The Study Of Biological Analytical Technology Using Functional Nucletic Acid As Sensing Element

Nucleic acid is the genetic material, which takes advantages of the hydrogen bonding, van der Waals, electrostatic and hydrophobic interactions and follows the Watson-Crick base pairing principle to form the double-stranded DNA. The characteristics based on its hybridization and PCR amplification are widely applied in genetic testing and analysis. compared to the technology for nucleic acid detection, the existing techniques for non-nucleic acid detection still has to face a lot of difficult issues, such as expensive equipment, low sensitivity, poor reproducibility, narrow analytical range, complicated operation and so on. Along with the continuously go deep into of the research of biosensors based on functional nucleic acid, detection techniques have been developed quickly for small organic molecules, inorganic ions, peptides, proteins, organelles and other non-nucleic acid molecules. Compared with the traditional detection methods, these techniques have proven to be simple and practical for routine detection with higher sensitivity, accurate and reliable result, lower detection limit, wider application scope, less interference, low operation costs, and simple operation. In this thesis, we employ three types of functional nucleic acids as sensing elements for the detection of non-nucleic acids:special base sequence of the DNA (mainly based on the principle of metal coordination chemistry), nucleic acid-based enzymes (Ribozyme and Deoxyribozyme), and nucleic acid aptamer. Via the mechanism of specifically biological recognition between the functional nucleic acid and target identification, the detection of targets can be achieved. Thus this sensing mechanism provides an innovative quantitative approach for a variety of non-nucleic acid molecules with high sensitivity and specificity. Therefore, we have developed a series of innovatively quantitative detection methods for metal ions and small molecules with high sensitivity and specifity, based on the functional nucleic acids as sensing elements.We have developed a highly sensitive and selective Hg2+ ion determination method at room temperature using thymine-Hg2+-thymine coordination chemistry and fluorescence polarization via gold nanoparticle enhancement. Our method demonstrates several analytical advantages. First, it has highly sensitivity with 0.2 ppb, which can be 2-3 orders of magnitude more sensitive than the other techniques. Second, it is highly selective, which allows detection of Hg2+ ion in the presence of an excess (1000-fold) of other metal ions in samples. Third, it takes only approximately 10 min to determine the concentration of mercury in aqueous media. Lastly, the assay can be carried out in 96- or 384-well plates, render it suitable for routine high-throughput applications. The method are also applied to dectect Cu2+ ion and Pb2+ ion based on metal ion-dependent DNA-cleaving DNAzyme.We have reported the development of a novel and versatile allosteric dual-DNAzyme unimolecular probe with simple and label-free design for Cu2+ ion detection. This unimolecular probe is based on a Cu2+ -specific DNA-cleaving DNAzyme and an HRP-mimicking DNAzyme, and it includes three main components. DomainⅠrepresents the substrate of DNA-cleaving DNAzyme using Cu2+ ion as specific cofactor. DomainⅡincludes the sequence of the HRP-mimicking DNAzyme, and DomainⅢrepresents the DNA-cleaving DNAzyme. In the absence of Cu2+ ion, these three domains act cooperatively in the conformation exhibiting active state of DNA-cleaving DNAzyme, and the resulting structure reveals higher stability than the G-quadruplex structure (active state of HRP-mimicking DNAzyme). Conversely, when the probe meets its target Cu2+ ion, the cleavage of substrate by DNA-cleaving DNAzyme will disturb the intramolecular DNA conformation, and this event results in an allosteric transformation from the active state of cleaving-DNAzyme to the active state of HRP-mimicking, giving, in turn, a colorimetric signal. Compared with other DNAzyme-based sensor designs, the allosteric dual-DNAzyme unimolecule strategy provides a robust and label-free probe construction by integrating DNAzyme, substrate, and signaling moiety into one molecule. This design utilizes intramolecular allosteric effect and signal amplification effect of HRP-DNAzyme such that, theoretically, the dual-DNAzyme unimolecule approach can be used for any cleaving-DNAzyme. The method exhibits a sensitivity of 1μM (65 ppb) Cu2+ ion in drinking water, which is much lower than the MAL (maximum allowable level) of～20μM (1.3 ppm) in the USA,～30μM (2.0 ppm) in the European Union and～15μM (1.0 ppm) in Canada.Bleomycins (BLMs) are widely used in combination of chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLMs contents in both pharmaceutical analysis and clinical sample, to make full use of therapeutic efficacy and to weaken their toxicity. We have developed a simple, rapid and convenient electrochemical detection of BLMs with high sensitivity and selectivity, based upon Fe(Ⅱ)·BLM-mediated DNA strand scission. A reported motif was employed as substrate probe for BLMs to self-assemble onto the gold electrode, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as signal reporter in stem-loop format. In the presence of Fe(Ⅱ)·BLM, the E-DNA sensor undergoes the irreversible cleavage event, which can be transduced into a measurable current decrease. The sensor equilibrium is relatively rapid and BLMs can be specifically detected within 30 min. Importantly, the proposed sensor achieves an excellent sensitivity as low as of picomolar detection limits (100 pM) and it is unaffected from nonspecific contaminants, as demonstrated by the investigation of BLMs in serum.Logic-gate operations displaying macroscopic outputs are promising systems for the development of intelligent soft materials that can perform effective functions in response to various input patterns. We have developed the general logic gate design based on an aptamer cross-linked hydrogel, in which trapped BSA-modified gold nanoparticles. A supramolecular hydrogel exhibits macroscopic gel sol behavior in response to two stimuli:ATP and cocaine. Competitive binding of the target (ATP or cocaine) to the aptamer causes the reduction of cross-linking density and therefore induces gel dissolution to release gold nanoparticle. On the basis of its multiple-stimulus responsiveness, we have sucessfully constructed gel-based supramolecular logic gates AND and OR types of stimulus-responsive gel sol behavior in the presence of combinations of the two stimuli.The ability to determine efficiently and accurately SNP genotypes and SNP allele frequencies in pooled DNA samples has great applications in molecular diagnostics, clinical genetic testing, pharmacogenomics, and whole-genome association studies. Here we propose two analytical method to estimate allele frequency of single nucleotide polymorphisms (SNPs) in pooled DNA samples. First we have developed a novel approach to SNP analysis in which allele-specific oligonucleotide hybridization is followed by non-gel capillary electrophoresis (ASOH-NGCE) in conjunction with laser-induced fluorescence (LIF). This allows for rapid multiplex genotyping and allele frequency estimation of SNPs. This method, based on site-separation of the hybridization duplexes, not only retains the simplicity and specificity of ASOH, but it also has the unique features of homogenous CE analysis. The method can be employed to carry out thermodynamics analysis of hybridization mixtures to estimate the allele frequencies in pooled DNAs. Second, we have developed a theoretical approach to estimate allele frequency of single nucleotide polymorphisms (SNPs) in pooled DNA samples. The approach is based on the physical principles of DNA immobilization and hybridization on solid surface using the Langmuir kinetic model and quantitative analysis of the allelic signals. It is inexpensive and highly efficient for detecting polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.