| With the rapid development of biological science, monitoring and detecting the distribution of intracellular biological molecules are critical for understanding their pathogenesis and diagnosing disease in its early stages. Due to the advantages of fluorescent biosensors, such as high sensitivity, excellent selectivity, low cost, simple operation, detection of targets in living cells with minimal damages and real-time spatial imaging, they have gained wide attention in practical applications.Desoxyribonucleic acid(DNA) has developed as functional nucleic acids (FNAs) with special features from a simple genetic information carrier. FNAs mainly include two types of nucleic acid molecules. The first type are aptamers, which are considered as alternatives of antibodies, have the ability to specifically bind a broad spectrum of analytes including metal ions, small molecules, proteins, drugs, and even whole cells. The other type are DNAzymes, have been demonstrated to perform catalytical reactions like protein enzymes with the help of cofactors. FNAs have the merits of high specificity, good stability, excellent biocompatibility, as well as rapid synthesis and easy modification, making them widely employed as new tools for molecular recognition in constructing fluorescent biosensors. Moreover, with the development of nanomaterials (such as quantum dots(QDs), gold nanoparticles (AuNPs), metal nanoclusters) and L-DNA, providing new sights for the biologic science research.Above aspects and many relevant documents considered, in this master thesis, we combine the DNAzyme, nanomaterials, as well as L-DNA with fluorescent detection to build several fluorescent biosensors for detection of biological molecules (metal ions and L-His). The major contents are as follows:(1) In chapter 2, by combining G-quadruplex formation with a catalytic DNAzyme, we report a label-free fluorescent biosensor. In the design of the substrate strand of the GR-5 DNAzymes, we employed a G-rich sequence to increase the fluorescence intensity of ZnPPIX and used it as signal reports. In the presence of Pb2+, the DNAzyme is activated to catalyze the cleavage of the substrate strand, and the G-rich sequence is released from the substrate strand resulting in the self-assembly of the G-quadruplex with the association of ZnPPIX, thus producing an increased fluorescence signal. Moreover, the DNAzyme and cofactors can undergo many cycles to trigger the cleavage of hairpin substrate strands and form lots of G-quadruplex. By this method, the biosensor achieves amplified detection of the target.(2) In chapter 3, catalytic molecular beacons that can specifically recognize cofactors and enhanced fluorescence of silver nanoclusters (AgNCs) can be gained when interact with G-rich sequences were combined to construct a label-free fluorescent biosensors, realizing the detection of Pb2+ and L-histidine with high sensitivity. In this design, the DNAzymes and cofactors can produce multiple enzymatic turnover. Therefore, lots of G-rich sequence released from the substrate strand, resulting enhanced fluorescence of silver nanoclusters (AgNCs) and finally providing a "turn-on" fluorescence response to target molecules. The proposed sensing platforms also show high sensitivity and selectivity to the cofactors.(3) In chapter 4, we constructed a sensitive biosensing platorm for the detection of Hg2+ or Ag+ by combining the "T-Hg2+-T" pairs, "C-Ag+-C" pairs with the principle of chiral recognition mechanism. The non-nature L-DNA is the mirror-image enantiomer of nature D-DNA, which has identical physical and chemical properties. Also, L-DNA can specifically recognize the achiral targets with similar sensitivity of traditional D-DNA. Hence, L-DNA probes were constructed to detect achiral molecules Hg2+ and Ag+. This design achieves high selectivity and good sensitivity with extra merits of excellent biostability, which can resist nuclease digestion in complex practical samples. This design provides new insight to construct biostable biosensors. |
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