| Recently, because of their high sensitivity, good selectivity, low cost and short time-consuming, biosensors have been widely applied to the detection of heavy metal ions, small biomoleculars and enzymes. The development of nanomaterials and functional nucleic acids provides the new strategies and platforms for the design of biosensing technology.As a part of the projects supported by the National Natural Science Foundation of China (No.21173071) and the Research Fund for the Doctoral Program of Higher Education of China (No.20114104110002)), in this doctoral thesis, we have developed a series of optical biosensors for the detection of heavy metal ions, small molecular, enzyme activity and drug screening by combining of the advantages of nanomaterials and functional nucleic acids. Compared with the traditional methods, the proposed detection methods are convenient, sensitive and cost-effective. The practicability of these developed methods was also verified. The detailed contents are described as follows:In the chapter2, we designed a highly selective and sensitive sensor for detection of Pb2+by using conjugated polymers and label-free oligonucleotides. This method is based on Pb2+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex with a water-soluble polythiophene derivative (PMNT) as "a polymeric stain" to transduce optical signal. We selected a specific sequence oligonucleotide, TBAA (5’-GGAAGGTGTGGAAGG-3’), which can form a G-quadruplex structure upon the addition of Pb2+. This strategy provided a promising alternative to Pb2+determination in the presence of Hg2+instead of the universal masking agent of Hg2+(such as CN-,SCN-). In the absence of Pb2+, PMNT and TBAA probe readily formed an electrostatic PMNT-TBAA complex in aqueous solution. In the complex, PMNT took a highly conjugated and planar conformation with a characteristic red color. In the present of Pb2+, the TBAA probe formed a G-quadruplex structure through the specific Pb2+binding G-quartets. PMNT wound on the surface of G-quadruplex through electrostatic interaction, resulting in the twisting of conjugated backbone. The color of PMNT-G-quadruplex is yellow. By this method, we could identify micromolar Pb2+concentrations within5min even with the naked eye. Furthermore, the detection limit was improved to the nanomolar range by the fluorometric method. We also successfully utilized this biosensor for the determination of Pb2+in tap water samples. In the chapter3, we designed a highly selective and sensitive sensor for detection of Pb2+by using graphene oxide (GO) and G-quadruplex DNA (G4). Based on Pb+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex and the remarkable difference in the absorbing affinity of GO with ssDNA and G-quadruplex, we constructed a GO-G4based fluorescence "turn-on" sensing system for rapid, sensitive and selective detection of Pb2+by using "mix-and-detect" assay format. We used the specific sequence G-rich ssDNA (TBAA) for the detection of Pb2+which was slected in chapter2. In the absence of Pb2+, the TBAA is in a flexible single strand state. The introduction of GO to the carboxy fluorescein (FAM)-labeled TBAA solution would result in strong binding between nucleotide bases and aromatic structure of GO via π-stacking, bringing the fluorophore into close proximity with the GO surface. Consequently, the fluorescence of FAM is quenched via energy transfer from dyes to GO. However, in the presence of Pb2+, the conformation of the TBAA is switched from a random coil to G-quadruplex complex. The introduction of GO into the sensor solution will result in weak quenching of the fluorescence of FAM due to the weak affinity of G-quadruplex complex to GO, and the fluorescence intensity should gradually increase with the increasing concentration of Pb2+. A detection limit of400pM for Pb2+ions was estimated. We also successfully utilized this biosensor for the determination of Pb2+in tap water and river water samples.In the chapter4, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which FAM was labeled at the3’termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity was greatly quenched after the addition of GO. The as-proposed method provides a low detection limit of0.6units mL-1for HIV-1RNase H activity analysis. Furthermore, this approach shows potential for high-throughput screening of HIV-1RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.In chapter5, we developed a novel molecular aptamer beacon biosensing strategy for high-sensitive detection of ATP based on Nicking Endonuclease (NEase)-assisted target recycling amplification. NEase is a special family of restriction endonucleases, which can recognize a specific sequence kown as a restriction site along a double-strand DNA and only cleave one strand of it, leaving a nick in the DNA. The NEase used here is Nt.CviPII, which recognizes a simple asymmetric sequence,5’-…↓CCA…-3’. In this assay, the aptamer of ATP was split into two subunits, which was named Aptl and Apt2, respectively. Aptl was designed to form a hairpin structure whose3’-terminal was modified a fluorophore. The Apt2was not labeled any dye molecule. The loop sequence of Aptl and Apt2were combined as the aptamer of ATP. In the absence of ATP, because of the mismatch bases, Apt2can not hybridize with the loop structure of Aptl. Aptl and Apt2were adsorbed onto the surface of graphene oxide through π-stacking interaction between the ring structrue in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. In the present of ATP, Apt2combined with ATP hybridized with Aptl to form double-stranded DNA structure. In this double-stranded DNA, it contains the cleavage sites of Nt.CviPII. The Nt.CviPII can recognize the specific nucleotide sequence and cleave the Aptl into two fragments. After nicking, the ATP and Apt2will dissociate from the probe-target complex. Then, ATP and Apt2can hybridize with another intact Aptl to form a new substrate for NEase and the cycle starts anew. Through such strand-scission cycle, only one target can trigger cleavage of a large quantity of Aptl, which can accumulate more short-strand DNA containing fluorophore. Because of the weak affinity between short-strand DNA with graphene oxide, the introduction of GO into the sensing solution will result in weak quenching of the fluorescence of FAM. After several cycles, it leads to significant amplification of the signal. The results revealed that this strategy offered a sensitive and selective method for the detection of ATP over the concentration range from10nM to1000nM with the detection limit of4nM. The sensitivity of assay is3orders magnitude better than previously reported methods. |
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