Gabriela Castillo

Electrochemical Aptasensor of Human Cellular Prion Based on Multiwalled Carbon Nanotubes Modified with Dendrimers: A Platform for Connecting Redox Markers and Aptamers

The present work aims to develop an electrochemical biosensor based on aptamer able to detect human cellular prions PrP(C) as a model biomarker of prion disease with high sensitivity. We designed the biosensor using multiwalled carbon nanotubes (MWCNTs) modified with polyamidoamine dendrimers of fourth generation (PAMAM G4) which in turn were coupled to DNA aptamers used as bioreceptors. Electrochemical signal was detected by a ferrocenyl redox marker incorporated between the dendrimers and aptamers interlayer. MWCNTs, thanks to their nanostructure organization and electrical properties, allow the distribution of aptamers and redox markers over the electrode surface. We demonstrated that the interaction between aptamers and prion proteins leads to variation in the electrochemical signal of the ferrocenyl group. High sensitivity with a detection limit of 0.5 pM and a wide linear range of detection from 1 pM to 10 μM has been demonstrated. Detection of PrP(C) in spiked blood plasma has been achieved in the same range of concentrations as for detection of PrP(C) in buffer. The sensor demonstrated a recovery of minimum 85% corresponding to 1 nM PrP(C) and a maximum of 127% corresponding to 1 pM PrP(C).

Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

Development of electrochemical aptasensor using dendrimers as an immobilization platform for detection of Aflatoxin B1 in food samples

This project focuses on the development of a DNA-aptamer based biosensor, which is referred to as an aptasensor, that would aid in the detection of mycotoxins in foodstuffs. We developed aptamer based biosensor for detection of Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus, species of fungi. The characterization of the sensor and detection of aflatoxin B1 was performed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). We tested an electrochemical aptasensor for AFB1 detection through the immobilization of DNA aptamers on the Poly (amido-amine) dendrimers of fourth generation (PAMAM G4) on gold electrode covered by cystamine. The DNA aptamers specific to AFB1 were attached on the PAMAM layer covalently by means of glutaraldehyde. The range of AFB1 concentrations was selected as target interval for further utilization of the biosensor in food samples. The aptasensor has been tested in certificated samples of peanuts as well as in spiked samples of peanuts demonstrating optimal response. The biosensor was also tested in other mycotoxins such as ochratoxin A (OTA) and aflatoxin B2 (AFB2), to investigate non-specific interaction.

Aptamer-based detection of thrombin by acoustic method using DNA tetrahedrons as immobilisation platform

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (KD) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by KD = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH3)6]3+ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.

High-Sensitive Impedimetric Aptasensor for Detection Ochratoxin A in Food

We report a high sensitive biosensor based on DNA aptamers for detection ochratoxin A (OTA). The thiolated DNA aptamers specific to OTA have been immobilised to a surface of gold electrode. The electrochemical impedance spectroscopy (EIS) at presence of redox probe [Fe(CN)6]−3/−4 has been used for determination of charge transfer resistance, Rct, following addition of OTA. We have shown that Rct increased with increasing OTA in a range of 0.1–100 nM. The sensor revealed high sensitivity (the limit of detection was 0.44 nM), selectivity and was regenerable. The sensor was validated in coffee extract.