Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor.

This paper describes for the first time the selection of aptamers selective to penicillin. Aptamers were selected using a specific process called Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique is based on the selection of DNA aptamers using penicillin G in solution while the ssDNA library is fixed on a support. One aptamer showing a good affinity to penicillin was finally selected and tested in electrochemical sensor configuration, using electrochemical impedance spectroscopy as detection technique. The developed aptasensor allowed the detection of penicillin in a wide concentration range, comprised between 0.4 and 1000µgL-1 Such performance was compatible with milk analysis, as the maximum residue limit tolerated in this matrix is 4µgL-1. The selectivity of the developed sensor was also studied, showing that the sensor was also able to bind other beta-lactam antibiotics, although with a weaker affinity. Finally the sensor was used for detection of penicillin G in milk. It was shown that a simple sample treatment with isopropanol followed by filtration was sufficient to eliminate matrix effects, allowing the determination of penicillin in milk at concentrations compatible with legislation requirements.

[1]  M. Mascini,et al.  Analytical applications of aptamers. , 2005, Biosensors & bioelectronics.

[2]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[3]  D. Patel,et al.  Structure, recognition and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs and aminoglycoside antibiotics. , 2000, Journal of biotechnology.

[4]  Sang Hyun Lee,et al.  Aptamers as functional nucleic acids:In vitro selection and biotechnological applications , 2003 .

[5]  B. Tavitian,et al.  Les aptamères ou l'évolution moléculaire dirigée : sélection et applications , 2006 .

[6]  Damià Barceló,et al.  Biosensors as useful tools for environmental analysis and monitoring , 2006, Analytical and bioanalytical chemistry.

[7]  M. del Valle,et al.  Signal amplification for thrombin impedimetric aptasensor: sandwich protocol and use of gold-streptavidin nanoparticles. , 2014, Biosensors & bioelectronics.

[8]  Longhua Guo,et al.  Electrochemiluminescence biosensor for ultrasensitive determination of ochratoxin A in corn samples based on aptamer and hyperbranched rolling circle amplification. , 2015, Biosensors & bioelectronics.

[9]  J. O. Tognolli,et al.  Application of Factorial Design Experiments to the Development of a Disposable Amperometric DNA Biosensor , 2011 .

[10]  T. Noguer,et al.  Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk. , 2016, Talanta.

[11]  J. Lis,et al.  New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization , 2014, Molecular therapy. Nucleic acids.

[12]  Jean-Louis Marty,et al.  Enzyme-Linked Aptamer Assays (ELAAs), based on a competition format for a rapid and sensitive detection of Ochratoxin A in wine , 2011 .

[13]  Jean-Louis Marty,et al.  Enzyme-linked immunosensor based on super paramagnetic nanobeads for easy and rapid detection of okadaic acid. , 2011, Analytica chimica acta.

[14]  M. Petz,et al.  Food processing effects on residues: penicillins in milk and yoghurt , 2003 .

[15]  Ilaria Palchetti,et al.  Electroanalytical biosensors and their potential for food pathogen and toxin detection , 2008, Analytical and bioanalytical chemistry.

[16]  R. Junqueira,et al.  Determination of Beta-Lactam residues in milk by high performance liquid chromatography , 2006 .

[17]  Ning Gan,et al.  A colorimetric aptasensor for chloramphenicol in fish based on double-stranded DNA antibody labeled enzyme-linked polymer nanotracers for signal amplification , 2015 .

[18]  R. Dietrich,et al.  Immunochemical detection of antibiotics and sulfonamides. , 1994, The Analyst.

[19]  Chunhai Fan,et al.  Aptamer-based biosensors , 2008 .

[20]  V. Remcho,et al.  Electrochromatographic retention studies on a flavin-binding RNA aptamer sorbent. , 2003, Analytical chemistry.

[21]  R. Žvirdauskienė,et al.  An evaluation of different microbial and rapid tests for determining inhibitors in milk , 2007 .

[22]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[23]  J. Lrudayaraj,et al.  Detection of glucose, galactose, and lactose in milk with a microdialysis-coupled flow injection amperometric sensor. , 2002, Journal of dairy science.

[24]  Åse Sternesjö,et al.  Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity , 2002 .

[25]  Jean-Louis Marty,et al.  Development of an oligosorbent for detection of ochratoxin A , 2011 .

[26]  M. J. Esplandiu,et al.  Impedimetric genosensors for the detection of DNA hybridization , 2006, Analytical and bioanalytical chemistry.

[27]  J. Baudart,et al.  Aptasensor and genosensor methods for detection of microbes in real world samples. , 2013, Methods.

[28]  Ian Ivar Suni,et al.  Electrochemical Impedance Biosensor for Glucose Detection Utilizing a Periplasmic E. coli Receptor Protein , 2005 .

[29]  S. d'Auria,et al.  A novel fluorescence polarization assay for determination of penicillin G in milk. , 2016, Food chemistry.

[30]  M. Sharman,et al.  Evaluation and validation according to international standards of the Delvotest® SP-NT screening assay for antimicrobial drugs in milk , 2008 .

[31]  Jing Chen,et al.  An aptamer-based signal-on bio-assay for sensitive and selective detection of Kanamycin A by using gold nanoparticles. , 2015, Talanta.

[32]  Giovanna Marrazza,et al.  Electrochemical and piezoelectric DNA biosensors for hybridisation detection. , 2008, Analytica chimica acta.

[33]  Regine Hakenbeck,et al.  Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x* , 2004 .

[34]  J. Miranda,et al.  Fast HPLC-MS/MS Method for Determining Penicillin Antibiotics in Infant Formulas Using Molecularly Imprinted Solid-Phase Extraction , 2015, Journal of analytical methods in chemistry.

[35]  J. Barbosa,et al.  Comparative study of the LC-MS/MS and UPLC-MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[36]  E. Usleber,et al.  Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment , 2011, Analytical and bioanalytical chemistry.

[37]  Nadia Nikolaus,et al.  Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics , 2012, Journal of analytical methods in chemistry.

[38]  M. Marco,et al.  Immunochemical detection of penicillins by using biohybrid magnetic particles , 2015 .

[39]  R. Stoltenburg,et al.  FluMag-SELEX as an advantageous method for DNA aptamer selection , 2005, Analytical and bioanalytical chemistry.

[40]  Sara Tombelli,et al.  New trends in affinity sensing: aptamers for ligand binding , 2003 .

[41]  S. Jayasena Aptamers: an emerging class of molecules that rival antibodies in diagnostics. , 1999, Clinical chemistry.

[42]  T. G. Drummond,et al.  Electrochemical DNA sensors , 2003, Nature Biotechnology.

[43]  L. Gámiz-Gracia,et al.  Trace determination of 10 beta-lactam antibiotics in environmental and food samples by capillary liquid chromatography. , 2009, Journal of chromatography. A.

[44]  C. Gaillard,et al.  Ethanol precipitation of DNA with linear polyacrylamide as carrier. , 1990, Nucleic acids research.

[45]  T. Noguer,et al.  Electrochemical DNA aptamer-based biosensor for OTA detection, using superparamagnetic nanoparticles , 2011 .

[46]  J. Riviere,et al.  Extralabel use of penicillin in food animals. , 2006, Journal of the American Veterinary Medical Association.

[47]  J. Barbosa,et al.  High resolution mass spectrometry in the identification of transformation products and metabolites from β-lactam antibiotics in thermally treated milk. , 2014, Journal of chromatography. A.

[48]  M. Serralheiro,et al.  Development of a New Amperometric Biosensor for Lactose Determination , 2003 .

[49]  Ailiang Chen,et al.  Replacing antibodies with aptamers in lateral flow immunoassay. , 2015, Biosensors & bioelectronics.