María Isabel Pividori

Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods

The present report reviews immobilisation techniques of purified oligonucleotides on electrochemical transducers and their corresponding detection techniques. Most of the literature reviewed was published in the 1990s. The immobilisation techniques of a DNA probe to the surface of an electrochemical transducer made from carbon, gold, platinum or polypyrrole, ranged from simple adsorption to covalent bonding. Recent efforts to couple the recognition layer containing the immobilised nucleic acid recognition layer with the electrochemical signal transducer are discussed. Special attention is given to hybridisation biosensing based on electroactive indicators.

Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors.

An immunoassay-based strategy for folic acid in vitamin-fortified milk with electrochemical detection using magneto sensors is described for the first time. Among direct and indirect competitive formats, best performance was achieved with an indirect competitive immunoassay. The immunological reaction for folic acid (FA) detection was performed, for the first time on the magnetic bead as solid support by the covalent immobilization of a protein conjugate BSA-FA on tosyl-activated magnetic bead. Further competition for the specific antibody between FA in the food sample and FA immobilized on the magnetic bead was achieved, followed by the reaction with a secondary antibody conjugated with HRP (AntiIgG-HRP). Then, the modified magnetic beads were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with electrochemical detection using magneto sensors was successfully evaluated using spiked-milk samples and compared with a novel magneto-ELISA based on optical detection. The detection limit was found to be of the order of microgl(-1) (13.1 nmoll(-1), 5.8 microgl(-1)) for skimmed milk. Commercial vitamin-fortified milk samples were also evaluated obtaining good accuracy in the results. This novel strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological and food samples.

Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles.

Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.

Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode

A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.

Magneto immunosensor for gliadin detection in gluten-free foodstuff: Towards food safety for celiac patients

Gliadin is a constituent of the cereal protein gluten, responsible for the intolerance generated in celiac disease. Its detection is of high interest for food safety of celiac patients, since the only treatment known until now is a lifelong avoidance of this protein in the diet. Therefore, it is essential to have an easy and reliable method of analysis to control the contents in gluten-free foods. An electrochemical magneto immunosensor for the quantification of gliadin or small gliadin fragments in natural or pretreated food samples is described for the first time and compared to a novel magneto-ELISA system based on optical detection. The immunological reaction was performed on magnetic beads as solid support by the oriented covalent immobilization, of the protein gliadin on tosyl-activated beads. Direct, as well as indirect competitive immunoassays were optimized, achieving the best analytical performance with the direct competitive format. Excellent detection limits (in the order of μg L(-1)) were achieved, according to the legislation for gluten-free products. The matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs (skimmed milk and beer), obtaining excellent recovery values in the results.

Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection.

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.

Classical dot-blot format implemented as an amperometric hybridisation genosensor.

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot-blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method--that increases the genosensor sensitivity--are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin-DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot-blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot-blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.

Electrochemical immunosensor for the diagnosis of celiac disease

A novel electrochemical immunosensing strategy for the detection of antibodies to tissue transglutaminase (tTG) in human serum is presented. The proposed immunosensor consists of the immobilization by physical adsorption of tTG from guinea pig liver on graphite-epoxy composite (GEC) electrodes. After the reaction with the human serum (containing the specific antibodies in the case of celiac disease), the electrode was incubated with different kinds of secondary labeled antibodies, namely, horseradish peroxidase (HRP)-conjugated goat antibodies to human whole immunoglobulins (Igs), to human IgG, and finally to human IgA. Among the different classes of antibodies in human serum toward tTG, the best results were achieved when anti-tTG IgA antibodies were investigated. In total, 10 positive and 10 negative serum samples were processed, obtaining a sensitivity of 70% and a specificity of 100% compared with the commercial enzyme-linked immunosorbent assay (ELISA) method performed in a hospital laboratory. This strategy offers great promise for a simple, cost-effective, and user-friendly analytical method that allows point-of-care diagnosis of celiac disease.

Magnetically separable polymer (Mag-MIP) for selective analysis of biotin in food samples.

This work presents an efficient method for the preparation of magnetic nanoparticles modified with molecularly imprinted polymers (Mag-MIP) through core-shell method for the determination of biotin in milk food samples. The functional monomer acrylic acid was selected from molecular modeling, EGDMA was used as cross-linking monomer and AIBN as radical initiator. The Mag-MIP and Mag-NIP were characterized by FTIR, magnetic hysteresis, XRD, SEM and N2-sorption measurements. The capacity of Mag-MIP for biotin adsorption, its kinetics and selectivity were studied in detail. The adsorption data was well described by Freundlich isotherm model with adsorption equilibrium constant (KF) of 1.46 mL g(-1). The selectivity experiments revealed that prepared Mag-MIP had higher selectivity toward biotin compared to other molecules with different chemical structure. The material was successfully applied for the determination of biotin in diverse milk samples using HPLC for quantification of the analyte, obtaining the mean value of 87.4% recovery.

Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H₂O₂ as substrates by square wave voltammetry was correlated with the ATG2. Graphite-epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%.