In-parallel polar monitoring of chemiluminescence emission anisotropy at the solid-liquid interface by an optical fiber radial array

Chemiluminescence (CL) detection is widely employed in biosensors and miniaturized analytical devices since it offers high detectability and flexible device design (there are no geometry requirements for the measurement cell, except the ability to collect the largest fraction of emitted photons). Although the emission anisotropy phenomenon for an emitting dipole bound to the interface between two media with different refractive index is well known for fluorescence, it is still poorly investigated for CL reactions, in which the excited-state reaction products can diffuse in solution before the photon emission event. In this paper, we propose a simple method for the realtime evaluation of the CL emission anisotropy based on a radial array of optical fibers, embedded in a poly(methyl methacrylate) semicylinder and coupled with a Charge-Coupled Device (CCD) camera through a suitable interface. The polar-time evolutions of the CL emission have been studied for catalyzing enzymes immobilized onto a solid surface (heterogeneous configuration) or free in solution (homogeneous configuration). Evidence of the anisotropy phenomenon is observed, indicating that the lifetime of the excited-state products of the enzyme-catalyzed reactions is shorter than the time required for their diffusion in solution at a distance at which the CL can be considered isotropic. These results open new perspectives in the development of CL-based miniaturized analytical devices

A Hetero-Bifunctional Spacer for the Smart Engineering of Carbon-Based Nanostructures

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. The cover picture shows a multifunctional platform based on carbon nanotubes, where a modular organic spacer acts as the anchoring site for controlled covalent functionalization of the surface. The combination of fluorescent dyes and post-derivatizable disulfide pendant arms capable of reacting with thiol end-capped (bio)molecules, generates optically traceable (bio)conjugates. The release of a pyridinic dye allows for a precise estimation of the functionalization loading through simple UV/Vis measurements. Details are given in the Full Paper by Giuliano Giambastiani etal. (DOI: 10.1002/cplu.201402391)

A Hetero-Bifunctional Spacer for the Smart Engineering of Carbon-Based Nanostructures

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Efforts have been made in recent years to develop novel functionalisation protocols aimed at imparting multimodality and improved properties to complex carbon-based nanostructures. The incorporation of cleavable bonds to the nanomaterial surface for the controlled release (or exchange) of specific molecules under appropriate chemical and biological settings is relatively unexplored. The design and synthesis of a hetero-bifunctional linker joining a "cleavable" disulfide moiety for the covalent anchoring of a wide range of thiol end-capped (bio)molecules and a "clickable" terminal acetylene group is described. The strategy is based on the well-established copper-mediated acetylene-azide coupling reaction between the acetylene linker and single-walled carbon nanotubes decorated with phenylazido pendant arms. As a result, easily "post-derivatisable" and traceable nanostructured platforms containing a linking group potentially available for a wide range of biological probes are prepared and completely characterised. Building on solid foundations: A hetero-bifunctional linker joining a "cleavable" disulfide moiety and a "clickable" terminal acetylene group was synthesized and used to decorate carbon nanotubes (CNTs). When used in combination with other selected terminal acetylene molecules, the linker can impart multimodality through a controlled click reaction to give carbon nanohybrids (see figure)

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide

Design Principles for Ligand-Sensing, Conformation-Switching Ribozymes

Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance

A Grafting Strategy for the Design of Improved G-Quadruplex Aptamers and High-Activity DNAzymes

Nucleic acid aptamers are generally obtained by in vitro selection. Some have G-rich consensus sequences with ability to fold into the four-stranded structures known as G-quadruplexes. A few G-quadruplex aptamers have proven to bind hemin to form a new class of DNAzyme with the peroxidase-like activity, which can be significantly promoted by appending an appropriate base-pairing duplex onto the G-quadruplex structures of aptamers. Knowing the structural role of base pairing, here we introduce a novel grafting strategy for the design of improved G-quadruplex aptamers and high-activity DNAzymes. To demonstrate this strategy, three existing G-quadruplex aptamers are chosen as the first generation. A base-pairing DNA duplex is grafted onto the G-quadruplex motif of the first generation aptamers. Consequently, three new aptamers with the quadruplex/duplex DNA structures are produced as the second generation. The hemin-binding affinities and DNAzyme functions of the second generation aptamers are characterized and compared with the first generation. The results indicate three G-quadruplex aptamers obtained by the grafting strategy have more excellent properties than the corresponding original aptamers. Our findings suggest that, if the structures and functions of existing aptamers are thoroughly known, the grafting strategy can be facilely utilized to improve the aptamer properties and thereby producing better next-generation aptamers. This provides a simple but effective approach to the design of nucleic acid aptamers and DNAzymes

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications