title: Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.
abstract: Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods.
authors.affiliation: Sección Departamental de Química Analítica, Universidad Complutense de Madrid, Pz Ramón y Cajal s/n, 28040 Madrid, Spain.
authors.forename: C Lorena
authors.initials: CL
authors.lastname: Manzanares-Palenzuela
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: de-Los-Santos-Álvarez
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Lobo-Castañón
authors.affiliation: Sección Departamental de Química Analítica, Universidad Complutense de Madrid, Pz Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: bealopru@ucm.es.
authors.forename: Beatriz
authors.initials: B
authors.lastname: López-Ruiz
mesh_terms: D001483:Base Sequence / Q000235:genetics*; D015374:Biosensing Techniques; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D030821:Plants, Genetically Modified; D013025:Glycine max / Q000235:genetics*; D013045:Species Specificity
chemical_list: D018744:DNA, Plant
keywords: Electrochemical hybridization assay; Enzymatic label; Genetically modified organisms; Multiplex quantification; Round-up Ready soybean