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Title: Photochrome aptamer switch assay (PHASA) for small molecule detection and quantification
Authors: Wu, Yuanyuan
Keywords: DRNTU::Engineering::Materials
Issue Date: 2017
Source: Wu, Y. (2017). Photochrome aptamer switch assay (PHASA) for small molecule detection and quantification. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: In order to detect small analytes with high specificity and efficiency that cannot otherwise be achieved by antibody–based immunoassays, the development of a new Photochrome Aptamer Switch Assay (PHASA) was explored. In this assay, aptamer which can be synthesized chemically and can bind small molecules specifically is utilized as the molecular recognition element. Stilbene compounds having a unique reporting power via an instant conformational change upon irradiation, and whose decay rate change depends on medium viscosity are used as the photochrome probe. Two scopes–competitive binding and adaptive binding were proposed and were applied on malachite green (MG)-MG aptamer model system. The competitive binding scope involves the competition for the aptamer binding pocket between MG analyte and stilbene-MG conjugate. The conjugate needs to possess both strong binding affinity and fluorescence decay. Four stilbene-MG conjugates: “fused” stilbene-MG, MG-CH2-NH-Stilb-OMe, MG-CH2-NAc-Stilb-OMe and MG-COO-Stilb-NO2 were synthesized. Their binding experiments with MG aptamer and decay kinetics measurements in MG aptamer binding buffer based aqueous mixtures were conducted. Results of the measured dissociation constant demonstrate that Kd(MG) (200 nM) < Kd(“fused” stilbene MG) (500 nM) < Kd(– CH2-NAc –) (1.7 µM) < Kd(– CH2-NH –) (2.4 µM) < Kd(– COO –) (6.6 µM). That is, “fused” stilbene-MG conjugate has the strongest binding affinity, and there is literally no binding of MG-COO-Stilb-NO2 to MG aptamer among the studied stilbene-MG conjugates. For the decay kinetics, the decay of both “fused” stilbene-MG and MG-COO-Stilb-NO2 conjugates are quenched while the decay of MG-CH2-NH-Stilb-OMe and MG-CH2-NAc-Stilb-OMe are preserved. Combining the binding affinity and decay kinetics, the MG-CH2-NAc-Stilb-OMe conjugate is the most promising candidate for PHASA competitive binding scope. The adaptive binding scope requires the conjugation between stilbene compounds and MG aptamer, and thiol-maleimide click chemistry was chosen as the cross-linker. The dissociation constant Kd value of four SH-modified MG aptamers are measured in the range of 700~900 nM, preserving their binding ability towards MG analyte. Thiol conjugation of stilbene maleimides proves to restore their quenched fluorescence decay in DMSO (v% ≤ 10%) and MG aptamer binding buffer solution mixtures, allowing subsequent detection based on decay rate change in stilbene-MG complex without further separation. Three stilbene maleamic acid compounds were also synthesized for better water solubility, their decay rates’ dependences on medium viscosity are confirmed and decay kinetics in DMSO and buffer solution mixtures are preserved instead of being quenched. Besides medium viscosity probing, further applications of studied stilbene compounds were also explored. The fluorescence decay rate of dimethylamino-stilbene-maleamic acid (DMASMA) is linearly quenched by EtOH in the concentration range of 0.004~0.1 mol L-1 where no effect from methanol and isopropanol (IPA) is observed. The photophysical properties of all three stilbene maleimic acids are sensitive to the solution pH. That is, the studied stilbene compounds can have promising applications in pH or EtOH sensing apart from medium viscosity probing.
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