A signal amplification system constructed by bi-enzymes and bi-nanospheres for sensitive detection of norepinephrine and miRNA.

Achieving the enhanced sensitivity and stability is always the pursuit for the fabrication of enzymatic biosensors. However, their sensitivity was still restricted by the fluctuant detection target (e.g. concentration), complex detection environment and limited recognition capability of enzymes. Herein, an effective and facile approach was designed to construct a bi-enzymatic and bi-nanospherical signal amplification system for fabrication of biosensors based on the designed polydopamine(PDA)-laccase@Au-glucose dehydrogenase. Therein, laccase-catalytic polymerized PDA nanoparticles (NPs) provided the supporting matrix for immobilization of laccase and AuNPs. The AuNPs with good conductivity and large surface area were used not only as a platform for enhanced loading capacity of glucose dehydrogenase but also as a conducting medium for electron transfer acceleration between enzymes and electrode. Moreover, the coordinated catalysis of bi-enzymes (laccase and glucose dehydrogenase) could avoid the fluctuated concentration of detection target (e.g. norepinephrine), while the application of bi-nanospheres loaded with large amount of enzymes could effectively amplify the signal of biosensors. Taking advantages of these merits, the as-prepared biosensors showed preeminent reproducibility, larger detection range from 0.5 nM to 0.5 μM, and lower detection limit of 0.07 nM (S/N = 3) for the norepinephrine detection. Besides, the constructed PDA-laccase@Au-glucose dehydrogenase was also successfully applied as the sensing probes for the detection of microRNA (miRNA), especially for single-nucleotide mismatched miRNA via specific recognition.