Surface chemistry is a fundamental aspect of the development of the sensitive biosensor based on microarray technology. Here, an advanced PNA-microarray system for the detection of miRNA, composed by a multilayered Si/Al/Agarose component, is described. A straightforward optical signal enhancement is achieved thanks to a combination of the Al film mirror effect and the positive interference for the emission wavelength of the Cy5 fluorescent label tuned by the agarose film. The PNA-microarray was investigated for the detection of miRNA_122, resulting in a sensitivity of about 1.75 μM and Limit of Detection in the range of 0.043 nM as a function of the capture probe sequence. The contribution, in terms of H-bonds amounts at 298 and 333 K, of the agarose coating to the dsPNA-RNA interactions was demonstrated by Molecular Dynamic simulations. These results pave the way for advanced sensing strategies suitable for the environmental monitoring and the public safety.
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