Valérie Pichon

Aptamer-based-sorbents for sample treatment—a review

To improve selectivity during sample pretreatment, various selective tools inducing a molecular recognition mechanism during the extraction procedure have been developed, such as sorbents constituted of immobilized antibodies, i.e., immunosorbents, or molecularly imprinted polymers. More recently, as an alternative to both previous approaches, aptamers immobilized onto a solid support, i.e., oligosorbents, were proposed. Thanks to the high affinity and high selectivity of the interaction that some aptamers offer toward some target analytes, they also provide powerful techniques that make selective extraction and the concentration of a target analyte from liquid matrices in one step or sample purification of extracts from solid matricesxa0possible. This review describes the development and the properties of these oligosorbents developed for different types of targets—pharmaceuticals, mycotoxins, proteins, cells, etc. After describing the immobilization procedures, we discuss different parameters characterizing the potential of aptamer-based supports as extraction sorbents. Close relations exist between extraction recoveries and the affinity and amounts of aptamers immobilized on the extraction device. In addition, analyte–aptamer interactions may be affected by matrix components and by additives in the samples. This may also lower extraction recoveries and affect the stability and the possible reusability of the aptamer-based sorbent. All these points are discussed and illustrated. Numerous examples of applications of these sorbents to the treatment of complex samples such as food samples, environmental samples, and biological fluids are also reported. Their association with analytical devices, from conventional to miniaturized analytical systems, is also discussed.

Miniaturized DNA aptamer-based monolithic sorbent for selective extraction of a target analyte coupled on-line to nanoLC

A complete characterization of a novel target-specific DNA aptamer-based miniaturized solid phase extraction (SPE)-sorbent coupled on-line to nanoLC is presented. A miniaturized oligosorbent (mOS) was prepared via the in situ sol-gel synthesis of a hybrid organic-inorganic monolith in 100xa0μm i.d. capillary columns using tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors, followed by covalent binding of a 5′-amino-modified DNA aptamer with a C12 spacer arm specific for a molecule of small molecular weight. Ochratoxin A (OTA), one of the most abundant naturally occurring mycotoxins, was chosen as model analyte to demonstrate the principle of such an approach. The mOS was coupled on-line to RP-nanoLC-LIF. Selective extraction of OTA on several mOSs was demonstrated with an average extraction recovery above 80xa0% when percolating spiked binding buffer and a low recovery on control monoliths grafted with a non-specific aptamer. Reproducibility of mOSs preparation was highlighted by comparing extraction yields. Otherwise, the mOSs demonstrated no cross-reactivity towards an OTA structural analogue, i.e., ochratoxin B. Due to the high specific surface area of the hybrid silica-based monolith, the coverage density of DNA aptamers covalently immobilized in the capillaries was very high and reached 6.27xa0nmolxa0μL−1, thus leading to a capacity above 5xa0ng of OTA. This miniaturized device was then applied to the selective extraction of OTA from beer samples. It revealed to be effective in isolating OTA from this complex matrix, thus improving the reliability of its analysis at the trace level.

Searching for a link between the L-BMAA neurotoxin and amyotrophic lateral sclerosis: a study protocol of the French BMAALS programme

Introduction Amyotrophic lateral sclerosis (ALS) is the most common motor neurone disease. It occurs in two forms: (1) familial cases, for which several genes have been identified and (2) sporadic cases, for which various hypotheses have been formulated. Notably, the β-N-methylamino-L-alanine (L-BMAA) toxin has been postulated to be involved in the occurrence of sporadic ALS. The objective of the French BMAALS programme is to study the putative link between L-BMAA and ALS. Methods and analysis The programme covers the period from 1 January 2003 to 31 December 2011. Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this period in the area under study (10 counties spread over three French regions) were collected. First, the standardised incidence ratio will be calculated for each municipality under concern. Then, by applying spatial clustering techniques, overincidence and underincidence zones of ALS will be sought. A case–control study, in the subpopulation living in the identified areas, will gather information about patients’ occupations, leisure activities and lifestyle habits in order to assess potential risk factors to which they are or have been exposed. Specimens of drinking water, food and biological material (brain tissue) will be examined to assess the presence of L-BMAA in the environment and tissues of ALS cases and controls. Ethics and dissemination The study has been reviewed and approved by the French ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-Mer IV). The results will be published in peer-reviewed journals and presented at national and international conferences.

Development of an analytical procedure for quantifying the underivatized neurotoxin β-N-methylamino-l-alanine in brain tissues

The cyanotoxin β-methylamino-l-alanine (BMAA) has received renewed attention as an environmental risk factor for sporadic cases of amyotrophic lateral sclerosis (ALS) (Nunn et al., Brain Res 410:375–379, 1987). The aim of the present study was to develop and to validate an analytical procedure that allows the quantification of native BMAA and of its natural isomer, 2,4 diaminobutyric acid (DAB), in brain tissues. An analytical procedure was previously reported by our group for the determination of underivatized BMAA in environmental samples. It included a step of sample clean-up by solid phase extraction (SPE) with a mixed-mode sorbent and the analyses were performed by LC/MS-MS using hydrophilic interaction chromatography and multiple reactions monitoring scan mode. As brain tissues have a higher lipid content, the crucial step of sample clean-up had been optimized by evaluating the efficiency of the addition of a liquid/liquid extraction step prior to the SPE procedure or alternatively, of washing steps to the SPE extraction procedure. The efficiency was checked by visualizing the complexity of the resulting chromatograms in LC/MS and their performance by using spiked brain samples. The optimized analytical procedure, including a washing step with cyclohexane to the SPE with a recovery yield close to 100xa0%, was validated using the total error approach and allowed the quantification of BMAA in a concentration level ranging from 20 to 1,500xa0ng/g in brain samples. Finally, the feasibility of implementation of this procedure was verified in human brain samples from two patients who died of ALS.

Molecularly imprinted polymers for the determination of organophosphorus pesticides in complex samples

Organophosphorus compounds constitute an important class of pesticides whose the toxicity of which arises from the inhibition of the acetylcholinesterase enzyme. They exhibit a wide range of physico-chemical properties, thus rendering their determination in complex oil samples particularly difficult. To facilitate their analysis at the trace level in various samples (environmental waters, soils, vegetables…), molecularly imprinted polymers (MIPs) that are synthetic polymers possessing specific cavities designed for a target molecule have been prepared. Often called synthetic antibodies, MIPs can replace antibodies in different application fields. Indeed, as immunosorbents, MIPs can be used as selective sorbents for the solid phase extraction of target analytes from complex matrices or as recognition elements in sensors. Their synthesis, characterization and use as selective sorbent for the selective recognition of organophosphorus pesticides have been already largely described and are summarized in this review.

Synthesis of a molecularly imprinted sorbent for selective solid-phase extraction of β-N-methylamino-L-alanine.

The aim of the work was to synthesize a molecularly imprinted material for the selective solid-phase extraction (SPE) of β-N-methylamino-L-alanine (L-2-amino-3-methylpropionic acid; BMAA) from cyanobacterial extracts. BMAA and its structural analogs that can be used as template are small, polar and hydrophilic molecules. These molecules are poorly soluble in organic solvents that are commonly used for the synthesis of acrylic-based polymers. Therefore, a sol gel approach was chosen to carry out the synthesis and the resulting sorbents were evaluated with different extraction procedures in order to determine their ability to selectively retain BMAA. The presence of imprinted cavities in the sorbent was demonstrated by comparing elution profiles obtained by using molecularly imprinted silica (MIS) and non-imprinted silica (NIS) as a control. The molecularly imprinted solid-phase extraction (MISPE) procedure was first developed in a pure medium (acetonitrile) and further optimized for the treatment of cyanobacterial samples. It was characterized by high elution recoveries (89% and 77% respectively in pure and in real media).The repeatability of the extraction procedure in pure medium, in real medium and the reproducibility of MIS synthesis all expressed as RSD values of extraction recovery of BMAA were equal to 3%, 12% and 5%, respectively. A MIS capacity of 0.34 µmol/g was measured. The matrix effects, which affected the quantification of BMAA when employing a mixed mode sorbent, were completely removed by adding a clean-up step of the mixed-mode sorbent extract on the MIS.

Potential of ion imprinted polymers synthesized by trapping approach for selective solid phase extraction of lanthanides

Ion imprinted polymers (IIPs) specific to lanthanides were synthesized using neodymium ions (Nd3+) as template ions. Nd3+ ions form binary complex ions with 5,7-dichloroquinoline-8-ol (DCQ) or vinylpyridine (VP), or ternary complex ions with both DCQ and VP in 2-methoxyethanol, before copolymerization in the presence of styrene and divinylbenzene as monomer and cross-linker, respectively. DCQ was expected to be trapped in the synthesized polymers pores. The template ion removal was then optimized. For the first time, the DCQ leakage was determined by HPLC-UV during the template removal and the sedimentation steps before solid-phase extraction (SPE) packing. It was observed that the trapped DCQ was unfortunately lost in significant amounts, up to 51%, and that this amount varied from one synthesis to another. The grinded and sieved polymers were next packed in SPE cartridges. The study of the SPE profiles obtained with the IIPs synthesized either with the binary or the ternary complex confirmed the prominent role of DCQ on the selectivity of an IIP by comparison with a non-imprinted polymer (NIP), i.e. a polymer synthesized under the same conditions as those of the IIP but without template ions. The influence of the porogenic solvent on the selectivity was also investigated by replacing 2-methoxyethanol by acetonitrile or dimethylsulfoxyde (DMSO). The polymers synthesized in DMSO led to the most repeatable results when elution solutions with a gradual decrease in pH were percolated through the cartridge. This is why DMSO was used to optimize the SPE protocol in order to maximize the difference of extraction yield between the IIP and the NIP, i.e. promoting a selective retention on the IIP. A value of about 30% was obtained for La3+, Ce3+, Nd3+, and Sm3+. Nevertheless, with the optimized SPE protocol, IIPs from different syntheses did not have the same SPE behavior, which may result from different random leakages of DCQ. This demonstrates for the first time the main limitation of the IIPs synthesized in bulk with the trapping approach for their use in SPE.

Selective solid phase extraction of lanthanides from tap and river waters with ion imprinted polymers

For the first time, an ion imprinted polymer (IIP) able to selectively extract simultaneously all the lanthanide ions was successfully synthesized in acetonitrile using Nd3+ as a template ion, methacrylic acid as a complexing monomer, and ethylene glycol dimethacrylate as a cross-linker. A non-imprinted polymer (NIP) was synthesized under the same conditions as those of the IIP, but in the absence of the template ion. After the removal of the template ions, grounding and sieving, the IIP particles were packed in solid phase extraction (SPE) cartridges. The selectivity of the IIP was evaluated by comparing its behavior with the one of the NIP. Each SPE step (percolation, washing, and elution) was optimized in order to find the best compromise between the selectivity and the extraction recoveries. Using the optimized SPE conditions, the extraction recoveries of eight lanthanide ions representative of the lanthanide family were higher than 77% with an average value of 83% with the IIP, whereas, in the case of the NIP, they ranged between 14 and 36% and they were below 3% for the interfering ions from alkali, transition, and post-transition metal families with the IIP. A first evaluation of the reproducibility of the SPE profiles was carried out by performing statistical tests on the data obtained with several cartridges filled with particles obtained from two different IIP and NIP syntheses. Promising results were obtained. The specific capacity, i. e. the adsorption capacity of Nd3+ ions by the specific cavities of the imprinted polymer, was about 9xa0mg of Nd3+ per gram of IIP (60xa0μmolxa0g-1), which is more than enough for the extraction of the lanthanide ions at trace levels. The breakthrough volume was about 1xa0mL per mg of IIP, leading to an enrichment factor of 15, which allows not only to selectively extract the lanthanides but also to concentrate them. Finally, the imprinted polymer was successfully used to selectively extract lanthanides from tap and river waters spiked at 1xa0μgxa0L-1.

Selective tools for the solid-phase extraction of Ochratoxin A from various complex samples: immunosorbents, oligosorbents, and molecularly imprinted polymers

The evolution of instrumentation in terms of separation and detection has allowed a real improvement of the sensitivity and the analysis time. However, the analysis of ultra-traces of toxins such as ochratoxin A (OTA) from complex samples (foodstuffs, biological fluids…) still requires a step of purification and of preconcentration before chromatographic determination. In this context, extraction sorbents leading to a molecular recognition mechanism appear as powerful tools for the selective extraction of OTA and of its structural analogs in order to obtain more reliable and sensitive quantitative analyses of these compounds in complex media. Indeed, immunosorbents and oligosorbents that are based on the use of immobilized antibodies and of aptamers, respectively, and that are specific to OTA allow its selective clean-up from complex samples with high enrichment factors. Similar molecular recognition mechanisms can also be obtained by developing molecularly imprinted polymers, the synthesis of which leads to the formation of cavities that are specific to OTA, thus mimicking the recognition site of the biomolecules. Therefore, the principle, the advantages, the limits of these different types of extraction tools, and their complementary behaviors will be presented. The introduction of these selective tools in miniaturized devices will also be discussed.

Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.