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–1 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.

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size—70 ± 12 µm; spot-to-spot distance—130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm−2 was observed, together with a limit of detection of about 19 target µm−2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.

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