| Nucleic acid aptamers (hereafter called aptamers) are single-stranded, small nucleic acids (typically 20-100 base pairs) engineered through repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). Aptamers often bind with high affinity to their molecular targets such as small molecules, metal ions, proteins, nucleic acids, and even cells, tissues and organisms. Therefore, aptamers have found important applications in modern biotechnology and pharmaceutical industry as they offer discriminate molecular recognition properties comparable to that of antibodies and enzymes. Compared with antibodies and enzymes, aptamers are advantageous as they are small molecules that can be engineered completely in test tubes, readily labeled and produced via chemical synthesis, and chemically stable and suitable for long-time storage. Thus aptamer-based biosensors afford easy and rapid determination of a wide range of targets with high sensitivity and stability and have been used in biomedical diagnostics and environmental monitoring. This thesis focuses on the development and application of aptamer-based biosensors and is consisted of four parts.The first part, chapter 1, of the thesis is a brief literature review that summarizes the characteristics of aptamers and their selection method. Meanwhile, the principles and characteristics of fluorescence and colorimetry along with their development in analytical chemistry are described. The concept of single nucleotide polymorhpisms, classification, testing and application are also discussed in this chapter. Finally, the aims of our research and the main contents of this thesis are described.In chapter 2, a novel label-free fluorescence method for the determination of lysozyme was developed based on aptamer/protein binding properties using LBA as recognition element and EB as fluorescent molecular switch. The fluorescence of EB is very weak in aqueous solution, In the absence of lysozyme, EB intercalated into the ds-DNA.As a result, the fluorescence intensity of EB is significantly enhanced. When lysozyme is added to the above system, it hybridizes with its aptamer, leading to the dissociation of ds-DNA and release of EB intercalated into ds-DNA. Consequently, fluorescence of EB is reduced. According to the fluorescence change, lysozyme was detected in the homogeneous solution. A linear relationship was established between the fluorescence change and lysozyme concentration over the range of 1×10-7 mol/L to 9×10-7 mol/L (R=0.9938) with a detection limit of 3×10-8 mol/L for lysozyme. The major advantage of this method is that it does not need labeling of DNA and target, offering a simple and rapid optical technique for the detection of lysozyme.In chapter 3, a simple and sensitive aptamer-based colorimetric sensing of lysozyme using unmodified gold nanoparticle (AuNP) probes was developed. It was found that in the absence of lysozyme, the unfolded LBA (ssDNA) would adsorb onto the AuNPs and enhance the AuNPs' stability against salt-induced aggregation and the red color of the AuNPs remains unchanged. In the prescence of lysozyme, the hybridization of lysozyme with its aptamer led to the LBA (ssDNA) far away from the AuNPs surface, resulting in salt-induced aggregation and color change from red to purple. The maximum adsorption wavelength shifted from 523 to 600 nm. The color of solution darkens as the concentration of lysozyme increases. At the same time, the adsorption ratio of A600/A523 increased. Under the optimal conditions, A600/A523 was found directly proportional to the concentration of lysozyme in the range of 1.0×10-9 mol/L to 7.0×10-8 mol/L (R=0.9854) with a detection limit of 3×10-10 mol/L. The results indicated that it offers a simple, rapid and easy method for the detection of lysozyme.In chapter 4, a new label-free fluorescence method for the detection of bleomycin (BLM) was developed using hairpin DNA containing C/C mismatch as recognition element and a small fluorescence molecule (ATMND) as fluorescence probe. In the absence of BLMs and Fe(II), ATMND is hydrogen bonded to an unpaired base in the stem of the hairpin DNA and quenches its fluorescence. Upon addition of Fe(Ⅱ)-BLM, the hairpin DNA undergoes irreversible cleavage by the free radicals produced from the interaction of Fe(Ⅱ)-BLM and molecular oxygen. As a result, ATMND is released from DNA duplex, recovering the fluorescence of ATMND. According to the fluorescence change before and after addition of Fe(II)-BLM, BLM was detected in homogeneous solution. Under optimal conditions, the (Fa-F)/F is linear with Fe(Ⅱ)-BLM concentration ranging from 2×10-8 mol/L to 9×10-7 mol/L (R=0.9931) and the detection limit is 7×10-9 mol/L. We also studied the effect of different metal ions on BLM-mediated DNA cleavage. Our data showed that the combination of Fe(Ⅱ)-BLM gave the best performance and afford the most significant increase in fluorescence. Thus a simple, rapid and sensitive method for the detection of BLM is developed. |
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