Rational design of a sensitive and enantiospecific electrocatalytically-amplified aptasensor for amphetamine-derivatives drugs – ECSTASE

The goal is to better understand and rationalize all of the parameters involved in the electrochemical detection and thus indirectly on the analytical performance of the aptacapteur using several techniques ranging from the SELEX approach, the chemical labeling of nucleic acids by redox compounds, to the functionalization of conductive surfaces and the modeling of the physical-chemical processes at the interface.

In the preliminary phase, the partners have investigated the chiral molecular recognition between the target and its aptamer from a kinetic point of view as well as from thermodynamics through the coupling of several techniques such as fluorescence polarization, electrochemical or the capillary electrophoresis. In addition to the wealth of information obtained, this work has led, for the first time, the development of a method for measuring enantiomeric excess (ee) by electrochemistry, balancing simplicity, speed, versatility (transferable to all analytes for which aptamer sequence is known) and high selectivity (ee measurement <0.1%).
Meanwhile, the study of molecular recognition for immobilized aptamer strand was initiated using powerful analytical methods such as surface plasmon resonance and quartz crystal microbalance coupled to electrochemistry. These results were used to assess the influence of the immobilization of the aptamer probe on its recognition properties and used directly in the design of electrochemical aptacapteur

The strategy use to study the molecular recognition between the target and the aptamer by different methodologies and techniques will allow to develop new analytical methodologies directly transferable to the development of devices for detecting small chiral organic molecules. Through this approach, we aim to provide the possibility an analytical tool that is simple, rapid, sensitive, potentially portable, and capable of performing enantioselective quantification of several amphetamine derivatives.

L. CHALLIER, C. FAVE, B. LIMOGES, D. MARCHAL, F. MAVRÉ, V. NOEL, B. SCHÖLLHORN, ELECTROCHEMICAL COMPETITIVE ASSAY AND USE THEREOF, Patent EP 12 165 696.1

L. CHALLIER, , F. MAVRÉ, J. MOREAU, C. FAVE, B. SCHOLLOHRN, D. MARCHAL, E. PEYRIN, V. NOEL, B. LIMOGES, Simple and Highly Enantioselective Electrochemical Aptamer-Based Binding Assay for Trace Detection of Chiral Compounds, Anal. Chem. 2012, 84 (12), pp 5415–5420.

J. MOREAU, C. FAVE, B. SCHÖLLHORN, L. CHALLIER, F. MAVRÉ, V. NOËL, B. LIMOGES, Fast and simple aptamer based electroanalytical method for the enantioselective recognition of small chiral molecules, ESEAC, Slovénie, 3-7 Juin 2012.

V. NOEL, L. CHALLIER, F. MAVRÉ, C. FAVE, D. MARCHAL, B. SCHÖLLHORN, B. LIMOGES, E. PEYRIN. Mise au point d’une méthodologie pour la détection électrochimique énantiosélective de cible chirale. Journées d’Electrochimie, Grenoble, juillet 2011.

The rapid diagnosis of amphetamine poisoning has become a major public health issue due to the increasing consumption of this class of illegal drugs. Such diagnoses are currently performed with the help of fast and qualitative immunological tests. However, these tests dramatically lack specificity and sensitivity. Therefore, there is an urgent need to develop new analytical tools that are superior to the existing immuno-techniques in terms of selectivity and sensitivity. In order to fill these requirements, we plan to replace both (i) the antibodies by aptamers that possess enantioselective recognition properties towards amphetamines, and (ii) the optical detection of an enzyme label by the electrochemical detection of a redox label that is designed to perform the electrocatalytic oxidoreduction of a substrate in solution upon the binding of the target molecule to the aptamer. Thanks to this original approach, we aim to provide a biosensor-like analytical platform that is easy to use, fast, sensitive, potentially field-portable, and fully integrated so that there is no need for additional reactants. We also envision an enantioselective quantification of amphetamine or methamphetamine products directly from undiluted biological samples. So far, no example of amphetamine aptasensors has been reported; neither have electrochemical aptasensors with enantioselective properties. Furthermore, among the diverse principles for electrochemical aptasensors, especially those relying on the detection of a redox label rising from the structural reorganization of the aptamer upon target binding, none is based on redox catalysis amplification as we propose. This amplification strategy is a strong point of the project since it would significantly improve the sensitivity of existing systems by a factor of 10 to 100. To achieve this goal, with multidisciplinary aspects, the project will rely on the close cooperation between five different teams with complementary skills located within three Universities: Paris Diderot University (Laboratoire d’Electrochimie Moléculaire LEM – UMR CNRS 7591 et ITODYS – UMR CNRS 7086), Joseph Fourier University in Grenoble (Département de Chimie Moléculaire, I2BM – UMR CNRS 5250 et Département de Pharmacochimie Moléculaire DPM – UMR CNRS 5063), and Victor Segalen University in Bordeaux (ARN : Régulations Naturelle et Artificielle ARNA – INSERM U869). This project is particularly ambitious since we wish to develop a well-controlled device with the aim to fully understand and rationalize the key parameters that govern the analytical performances, as well as to predict and optimize its selectivity, sensitivity and detection limit. For the success of the project, the skills and capabilities from the different partners will all be required. Strong efforts will be needed for the amphetamine enantio-selective aptamer selection using the SELEX method as well as for the systematic measurement of thermodynamic and kinetic parameters relative to the bimolecular recognition whether it is happening in solution or at the electrode surface. These data will be very useful to better understand the effect of immobilization on the binding properties and affinity of the aptamer, and also the possible necessity to play on the nature of the link enabling the attachment of the aptamer to the electrode surface.