusters (Cu NCs) exhibit peroxidase (POD) activity and red fluorescence under optimal pH 3–4 conditions. However, the surface engineering by the Mo(VI) doping and imidazole (IMZ) coordination onto the Cu NCs surface (Mo-IMZ-Cu NCs) can significantly improve the POD-like activity of Cu NCs within a wide pH range (pH 3–9), which broke the pH limitation. In addition, the fluorescence intensity was also greatly enhanced. Mechanism studies suggest that the introduction of Mo(IV) accelerated the electron transfer in the system, which not only greatly boosted the POD activity of Cu NCs but also enhanced the fluorescence of Cu NCs. More interestingly, the POD activity and fluorescence of Mo-IMZ-Cu NCs can be remarkably suppressed by the antibiotic kanamycin (Kana) selectively due to the formation of hydrogen bonding and electrostatic interaction between Kana and Mo-IMZ-Cu NCs, which leads to the aggregation of Mo-IMZ-Cu NCs into a network structure. On the basis of this phenomenon, an enzymatic colorimetry/fluorescence dual-modal platform was explored for the sensitive and selective detection of Kana, with a linear range of 1.5–15,000 nM and a detection limit (LOD) of 0.413 nM (S/N = 3) for the colorimetry mode, and a linear range of 1–10,000 nM and an LOD of 0.174 nM for the fluorimetric assay. Moreover, the as-proposed dual-modal sensor was capable of detecting Kana in real samples. Therefore, this work provides a feasible route to design nanozymes with high activity in a broad pH range, facilitating wide potential applications, including development of bioanalytical techniques.

As the subunit of the SARS-CoV-2 spike protein (SARS-CoV-2 SP), the receptor-binding domain (RBD) can specifically bind to human angiotensin-converting enzyme 2 (ACE2), enabling viral infection of host cells. Therefore, it is of utmost significance to explore probes that can bind to the conserved epitope of the SARS-CoV-2 RBD with good affinity, so as to recognize various SARS-CoV-2 variants for diagnosis, vaccination, and the development of new detection methods. Herein, we biopanned peptide RN3 (sequence: YSIDWVFHHPML) by phage display, which can bind to the SARS-CoV-2 RBD with excellent affinity and selectivity. Molecular dynamics simulation and molecular docking verified that the His8 and Met11 residues in peptide RN3 are bound to the Asn487 and Tyr489 residues of the conserved epitope of the SARS-CoV-2 RBD subunit on the SARS-CoV-2 SP (His8-Asn487 and Met11-Tyr489, respectively) by hydrogen bonding. Alanine scanning confirmed that Met11 of peptide RN3 is the key amino acid for binding to the SARS-CoV-2 SP. The median inhibition concentration for peptide RN3 inhibiting the SARS-CoV-2 SP binding to ACE2 was 37 nM. Subsequently, phage RN3 was used as a capture probe, the SARS-CoV-2 SP-specific binding peptide Pn (sequence: WNLDLSQWLPPMGGGSKKKC) as a detection probe, and Au@Pd NP-based peroxidase-mimicking nanozyme for signal amplification. A phage RN3/antigen/peptide Pn sandwich ELISA was established for the selective detection of SARS-CoV-2 SP with a linear range of 5–1000 pg/mL and a limit of detection of 2.93 pg/mL. This method was applied to double-blind testing of 13 randomized clinical samples, which could distinguish between positive and negative samples. The detected results are well consistent with those of the gold-standard RT-PCR method. Thus, this study proposed phage-displayed dual peptides to construct a reliable and inexpensive ELISA for screening SARS-CoV-2 infection. A similar strategy can be extended to study other pathogens.

Enzymatic biofuel cell (EBFC) is an electrochemical device that uses enzymes to convert the chemical energy of renewable biomass into electrical energy. However, improving the EBFC performance, such as the cost-effectiveness, operational stability, output power density (P_max), and open circuit voltage (OCV), remains challenging. Especially, the activity and stability of enzymes, substrate tolerance, cost, and immobilization strategy are crucial in developing EBFCs. Herein, we report on MnO₂ nanosheets (MnO₂ NSs) prepared via facile chemical precipitation, exhibiting outstanding laccase-mimicking activity with good stability and excellent resistance to halide ions. Then, for the first time, the laccase-like nanozyme could replace natural laccase as a cathode catalyst for efficient oxygen reduction reaction under nearly neutral pH condition, which exhibits an onset potential of 841 mV vs NHE in O₂ reduction mediated by 2,2-bisazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt. In addition, cascade enzymes of polyphosphate glucokinase (PPGK) and glucose 6-phosphate dehydrogenase (G6PDH) were successfully displayed on the Escherichia coli (E. coli) surface. The resultant PPGK-E. coli can catalyze the oxidation of glucose to glucose-6-phosphate (G6P), which can be subsequently oxidized by G6PDH-E. coli in the presence of the coenzyme NAD⁺ efficiently and converted to NADH. Finally, a dual-chamber glucose/O₂ EBFC was constructed with PPGK-E. coli/G6PDH-E. coli (2:1) as the anodic catalyst and an MnO₂ NS-based cathodic catalyst. The bacterial surface-displayed enzymes were kept in an anodic solution, which could not only retain the enzyme activity but also ensure mass transfer efficiency, thereby greatly enhancing the enzyme's inherent catalytic potential. The as-developed EBFC achieved an OCV of 0.68 V and a P_max of 32.2 μW/cm² as well as good operational stability. The strategies proposed provide new ideas for constructing high-performance EBFCs using other nanozymes and biocatalysts, which may provide new avenues for the development and utilization of sustainable energy for promising applications in wearable devices or in self-powered sensors.