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Title: Reversible plasmonic biosensors based on aptamers and hydrogels
Authors: Khulan, Sergelen
Keywords: DRNTU::Science::Medicine::Biosensors
Issue Date: 27-Aug-2018
Source: Khulan, S. (2018). Reversible plasmonic biosensors based on aptamers and hydrogels. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: This thesis presents several interlinked projects aimed at sensitive detection of chemical and biological species by the use of surface plasmon resonance-based biosensors. This optical technique takes advantage of electromagnetic field confined at the nanoscale that is generated by the coupling light to the collective oscillation of charge density at the surface of metallic films and metallic nanoparticles. In particular, the thesis utilizes surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and surface plasmon resonance (SPR) biosensors for sensitive readout of heterogeneous assays on gold sensor surface. It focuses on several important aspects that are essential to unlock the potential of optical biosensors in the emerging field of biomedical sciences that require detection of species serving as biomarkers or drugs, in close contact with the human body, outside of specialized laboratories. Specially,it aims at optical biosensor systems that hold potential for continuous monitoring of compounds in complex liquid samples and the following three projects have been pursued.Firstly, fluorescence assay with weak affinity recognition elements that reversibly bind target analyte has been employed. In order to compensate for low fluorescence signal associated with the affinity binding of target analyte at the sensor surface, the SPFS was implemented. A fluorophore-labeled hairpin aptamer structure was designed for reversible real-time biosensor, which was demonstrated for a model analyte –adenosine triphosphate. The sensing concept relies on resonant fluorescence energy transfer between the fluorophore label and metal that occurs at short distances. The aptamer hairpin is opened and closed by the specific capture of target analyte which is translated to strong variations in fluorescence signal intensity amplified by the intense surface plasmon field. A fully reversible sensor was achieved with up to 23-fold increased fluorescence signal when target analyte was captured. In addition to the aptamer hairpin, split sequences were implemented in a sandwich-type assay for the same analyte. The lack of interaction of the aptamer split sequences in the absence of the analyte allowed for a highly reduced background interference. The sensor demonstrated full reversibility that allowed multiple rounds of detection on the same sensor chip, with time resolution of several minutes for ligands with equilibrium affinity binding constants at around mM concentration. Secondly, hydrogel materials were employed for the construction of a biointerface that is resistant to fouling from blood serum. It was utilized in the form of thin surface-attached layers and free-standing membranes spanning above gold-coated surface plasmon resonance-based biosensors. The hydrophilic nature of the used hydrogels provided efficient means to repel the unspecific sorption from serum, as demonstrated by optical waveguide spectroscopy method. In addition, the interaction of biomolecules with the poly-(N-isopropyl)acrylamide – based hydrogel used was thoroughly investigated by fluorescence correlation spectroscopy. These materials were tailored for filtering applications as the permeability of hydrogels can be efficiently controlled by tuning the pore size. A thermo-responsive hydrogel was used for dynamic switching of the membrane with about micrometer thickness between its closed and permeable states. Thirdly, a thin hydrogel layer was employed as an efficient 3D affinity binding matrix that takes advantage of its large surface area. This material was employed in a fluorescence assay that relies on horse-radish peroxidase label and tyramide-based enzymatic fluorescence signal amplification. On 2D surface architectures, self-quenching occurs which limits the performance characteristics of this approach. The use of 3D hydrogel architecture offers means to overcome this limitation and is demonstrated to provide 2 orders of magnitude increase in the fluorescence signal intensity compared to conventional fluorophore-labeled detection scheme.
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