Highly sensitive amperometric detection of genomic DNA in animal tissues

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase– avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 µl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT–PCR mixture.     

Highly sensitive amperometric immunosensor for the detection of Escherichia coli

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH₂-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl β-d-galactopyranoside (PAPG), the bacterial enzyme, β-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10⁴ cfu/ml (phenyl phosphate system) or 10⁵ cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1–5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Highly sensitive amperometric biosensors for phenols based on polyaniline-ionic liquid-carbon nanofiber composite

A novel polyaniline-ionic liquid-carbon nanofiber (PANI-IL-CNF) composite was greenly prepared by in situ one-step electropolymerization of aniline in the presence of IL and CNF for fabrication of amperometric biosensors. The scanning electron micrographs confirmed that the PANI uniformly grew along with the structure of CNF and the PANI-IL-CNF composite film showed a fibrillar morphology with the diameter of around 95 nm. A phenol biosensor was constructed by immobilizing tyrosinase on the surface of the composite modified glassy carbon electrode via the cross-linking step with glutaraldehyde. The biosensor exhibited a wide linear response to catechol ranging from 4.0 x 10(-10) to 2.1 x 10(-6)M with a high sensitivity of 296+/-4 AM(-1)cm(-2), a limit of detection down to 0.1 nM at the signal to noise ratio of 3 and applied potential of -0.05 V. According to the Arrhenius equation, the activation energy for enzymatic reaction was calculated to be 38.8 kJmol(-1) using catechol as the substrate. The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 1.44, 1.33, 1.16, 0.65 microM for catechol, p-cresol, phenol, m-cresol, respectively. The functionalization of CNF with PANI in IL provided good biocompatible platform for biosensing and biocatalysis.     

Single-cell immunosensors for protein detection

A single-cell detector is described that combines the natural signal amplification of whole-cell biosensors with the flexibility and specificity of immunological recognition. An immune cell that expresses receptors for the constant region of immunoglobulin G (IgG) is loaded with a Ca(2+)-indicating dye and with antibodies directed against the protein of interest. Introduction of a multivalent protein antigen causes cross-linking of the receptors, which results in a detectable increase in the concentration of cytosolic Ca(2+). Some immune cell lines respond to stimulation with oscillations in their cytosolic Ca(2+) levels that complicate their use as detectors. The human monocytic cell line U-937, when treated with the cytokine interferon-gamma, produces a large, short-lived Ca(2+) signal in response to cross-linking of its high-affinity IgG receptors. U-937 was therefore chosen for development as an immunity-based detector. Human and rabbit antibodies are found to effectively stimulate the cell, causing a prompt and transient response. The cell is able to respond to repeated stimulation, though the response diminishes during rapid stimulation. Ovalbumin can be detected in micromolar concentrations. Possible fundamental constraints on the size of a detectable analyte are discussed.     

Novel amperometric immunosensors based on iridium oxide matrices

Novel immunosensors based on antibodies immobilized in electrochemically grown iridium oxide (IrOx) thin film matrices have been developed. Antibody loading in the oxide was evaluated using a non-competitive electrochemical immunoassay for IgG. Anti-IgG loading in the oxide was found to be dependent on the concentration of anti-IgG present in the oxide growth step, with 400 microg/ml anti-IgG producing maximum amperometric responses.To study the potential analytical properties of the matrix, the dose-response behavior of the sensors was determined using optimized alkaline phosphatase-linked IgG immunoassay. Hydroquinone diphosphate (HQDP) was used as enzyme substrate and the oxidation of hydroquinone was detected amperometrically at +420 mV. The sensors displayed a linear dose-response behavior for IgG concentrations between 10 and 200 ng/ml, saturating above 600 ng/ml, and had a low detection limit of 8 ng/ml.Finally, the method was used to produce sensors containing immobilized anti-transferrin. Using a non-optimized electrochemical immunoassay for human transferrin (HT), dose-response behavior was observed for HT concentrations between 100 and 600 ng/ml.The results presented in this paper show that IrOx matrices represent a new method for immunosensor fabrication. The oxide acts as a hydrophilic, highly porous, three-dimensional matrix that can immobilize antibodies and retain their activity. The method is attractive because it offers the potential for high antibody loadings and is suitable for mass production of sensors in an easy and economical manner.     

New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors

Polysulfone membrane is used for the first time for the preparation of electrochemical immunosensors. A disposable immunosensor based on a porous conductor polymer graphite-polysulfone-electrode has been developed using a phase inversion technique for the determination of anti-rabbit IgG (anti-RIgG) as a model analyte. To construct the sensor, a conductor membrane was deposited on the surface of working graphite-epoxy composite (GEC) electrode. The membrane was characterized by SEM. This sensor was based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). Incubation parameters were optimized in this work. The immunological reaction was detected using an enzymatic-labeling procedure (HRP enzyme) combined with the amperometric detection using H(2)O(2) as substrate and hydroquinone as mediator. This sensor shows stability during a week and a good reproducibility. The current was monitored amperometrically at -0.1 V versus SCE and this method showed a linear range of the anti-RIgG from 1 to 6 microg/ml. The detection limit was determined to be 0.77 microg/ml.     

Highly sensitive immunoadsorption procedure for detection of low-abundance proteins

A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23 degrees C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50% depending on the antigen and antibody. Chaotropic agents such as 2 M urea, 0.2 M KOH, and 3.5 M MgCl2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor.     

Highly sensitive chemiluminescent method for the detection of cell contamination

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.     

Highly sensitive feature detection for high resolution LC/MS

Background  

Liquid chromatography coupled to mass spectrometry (LC/MS) is an important analytical technology for e.g. metabolomics experiments. Determining the boundaries, centres and intensities of the two-dimensional signals in the LC/MS raw data is called feature detection. For the subsequent analysis of complex samples such as plant extracts, which may contain hundreds of compounds, corresponding to thousands of features – a reliable feature detection is mandatory.     

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.     

Rapid and sensitive detection of microcystin by immunosensor based on nuclear magnetic resonance

A stable and sensitive toxin residues immunosensor based on the relaxation of magnetic nanoparticles was developed. The method was performed in one reaction and offered sensitive, fast detection of target toxin residues in water. The target analyte, microcystin-LR (MC-LR) in Tai lake water, competed with the antigens on the surface of the magnetic nanoparticles and then influenced the formation of aggregates of the magnetic nanoparticles. Accordingly, the magnetic relaxation time of the magnetic nanoparticles was changed under the effect of the target analyte. The calibration curve was deduced at different concentrations of the target analyte. The limit of detection (LOD) of MC-LR was 0.6 ng g⁻¹ and the detection range was 1–18 ng g⁻¹. Another important feature of the developed method was the easy operation: only two steps were needed (1) to mix the magnetic nanoparticle solution with the sample solution and (2) read the results through the instrument. Therefore, the developed method may be a useful tool for toxin residues sensing and may find widespread applications.     

Bienzyme HRP-GOx-modified gold nanoelectrodes for the sensitive amperometric detection of glucose at low overpotentials

Gold nanotubular electrode ensembles were prepared by using electroless deposition of the metal within the pores of polycarbonate track-etched membranes. Mono-enzyme (GOx) and monolayer/bilayer bienzyme (GOx/HRP) bioelectrodes were prepared by immobilizing the enzymes onto gold nanotubes surfaces modified with mercaptoethylamine. Batch amperometric responses to glucose for the different bioelectrodes were determined and compared. The response of the two geometries (monolayer and bilayer) of the bienzyme electrodes was shown to vary with regard to sensitivity at detection potentials above 0V. On the contrary, at detection potentials below 0V, no noticeable influence of the configuration of the bienzyme on the response intensity was observed. The mono-enzyme (650 microAmM-1 in benzoquinone (BQ) at -0.8 V versus Ag/AgCl) and the two bienzyme bioelectrodes (+/-400 microAmM-1 in hydroquinone (H2Q) at -0.2V versus Ag/AgCl) display remarkable sensitivities compared to a classical GOx-modified gold macroelectrode (13 microAmM-1 in BQ at -0.8 V versus Ag/AgCl). A remarkable feature of the bienzyme electrodes is the possibility to detect glucose at very low applied potentials where the noise level and interferences from other electro-oxidizable compounds are minimal. Another important characteristic of the monolayer bienzyme electrode is the possible existence of a direct electronic communication between HRP and the transducer surface.     

Nanostructured cantilevers as nanomechanical immunosensors for cytokine detection

The use of microfabricated cantilevers as immunosensors for the detection of human interleukin-1 beta (HIL1-β), one of the proinflammatory cytokines, is demonstrated. Cytokines are important biomarkers to monitor the effect of drugs influencing the immune system or inflammation. Nanostructured microcantilevers (MCs) functionalized with anti-human interleukin-1 beta (anti HIL1-β) antibody (Ab) are used for achieving immunonanome-chanical responses. Antibody-antigen binding interaction induces an apparent surface stress, thereby causing static bending of the MC that is detected in this work by an optical beam bending technique. Specific binding of HIL1-β to surface-bound Ab produces substantial compressive surface stress, which enables its detection in presence of other interfering proteins. Combination of biospecificity of antibodies with the sensitivity of nanomechanical surface stress detection using MCs with one side nanostructured allows detection of cytokine HIL1-β in the ppb range. The nanostructured immuno-MCs, when compared to the smooth surface MC, exhibit higher responses and yet better reversibility. The detection of HIL1-β shows good measurement reproducibility (CV=10%) in the same day tested via three replicate consecutive measurements of a solution of 0.5 ppm of HIL1-β. Also, a reasonable stability of the Ab functionalized MC within 7 d of functionalization is observed. It can be envisioned that differentially functionalized MC arrays can be made in a similar fashion.     

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.     

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii

Background  

Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome.