"Dipstick" Colorimetric Detection of Metal Ions Based on Immobilization of DNAzyme and Gold Nanoparticles onto a Lateral Flow Device.

Real-time, on-site detection and quantification of different trace analytes is a challenge that requires both searching a general class of molecules to recognize a broad range of contaminants and translating this recognition to easily detectable signals. Functional nucleic acids, which include DNAzymes (DNA with catalytic activity) and aptamers (nucleic acids that bind an analyte), are ideal candidates for the target recognition. These nucleic acids can be selected by a combinatorial biology method called in vitro selection to interact with a particular analyte with high specificity and sensitivity. Furthermore, they can be incorporated into sensors by attaching signaling molecules. Due to the high extinction coefficients and distance-dependent optical properties, metallic nanoparticles such as the commonly used gold nanoparticles have been shown to be very attractive in converting analyte-specific functional DNA into colorimetric sensors. DNAzyme directed assembly of gold nanoparticles has been used to make colorimetric sensors for metal ions such as lead, uranium, and copper. To make the operation even easier and less vulnerable to operator's errors, dipstick tests have been constructed. Here, we describe protocols for the preparation of DNAzyme-linked gold nanoparticles (AuNP) that are then immobilized on to lateral flow devices to make easy-to-use dipstick tests for metal ions.

The importance of peripheral sequences in determining the metal selectivity of an in vitro-selected Co(2+) -dependent DNAzyme.

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.

Molecular diagnostic and drug delivery agents based on aptamer-nanomaterial conjugates.

Recent progress in an emerging area of designing aptamer and nanomaterial conjugates as molecular diagnostic and drug delivery agents in biomedical applications is summarized. Aptamers specific for a wide range of targets are first introduced and compared to antibodies. Methods of integrating these aptamers with a variety of nanomaterials, such as gold nanoparticles, quantum dots, carbon nanotubes, and superparamagnetic iron oxide nanoparticles, each with unique optical, magnetic, and electrochemical properties, are reviewed. Applications of these systems as fluorescent, colorimetric, magnetic resonance imaging, and electrochemical sensors in medical diagnostics are given, along with new applications as smart drug delivery agents.

Easy-to-use dipstick tests for detection of lead in paints using non-cross-linked gold nanoparticle-DNAzyme conjugates.

Easy-to-use dipstick tests for lead have been developed by immobilizing nanoparticle-DNAzyme conjugates on lateral flow devices and their application for detecting lead in paints is demonstrated.

Nanoparticles/Dip stick.

Aptamers are single-stranded nucleic acids or peptides that can bind target molecules with high affinity and specificity. The conformation of an aptamer usually changes upon binding to its target analyte, and this property has been used in a wide variety of sensing applications, including detections based on fluorescence, electrochemistry, mass, or color change. Because native nucleic acids do not possess signaling moieties required for most detection methods, aptamer sensors usually involve labeling of external signaling groups. Among the many kinds of labels, inorganic nanoparticles are emerging as highly attractive candidates because some of their unique properties. Here, we describe protocols for the preparation of aptamer-linked gold nanoparticles (AuNPs) that undergo fast disassembly into red dispersed nanoparticles upon binding of target analytes. This method has been proven to be generally applicable for colorimetric sensing of a broad range of analytes. The sample protocols have also been successfully applied to quantum dots and magnetic nanoparticles. Finally, to increase the user friendliness of the method, the sensors have been converted into simple dipstick tests using lateral flow devices.

Activity, folding and Z-DNA formation of the 8-17 DNAzyme in the presence of monovalent ions.

The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K(+), Rb(+), or Cs(+) or in the presence of 80 mM, [Co(NH(3))(6)](3+). Only 4 M Li(+), NH(4)(+) and, to a lesser extent, Na(+) conferred detectable activity. The observed rate constants (k(obs) approximately 10(-3) min(-1) for Li(+) and NH(4)(+)) are approximately 1000-fold lower than that in the presence of 10 mM Mg(2+), and approximately 200,000-fold slower than that in the presence of 100 microM Pb(2+). Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within approximately 10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb(2+) sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (K(d) approximately 0.35 M for Rb(+) and Cs(+)), while those that confer DNAzyme activity possess stronger affinity (K(d) approximately 0.22 M for Li(+), Na(+) and NH(4)(+)). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb(2+); higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn(2+) and Mg(2+); the K(d) values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb(2+). These results indicate that Pb(2+)-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.

MRI detection of thrombin with aptamer functionalized superparamagnetic iron oxide nanoparticles.

Design of smart MRI contrast agent based on superparamagnetic iron oxide nanoparticles and aptamers has been described for the detection of human alpha-thrombin protein. The contrast agent is based on the assembly of the aptamer functionalized nanoparticles in the presence of thrombin. A detectable change in MRI signal is observed with 25 nM thrombin in human serum. Changes were neither observed with control analytes, streptavidin, or bovine serum albumin, nor with inactive aptamer functionalized nanoparticles.