In situ DNA synthesis in porous silicon for biosensing applications

Abstract

A bottom up approach to functionalizing high quality porous silicon optical structures with nucleic acid bioreceptors is presented in this dissertation. The solid-phase synthesis method using phosphoramidite protected nucleic acids is applied for the first time in porous silicon waveguides to achieve DNA attachment within the pores. Biomolecule attachment is monitored by coupling light into the waveguide to probe changes in the effective refractive index of the optical structure. We show herein that the in situ DNA synthesis method achieves a higher surface coverage with bioreceptors than the traditional infiltration of pre-synthesized DNA strands into mesoporous silicon structures. With the in situ approach, DNA conformation, flexibility, and length play little role in DNA bioreceptor density within the substrate.

The increased sensitivity resulting from in situ preparation of DNA functionalized porous silicon waveguide sensors has been demonstrated for 8-, 16-, and 24mer DNA oligo receptors and complementary nucleic acid targets, with the lowest detection limits in the nanomolar range. Functionalization of the porous silicon with a two-component silane monolayer, only one component of which is active for in situ DNA synthesis, allows for precise control of the synthesized DNA surface density. Tuning of the DNA density in the pores enables improved biosensor sensitivity by maximizing the number of bioreceptors that can capture target molecules without being impeded by steric crowding. Using synthesized DNA oligos in porous silicon as aptamers, highly selective detection of small molecule targets other than complementary DNA molecules is possible. This work demonstrates for the first time the optical measurement of DNA aptamer-based capture of small molecules in a porous silicon waveguide. Selective detection of the small molecules adenosine and ochratoxin A is described, providing evidence that DNA aptamers retain their functionality within the mesoporous substrate. This first demonstration of DNA aptamer-based sensing within porous silicon may be expanded to other small molecule targets of interest, combining the high selectivity of aptamer detection schemes with the sensitivity and filtering capabilities afforded by porous silicon waveguide sensors.