Table 2.

Working protocols evaluated to prepare the amperometric biosensor for the determination of miRNA-21.

Incubation Steps (30 min Each)	Protocol	Total Assay Time, min
2	(1) b-DNACp immobilization onto Strep-MBs→b-DNACp-MBs; (2) Simultaneous hybridization of the target miRNA with the b-DNACp-MBs and labeling of the b-DNACp–miRNA heteroduplexes, immobilized onto the MBs with an anti-DNA–RNA hybrid antibody and ProtA–PolyHRP40 mixture solution pre-incubated for 1 h.	60
3	(1) b-DNACp immobilization onto Strep-MBs→b-DNACp-MBs; (2) Hybridization of the target miRNA with the b-DNACp-MBs; (3) Recognition of the b-DNACp–miRNA heteroduplexes immobilized onto the MBs by the anti DNA–RNA hybrid antibody and ProtA–PolyHRP40 mixture solution.	90
4	(1) b-DNACp immobilization onto Strep-MBs→b-DNACp-MBs; (2) Hybridization of the target miRNA with the b-DNACp-MBs; (3) Recognition of the b-DNACp–miRNA heteroduplexes immobilized onto the MBs by the anti DNA-RNA hybrid antibody; (4) Labeling of the anti DNA–RNA hybrid antibody with ProtA–PolyHRP40.	120