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A biosensor structure comprising silicon nitride (Si3N4) micrograting arrays coated with a spin-on-glass (SOG) material was investigated. This grating structure was located on a silicon groove, which was etched by a deep reactive ion etching (DRIE) process. The biosensor was used as a specific detector of DNA molecules and antibody--antigen interactions. In our DNA sensing experiments, the first step was the activation of the grating surface with amine functional groups, followed by attachment of a 23-base oligonucleotide probe layer for hybridization with a complementary target DNA. The sensing device was tested for detecting specific antigen/antibody interactions for human serum albumin (HSA) and antigen bovine serum albumin (BSA). The readout system consisted of a white light lamp that illuminated a small spot on the grating surface at normal incidence through a fiber optic probe with a spectrometer used to collect the reflected light through a second fiber. We show that these sensing devices have the capability to detect DNA as well as antigen--antibody binding for HSA. The detection sensitivity for HSA was better than that for DNA mainly owing to the larger size and concomitant refractive index changes upon binding to the sensor. We show that it is possible to quantify the amount of biomolecules bound to the grating surface by measuring the wavelength shift of the reflectance spectra upon exposure to the samples.

Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2+and Na+concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34±2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32±1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the KDvalue and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+]=25mM vs. 125mM, indicating Mg2+and Na+synergy in the RNA folding process.

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