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.

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.

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.

Electrochemical genosensors for biomedical applications based on gold nanoparticles.

Two gold nanoparticles-based genomagnetic sensors designs for detection of DNA hybridization are described. Both assays are based on a magnetically induced direct electrochemical detection of gold tags on magnetic graphite-epoxy composite electrodes. The first design is a two strands assay format that consists of the hybridization between a capture DNA strand which is linked with paramagnetic beads and another DNA strand related to BRCA1 breast cancer gene used as a target which is coupled with streptavidin-gold nanoparticles. The second genomagnetic sensor design is a sandwich assay format with more application possibilities. A cystic fibrosis related DNA strand is used as a target and sandwiched between two complementary DNA probes: the first one linked with paramagnetic beads and a second one modified with gold nanoparticles via biotin-streptavidin complexation reactions. The electrochemical detection of gold nanoparticles by differential pulse voltammetry was performed in both cases. The developed genomagnetic sensors provide a reliable discrimination against noncomplementary DNA as well against one and three-base mismatches. Optimization parameters affecting the hybridization and analytical performance of the developed genosensors are shown for genomagnetic assays of DNA sequences related with the breast cancer and cystic fibrosis genes.

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Electrochemical biosensing of pesticide residues based on affinity biocomposite platforms.

A novel and very sensitive electrochemical immunosensing strategy for the detection of atrazine based on affinity biocomposite transducers is presented. Firstly, the graphite-epoxy composite transducer was bulk-modified with different universal affinity biomolecules, such as avidin and Protein A. Two strategies for the immobilization of the anti-atrazine antibodies on both biocomposite transducers were evaluated: 'wet-affinity' and 'dry-assisted affinity' immobilization. Finally, the performance of a novel anti-atrazine immunocomposite bulk-modified with anti-atrazine antibodies was also evaluated. The better immobilization performance of the anti-atrazine antibodies was achieved by 'dry-assisted affinity' immobilization on Protein A (2%) graphite-epoxy biocomposite (ProtA(2%)-GEB) as a transducer. The immunological reaction for the detection of atrazine performed on the ProtA(2%)-GEB biosensors is based on a direct competitive assay using atrazine-HRP tracer as the enzymatic label. The electrochemical detection is thus achieved through a suitable substrate and a mediator for the enzyme HRP. This novel strategy was successfully evaluated using spiked orange juice samples. The detection limit for atrazine in orange juices using the competitive electrochemical immunosensing assay was found to be 6 x 10(-3) microgL-1 (0.03 nmolL-1) thus this biosensing method accomplishes by far the LODs required for the European Community directives for potable water and food samples (0.1 microgL-1). This strategy offers great promise for rapid, simple, cost effective, and on-site biosensing of biological, food, and environmental samples.

Electrochemical magneto immunosensing of antibiotic residues in milk.

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.

Electrochemical biosensing based on universal affinity biocomposite platforms.

Rigid conducting biocomposites are versatile and effective transducing materials for the construction of a wide range of amperometric biosensors such as immunosensors, genosensors and enzymosensors, particularly if the transducer is bulk-modified with universal affinity biomolecules. The strept(avidin)-graphite-epoxy biocomposite could be considered as an universal immobilization platform whereon biotinylated DNAs, oligonucleotides, enzymes or antibodies can be captured by means of the highly affinity (strept)avidin-biotin reaction. Universal affinity biocomposite-based biosensors offer many potential advantages compared to more traditional electrochemical biosensors commonly based on a biologically surface-modified transducer. The integration of many materials into one matrix is their main advantage. As biological bulk-modified materials, the conducting biocomposites act not only as transducers, but also as reservoir for the biomaterial. After its use, the electrode surface can be renewed by a simple polishing procedure, establishing a clear advantage of these approaches relative to classical biosensors and other common biological assays. Moreover, the same material is useful for the analysis of many molecules whose determinations are based on genetic, enzymatic or immunological reactions. The different strategies for electrochemical genosensing, immunosensing and enzymosensing, all of them being dependent on the presence of a redox enzyme marker for the generation of the electrochemical signal, based on this universal affinity biocomposite platform are all presented and discussed.

CD4 quantification based on magneto ELISA for AIDS diagnosis in low resource settings.

The Acquired Immune Deficiency Syndrome (AIDS) affects the life of millions of people around the world. Although rapid and low cost screening tests are widely available for the diagnosis of HIV infection, the count of CD4+ T lymphocytes remains a drawback in the areas mostly affected by the HIV, being this control imperative for assessing the deterioration of the immunological system and the progression towards AIDS, when the counting of cells falls down 200cellsµL(-1). This paper describes a high-throughput, simple and rapid method for CD4+ T lymphocytes quantification, directly in whole blood, based on a magneto ELISA. The CD4 cells are separated and preconcentrated from whole blood in magnetic particles, and labeled with an enzyme for the optical readout performed with a standard microplate reader. The magneto ELISA is able to reach the whole CD4 counting range of medical interest, being the limit of detection as low as 50 CD4+ cells per µL of whole blood, without any pretreatment. This method is a highly suitable alternative diagnostic tool for the expensive flow cytometry at the community and primary care level, providing a sensitive method but by using instrumentation widely available in low-resource settings laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.

Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis.

The determination of phenolic compounds is significant given its toxicity, even at very low concentration levels. Amperometric determination of phenols is a simple technique available. Direct oxidation of phenols can be used, but another possibility is the use of polyphenol oxidase (tyrosinase) enzyme biosensors that oxidises the phenolic compounds into their corresponding quinones. Reduction of the resulting quinones accomplishes the amplification of the amperometric signal, as long as the result of the reduction process is the corresponding cathecol, this being able to be oxidised again by the polyphenol oxidase immobilized on the surface of the biosensor. In this communication, simultaneous determination of different phenols was carried out combining biosensor measurements with chemometric tools, in what is known as electronic tongue. The departure information used was the overlapped reduction voltammogram generated with the amperometric biosensor based on polyphenol oxidase. Artificial Neural Networks (ANN) were used for extraction and quantification of each compound. Phenol, cathecol and m-cresol formed the three-analyte study case resolved in this work. Good prediction ability was attained, and so, the separate quantification of these three phenols was accomplished.

Electrochemical magneto-actuated biosensor for CD4 count in AIDS diagnosis and monitoring.

The counting of CD4(+) T lymphocytes is a clinical parameter used for AIDS diagnosis and follow-up. As this disease is particularly prevalent in developing countries, simple and affordable CD4 cell counting methods are urgently needed in resource-limited settings. This paper describes an electrochemical magneto-actuated biosensor for CD4 count in whole blood. The CD4(+) T lymphocytes were isolated, preconcentrated and labeled from 100 µL of whole blood by immunomagnetic separation with magnetic particles modified with antiCD3 antibodies. The captured cells were labeled with a biotinylated antiCD4 antibody, followed by the reaction with the electrochemical reporter streptavidin-peroxidase conjugate. The limit of detection for the CD4 counting magneto-actuated biosensor in whole blood was as low as 44 cells µL(-1) while the logistic range was found to be from 89 to 912 cells µL(-1), which spans the whole medical interest range for CD4 counts in AIDS patients. The electrochemical detection together with the immunomagnetic separation confers high sensitivity, resulting in a rapid, inexpensive, robust, user-friendly method for CD4 counting. This approach is a promising alternative for the costly standard flow cytometry and suitable as diagnostic tool at decentralized practitioner sites in low resource settings, especially in less developed countries.

Renewable Protein A modified graphite-epoxy composite for electrochemical immunosensing.

A novel rigid and renewable transducing material for electrochemical immunosensing, based on Protein A bulk-modified graphite-epoxy biocomposite (ProtA-GEB) is reported. Protein A is able to bind to the Fc region of antibodies and provide an affinity matrix for antibody immobilisation onto the transducer. The rigid conducting biocomposite acts not only as a transducer, but also as a reservoir for protein A. After use, the electrode surface can be renewed by a simple polishing procedure, highlighting a clear advantage of this new approach with respect to classical immunoassays. The performance of ProtA-GEB transducers was compared with surface-modified transducers based on a simple dry adsorption procedure, where both Protein A and directly the antibody were adsorbed onto the surface of graphite-epoxy composite (ProtA/GEC and IgG/GEC, respectively). The application of the new biocomposite material in electrochemical immunosensing was studied using a model competitive immunoassay. The immunological reaction was detected using an enzymatic-labeling procedure together with the amperometric detection through a suitable substrate (H(2)O(2)) for the enzyme (HRP). The enzymatic labelling was performed using a two-step procedure based on the biotin/streptavidin interaction as well as a one-step procedure using an antibody labelled with the enzyme. Electrochemical and microscopic characterisation of ProtA-GEB transducer, optimisation of the immunosensor design as well as the stability of this material are also reported.

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.

Development of electrochemical immunosensing systems with renewable surfaces.

The repeated use of immunochemically modified solid phases in electrochemical immunosensor analysis is the driving interest of this work. Two new strategies have been developed. One of these strategies is aimed at the development of a manual methodology. It comprises the construction of amperometric immunosensors based on rigid biocomposites. These biocomposites are formed by a conducting polymer composite matrix that acts as a reservoir of an immobilized immunologic material. The surface of the biocomposite can be renewed by a simple polishing procedure. The second strategy involves the design of an automatic methodology. It features an immunochemical analytical system using flow injection techniques. The potentiometric detection uses a solid phase formed by immunologic reagents immobilized in magnetic particles. These particles are fixed to the sensor with the use of a magnetic field. The renewal of the reactive surface is achieved by the release and activation of the restraining magnetic field and the manipulation of the flow. The analytical properties of these immunosensors were evaluated measuring RIgG using a competitive technique and measuring GaRIgG with a sandwich methodology. The labelling enzymes of the immunoconjugates were peroxidase in amperometric measurements and urease in potentiometric measurements.

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.

Determination of lysine in pharmaceutical samples containing endogenous ammonium ions by using a lysine oxidase biosensor based on an all-solid-state potentiometric ammonium electrode.

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites.

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

Covalent binding of urease on ammonium-selective potentiometric membranes.

As part of the development of disposable urea bioselective probes, the covalent binding of urease on ammonium-selective potentiometric membranes has been assessed. Nonactin/bis(1-butylpentyl)adipate/poly(vinylchloride) (PVC) membranes, directly applied to an internal solid contact (conductive epoxy-graphite composite), has been used as a support for covalent immobilization of urease. Two types of all-solid-state construction process have been assayed: thin layers of cellulose acetate (CA) were coated on the PVC ammonium-selective membranes (type 1) and blends of PVC and CA at various ratios were used as ammonium-selective membrane matrices (type 2). Urease was covalently attached to CA via aldehyde groups. These groups were created on the polysaccharide with sodium periodate to which the enzyme was immobilized through a spacer (hexamethylenediamine). The viability of both types of probe for the determination of ammonium ions was assessed after each step of the activation process. Results indicated that type 2 potentiometric probes are altered after the treatment with sodium periodate. Good results were obtained with type 1 probes. Their dynamic concentration range of response to urea was from 2 x 10(-5) to 0.01 M with a sensibility of 50 mV/decade.

Photosensitive polyurethanes applied to the development of CHEMFET and ENFET devices for biomedical sensing.

Chemical microsensors based on ion-selective field effect transistor (ISFET) transducers with ion-selective and enzymatic membranes have been fabricated. In this case, photolithographically patterned membranes based on acrylated urethanes have been developed and applied onto the gate area of ISFET chips. Aliphatic urethane diacrylate has been used for K+ and NH+4 membranes, while a photocurable hydrogel formulation based on other type of acrylated urethane has been optimized for urea-FET sensors. Resulting potassium and ammonium sensors show similar performances to those found when PVC membranes are employed. An integrated packaging process for ISFET-based sensors has been developed giving the possibility of carrying out most of the encapsulation on wafer level. For this purpose, a photocurable polyurethane encapsulant formulation has been optimized to be microstructured by photolithography. Finally, a preliminary study of biocompatibility of photosensitive formulations containing urethane oligomers has been performed in order to examine future applications in biomedical and clinical analysis.

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.