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Size‐dependent optical absorption of semiconductive (2H) layered molybdenum disulfide (MoS2), exhibiting great discrimination abilities to single‐ and double‐stranded DNA (ssDNA) and (dsDNA), is studied. In the presence of high concentration of salt, layered MoS2 trends to aggregate rapidly, leading to the increases of sizes in both vertical and lateral dimensions of the nanosheets, which results from the interplay between van der Waals attraction and electrical double‐layer repulsion. Meanwhile, the aggregation behavior of layered MoS2 is remarkably inhibited by the synergistic effects of DNA oligonucleotides. ssDNA can adsorb on the surface of layered MoS2, resulting in a great dispersion, even in the presence of high concentration of salt, while the dispersion behavior is weakened when ssDNA is replaced by dsDNA. Whereas compared to graphene with zero bandgap energy, layered MoS2, with semiconductive properties, exhibits great characteristic optical absorption in visible wavelength region devoted to exploring the aggregation behavior of layered MoS2. Therefore, DNA oligonucleotides induced size control of layered MoS2, contributing to the regular change of its characteristic absorption in visible region, is considered a label‐free bioassay for the detection of single‐nucleotide polymorphism. Due to its easy operation and high specificity, it is expected that the proposed assay holds great promise for further applications.

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