Hybridization of two or more DNA or RNA strands is well documented for the process taking place with all strands free in solution or when one strand is immobilized on a substrate. This study contributes to the investigation of the hybridization process when two single DNA strands (ssDNA) are in close proximity. We took advantage of an X sensor in which hybridization of four DNA strands enables the formation of a DNA four-way junction (crossover or X) structure. We immobilized multiple layers of crossover structures to study its hybridization being triggered by short ssDNA coming from solution and further investigate how many layers of these structures can hybridize by the addition of only one ssDNA (called input). Using a molecular beacon as reporter, we combined crossover DNA strands that recognize the reporter sequence at one side and at the other, the sequence of its input or downward crossover layer. Fluorescent signal was detected by separation of the molecular beacon’s fluorophore and quencher, as it hybridizes with the system of layers. Immobilization of the X structures into the scaffold proved to increase their communication, in comparison to being free in solution. This evidence gives us significant information for the communication of hybridized layers in a localized system, showing a promising standard for development of multilayered logic gates. The potential of these crossover DNA strands using X structure include applications in the future of biological systems, nanotechnology, and target DNA recognition for its ability to quickly recognize a signal and propagate it through extended DNA nanostructure in a controlled manner.