The Applications Of DNA Probes In The Detection Of Biomolecules And Ions

DNA probes are suitable for the detection of target DNA sequences andnon-nucleic acid analytes including proteins, small molecules or metalions due to their ability to specifically bind to analytes. DNA sequenceslabelled with fluorescent dyes or nanoparticles are usually used in theapplications of DNA sensors for the determination of analytes. Suchfluorescence assays are often highly sensitive and selective, thus thedevelopment of fluorescence DNA probes has attracted great attentions inrecent years.In most cases, the probe sequence was labelled with a fluorophore anda quencher, and the fluorescence intensity was altered through F rsterresonance energy transfer (FRET). The complex structure of DNA probeswould increase the difficulty and the cost of the probe preparation. Here,to overcome this problem, we respectively developed various DNA probesinvolved in the detection systems for a series of biomolecules and ions.These detection systems included the fluorescence marked DNA probe withgraphene oxide (GO) system, the single fluorescence marked DNA probesystem and the unmarked DNA probe with Cadmium Telluride quantum dots（CdTe QDs）system.In chapter one, we introduced the components of the DNA sensor, andthe applications of DNA probes in different analysis techniques.In chapter two, we introduced a novel sensing strategy for recognitionof Staphylococcus aureus (S. aureus) DNA sequence by utilizing GO as thequencher and dye-labelled single-stranded DNA (ssDNA) as the probe. Inthis experiment, the probe DNA hybridized with S. aureus DNA prior to theaddition of GO. Since ssDNA can bind to GO with significantly higheraffinity than double-stranded DNA (dsDNA), ssDNA would be adsorbed ontothe GO sheet after the addition of GO, leading to fluorescence quenching;while the formed dsDNA in the presence of target DNA could hardly be adsorbed by GO, leading to low quenching efficiency. As a result, we wereable to detect S. aureus DNA specifically based on the fluorescencechanges of the probe.In chapter three, we introduced a new assay strategy to detect Pb2+with a dye-llabeled AGRO100aptamer as the probe. AGRO100is a guanine（G）-rich oligonucleotide that can form a stable intermolecularG-quadruplex structure. In our method, dye-labelled AGRO100aptamers ina flexible single stranded state could be adsorbed onto the GO sheet inthe absence of Pb2+, and led to obvious fluorescence quenching as a resultof FRET. When Pb2+was added, the conformation of AGRO100was switched toa special intermolecular G-quadruplex/Pb2+complex, which had a weakaffinity with GO, and the fluorescence was restored. By measuring thefluorescence intensity changes, the concentration of Pb2+ions wasdetermined.In chapter four, a fluorescent sensing method for the detection ofhemoglobin was developed based on a fluorescent dye-labelled G-richoligonucleotide sequence. In the presence of K+, hemin could bind to thisG-rich probe to form the G-quadruplex/hemin complex and promote theelectron transfer from the dye to hemin, which led to fluorescencequenching of the probe. By measuring the fluorescence intensity, we wereable to detect hemin quantificationally. One hemoglobin moleculecontained four heme molecules which would be released and oxidized whenhemoglobin was cleaved by trypsin under proper conditions. The formedhemin was able to bind to the probe and the G-quadruplex/hemin complexwas formed, resulting in fluorescence quenching. By measuring thefluorescence intensity, we were able to detect hemoglobinquantificationally.In chapter five, we utilized the dye-labeled DNA sequence withnegative charge as a fluorescence probe for the trace detection of protamine, heparin and trypsin. Such a DNA probe could effectively bindto the positively charged protamine due to the electrostatic attraction,facilitating the electron transfer from the dye to protamine, and thefluorescence was effectively quenched. However, in the presence ofheparin, protamine preferred to bind to heparin instead of DNA due to thestronger affinity of heparin to protamine, and the fluorescence could berestored. Trypsin could cleave exclusively arginine and lysine residues.And the protamine which is rich in arginine could be hydrolyzed after theaddition of trypsin to the mixture of DNA and protamine, thus thefluorescence could be recovered. Therefore, we proposed a route to detectprotamine, heparin and trypsin.In chapter six, we demonstrated a label-free fluorescence method forthe trace determination of Ag+and S2-based on the electrostaticinteraction between3-Mercaptopropyl acid (MPA) stabilized CdTe QDs,cationic surfactant cetyltrimethylammonium bromide (CTAB) and cytosine（C）-rich ssDNA. C-ssDNA could bind to CTAB-capped CdTe QDs. Cytosinebases can interact with Ag+to form C-Ag+-C base pairs. The C–Ag+–Ccoordination would force the C-ssDNA to fold into a hairpin structurewhich has a more negative charge density, promoting the electron transfer(ET) from QDs to the coordinated Ag+. That would lead to the fluorescencequenching. S2-could react with Ag+to form stable Ag2S, interrupting theformation of C-Ag+-C base pairs, and the fluorescence was recovered. Bymeasuring the fluorescence intensity, we were able to detect Ag+and S2-quantificationally.