Xu Wu

Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Julia Xiaojun Zhao

Abstract

In the first project, a simple, rapid, and reversible fluorescent DNA INHIBIT logic gate has been developed for sensing mercury (Hg2+) and iodide (I-) ions based on a molecular beacon (MB). In this logic gate, a mercury ion was introduced as the first input into the MB logic gate system to assist in the hybridization of the MB with an assistant DNA probe through the thymine–Hg2+–thymine interaction, which eventually restored the fluorescence of MB as the output. With this signal-on process, mercury ions can be detected with a limit of detection as low as 7.9 nM. Furthermore, when iodide ions were added to the Hg2+/MB system as the second input, the fluorescence intensity decreased because Hg2+ in the thymine–Hg2+–thymine complex was grabbed by I- due to a stronger binding force. Iodide ions can be detected with a limit of detection of 42 nM. Meanwhile, we studied the feasibility and basic performance of the DNA INHIBIT logic gate, optimized the logic gate conditions, and investigated its sensitivity and selectivity. The results showed that the MB based logic gate is highly selective and sensitive for the detection of Hg2+ and I- over other interfering cations and anions.

In the second project, an ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3’-5’ exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL-1 with a limit of detection of 0.01 U mL-1. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples.

In the third project, A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5´-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because less affinity with ssDNA than dsDNA. The decrease extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U/mL and good linear correlation between 0.25-1.00 U/mL (R2=0.9896). In this assay, no label was needed for the fluorescence detection. Moreover, the inhibition behaviours of the T4 PNK’s inhibitors were evaluated by this assay. The result indicated a potential of using this assay for monitoring of phosphorylation-related process.

In the fourth project, a facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under irradiation with ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range 800–850 nm with an excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing a significant advantage for the sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as the quantum yields, fluorescence life times, and photostability, were measured and the fluorescence quantum yield was as high as 54.5%. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs are chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to graphene sheets and carbon nanotubes. Coupled with 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 mM.

In the fifth project, a general, environmental-friendly, one-pot method for the fabrication of reduced graphene oxide (RGO)/metal (oxide) (e.g. RGO/Au, RGO/Cu2O, and RGO/Ag) nanocomposties was developed using glucose as the reducing agent and stabilizer. The RGO/metal (oxide) nanocomposites were characterized using STEM, FE-SEM, EDS, UV-vis absorption spectroscopy, XRD, FT-IR and Raman spectroscopy. The reducing agent, glucose, not only reduced GO effectively to RGO, but it also reduced the metal precursors to form metal (oxide) nanoparticles on the surface of RGO. Moreover, the RGO/metal (oxide) nanocomposites were stabilized by gluconic acid on the surface of RGO. Finally, the developed nanomaterials were successfully applied to simultaneous electrochemical analysis of L-ascorbic acid (L-AA), dopamine (DA) and uric acid sing RGO/Au nanocomposite as an electrode catalyst.

In the sixth project, a reduced graphene oxide/silver nanoparticle (RGO/Ag) nanocomposite using glucose as the environmental-friendly reducing agent was developed. The antibacterial activity of RGO/Ag nanocomposite was carefully investigated using Escherichia coli (E. coli) and Klebsiella pneumoniae (Kp) as bacterial models. We found that, compared with AgNPs, graphene oxide (GO) and RGO, RGO/Ag nanocomposite had higher antibacterial efficiency. Furthermore, under the near-infrared (NIR) irradiation, RGO/Ag nanocomposite demonstrated enhanced synergetic antibacterial activity through the photothermal effect. Almost 100 % of E. coli and Kp were killed by the treatment of 15 µg/mL and 20 µg/mL, respectively, with NIR irradiation. Moreover, the membrane integrity assay and ROS species assay demonstrated that RGO/Ag nanocomposite under NIR irradiation caused the cell membranes disruption and generation of ROS species, providing other possible mechanisms for their high antibacterial activity besides photothermal effect.

In the seventh project, a rigid distance spacer, silica shell, was used between GO and dyes in this work to elucidate the quenching ability of GO. First, an organic dye was doped in silica nanoparticles, followed by the modification of another layer of silica shell with a different thickness. Due to the electrostatic interaction between GO and positively charged silica nanoparticles, GO wrapped the silica nanoparticles when they were mixed together. Therefore, the distance between GO and organic dyes was adjusted by the thickness of the silica shell. The quenching efficiency of GO to two different organic dyes, including Tetramethylrhodamine (TAMRA) and Tris(bipyridine)ruthenium(II) chloride (Rubpy), was measured at various distances. This quenching ability investigation of GO to dyes with distance-dependent manner would provide a guideline for the design of the fluorescent functional composite using GO in the future.

In the eighth project, we characterized the antibacterial activity of GO in both cell culture and animal models. Klebsiella pneumoniae (Kp) is one of the most common multidrug resistant (MDR) pathogens in causing persistent nosocomial infections and is very difficult to eradicate once established in the host. First, we demonstrated that GO exerted direct killing of Kp in agar dishes and afforded the protection of alveolar macrophages (AM) from Kp infection in culture. We then evaluated the mortality, tissue damage, polymorphonuclear neutrophil (PMN) penetration, and bacterial dissemination in Kp-infected mice. Our results revealed that GO can counteract the invasive ability of Kp in vivo, resulting in lessened tissue injury, significant but subdued inflammatory response, and prolonged mouse survival. These findings indicate that GO may be an alternative agent for controlling MDR pathogens in clinics.

Download

Share

COinS