Horseradish peroxidase-encapsulated DNA nanoflowers: An innovative signal-generation tag for colorimetric biosensor

ABSTRACT Herein a versatile colorimetric biosensing platform was designed for sensitive, specific, and rapid screening of cancer-derived exosomes (HepG2 cell-derived) by introducing horseradish peroxidase -encapsulated DNA nanoflowers (HRP-DF) as the biorecognition elements and signal-generation tags. HRP was concurrently associated with the growing ultralong-chain DNA and the magnesium pyrophosphate crystals (Mg2PPi) during the rolling circle amplification (RCA), thereby ultimately leading to the direct fixation of HRP in DFs. For demonstration, a linear DNA circular molecule encoding the complementary sequence of CD63 aptamer (a nucleic acid sequence that can highly bind to exosomes) was used as a starting amplification template to obtain HRP-DF with the high biorecognition ability of exosomes. Upon addition of target exosomes, a sandwiched reaction was carried out between the cholesterol-modified DNA probes-conjugated magnetic bead (MB) and the HRP-DFs, accompanying formation of ternary complexes (MB-exosomes-HRP-DF). After simple magnetic separation, the HRP carried on the ternary complexes (MB-exosomes-HRP-DF) initiated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, nearly colorless) into green-colored oxidized ABTS in the presence of hydrogen peroxide (H2O2). The obvious color change (from nearly colorless to dark green) of ABTS-H2O2 system can be easily observed with the naked eye and accurately monitored by UV-visible spectrometry, which was also proportional to the concentration of exosomes. Impressively, HRP-DF-based biosensing platform exhibited satisfactory colorimetric responses toward target exosomes within the working range from 5.0 × 103 to 5.0 × 106 particles/μL at a low detection limit of 3.32 × 103 particles/μL. Combined with a one-step sandwich reaction, magnetic separation and HRP-DF-based color-changing, this system had the advantages of acceptable accuracy, strong anti-interference ability and good reproducibility.