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An electrochemical DNAzyme sensor for sensitive and selective detection of lead ion (Pb2+) has been developed, taking advantage of catalytic reactions of a DNAzyme upon its binding to Pb2+ and the use of DNA-Au bio-bar codes to achieve signal enhancement. A specific DNAzyme for Pb2+ is immobilized onto an Au electrode surface via a thiol-Au interaction. The DNAzyme hybridizes to a specially designed complementary substrate strand that has an overhang, which in turn hybridizes to the DNA-Au biobar code (short oligonucleotides attached to 13 nm gold nanoparticles). A redox mediator, Ru(NH3)63+, which can bind to the anionic phosphate of DNA through electrostatic interactions, serves as the electrochemical signal transducer. Upon binding of Pb2+ to the DNAzyme, the DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal of the substrate strand along with the DNA bio-bar code and the bound Ru(NH3)63+ from the Au electrode surface. The release of Ru(NH3)63+ results in lower electrochemical signal of Ru(NH3)63+ confined on the electrode surface. Differential pulse voltammetry (DPV) signals of Ru(NH3)63+ provides quantitative measures of the concentrations of Pb2+, with a linear calibration ranging from 5 nM to 0.1 μM. Because each nanoparticle carries a large number of DNA strands that bind to the signal transducer molecule Ru(NH3)63+, the use of DNA-Au bio-bar codes enhances the detection sensitivity by five times, enabling the detection of Pb2+ at a very low level (1 nM). The DPV signal response of the DNAzyme sensor is negligible for other divalent metal ions, indicating that the sensor is highly selective for Pb2+. Although this DNAzyme sensor is demonstrated for the detection of Pb2+, it has the potential to serve as a general platform for design sensors for other small molecules and heavy metal ions. [ABSTRACT FROM AUTHOR]

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nueclic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptamenc system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a selt-blocked tluoresceflt bifunctional oligonudeotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 x 10-10 M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme ass proof of concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis. [ABSTRACT FROM AUTHOR]

It is well-known that Zr4+ could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (-PO32--Zr4+-PO32--). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr4+ via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr4+ to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a "turn-on" excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr4+. This allows detection of Zr4+ with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr4+ in river water samples with satisfactory result. [ABSTRACT FROM AUTHOR]

This work reports the development of a new molecular beacon-based junction sensing system with highly Sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNAcleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system. [ABSTRACT FROM AUTHOR]

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical. [ABSTRACT FROM AUTHOR]

A novel electrochemical sensor was developed for sensitive and selective detection of mercury(II), based on thymine-Hg2+-thymine (T-Hg2+-T) coordination diemistiy. This strategy exploited the cooperativity of proximate poly-T oligonucleotides in coordination with Hg2+. Ferrocene (Fc)-tagged poly-T oligonucleotides were immobilized on the electrode surface via self-assembly of the terminal thiol moiety. In the presence of Hg2+, a pair of poly-T oligonucleotides could cooperatively coordinate with Hg2+, which triggered a conformational reorganization of the poly-T oligonucleotides from flexible single strands to relatively rigid duplex like complexes, thus drawing the Fe tags away from the electrode with a substantially decreased redox current. The response characteristics of the sensor were thoroughly investigated using capillary electrophoresis and electrochemical measurements. The results revealed that the sensor showed a sensitive response to Hg2+ in a concentration range from 1.0 nM to 2.0 μM, with a detection limit of 0.5 nM. Also, this strategy afforded exquisite selectivity for Hg2+ against a reservoir of other environmentally related metal ions, compared to existing anodic stripping voltammetry (ASV) techniques. In addition, this sensor could be implemented using minimal reagents and working steps with excellent reusability through mild regeneration procedure. It was expected that this cost-effective electrochemical sensor might hold considerable potential in on-site applications of Hg2+ detection. [ABSTRACT FROM AUTHOR]