High-Performance Biosensing Platforms Based on Enzyme-Linked Nucleic Acid Amplification Regulated by Synergistic Allosteric Hairpin Catalysis of Bimetallic Nanozymes and Its Mechanisms

Sugar cane smut disease can greatly decrease both the production and quality of sugar cane, and its early diagnosis is an effective strategy to ensure the quality and increase the income of sugar cane. Therefore, developing high-precision detection methods has major implications for the actual production of sugar cane. Herein, we synthesize bimetallic nanozymes Fe₃O₄@AuNPs with excellent glucose oxidase-like activity and nitrogen-doped graphdiyne (N-GDY) with excellent conductivity and interfacial loading capacity, which are used as catalysts for biofuel cells and flexible electrode substrates, respectively. An allosteric hairpin-regulated enzymatic cascade nucleic acid amplification strategy is employed to construct a novel biosensing platform for precise and highly sensitive analysis of the pathogen causing sugar cane smut disease, and the catalytic mechanism of the nanozymes is studied. The sugar cane smut pathogen can specifically cause the complementary region of the allosteric hairpin to migrate to form a new functional hairpin. Under the promotion of enzymes, a dual nucleic acid amplification occurs using the new functional hairpin as a template and outputs a large amount of double-stranded products, which are captured by the RCA long chain on the biocathode. At the cathode, DNA double strands are capable of holding a large quantity of Ru[(NH₃)₆]³⁺ through electrostatic attraction. The nanozymes on the anode can catalyze the oxidation of glucose to produce electrons, and AuNPs/N-GDY can efficiently transfer electrons to the cathode to obtain a strong open-circuit voltage signal, which exhibits a strong linear correlation to the pathogen in the range of 0.0001-10000 pM, with a detection limit of 53.29 aM (S/N = 3). The sensing platform offers a reliable method that allows highly precise and accurate detection of sugar cane smut disease and has great application and development potential for early identification of smut and on-site rapid detection.