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Hybrid systems consisting of nucleic-acid-functionalized silver nanoclusters (AgNCs) and graphene oxide (GO) are used for the development of fluorescent DNA sensors and aptasensors, and for the multiplexed analysis of a series of genes of infectious pathogens. Two types of nucleic-acid-stabilized AgNCs are used: one type includes the red-emitting AgNCs (616 nm) and the second type is near-infrared-emitting AgNCs (775 nm). Whereas the nucleic-acid-stabilized AgNCs do not bind to GO, the conjugation of single-stranded nucleic acid to the DNA-stabilized AgNCs leads to the adsorption of the hybrid nanostructures to GO and to the fluorescence quenching of the AgNCs. By the conjugation of oligonucleotide sequences acting as probes for target genes, or as aptamer sequences, to the nucleic-acid-protected AgNCs, the desorption of the probe/nucleic-acid-stabilized AgNCs from GO through the formation of duplex DNA structures or aptamer–substrate complexes leads to the generation of fluorescence as a readout signal for the sensing events. The hybrid nanostructures are implemented for the analysis of hepatitis B virus gene (HBV), the immunodeficiency virus gene (HIV), and the syphilis (Treponema pallidum) gene. Multiplexed analysis of the genes is demonstrated. The nucleic-acid-AgNCs-modified GO is also applied to detect ATP or thrombin through the release of the respective AgNCs-labeled aptamer–substrate complexes from GO. [ABSTRACT FROM AUTHOR]

The Zn2+-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal auto-catalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10-11 M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis. [ABSTRACT FROM AUTHOR]

A systematic study of the amplified optical detection of DNA by Mg2+-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10-9 M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg2+-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10-14 M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene. [ABSTRACT FROM AUTHOR]

The fluorescent dyes methylene blue, MB+, and thionine, Th+, can be trapped in the pores of mesoporous silica, MP-Si02, by means of functional nanostructures consisting of the Mg2+- or Zn2+-dependent DNAzyme sequences. In the presence of Mg2+ or Zn2+ ions the respective DNAzymes are activated, leading to the specific cleavage of the respective caps, and the selective release of MB+ or Th+. The enlargement of the conserved loop domains of the Mg2+- or Zn2+-dependent DNAzyme sequences with foreign nucleotides prohibits the formation of active DNAzymes and eliminates the release of the respective dyes. This is due to the flexibility of the loops that lacks affinity for the association of the ions. The insertion of aptamer sequences (e.g., the adenosine-5'-triphosphate (ATP) aptamer) or ion-binding sequences (e.g., T-rich Hg2+ ion-binding domains) as foreign components to the loop regions allows the formation of active Mg2+- or Zn2+-dependent DNAzyme structures through the cooperative formation of aptamer-ATP complexes or T-Hg2+-T bridges. These aptamer--substrate complexes or T-Hg2+-T bridges allosterically stabilize and activate the DNAzymes, thus allowing the selective release of the fluorescent substrates MB+ or Th+. The metal ion-driven DNAzyme release of substrates from the pores of MP-SiO and particularly the allosteric activation of the DNAzymes through cooperative aptamer--substrate complexes or metalion bridges, has important future nanomedical implications for targeted release of drugs. This is demonstrated with the triggered release of the anticancer drug, doxorubicin, by the Mg2+-DNAzyme-locked pores or by the aptamer-ATP complex-triggered activation of the Mg2+-dependent DNAzyme. [ABSTRACT FROM AUTHOR]