Cristina Ocaña

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15-2.5 ng/mL, with an acceptable recovery percentage (91-95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up.

Signal amplification for thrombin impedimetric aptasensor: Sandwich protocol and use of gold-streptavidin nanoparticles

In this work, we report a highly specific amplification strategy demonstrated for the ultrasensitive biosensing of thrombin with the use of gold-streptavidin nanoparticles (strep-AuNPs) and silver reduction enhancement. The biotinylated aptamer of thrombin was immobilized onto an avidin-graphite epoxy composite (AvGEC) electrode surface by affinity interaction between biotin and avidin; electrochemical impedance measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The change in interfacial charge transfer resistance (Rct) experimented by the redox marker, was recorded to confirm aptamer complex formation with target protein, thrombin (Thr), in a label-free first stage. A biotinylated second thrombin aptamer, with complementary recognition properties was then used in a sandwich approach. The addition of strep-AuNPs and silver enhancement treatment led to a further increment of Rct thus obtaining significant signal amplification. The AptThrBio1-Thr-AptThrBio2 sandwich formation was inspected by confocal microcopy after incubation with streptavidin quantum dots. In order to visualize the presence of gold nanoparticles, the same silver enhancement treatment was applied to electrodes already modified with the nanoparticle-sandwich conjugate, allowing direct observation by scanning electron microscopy (SEM). Results showed high sensitivity and selectivity for thrombin detection, with an improvement from ca. 4.7 pM in a simple assay to 0.3 pM in the amplified reported scheme.

Label free aptasensor for Lysozyme detection: A comparison of the analytical performance of two aptamers

This work presents a comparison of two different aptamers (Apts) (COX and TRAN) for the detection of a ubiquitous protein Lysozyme (Lys) using Apt-based biosensors. The detection is based on the specific recognition by the Apt immobilized on screen printed carbon electrodes (SPCEs) via diazonium coupling reaction. The quantitative detection of Lys protein was achieved by electrochemical impedance spectroscopy (EIS). A very good linearity and detection limits for the quantitation of Lys were obtained from 0.1 to 0.8 μM and 100 nM using Apt COX and from 0.025 to 0.8 μM and 25 nM using Apt TRAN respectively. The obtained results showed that the developed aptasensors exhibit good specificity, stability and reproducibility for Lys detection. For real application, the aptasensors were tested in wine samples and good recovery rates were recorded in the range from 94.2 to 102% for Lys detection. The obtained recovery rates confirm the reliability and suitability of the developed method in wine matrix. The developed method could be a useful and promising platform for detection of Lys in different applications.

A Reusable Impedimetric Aptasensor for Detection of Thrombin Employing a Graphite-Epoxy Composite Electrode

Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]4− using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

Impedimetric Aptasensors Using Nanomaterials

Abstract This chapter describes aptamer biosensors that employ electrochemical impedance signal as transduction principle. With this idea, detection of substances other than nucleic acids through specific conformer folding region interacting with a complementary substance is the starting point to develop these affinity sensors. Substances detected can be ions, small organic molecules, proteins, even microorganisms, or cells. Electrochemical impedance spectroscopy permits, then, a labeless detection, by simple use of a redox probe. As current topic, it will focus on the use of nanocomponents to improve sensor performance, mainly carbon nanotubes integrated in the sensor platform, graphene or nanoparticles, for signal amplification. Different formats and variants available for analytical applications will be reviewed.