Quantifying and Controlling DNA Probe Density on the Surface of Silicon Nitride Optical Waveguides.

Photonic biosensors offer a label-free, sensitive, and cost-effective means of detecting pathogens and biomarkers, such as methylated DNA, in liquid biopsy samples. However, challenges persist in controlling and quantifying the surface density of probes and complementary targets, which is essential to achieve optimal sensitivity. To address these issues in DNA detection, the surfaces of asymmetric Mach-Zehnder interferometer (aMZI) waveguide sensors were functionalized using two approaches to achieve density-controlled probe-DNA surfaces. In one method, varying ratios of BSA and biotinylated BSA were incubated on each sensor surface, followed by neutravidin and biotinylated probe DNA (pDNA), allowing for controlled surface coverage on each aMZI sensor. A second approach involved direct binding of amino-pDNA, mixed with nonprobe DNA, to the carboxylated aMZI surface after EDC-NHS activation. Target-DNA (tDNA) hybridization was then introduced at different concentrations to assess the effect of surface density on binding. A quantification method was developed to account for the molecular mass density, enabling the estimation of real-time signal responses during both protein functionalization and DNA binding steps. Results showed that higher tDNA solution concentrations exhibited a strong dependence on surface coverage, while lower concentrations showed a minimal dependence. Fluorescence spectroscopy, using fluorescently labeled tDNA, confirmed a direct linear correlation between the surface density and fluorescence intensity, offering a simpler yet robust method for quantitative surface characterization. This correlation provides an alternative method for estimating surface density without the need for laborious characterization. This study contributes to the development and understanding of photonic biosensing techniques for biomarker detection in liquid biopsy samples.