Fabrication and Characterization of a Test Platform Integrating Nanoporous Structures With Biochemical Functionality

The application of solid-state nanopore technology for biosensing is a rapidly developing area of research with high commercial potential. Different synthetic materials, including silicon nitride, alumina, and polymers, are employed to fabricate single and multiple pores and offer a good platform for selective biomolecule detection. Two solid-state pore arrays, one with integrated silicon microfluidic system, were considered and an immobilization strategy suitable for detecting a single-stranded DNA (ssDNA) sequence was investigated. For the silicon nitride pores, a modification method based on the use of 3-aminopropyltriethoxysilane for silanization and 1,4-phenylene diisothiocyanate for amine crosslinking was applied to immobilize 100-nM ssDNA (amine C6) and a 100-nM limit of detection for complementary to probe ssDNA (Cy5) was estimated. The polycarbonate pores (the second type of the pore arrays) underwent surface modification based on an oxidation reduction reaction using sodium periodate and sodium borohydride and was used to immobilize 10-nM ssDNA and an estimated 100-nM limit of detection was also achieved. Linear sweep voltammetry was used to characterize the pores and a current potential profile was obtained after both immobilization of probe ssDNA and hybridization of complementary to probe ssDNA on the modified pore array surface. A decrease in current amplitude was measured after surface modification of both pore arrays, and this was attributed to the appearance of an additional layer on the pore surface reducing the pore opening and hindering the current flow. The hybridization event was also supported by contact angle measurements, where an increase in hydrophilicity was recorded at the different surface modification steps that were applied to produce the biofunctionalized nanopore. In addition, fluorescence was observed on the surfaces after hybridization, through incorporation of a CY5 fluorescent tag attached on the 5’ end of the complementary to probe DNA. These results show the potential to use both silicon nitride and polycarbonate nanopores in DNA detection applications.