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.     

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.     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

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.     

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.     

Nanogold particle-enhanced oriented adsorption of antibody fragments for immunosensing platforms

A general design strategy for immunosensing platforms has been proposed on the basis of Nanogold particle-enhanced oriented adsorption of antibody fragments. Quartz crystal microbalance (QCM) as a model transducer was fabricated with plasma-polymerized film (PPF) of n-butyl amine and then with nanogold particles resulting in a PPF-nanogold adsorption procedure for half-IgG fragments obtained by reduction of intact immunoglobulin (IgG). Thermodynamic studies reveal that the proposed procedure is superior to the traditional oriented ones in that it created immunosurface of increased antibody surface density (amount) and antigen binding constants. Sensors produced according to the new immobilization procedure exhibit better immunosensing performances including high sensitivity, fast response rate, and favorable operational stability etc. This Nanogold particle-enhanced immobilization technique may be tailored as a promising alternative for various immunosensing platforms in solid-phase immunoassay and affinity chromatography.     

Immunosensors for detection of pesticide residues

Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis.     

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 arrays coupled with magnetic separators for immunochemistry

Electrochemical methods are increasingly applied to immunoassays, because they overcome problems associated with other modes of detection. In particular, with respect to conventional immunoassays, electrochemical immunosensors show versatility, reliability, and fast analysis time. In immunosensor strategy, the antigen or antibody can be immobilized directly onto the surface of the electrochemical transducer that will finally be used to reveal the amount of the affinity reaction. However, the use of the electrode surface as a solid phase as well as an electrochemical transducer presents some problems: a shielding of the surface by biospecifically bound antibody molecules can cause hindrance in the electron transfer, resulting in a reduced voltammetric signal. Thus, as an alternative solid phase, magnetic beads because of their low toxicity and high biocompatibility have gained much attention in chemistry, associated with various analytical techniques, due to their suitability for immobilization of biomolecules. Magnetic micro- or nanobeads can be separated easily and quickly by magnetic forces and will be used together with bioaffine ligands, e.g., antibodies or proteins with a high affinity to the target. The special advantages of magnetic separation techniques are the fast and simple handling of a sample vial and the opportunity to deal with large sample volumes without the need for time-consuming centrifugation steps. This also makes biomagnetic separation ideal for automated assay/analysis systems which will play a very important role in the near future. This review presents some examples of immunochemical assay developed using magnetic beads as a solid phase coupled with electrochemical detection techniques, in particular, using electrochemical arrays as transducers. Applications related to static measurements, together with in-flow detection systems are presented.     

An immunoassay for atrazine using tunable immunosorbent

A novel, simple, economical, and environmentally friendly tunable immunosorbent-based immunoassay for sensitive and selective determination of atrazine is reported. Tunable immunosorbents consisting of a fusion between an elastin-like polypeptide made up of 77 repeating units of the pentapeptide VPGVG and a single-chain Fv of an anti-atrazine antibody were synthesized biologically and purified by temperature-triggered phase transition. A competitive immunoassay based on the competition of atrazine-horseradish peroxidase and atrazine was established with IC(50) and lower detection limit of 0.16 and 0.01 ppb, respectively. Excellent recoveries (mean values ranging between 92 and 104%) were demonstrated in simulated atrazine-contaminated water samples.     

Detection of pesticide residues using an immunodevice based on negative dielectrophoresis

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 microm diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgG) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 microgL(-1) and in pre-treated wine samples of 6.8 microg L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 microg L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples.     

Bioelectrocatalytic signaling from immunosensors with back-filling immobilization of glucose oxidase on biorecognition surfaces

We report a novel method of electrochemical signaling from antigen-antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4-dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen-functionalized electrode was fabricated and an anti-DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back-filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme-labeled antibody. The non-labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate-treated GOX) reacted and "back-filled" the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and approximately 6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti-DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10(-4) to 0.1 g/L protein and a limit of detection around 2 x 10(-5) g/L.     

A new immunosensing method by galactose oxidase-mediated electrocatalysis using a virtual beaker array

We report a novel and convenient method for the determination of glycoproteins, especially antibodies, using galactose oxidase (GAO) on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in carbohydrate chains of glycoproteins. GAO converts galactose residues to their corresponding aldehyde and H₂O₂, the latter being electroactive and quantifiable by DC amperometry. The method does not require processes such as antibody labeling or the use of enzyme-tagged secondary antibodies. For an array-type immunosensing, the platform surface for antigen immobilization was specially designed by using differentiated surface wetting property of hydrophobic and hydrophilic patterns. We patterned the hydrophobic surface of the poly(dimethylsiloxane) substrate by microcontact printing with the poly(amidoamine) dendrimer ink, providing hydrophilic patterns on a hydrophobic base substrate. By applying aqueous solution on the patterned surface, an array of free-standing water droplets was made. With the prepared virtual beaker array, electrochemical immunosensing was performed by using anti-dinitrophenyl-IgG as a model target protein. From immunoassay with GAO-mediated electrocatalysis, a good correlation in amperometric signal with the target IgG was registered. The total assay time was about 20 min, including antibody recognition and signal registration.     

Ionic liquid-carbon composite glucose biosensor

The use of ionic liquids that are solid at room temperature such as n-octyl-pyridinium hexafluorophosphate (nOPPF(6)) is shown to be advantageous in the fabrication of new form of biocomposite materials with attractive performance over other types of composites and pastes involving non-conductive binders. The resulting IL/graphite material brings new capabilities for electrochemical devices by combining the advantages of ILs and "bulk" composite electrodes. The electrocatalytic properties of the ILs are not impaired by their association with the graphite powder. The marked electrocatalytic activity towards hydrogen peroxide permits effective amperometric biosensing of glucose in connection with the incorporation of glucose oxidase within the three-dimensional IL/graphite matrix. The accelerated electron transfer is coupled with low background current and improved linearity. The advantages of these IL-based biocomposite devices are illustrated from comparison to conventional mineral oil/graphite biocomposite. The influence of the IL and glucose oxidase (GOx) loading upon the amperometric and voltammetric data, as well as the electrode capacitance and resistance, is examined. The preparation of IL/graphite composites overcomes a major obstacle for creating IL-based biosensing devices and expands the scope of IL-based electrochemical devices.     

胶体金修饰纳米免疫传感器的制备与性能测定

基于原子力显微术,利用电化学、胶体金修饰等,进行与生物分子的结构与功能相关的免疫识别研究。利用分子自组装技术,设计出胶体金修饰CD29免疫传感器,并将原子力显微镜(AFM)针尖修饰CD29后,利用力曲线模式,对免疫传感器进行分子识别及活性点分析。CD29免疫传感器的活性点分析表明,只有62.5%的表面区域有明显力的黏附性,即活性部位,其余部分无活性。通过AFM扫描表面,发现抗体在表面聚集成团状,失去蛋白分子的原有结构,且将活性部位隐藏于内部。推断出这可能是导致蛋白失活的主要原因。     

Gold nanoparticle-based signal amplification for biosensing

Colloidal gold nanoparticles (AuNPs), with unique properties such as highly resonant particle plasmons, direct visualization of single nanoclusters by scattering of light, catalytic size enhancement by silver deposition, conductivity, and electrochemical properties, are very attractive materials for several applications in biotechnology. Furthermore, as excellent biological tags, AuNPs can be easily conjugated with biomolecules and retain the biochemical activity of the tagged biomolecules, making AuNPs ideal transducers for several biorecognition applications. The goal of this article is to review recent advances of using AuNPs as labels for signal amplification in biosensing applications. We focus on the signal amplification strategies of AuNPs in biosensing/biorecognition, more specifically, on the main optical and electrochemical detection methods that involve AuNP-based biosensing. Particular attention is given to recent advances and trends in sensing applications.     

A bio-inspired support of gold nanoparticles-chitosan nanocomposites gel for immobilization and electrochemical study of K562 leukemia cells

A novel nanocomposites gel was prepared by neutralizing a designer nanocomposites solution of chitosan encapsulated gold nanoparticles formed by reducing in situ tetrachloroauric acid in chitosan. The bio-inspired gel was designed for immobilization and electrochemical study of cells and monitoring adhesion, proliferation, and apoptosis of cells on electrodes. Using K562 leukemia cells as a model, an impedance cell sensor was constructed. The methods for preparation of the gel and immobilization of cells were simple and "green". The nanocomposites gel showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells. The living cells immobilized on glassy carbon electrode exhibited an irreversible voltammetric response and increased the electron-transfer resistance with a good correlation to the logarithmic value of concentration ranging from 1.34 x 10(4) to 1.34 x 10(8) cells mL-1 with a limit of detection of 8.71 x 10(2) cells mL-1 at 10sigma. This work implied that the nanocomposites gel based on biopolymer and nanoparticles possessed potential applications for biosensing and provided a new avenue for electrochemical investigation of cell adhesion, proliferation, and apoptosis.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Amperometric micro-immunosensor for the detection of tumor biomarker

In this paper, a highly sensitive, reagentless, electrochemical strategy is reported for the detection of a cancer biomarker-Vascular Endothelial Growth Factor (VEGF). Disc shaped carbon fiber microelectrodes were used as the immunosensor platform. Ferrocene monocarboxylic acid labeled anti-VEGF was covalently immobilized on the microelectrode surface using a Jeffamine cross-linker. The formation of immunocomplexes leads to a decrease in the electrochemical signal of ferrocene monocarboxylic acid owing to increased spatial blocking of microelectrode surface. These signal changes enable quantitative detection of VEGF in solution. Voltammetric measurements were conducted to evaluate the interfacial immunoreactions and to quantitatively detect VEGF biomarker. The proposed immunosensing strategy allows a rapid and sensitive means of VEGF analysis with a limit of detection of about 38 pg/mL. This opens up the possibility of employing these electrodes for various single cell analysis and clinical applications. Further, experimental conditions such as concentration of the immobilized antibodies and incubation period were optimized. Following this, the stability and specificity of the immunosensors were also evaluated.     

Optical probes and transducers

Biosensors are by definition a combination of a biological receptor compound and a physical or physicochemical transducer. Therefore, the transducing structure is a critical part of every biosensor. In the development of new and improved biosensing layers the importance of the transducing structure is not restricted to the substrate to which biological structures have to be coupled. A field of even greater importance is the use of transducers as probes providing information on the structure and function of biosensing layers, and their relation to a transducer surface.

The aim of this paper is to give an overview on optical transducer principles and optical (surface) analytical techniques relevant as part of biosensing structures as well as probes in the development and optimisation of biosensing layers. Categories discussed are basic optical effects, materials involved, surface chemistry, the principal and technological limits of spatial resolution, and sensitivity. The intimate relation between the spatial resolution of a probe, the resulting size of interaction areas, and the feasibility of array structures is pointed out.

Two interferometric methods are presented in principle, and their application to biosensing and some results are discussed in detail. The necessity to characterise receptor layers to get detailed information about the interaction process is pointed out. The close relationship between optimal characterisation of layers by selection of adequate probe technologies and improvement of probe performance, and the development of new biosensing layers is discussed. Finally an outlook is given for future aspects of improved spatial resolution and multianalyte detection.