Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

Nile blue as reporter dye in salt aggregation based-colorimetric aptasensors for peptide, small molecule and metal ion detection.

Herein, we report a novel approach for the design of a colorimetric aptasensor, relying on a Dye Salt Aggregation-based Colorimetric Oligonucleotide assay (DYSACO assay). This method is based on the use of an intercalating agent, Nile Blue (NB), whose aggregation capacities (and thus modification of its absorption spectrum) are drastically amplified by adding salts to the working solution. The presence of an aptamer could protect NB from such aggregation process due to its intercalation into double-stranded DNA and/or interaction with nucleobases. In response to the addition of the specific ligand, the competition between NB and the target for binding to the aptamer occurs, resulting in an increase in the dye salt aggregation and then in the blue-to-blank color change of the solution. The proof-of-principle was demonstrated by employing the anti-l-tyrosinamide aptamer and the assay was successfully applied to the trace enantiomer detection, allowing the detection of an enantiomeric impurity down to approximately 2% in a non-racemic sample. Through a reversed mechanism based on the increased capture of NB by DNA upon analyte binding, the sensing platform was further demonstrated for the Hg(II) detection. Water samples of different origin were spiked with Hg(II) analyte at final range concentrations comprised between (0.5-15 µM). An excellent overall recovery of 122 ± 14%; 105 ± 14%; 99 ± 9%; was respectively obtained from river, tap and mineral water, suggesting that the sensor can be used under real sample conditions. The assay was also shown to work for sensing the ochratoxin A and d-arginine vasopressin compounds, revealing its simplicity and generalizability potentialities.

Quantification of Ions and Organic Molecules, in Nanoliter Samples, in the Absence of Reference Materials.

A new analytical methodology, using capillary electrophoresis in indirect UV absorbance mode, is developed for the quantification of analytes in the absence of reference materials. The methodology allows the quantification of organic molecules and/or small ions, anionic or cationic, absorbing or not in the UV range, carrying either one or two electric charges. Two methods of data processing were compared. The first is based on the use of a dynamic simulator of electromigration, and the second uses the Kohlrausch regulating function combined with the electroneutrality equation. The experimental conditions presented in this work allow a precise quantification of anions having electrophoretic mobilities (µep) between -22.71 and -36.92 × 10-9 m2 V-1 s-1 and cations with µep between +30.59 and +63.60 × 10-9 m2 V-1 s-1 with percent relative errors lower than -5.52%. The effect of the integration errors on the reliability of the results is discussed in detail.

Detection of small molecules by fluorescence intensity using single dye labeled aptamers and quencher transition metal ions.

We describe herein an aptamer-based sensing approach that signal the presence of small-molecule targets when fluorescent DNA probes are challenged with the Ni2+ or Co2+ quencher metal ions. Functional oligonucleotides targeting L-tyrosinamide (L-Tym), adenosine (Ade) or cocaine (Coc) were end-labeled by the Texas-Red fluorophore. A fluorescence quenching occurred upon association of these transition metal ions with the free conjugates. The formation of the target-probe complex, by the way of variations in the overall binding of quencher metal ions along the DNA strands, led to a partial restoration (for the Ade and Coc systems) or a further attenuation (for the L-Tym system) of the fluorescence intensity. The absolute signal gain varied from 40 to 180% depending on the target-probe pair investigated. The approach was also used to detect the compound Ade in a spiked biological matrix in 1 min or less. The transition metal ion-based quenching strategy is characterized by its very simple implementation, low cost, and rapid signaling.

Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration.

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

Anti-pesticide DNA aptamers fail to recognize their targets with asserted micromolar dissociation constants.

Herein, we originally aimed at developing fluorescence anisotropy biosensor platforms devoted to the homogeneous-phase detection of isocarbophos and phorate pesticides by using previously isolated DNA aptamers. To achieve this, two reporting approaches displaying very high generalizability features were implemented, based on either the complementary strand or the SYBR green intercalator displacement strategies. Unfortunately, none of the transduction methods led to phorate-dependent signals. Only the SYBR green displacement method provided a small output in the presence of isocarbophos, but at an analyte concentration greater than 100 µM. In order to identify the origin of such data, isothermal titration calorimetry (ITC) experiments were subsequently performed. It was shown that aptamers bind neither isocarbophos nor phorate in free solution with the claimed micromolar dissociation constants. This work puts forward some doubts about the previously described aptasensors that rely on the use of these functional DNA molecules. It also highlights the need to carefully investigate the binding capabilities of aptamers after their isolation and to include appropriate control experiments with scrambled or mutated oligonucleotides.

Aptamer Switches Regulated by Post-Transition/Transition Metal Ions.

We introduced an aptamer switch design that relies on the ability of post-transition/transition metal ions to trigger, through their coordination to nucleobases, substantial DNA destabilization. In the absence of molecular target, the addition of one such metal ion to usual aptamer working solutions promotes the formation of an alternative, inert DNA state. Upon exposure to the cognate compound, the equilibrium is shifted towards the competent DNA form. The switching process was preferentially activated by metal ions of intermediate base over phosphate complexation preference (i.e. Pb2+ , Cd2+ ) and operated with diversely structured DNA molecules. This very simple aptamer switch scheme was applied to the detection of small organics using the fluorescence anisotropy readout mode. We envision that the approach could be adapted to a variety of signalling methods that report on changes in the surface charge density of DNA receptors.

Kissing interactions for the design of a multicolour fluorescence anisotropy chiral aptasensor.

The development of enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities. Herein, we demonstrated that the previously reported aptamer kissing complex (AKC) assay strategy can be implemented for designing a chiral tool that allows both the simultaneous enantiomer quantification and the enantiopurity analysis. D- and L-arginine vasopressin (AVP) were employed as model enantiomeric targets. The D- and L-AVP engineered aptamers (aptaswitch) were used as recognition units whereas the Fluorescein or Texas Red labelled D- and L-hairpin probes (aptakiss) served as probes of the enantiomer-dependent AKC formation. The orthogonal fluorescence anisotropy signaling scheme at two different emission wavelengths permitted the concomitant sensing of the AVP enantiomers in a single sample, under a high-throughput microplate format. It was also shown that the AKC-based enantioselective sensor allowed the enantiomeric impurity detection at a level as low as 0.01%.

Capillary electrophoretic apparatus for the endpoint detection in microtitration methods.

Titration methods are routinely used in the laboratories for the quantification of acids and bases, for the complexometric determination of metal ions and for the ion-pair titrations of drugs in pharmaceutical control. They also find application in a wide variety of chemical and biochemical studies. However, conventional titration methods (CTM) require large amounts of samples that are not always available. In absence of micro-titrator devices, the application of these methods for expensive samples and for small batch sizes is not possible. In this work, it was demonstrated that the commercial capillary electrophoretic apparatus (CEa) can be used, in a quick and easy way, for the end-point detection in a microtitration process. The proposed methodology exploits the change of the solutions conductivity during the titrations. The equivalent points can be easily located by plotting the change in electrical current as a function of the titrant volume added. More interestingly, only 1.1-1.5 mL of analyte solutions are required to establish the titration curves. The advantages and the limitations of the procedure are discussed in detail.

Photochemistry on the Space Station-Aptamer Resistance to Space Conditions: Particles Exposure from Irradiation Facilities and Real Exposure Outside the International Space Station.

Some microarray-based instruments that use bioaffinity receptors such as antibodies or aptamers are under development to detect signatures of past or present life on planetary bodies. Studying the resistance of such instruments against space constraints and cosmic rays in particular is a prerequisite. We used several ground-based facilities to study the resistance of aptamers to various types of particles (protons, electrons, neutrons, and carbon ions) at different energies and fluences. We also tested the resistance of aptamers during the EXPOSE-R2 mission outside the International Space Station (ISS). The accumulated dose measured after the 588 days of this mission (220 mGy) corresponds to the accumulated dose that can be expected during a mission to Mars. We found that the recognition ability of fluorescently labeled aptamers was not significantly affected during short-term exposure experiments taking into account only one type of radiation at a time. However, we demonstrated that the same fluorescent dye was significantly affected by temperature variations (-21°C to +58°C) and storage throughout the entirety of the ISS experiment (60% of signal loss). This induced a large variability of aptamer signal in our analysis. However, we found that >50% of aptamers were still functional after the whole EXPOSE-R2 mission. We conclude that aptamer-based instruments are well suited for in situ analysis on planetary bodies, but the detection step requires additional investigations.

Aptamer Efficacies for In Vitro and In Vivo Modulation of αC-Conotoxin PrXA Pharmacology.

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins.

Herein, we report for the first time the isolation of DNA aptamers directed against the whole tau protein, an important Alzheimer's disease (AD) biomarker. Non-SELEX approach based on the capillary electrophoresis partitioning technique was employed to isolate a high-affinity DNA sequence pool towards the target in only three rounds and one working day. High-throughput sequencing was next performed and the recognition ability of five selected aptamers was preliminary evaluated by surface plasmon resonance using the protein target immobilized on the chip. Finally, the analytical potential of the most affine aptamer was demonstrated through the design of a homogeneous-phase fluorescence anisotropy assay. This DNA aptamer was found to be able to recognize not only the whole τ-441 but also the τ-381, τ-352, τ-383 isoforms. The sensing platform allowed the determination of these four targets with a detection limit of 28 nM, 3.2 nM, 6.3 nM and 22 nM, respectively.

Panoply of Fluorescence Polarization/Anisotropy Signaling Mechanisms for Functional Nucleic Acid-Based Sensing Platforms.

Fluorescence polarization/anisotropy is a very popular technique that is widely used in homogeneous-phase immunoassays for the small molecule quantification. In the present Feature, we discuss how the potential of this signaling approach considerably expanded during the last 2 decades through the implementation of a myriad of original transducing strategies that use functional nucleic acid recognition elements as a promising alternative to antibodies.

Mirror-image aptamer kissing complex for arginine-vasopressin sensing.

The recently reported aptamer kissing complex (AKC) strategy has allowed for the development of a new kind of sandwich-like sensing tools. Currently AKC assays have been only applied to low molecular weight molecules and their functionality in complex matrices remains challenging. The objective of the present study broken down into two sub-aims; exploring the propensity to broaden the scope of detectable analytes and designing a more robust system for potential applications to realistic samples. An all L-configuration aptaswitch module derived from a hairpin spiegelmer specific to a larger target, i.e. the arginine-vasopressin (AVP) hormone, was elaborated. The target-induced AKC formation in presence of a specific mirror-image RNA hairpin (L-aptakiss) probe were analyzed by using fluorescence anisotropy. The mirror-image kissing complex was successfully formed when the L-AVP target bound to the engineered L-aptaswitch element. It was also established that the use of methanol as cosolvent significantly improved the assay sensitivity through the stabilization of the ternary complex. Finally, the capability of the mirror-image method to operate in 10-fold diluted, untreated human serum was illustrated. The current work revealed that the AKC concept can be expanded to a wider range of targets and converted to a L-configuration sensing platform especially suitable for bioanalysis purposes.

Inline Coupling of Electrokinetic Preconcentration Method to Taylor Dispersion Analysis for Size-Based Characterization of Low-UV-Absorbing Nanoparticles.

The inline coupling of the field-amplified sample injection (FASI) to Taylor dispersion analysis (TDA) was used to characterize low-UV absorbing carboxylated silica nanoparticles (cNPs). The hydrodynamic diameters (Dh) were measured by using a commercial capillary electrophoresis instrument. The proposed methodology did not require any complicated instruments or chromophoric dye to increase the detection sensitivity. A practical method based on a half-Gaussian fitting was proposed for the data processing. The results obtained by this method were compared with those derived from dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. From these results, it appeared that the size derived by TDA is in excellent agreement with those measured by DLS and TEM, as demonstrated by stable nanoparticles with narrow size distributions. Intermediate precision relative standard deviations less than 5% were obtained by FASI-TDA. The effect of the FASI-induced cNP peak dispersion on the reliability of the results was discussed in detail.

Linear Chain Formation of Split-Aptamer Dimers on Surfaces Triggered by Adenosine.

The detection of small molecules impacts various fields; however, their small size and low concentration are usually the cause of limitations in their detection. Thus, the need for biosensors with appropriate probes and signal amplification strategies is required. Aptamers are appropriate probes selected specifically against small targets such as adenosine. The possibility to split aptamers in parts led to original amplification strategies based on sandwich assays. By combining the self-assembling of oligonucleotide dimers with split-aptamer dangling ends and a surface plasmon resonance imaging technique, we developed an original amplification approach based on linear chain formation in the presence of the adenosine target. In this article, on the basis of sequence engineering, we analyzed its performance and the effect of the probe grafting density on the length of the chains formed at the surface of the biosensor.

Efficient functional neutralization of lethal peptide toxins in vivo by oligonucleotides.

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.

Aptamers in Bordeaux, 24-25 June 2016.

The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of "Aptamers in Bordeaux" will take place on September 2017 (http://www.aptamers-in-bordeaux.com/).