Characterization of DNA-Functionalized Surfaces by Fluorescence MicroscopyPublic Deposited
In the field of clinical diagnostics, biosensors are used to identify genetic disorders by hybridizing a surface-bound DNA sequence with the patient’s DNA. To further improve biosensor design, two nonlinear optical techniques, sum frequency generation (SFG) and second harmonic generation (SHG), were used to probe the orientation, chirality, and surface charge density of the surface-bound DNA strands. In this system, short DNA oligonucleotides were attached to a N-hydroxysuccinimide-ester (NHS) functionalized fused-quartz surface via an amide bond. However, the reaction steps involved in preparing these surfaces have not been fully optimized for the SHG and SFG experiments. In this current study, fluorescence confocal microscopy was used to image fluorescently labeled single-strand DNA (ssDNA) and its labeled complementary sequence. The optimum time duration required for hybridization was found to be 2 hr. Using contact angle, the minimum NHS linker concentration required for high surface coverage was found to be 1 mg/mL NHS linker in toluene, and an optimal rinsing method to minimize aggregates and remove unreacted silane was also determined. These optimizations of the experimental procedure will help to conserve valuable time and materials during the DNA surface-functionalization process to prepare substrates for SHG and SFG experiments.