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Based on the mesoporous silica nanoparticles (MSN), a novel, simple and label-free aptamer biosensor was designed for the detection of aflatoxin B₁ (AFB₁). Here, the aptamers were used as molecular recognition probes and “gated molecules” while Rh6G was loaded into the interior of the particles as the signal probe. In the absence of AFB₁, the “gate” was closed to prevent the leakage of the signal probe because of the immobilization of aptamers on the surface of MSN-NH₂. With the presence of AFB₁, the “gate” could be opened to release the signal probe for the specifical binding of aptamers to AFB₁. Our results showed that the fluorescence intensity was positively correlated with the concentration of AFB₁ (0.5–50 ng mL⁻¹), with the detection limit as low as 0.13 ng mL⁻¹. What's more, this design provides a new approach for rapid, sensitive and selective detection based on aptamers and it could be …