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The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg2+-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg2+-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM). [ABSTRACT FROM AUTHOR]

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 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]