Synthesis Of Aptamer Bio-dots And Their Application Research In Detecting Thrombin And Methionine

Bio-dots have great potentials of broad biomedical and optical applications due to their stable fluorescence, high biocompatibility, low cytotoxicity, and good solubility. Bio-dots have be proven to be promising substitutes of the heavy- metal-containing semiconductor QDs and Carbon quantum dots(CQDs). In this work, a novel class of fluorescent thrombin-binding aptamer(TBA) bio-dots is first synthesized by a green hydrothermal treatment. On the basis of characterization of TBA bio-dots(TBA-dots), the novel biosensors for detection of thrombin with multiple signals and detection of methionine high selectively are constructed. This thesis mainly contains three aspects of research work:(1) A novel class of bio-dots named TBA-dots is first synthesized that is derived from TBA via TBA self-assembly and emits a bright blue fluorescent light. The TEM image of TBA-dots shows that TBA-dots are spherical in shape with an average size of 1.6 ± 0.2 nm. The AFM image of TBA-dots shows a typical topographic height of approximately 1.8 nm. We employ XPS to explore the composition and surface chemical states of TBA-dots. It turns out that the composition of TBA-dots is close to that of TBA. The newly formed O-N bond indicates that TBA-dots form sp2 carbon-like centers as the luminescence centers or chromophores. Moreover, FTIR characterization shows the existence of abundant amine groups on the surface of TBA-dots. The optical properties of TBA-dots are inspected by UV–vis absorption spectra and fluorescence spectra. TBA-dots exhibit an absorption band located in the range of 320 to 420 nm which is a new peak of bio-dots. The maximum emission is located at 425 nm with a 320 nm excitation and there is no remarkable shift of the maximum emission peak, indicating both the size and the surface state of the sp2 carbon-like centers in the TBA-dots should be uniform.(2) TBA-dots could assemble onto the surface of AuNPs through Au-N bond since there are abundant amine groups on the surface of TBA-dots. Plentiful TBA-dots would induce the aggregation of AuNPs since that plentiful TBA-dots adsorb onto the surface of AuNPs to form a “shell” which reduces the electrostatic repulsion between adjacent AuNPs. Meanwhile, the fluorescence of TBA-dots would be quenched by AuNPs via fluorescence resonance energy transfer(FRET). O n the basis of this phenomenon, a novel TBA-dots/AuNPs nanosensor with colorimetric, fluorometric and light-scattering signals for detection of thrombin is developed. In the presence of thrombin, thrombin serves on bridge that links the TBA-dots/AuNPs together since it includes two binding sites for the aptamers, inducing more AuNPs aggregating. Thus, thrombin can be determined based on the aggregation degree of AuNPs with colorimetric and light scattering signals. Meanwhile, the FRET efficiency is decreased because of reduced overlap between the emission spectrum of TBA-dots and the absorbance spectrum of AuNPs, and the fluorescene recovery of TBA-dots occurs. Therefore, thrombin can be determined based on the fluorescene recovery degree of TBA-dots with fluorometric signals. This is the first time to construct a biosensor based on aptemer bio-dots. O ur results show that the TBA-dots/AuNPs nanosensor with multiple signals allows the detection of thrombin as low as 0.59 nM and performs with high selectivity.(3) On the basis of the phenomenon that abundant TBA-dots adsorb onto the AuNPs surface forming a “shell” and then induce the aggregation of AuNPs, a novel colorimetric biosensor for selective detection of methionine is developed. Since that methionine is one of sulfer-containing amino acids, methionine could prior adsorb onto the surface of AuNPs through stronger Au-S bond in the presence of TBA-dots. As a result, methionine could effectively prevent the bio-dots assembly- induced aggregation of AuNPs. Thus, methionine can be determined based on the dispersion degree of AuNPs with colorimetric and light scattering signals. In particular, this colorimetric biosensor has high resistance to the interference of biothiols(cysteine, homocysteine, and glutathione). It allows the detection of methionine as low as 2.6 nM, which is lower than those of other methods. Moreover, its applicability for detection of methionine in human serum has been demonstrated.