Single molecule imaging of DNA on nanogap embedded plasmonic gratings with enhanced fluorescence and improved level of detection
Authors:   , Pathak Avinash 
Published: December 2023

One of the most commonly used detection tools in clinical diagnostics, life science research, chemical, and biological sensors is fluorescence. Because of its cost effective, sensitive, and specific nature fluorescence has emerged as one of the key techniques used for detection of analytes in many biological applications like cell imaging, DNA amplification, and sequencing etc. However, there has been a growing need to detect weak signals and improve the contrast levels in fluorescence images. This is essential for detecting single fluorophores and improving the limit of detection for fluorescence based biosensing. In recent years, researchers have developed many methods like total internal reflection microscopy (TIRF), confocal microscopy, and two-photon excitation microscopy (TPEM), which improve the fluorescence emission, thereby increasing the detection sensitivity. To further enhance the output of this technique, a number of nano-patterned structures like plasmonic gratings, photonic crystals and metallic bowtie nano-antennas have been developed. These nano-structures enhance the electric field intensity which can be used to couple light to the surface-bound fluorescent dye molecules thereby providing extreme signal amplification necessary for detecting low quantities of biomolecules tagged with fluorophores. In this thesis, a cheap and simple fabrication technique is presented for producing polymethylsilsesquioxane (PMSSQ) based gratings embedded with nano-gaps using micro-contact printing where a commercially available high-definition (HD) DVD-R is used as the mold to obtain the starting PDMS stamps. The nano-gaps are formed spontaneously within the grating structure as a result of tensile strain in the elastomeric PDMS stamp during the printing process. This method overcomes all the shortcomings of conventional lithography, e-beam lithography and reactive ion-etching (RIE) procedures, as the nano-structured substrates can be fabricated relatively quickly, easily and at a low production cost. These nano-gap embedded PMSSQ gratings can then be used as a base pattern for depositing metal layers to form plasmonic gratings. Using this approach, nano-gap embedded silver gratings were fabricated for enhanced fluorescence emission. Because of the enhancement on these nano-structured substrates, several applications can be thought of in bio-sensing area as it can lower the detection limit of analytes, single molecule imaging, and other diffraction-limited optics. In addition, this technique coupled with another deposition regime, called GLAD (Glancing Angle Deposition) produces further improvement of fluorescence and level of detection. Glancing angle deposition or GLAD is a well-known technique that is most often utilized to produce very unique surface features by extremely simple techniques. In most cases, little or no lithographic process is needed in order to produce columnar structures on surfaces when GLAD is used by itself. In its most basic form, GLAD represents the deposition of metallic vapors or metallic atoms at an extremely oblique angle with respect to the surface over which such deposition takes place. In addition, several other techniques such as a horizontal or vertical rotation may be combined with the angled deposition process to produce features which are symmetric and repeatable. This not only produces a more streamlined process for the fabrication of such devices, but also offers tunability to ensure the highest improvement in the level of detection of a given species.