| As a kind of novel specific molecule tools, The molecular recognition formats of aptamer-target and antibody-antigen are similar. Further, the aptamers have several advantages over antibodies. Aptamers have been developed for a wide range of targets, including toxins and substances with low or no immunogenicity. Acting as nucleic acids, the aptamers with extreme stability can be easily regenerated in minutes after denaturation, conveniently modified or labeled by functional groups, and suitably adopted in nanodevices. As a result, aptamers are emerging as a kind of novel recognition molecules in analytical techniques.Signal amplification, especially ribozyme combined with other analysis technology of signal amplifier technique, due to its simple, sensitivity and specificity, relatively short response time, in recent years in the analysis of trace substances have developed rapidly.In this thesis, potassium ions was studied as a model to develop new small molecular or protein detection methods using aptamers and fluorescence probes; . Based ribozyme amplification, we devised a new method of the detection Single Nucleotide Polymorphism, This thesis is composed of the following three parts:1. An aptamer-based and pyrene-labeled fluorescent biosensor for homogeneous detection of potassium ionA simple and homogeneous method based on aptamer and pyrene moieties for the detection of K+ was developed. The aptamer was labeled by pyrene moiety at 3'end as molecular recognition element. In the presence of K+, the complementary oligonucleotide labeled by pyrene moieties at 5'end was displaced from aptamers, which was accompanied by dramatic decrease of the excimer fluorescence of pyrenes. However, no excimer fluorescence decreased in the absence of the target. With optimum conditions, relative changes of the pyrene excimer fluorescence intensity were proportional to the concentration of K+ in the range of 6.3×10?4 to 1.0× 10?2M with a detection limit of 5.0×10–4M. Moreover, in the presence of Na+, NH4+, Mg2+ and Ca2+ cations of biological fluid, this method was able to detect K+ with high selectivity.2. An aptamer-based fluorescent Biosensor for potassium ion detection using a pyrene labeled molecular beaconsA novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probe for the determination of potassium ions (K+) was developed in the present work. An unmodified aptamer of K+ was used as molecular recognition element and a partially complementary oligonucleotide with the aptamer was used to transduce the signal. The complementary oligonucleotide was labeled by pyrene moieties at both termini to exploit the pyrene excimer (excited state dimer) emission for the transduction of the binding event of K+ with aptamer. In the presence of targets, the complementary oligonucleotides were displaced from aptamers by targets, which was accompaned by excimer fluorescence of pyrenes as a results of the hybridization of the complimentary ends of the oligonucleotide to bring pyrene moieties into close proximity, whereas it gave only monomer emission in the absence of targets. With the excimer light-switching of the pyrene reducing the background signal from the probe itself, the relative fluorescence intensity was proportional to the concentration of K+ in the range of 6.0×10?4 to 2.0×10?2 M and a detection limit of 4.0×10–4 M was achieved. Moreover, our method was able to detect K+ with high selectivity in the presence of Na+,NH4+,Mg2+ and Ca2+ ions of biological fluids.3. Enzyme-based of signal aplification and the detection of single nucleotide polymorphisms by chemiluminescenceT4 DNA ligase can link da-DNA, and Klenow Fragment exo- polymerase makes DNA polymerized from 5'→3'. Based on the specific nature of the enzyme,make the single nucleotide polymorphisms as the connection sites of T4 DNA ligase.If is mutation, T4 DNA ligase make ds-DNA connect, and the Klenow fragment of DNA polymerase makes DNA polymerized from 5'→3'. Finally, chemilumin- escence detection signal. Using this method, highly selectivity of single nucleotide polymorphisms, and the detection limit of 5.0×10-16 M. |
