Photochemically-activated electrodes: application in design of reversible immunosensors and antibody patterned interfaces (original) (raw)

Probing antigen–antibody interactions on electrode supports by the biocatalyzed precipitation of an insoluble product

Electroanalysis, 2000

The ampli®ed sensing of an antibody by an antigen monolayer-functionalized transducer by using a biocatalyzed precipitation of an insoluble product on the transducer is addressed. Faradaic impedance spectroscopy, cyclic voltammetry and microgravimetric, quartz crystal microbalance analyses are used to probe the precipitation of the insoluble product on the transducer. A dinitrophenyl, DNP, antigen monolayer is assembled on a Au-electrode, or a Au-quartz crystal, as a sensing interface for the dinitrophenyl-antibody, DNP-Ab. An anti-Fc-antibody-HRP conjugate is used as a biocatalytic probe for the formation of the antigen/DNP-Ab complex on the transducer. Biocatalyzed oxidation of 4-chloronaphthol by H 2 O 2 in the presence of the anti-Fc-antibody-HRP conjugate yields an insoluble product on the transducer. Formation of the insoluble ®lm on the electrode results in the increase of interfacial electron-transfer resistances detected by impedance spectroscopy or cyclic voltammetry. The precipitate formation also results in the mass increase of the modi®ed Au-quartz crystal detected as a frequency change of the piezoelectric transducer. The DNP-Ab is easily sensed at a sensitivity that corresponds to 0.5 ng mL 71 (3610 712 M).

Application of redox enzymes for probing the antigen-antibody association at monolayer interfaces: Development of amperometric immunosensor electrodes

Analytical …, 1996

Insulation of the electrical contact between a redox protein and an electrode surface upon association of an antibody to an antigen monolayer assembled on the electrode is used to develop immunosensor devices. In one configuration, a mixed monolayer consisting of the NE-(2,4dinitrophenyl)lysine antigen and ferrocene units acting as electron transfer mediators is applied to sense the dinitrophenyl antibody (DNP-Ab) in the presence of glucose oxidase (GOx) and glucose. In the absence of DNP-Ab, the mixed monolayer electrode stimulates the mediated electrocatalyzed oxidation of glucose that results in an amplified amperometric response. Association of the DNP-Ab to the modified electrode blocks the electrocatalytic transformation. The extent of the electrode insulation by the DNP-Ab is controlled by the Ab concentration in the sample. In the second configuration, a NE-(2,4dinitrophenyl)lysine antigen monolayer assembled on a Au electrode is applied to sense the DNP-Ab in the presence of a redox-modified GOx, exhibiting electrical communication with the electrode surface. Two kinds of redox-modified "electrically wired" GOx are applied: GOx modified by N-(ferrocenylmethyl)caproic acid, Fc-GOx, and a novel electrobiocatalyst generated by reconstitution of apo-GOx with a ferrocene-modified FAD semisynthetic cofactor. Electrocatalytic oxidation of glucose by the electrically wired biocatalysts proceeds in the presence of the antigen monolayer electrode, giving rise to an amplified amperometric signal. The electrocatalytic transformation is blocked upon association of the DNP-Ab to the monolayer electrode. The extent of electrode insulation toward the bioelectrocatalytic oxidation of glucose is controlled by the DNP-Ab concentrations in the samples. The application of biocatalysts for amperometric sensing of antigen-antibody interactions at the electrode surface makes the electrode insensitive to microscopic pinhole defects in the monolayer assembly. The antigen monolayer electrode is applied to sense the DNP-Ab in the concentration range 1-50 µg mL -1 .

Characterization of surface modification on self-assembled monolayer-based piezoelectric crystal immunosensor for the quantification of serum α-fetoprotein

Journal of Materials Science: Materials in Medicine, 2011

Self-assembled monolayers (SAMs) on coinage metallic material can provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. Recently, a bio-sensing system has been produced by analysis of the attachment of antibody using alkanethiols, to form SAMs on the face of Au-quartz crystal microbalance (QCM) surfaces. In this study, the attachment of anti-afetoprotein monoclonal antibody to a SAMs surface of 11-mercaptoundecanoic acid was achieved using watersoluble N-ethyl-N 0 -(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agents. Surface analyses were utilized by X-ray photoelectron spectroscopy and atomic force microscopy. The quantization of immobilized antibody was characterized by the frequency shift of QCM and the radioactivity change of 125 I labeled antibody. The limit of detection and linear range of the calibration curve of the QCM method were 15 ng/ml and 15-850 ng/ml. The correlation coefficients of a-fetoprotein concentration between QCM and radioimmunoassay were 0.9903 and 0.9750 for the standards and serum samples, respectively. This report illustrates an investigation of SAMs for the preparation of covalently immobilized antibody biosensors.

Immobilization of Antibodies on Biosensing Devices by Nanoarrayed Self-Assembled Monolayers

Langmuir, 2006

This work presents an original and straightforward technique for antibody immobilization onto a surface, keeping the antibody in a biologically reactive configuration. Self-assembly of molecular monolayers and plasma-based colloidal lithography were combined to create chemical nanopatterns on the surface of a biosensing device. This technique was employed to create an array of 100 nm wide motifs having a hexagonal 2-D crystalline structure, characterized by COOH-terminated nanospots in a CH 3 -terminated matrix. The quality control of the chemical nanopattern was carried out by combining atomic force microscopy, ellipsometry, and contact angle measurements. Enzyme-linked immunosorbent assay experiments were set up showing that the COOH/CH 3 nanopatterned surface constrains the immobilization of the antibodies in a biologically reactive configuration, thus significantly improving the device performances as compared to those of more conventional nonpatterned COOH-terminated or CH 3 -terminated surfaces. Figure 4. (a) AFM picture of the HDT (CH 3 )-covered sample after the ELISA procedure (vertical scale [0, 23 nm]). (b) AFM picture of the MHD (COOH)-covered sample after the ELISA procedure (vertical scale [0, 23 nm]). (c) AFM picture of the nanopatterned (COOH/CH 3 ) sample after the ELISA procedure (vertical scale [0, 106 nm]). Insets: respective FFT of the h(x,y) functions.

A comparative evaluation of molecular recognition by monolayers composed of synthetic receptors or oriented antibodies

Biosensors and Bioelectronics, 2008

Recombinant anti-morphine Fab fragments have been immobilised on gold by covalent attachment through the free thiol groups of the fragment. The antibody fragments were intercalated with a non-ionic hydrophilic polymer in order to suppress non-specific binding of interfering substances. The antibodies are oriented on the surface due to the thiol groups of the antibody and the layer shows a high response to antigen. Non-specific binding of bovine serum albumin is moreover very low because of the repellent polymer. Synthetic receptors composed of an imprinted self-assembled monolayer made from lipoates and the template, morphine, exhibit the same binding response to the antigen, morphine as the site-specific oriented antibody monolayer. A similar binding curve could be obtained as that for binding of morphine to an antibody Fab fragment/polymer layer-indicating that synthetic receptors produced are comparable to those of antibody layers. Concentrations down to 0.1 ng/ml have been measured with surface plasmon resonance.

Antibody orientation on biosensor surfaces: a minireview

The Analyst, 2013

Detection elements play a key role in analyte recognition in biosensors. Therefore, detection elements with high analyte specificity and binding strength are required. While antibodies (Abs) have been increasingly used as detection elements in biosensors, a key challenge remainsthe immobilization on the biosensor surface. This minireview highlights recent approaches to immobilize and study Abs on surfaces. We first introduce Ab species used as detection elements, and discuss techniques recently used to elucidate Ab orientation by determination of layer thickness or surface topology. Then, several immobilization methods will be presented: non-covalent and covalent surface attachment, yielding oriented or random coupled Abs. Finally, protein modification methods applicable for oriented Ab immobilization are reviewed with an eye to future application.

Characterization of Covalently Bound Anti-Human Immunoglobulins on Self-Assembled Monolayer Modified Gold Electrodes

Advanced Biosystems

The attachment of biological recognition sites is essential for the successful development of reliable biosensors being able to detect target analytes at low concentrations. In addition, the immobilization strategy can affect biological systems and the processes associated with key relevant events such as biorecognition itself. The selected biofunctionalization protocol depends on several factors involving biomolecules properties, nature of the immobilization surface, sample matrix, and buffer medium. It also impacts on the sensor's analytical performance metrics such as the sensitivity, selectivity, and reproducibility. Ideally, the bioactive binding sites should be immobilized onto surfaces with controlled density and proper orientation in order to be accessible for reacting with the ligand. For a reproducible assay, bioreceptors activity and surface coverage should remain unchanged from one batch to the other. Up to date biomolecule immobilization involving silanized layers, polymer membranes, Langmuir-Blodgett films, protein A, and self-assembled monolayer (SAM), has been achieved on various substrates, ensuring also the correct orientation of the recognition site without loss of activity and high loading of the sensor surface in order to gain maximum signal as well as selective analyte detection. [1] All the above reveal the importance of investigating the surface chemistry of biomodified materials, including noble metals, nanoparticles, metal oxides, polymers etc., for the optimization of the incorporation of biomolecules in miniaturized electronic sensors. Especially, the incorporation of biorecognition sites on inert metal surfaces such as gold gives rise to biosensors taking advantage of different physical quantifiable signals including electrochemical sensing, [2-4] surface plasmon resonance (SPR), [5-8] or the lately gaining significant attention electrolyte gated thin film transistors (EG-TFTs) [9-13] based biosensors. Biorecognition elements (e.g., proteins, DNA, cells, etc.) have been largely immobilized on active gold sensor surfaces via weak physical (ionic, hydrophobic) or strong chemical (through thiol chemistry, bifunctional linkers or adapter mole cules) interactions. [14] Herein, a Bioconjugated gold surfaces constitute interesting platforms for biosensing applications. The immobilization of antibodies such as anti-immunoglobulin G and M (anti-IgG and anti-IgM) on gold electrodes via self-assembled monolayers (SAMs) is here studied as a model system for further immunoassays development. The biolayer is characterized by means of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), a dedicated thin-film transistor (TFT)-based platform and electrochemical surface plasmon resonance (EC-SPR). XPS analysis confirms the presence of all the chemical species involved in the fabrication process as well as the covalent attachment of the antibodies with high reproducibility. Visualization of the biolayer topography by AFM shows nanostructures with a thickness consistent with the actual size of the protein, which is also verified by SPR measurements. EC-SPR allows taking advantage of complementary electrochemical and optical signals during the functionalization steps. Moreover, the functionalization of gold leads to a change in the work function, which is demonstrated in an electrolyte gated thin-film transistor configuration. Such configuration enables also to evaluate the electrostatic changes occurring on the gate that are connected with the threshold voltage shifts. The data support that functional biomodified gold surfaces can be reproducibly prepared, which is a prerequisite for further biosensor development.

Immobilization of Antibodies on a Photoactive Self-Assembled Monolayer on Gold

Langmuir, 1996

This paper presents a strategy for immobilizing biomolecules on a photoactivable surface. A selfassembled monolayer is prepared by adsorbing an ω-functionalized dialkyl disulfide on gold. Functional groups of this monolayer are converted in two steps into a benzophenone derivative with an overall yield of 50 (10%. Several independent techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiolabel assay, and autoradiography) characterize the reaction and photoimmobilization of antibodies on this surface. The photoimmobilized antibodies cover the surface as a homogeneous and dense monolayer that could not be disrupted by vigorous washing with the detergent Tween 20. Immunoassays demonstrated specific recognition of the immobilized immunoglobulins as measured by their complexation with alkaline phosphatase-linked antibodies. The method of photoimmobilization used here leads to a homogeneous single layer of IgGs, in which the proteins maximize their contact with the surface. Residual adsorption of IgG on the nonirradiated surface of benzophenone remains one limitation of this approach. Progressively higher coverages of IgGs on the surface did not lead to strictly proportional changes of the biological activity of these surfaces, probably because of interactions between the IgGs in the film. This method of photoimmobilization is nonetheless useful as an experimental system to immobilize other proteins because it is simple, flexible, and efficient.

Assembly of functionalized monolayers of redox proteins on electrode surfaces: novel bioelectronic and optobioelectronic systems

Biosensors and …, 1997

Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid.

Association of Anti-Dinitrophenyl Antibody onto a Patterned Organosiloxane Antigen Monolayer Prepared by Microcontact Printing: An AFM Characterization

Langmuir, 1999

A patterned array consisting of a microstructured layer of octadecyltrichlorosilane (OTS) and (3-((2,4dinitrophenyl)amino)propyl)triethoxysilane (DNP analogue) was assembled onto a Si wafer using the microcontact printing method. The microstructured, patterned support was imaged by AFM, using a bare Au tip, a hydrophobic alkyl mercaptan-functionalized Au tip, and a hydrophilic hydroxyalkyl mercaptanmodified Au tip. The lateral friction forces between the tip and the patterned surface are controlled by hydrophobic-hydrophobic and hydrophobic-hydrophilic interactions and eventually H bonds between the tip and the functionalized surface. The (3-((2,4-dinitrophenyl)amino)propyl)triethoxysilane domains of the microstructured surface act as antigens for the anti-dinitrophenyl antibody (anti-DNP-Ab). Interaction of the patterned support with the DNP-Ab solution yields an overall hydrophilic interface due to the association of the antibody to the entire support. Analysis of the adhesive and friction interactions between the Au tip and the DNP-Ab associated with the OTS and DNP analogue regions, and the roughness factors of the respective domains that include the DNP-Ab, enables us to conclude that the DNP-Ab associates nonspecifically to the OTS sites, while specific binding of the DNP-Ab occurs on the DNP antigen regions.