Non-nucleic Acid Targets Research Articles

Novel Nanopore Dielectrophoresis Mechanism for Selective Microrna Detection in Clinical Set

The nanopore can electrophoretically trap single DNA/RNA molecules for genetic/epigenetic detections. However, low selectivity for complex samples (extracts from plasma) remains the challenge to clinical applications. We report an novel biophysical mechanism--Carrier-guided-Nanopore-Dielectrophoresis(CND)--for selective nucleic acids detection. We invented a polycationic micro-carrier. Upon hybridization with the target, the target•carrier forms a moment-tunable dipole, which can be attracted into the nanopore by dielectrophoresis from a huge electric field gradient (107V/m-per-nm) outside the nanopore entrance. In contrast, any non-target species without carrier hybridization carries negative charge and would electrophoretically migrate away from the nanopore. Consequently only the target•carrier nanopore signatures can be identified; any interference signal from non-target nucleic acids is completely eliminated. Unlike electrophoresis that lacks selectivity, the nanopore dielectrophoresis can selectively drive target nucleic acids of any size by using a universal micro-carrier. This represents the first and substantial step in translating the nanopore-sensor into a clinically-usable tool for molecular diagnostics. We demonstrate how to utilize this mechanism to accurately quantify cancer-derived microRNA biomarkers in patient plasma.Nat.Nanotech._DOI:10.1038/NNANO.2011.147.ACS_Nano_DOI:10.1021/nn305789zView Large Image | View Hi-Res Image | Download PowerPoint Slide

In situ growth of positively-charged gold nanoparticles on single-walled carbon nanotubes as a highly active peroxidase mimetic and its application in biosensing

We prepared a new positively-charged gold nanoparticle ((+)AuNPs)–single-walled carbon nanotubes (SWNTs) nanohybrids. The results showed that small (+)AuNPs dispersed uniformly on the surface of SWNTs. Importantly the resulting nanohybrids exhibited fascinating peroxidase-like activity, which can catalyze oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to develop a blue color in aqueous solution. Furthermore, single-stranded DNA (ss-DNA) can resist salt-induced (+)AuNPs–SWNTs nanohybrids aggregation, whereas double-stranded DNA (ds-DNA) can not inhibit salt-induced nanohybrids aggregation. Based on these unique properties of the (+)AuNPs–SWNTs nanohybrids, we developed a label-free colorimetric method for DNA hybridization detection. The response to target DNA concentration was linear in the range of 0.025–0.5μM with a detection limit of 2nM (3σ). Based on the specific recognition of aptamer, the method can be extended to detect non-nucleic acid targets such as cocaine. The present limit of detection for cocaine is 2nM. The method offers the advantages of simple, cheap, rapid and sensitive.

Peptide and oligonucleotides aptamers as new ligands for analytical chemistry

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets. Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process. Recently Aptamers from peptides libraries have been realized with similar techniques in order to obtain structures with much varied receptors points and higher analytical capabilities. Some experimental results will be reported and discussed.

A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA–RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3′-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

Specific detection of Shigella sonnei by enzyme-linked aptamer sedimentation assay

Development of potent new anti-Shigella agents for rapid and specific detection and treatment is of great importance. Aptamers, nucleic acid oligomers capable of specific binding to a wide range of non-nucleic acid targets, may be of value for this purpose. In the present study, we used a Systematic Evolution of Ligands by Exponential enrichment (SELEX) process to select DNA aptamers that bind to whole S. sonnei cells. The resulting aptamers exhibited specificity in binding only to S. sonnei cells. Five unique DNA sequences were isolated from the aptamer cocktail by cloning, among which ASA4 showed the highest affinity. © 2011 Progress in Biological Sciences, Vol. 1, No.1, 11-15.

Detection of Non-Nucleic Acid Targets with an Unmodified Aptamer and a Fluorogenic Competitor

Aptamers are oligonucleotides that can bind to various non-nucleic acid targets, ranging from proteins to small molecules, with a specificity and affinity comparable to that of antibodies. Most aptamer-based detection strategies require modification on the aptamer, which could lead to a significant loss in its affinity and specificity to the target. Here we reported a generic strategy to design aptamer-based optical probes. An unmodified aptamer specific to the target and a fluorogenic competitor complementary to the aptamer are utilized for target recognition and signal generation, respectively. The competitor is a hairpin oligonucleotide with a fluorophore attached on one end and a quencher attached on the other. When no target is present, the competitor binds to the aptamer. However, when the target is introduced, the competitor will be displaced from the aptamer by the target, thus resulting in a target-specific decrease in fluorescence signal. Successful application of this strategy to different types of targets (small molecules and proteins) as well as different types of aptamers (DNA and RNA) has been demonstrated. Furthermore, a thermodynamics-based prediction model was established to further rationalize the optimization process. Due to its rapidness and simplicity, this aptamer-based detection strategy holds great promise in high throughput applications.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets. Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.

High-throughput techniques for analyzing complex bacterial communities.

A more complete understanding of microbial diversity and the environmental processes they control will require much more than a biotic inventory. It will require a deeper understanding of the basic features of systems organization and inter-population interactions. Communities, not total biomass, control net process rates driving the biogeochemical cycles sustaining the biosphere. Although the general patterns of macroorganismal diversity are relatively well known, spatial and temporal patterns of microorganismal diversity are essentially unknown. Having tools capable of resolving these patterns is a prerequisite to developing an understanding of the relationship between community structure and function. This talk discusses conceptual and technical developments that now provide the framework for systematically resolving temporal and spatial patterns of microorganisms and relating those patterns to processes at local and system levels. Of particular emphasis will be ongoing studies using highly parallel analyses with DNA microarrays for intensive monitoring of microbial populations in environmental systems. Although microarray technology is reasonably well established for studies of model organisms in well-defined laboratory settings, the application of this technology to environmental systems of uncharacterized diversity imposes additional demands on implementation; in particular, the requirement for optimized discrimination between target and non-target nucleic acids in complex, and undefined, mixtures. To increase the resolving power (information content) of our DNA microarray format, we are investigating the use of thermal dissociation of hybrids immobilized on individual array elements to resolve target and non-target sequences that differ by a single nucleotide. These studies, combined with specialized algorithms for optimizing the readout of the microarray should serve for informed environmental application. Initial studies have validated the general approach for analyses of sediment systems.

Selecting nucleic acids for biosensor applications.

In vitro selection can be used to generate nucleic acid binding species (aptamers) and catalysts (ribozymes) that can recognize a variety of molecules. Because nucleic acid function is largely derived from readily tabulated secondary structures, it has proven possible to engineer aptamers and ribozymes to function as biosensors. Labeling nucleic acids with reporter molecules has yielded simple antibody substitutes, but by relying on ligand-dependent conformational changes it has also proven possible to generate biosensors that can recognize and specifically report the presence of ligands in homogenous solution. It may prove possible to generate signaling aptamers and allosteric ribozymes (aptazymes) that are responsive to a large fraction of an organismal proteome or metabolome using automated methods. Nucleic acid biosensor arrays for non-nucleic acid targets could likely be generated with the same facility as DNA chips.

Adapting selected nucleic acid ligands (aptamers) to biosensors.

A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.