Functional Molecular Interfaces for Impedance-Based Diagnostics.

In seeking to develop and optimize reagentless electroanalytical assays, a consideration of the transducing interface features lies key to any subsequent sensitivity and selectivity. This review briefly summarizes some of the most commonly used receptive interfaces that have been employed within the development of impedimetric molecular sensors. We discuss the use of high surface area carbon, nanoparticles, and a range of bioreceptors that can subsequently be integrated. The review spans the most commonly utilized biorecognition elements, such as antibodies, antibody fragments, aptamers, and nucleic acids, and touches on some novel emerging alternatives such as nanofragments, molecularly imprinted polymers, and bacteriophages. Reference is made to the immobilization chemistries available along with a consideration of both optimal packing density and recognition probe orientation. We also discuss assay-relevant mechanistic details and applications in real sample analysis.

Anisotropic silver nanoparticles sensitized by oxidized lipoproteins for detection of appropriate antibodies.

Antibodies to oxidized lipoproteins are useful markers for diagnosis and prognosis of some heart diseases. Low density lipoproteins (LDL) were purified from human plasma and were oxidized by CuCl2. They were immobilized on anisotropic silver nanoparticles with tunable plasmon resonance band and were applied for detection of appropriate antibodies in sera of patients with stenosis. The presence of such antibodies brings to significant shifting of plasmon resonance band to long-wave region in comparison with control sera. This approach permits to detect such antibodies by rapid method with one step procedure.

Optimización del proceso de inmovilización de anticuerpos en inmunobiosensores / Optimization of the antibody immobilization process in immunobiosensors

Antecedentes: La detección rápida y específica de agresivos biológicos es fundamental en diversos campos como control ambiental, diagnóstico clínico, industria alimentaria, seguridad y defensa. La especificidad de la unión antígeno-anticuerpo es empleada en multitud de biosensores, como equipos de identificación de agentes de guerra biológicos, pero el cómo se una ese anticuerpo en la superficie del biosensor, en términos de densidad, orientación, y estabilidad determinará la capacidad diagnóstica del dispositivo. Objetivo: Desarrollo de procesos de inmovilización de anticuerpos en superficies planares que permitan una unión antígeno-anticuerpo eficiente, para su posterior uso en dispositivos inmunológicos de sensado. Material y Métodos: Se ensayaron tres métodos de inmovilización del anticuerpo-fluoresceína sobre una membrana Zprobe: adsorción pasiva, unión covalente con glutaraldehído (0,5 %) y unión orientada con proteína mediadora A/G (5 y 10 µg). Se seleccionó albúmina sérica bovina-ficoeritrina como simulante de toxina proteica. Resultados: El porcentaje de retención del anticuerpo inmovilizado durante el proceso de inmunocaptura fue similar en los métodos ensayados. La densidad del anticuerpo inmovilizado fue mayor en la inmovilización con glutaraldehído y menor con proteína A/G. Sin embargo, respecto a la eficiencia de la inmunocaptura del antígeno, la inmovilización del anticuerpo con glutaraldehído fue la menos eficiente frente a la inmovilización con proteínas A/G, que resultó ser la más eficaz. Conclusiones: La utilización de glutaraldehído en la inmovilización del anticuerpo, aunque incrementa la densidad de unión del mismo sobre una membrana Zprobe, interfiere en el proceso de inmunodetección antigénica, mientras que el uso de la proteína mediadora A/G permiten un sistema de inmunocaptura más eficiente, con una menor densidad de anticuerpo inmovilizado

Antecedent: A quick and specific detection of biological warfare agent is the keystone in several fields like environmental control, clinic diagnostic, food industry, security and defence. The specificity of antigen-antibody binding is used in a multitude of biosensors like biological warfare-agent detection equipment. However, how the antibody is attached to the biosensor surface, in terms of density, orientation and stability, will determine the diagnosis capability of the device. Aim: the development of antibodies immobilization proceedings in planar surface for an efficient antigen-antibody reaction to be used in immunological sensing devices. Material and Methods: three immobilization methods of fluorescein labelled antibody were assayed on Zprobe membrane: passive adsorption, covalent bond by glutaraldehyde, well-oriented immobilization by the intermediate protein A/G. Bovine serum albumin labelled with R-phycoerytrin was selected as toxin surrogate. 0,5 % glutaraldehyde and A/G chimeric protein (5 and 10 µg) were used as immobilization reactive. Results: immobilized antibody retention during the immunocapture process was similar between all the assayed immobilization methods. The immobilized antibody density by glutaraldehyde was higher than that by protein A/G. However, with regard to the antigenic immune-capture efficiency antibody immobilization by glutaraldehyde was the less efficient than immobilization by protein A/G. Conclusions: Antibody immobilization by glutaraldehyde, in spite of increasing the retained antibody density on the Zprobe membrane, interferes in the antigenic immunodetection whereas the intermediate protein A/G improves it, allowing a very efficient immunocapture system with less antibody density

Dual targeting improves capture of ultrasound microbubbles towards activated platelets but yields no additional benefit for imaging of arterial thrombosis.

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewisa individually (MBsLea) or dually with sLea and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MBDual). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MBControl. While MBsLea rolled slowly on the platelets' surface, MBDual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MBsLea and MBDual revealed a higher ultrasound mean acoustic intensity than MBControl (p < 0.05), however MBDual demonstrated no additional increase in mean signal intensity as compared to MBsLea. The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p < 0.05). While dual targeting of MB using fast binding carbohydrate polymers and specific antibodies is a promising strategy to support adhesion to activated platelets under arterial shear stress, these advantages seem not readily translatable to in vivo models.

Multifunctional substrate of label-free electrochemical immunosensor for ultrasensitive detection of cytokeratins antigen 21-1.

Poly(thionine)-Au, a novel multifunctional substrate with excellent redox signal, enzyme-like activity, and easy antibody immobilisation, was synthesised using HAuCl4 as the oxidising agent and thionine as the monomer. The prepared poly(thionine)-Au composite exhibited an admirable electrochemical redox signal at -0.15 V and excellent H2O2 catalytic ability. In addition, gold nanoparticles in this composite were found to directly immobilise antibodies and further improve conductivity. In addition, a label-free electrochemical immunosensor was developed using poly(thionine)-Au as the sensing substrate for ultrasensitive detection of cytokeratin antigen 21-1 (CYFRA 21-1), an immunoassay found in human serum. The prepared immunosensor showed a wide liner range from 100 ng mL-1 to 10 fg mL-1 and an ultralow detection limit of 4.6 fg mL-1 (the ratio of signal to noise (S/N) = 3). Additionally, this method was used to analyse human serum samples and yielded results consistency with those of ELISA, implying its potential application in clinical research. The poly(thionine)-Au composite can be easily extended to other polymer-based nanocomposites, which is significant for other electrochemical immunoassays.

Quantification of antibody coupled to magnetic particles by targeted mass spectrometry.

Quantifying the amount of antibody on magnetic particles is a fundamental, but often overlooked step in the development of magnetic separation-based immunoaffinity enrichment procedures. In this work, a targeted mass spectrometry (MS)-based method was developed to directly measure the amount of antibody covalently bound to magnetic particles. Isotope-dilution liquid chromatography-tandem MS (ID-LC-MS/MS) has been extensively employed as a gold-standard method for protein quantification. Here, we demonstrate the utility of this methodology for evaluating different antibody coupling processes to magnetic particles of different dimensions. Synthesized magnetic nanoparticles and pre-functionalized microparticles activated with glutaraldehyde or epoxy surface groups were used as solid supports for antibody conjugation. The key steps in this quantitative approach involved an antibody-magnetic particle coupling process, a wash step to remove unreacted antibody, followed by an enzymatic digestion step (in situ with the magnetic particles) to release tryptic antibody peptides. Our results demonstrate that nanoparticles more efficiently bind antibody when compared to microparticles, which was expected due to the larger surface area per unit mass of the nanoparticles compared to the same mass of microparticles. This quantitative method is shown to be capable of accurately and directly measuring antibody bound to magnetic particles and is independent of the conjugation method or type of magnetic particle. Graphical Abstract Schematic illustration of the isotope-dilution mass spectrometry-based workflow to directly measure antibody bound to magnetic particles (MP).

Polydimethylsiloxane microfluidic chemiluminescence immunodevice with the signal amplification strategy for sensitive detection of human immunoglobin G.

A polydimethylsiloxane (PDMS) microfluidic chemiluminescence (CL) immunodevice for sensitive detection of human immunoglobin G (IgG) with the signal amplification strategy was developed in this work. The immunodevice was prepared by covalently immobilizing capture antibodies (Abs) on the silanized microchannel of microfluidic chip. Gold nanoparticles (AuNPs) functionalized with a high molar ratio of horseradish peroxidase (HRP) were used as an Ab label for signal amplification. Using a sandwich immunoassay, the multi-HRP conjugated AuNPs can catalyze the luminol-H2O2 CL system to achieve the high sensitivity. In addition, the double spiral flow-channel was adopted here, which can still contribute to the high sensitivity. Based on signal amplification strategy, the performance of human IgG tests revealed a lower detection limit (DL) of 0.03ng/mL and showed an increase of 7.4-fold in detection sensitivity compared to a commercial Ab-HRP conjugation. This microfluidic immunodevice can provide an alternative approach for sensitive detection of human IgG in the field of clinic diagnostic and therapeutic.

Noncovalently functionalized monolayer graphene for sensitivity enhancement of surface plasmon resonance immunosensors.

A highly efficient surface plasmon resonance (SPR) immunosensor is described using a functionalized single graphene layer on a thin gold film. The aim of this approach was two-fold: first, to amplify the SPR signal by growing graphene through chemical vapor deposition and, second, to control the immobilization of biotinylated cholera toxin antigen on copper coordinated nitrilotriacetic acid (NTA) using graphene as an ultrathin layer. The NTA groups were attached to graphene via pyrene derivatives implying π-π interactions. With this setup, an immunosensor for the specific antibody anticholera toxin with a detection limit of 4 pg mL(-1) was obtained. In parallel, NTA polypyrrole films of different thicknesses were electrogenerated on the gold sensing platform where the optimal electropolymerization conditions were determined. For this optimized polypyrrole-NTA setup, the simple presence of a graphene layer between the gold and polymer film led to a significant increase of the SPR signal.

Microfluidic immunoassay with plug-in liquid crystal for optical detection of antibody.

Recent advance in liquid crystal (LqC) based immunoassays enables label-free detection of antibody, but manual preparation of LqC cells and injection of LqC are required. In this work, we developed a new format of LqC-based immunoassay which is hosted in a microfluidic device. In this format, the orientations of LqC are strongly influenced by four channel walls surrounding the LqC. When the aspect ratio (depth/width) of the channel is smaller than 0.38, LqC orients homeotropically inside the microchannel and appears dark. After antigens bind to immobilized antibodies on the channel walls, a shift of the LqC appearance from dark to bright (due to the disruption of LqC orientation) can be visualized directly. To streamline the immunoassay process, a tubing cartridge loaded with a sample solution, washing buffers and a plug of LqC is connected to the microfluidic device. By using pressure-driven flow, the cartridge allows antigen/antibody binding, washing and optical detection to be accomplished in a sequential order. We demonstrate that this microfluidic immunoassay is able to detect anti-rabbit IgG with a naked-eye detection limit down to 1 µg mL(-1). This new format of immunoassay provides a simple and robust approach to perform LqC-based label-free immunodetection in microfluidic devices.

Characterisation of a label-free biosensor based on long period grating.

Optical fibre gratings, especially long period gratings, have been recently proposed as optical devices for biochemical sensing. A biochemical interaction along the grating portion induces a refractive index change and hence a change in the fiber transmission spectrum. This provides an alternative methodology with respect to other label-free optical approaches, such as surface plasmon resonance, interferometric configurations and optical resonators. The fibre biofunctionalization has been carried out by means of a novel chemistry using Eudragit L100 copolymer as opposed to the commonly used silanization procedure. Antigen-antibody interaction has been analysed by means of an IgG/anti-IgG bioassay. The biosensor was fully characterised, monitoring the kinetics during the antibody immobilization and the antigen interaction and achieving the calibration curve of the assay. A comparison of the biosensor performance was made by using two different long period gratings with distinct periods.

Spatially-resolved EELS analysis of antibody distribution on biofunctionalized magnetic nanoparticles.

Spatially resolved electron energy loss spectroscopy (SR-EELS) using scanning transmission electron microscope (STEM) allows the identification and determination of the spatial distribution of the components/elements of immuno-functionalized core-shell superparamagnetic magnetite nanoparticles. Here, we report that SR-EELS measurements allow the direct identification and study of the biological moieties (protein G and anti-HRP antibody) in complex bionanocarriers of relevance for biomedical applications. Our findings show that the biomacromolecules are located on specific areas on the nanoparticles' surface. In addition, efficiency of this functionalization was evaluated by means of biochemical techniques.

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry.

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.

Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production.

The present report describes the integration and application possibilities of a new microarray concept based on adhesive surface. The method was shown to enable the straightforward production of 384 and 1536-well plates modified with 100 and 25 spots per well, respectively. Such in-well densities were only possible thanks to the fabrication process which implies first the deposition of the microarray on a flat adhesive surface and then its assembly with bottomless 384 or 1536-well plates. The concept was also confronted to various applications such as oligonucleotide detection, localised cell culture onto spotted adhesion proteins and immobilisation of peptide or active antibodies for immunoassays. In the particular case of immunotesting, the study focused on liver diseases diagnosis and more particularly on the detection of either one liver cancer marker, the alpha-fetoprotein, or the detection of Hepatitis C Virus infection. In every cases, interesting performances were obtained directly in crude patient serum, proof of the robust and generic aspect of the platform.

Human chorionic gonadotropin interactions with immobilized anti-hCG studied by quartz-crystal microbalance with dissipation monitoring.

Previous studies have found that commercial human pregnancy tests are often too insensitive to function to their advertised >99% accuracy. Improper orientation of proteins used for recognition of ligands in sensors can often prevent the binding site from being available to the ligand, thereby decreasing sensor sensitivity. We have developed a simple method for the immobilization of anti-human chorionic gonadotropin on a sensor surface that maximizes its sensitivity by ensuring ligand binding sites are exposed and densely packed. This surface also has an improved regenerative capacity over previously reported human chorionic gonadotropin sensors, retaining 99% of initial sensitivity after six regeneration cycles with 8M urea.

Biointeraction analysis of immobilized antibodies and related agents by high-performance immunoaffinity chromatography.

A method is described based on high-performance immunoaffinity chromatography for examining the interactions of immobilized antibodies or related binding agents with their targets. It is shown how this method can be used to obtain information on the binding, elution and regeneration kinetics of immobilized binding agents, such as those used with immunoaffinity supports. The theory behind this approach is briefly described and it is demonstrated how both the kinetic and thermodynamic properties of a biointeraction can be determined experimentally through this method. Several applications are used to illustrate this technique, including antibody-antigen interactions and the binding of aptamers with their targets in the presence of silica-based supports. The same approach can be adapted for use with other types of targets, binding agents and support materials.

A novel biosensor based on serum antibody immobilization for rapid detection of viral antigens.

In this paper, we represent a label-free biosensor based on immobilization of serum antibodies for rapid detection of viral antigens. Human serum containing specific antibodies against Japanese encephalitis virus (JEV) was immobilized on a silanized surface of an interdigitated sensor via protein A/glutaraldehyde for electrical detection of JEV antigens. The effective immobilization of serum antibodies on the sensor surface was verified by Fourier transform infrared spectrometry and fluorescence microscopy. The signal of the biosensor obtained by the differential voltage converted from the change into non-Faradic impedance resulting from the specific binding of JEV antigens on the surface of the sensor. The detection analyzed indicates that the detection range of this biosensor is 1-10 µg/ml JEV antigens, with a detection limit of 0.75 µg/ml and that stable signals are measured in about 20 min. This study presents a useful biosensor with a high selectivity for rapid and simple detection of JEV antigens, and it also proposes the biosensor as a future diagnostic tool for rapid and direct detection of viral antigens in clinical samples for preliminary pathogenic screenings in the case of possible outbreaks.

Assessing antibody microarrays for space missions: effect of long-term storage, gamma radiation, and temperature shifts on printed and fluorescently labeled antibodies.

Antibody microarrays are becoming frequently used tools for analytical purposes. A key factor for optimal performance is the stability of the immobilized (capturing) antibodies as well as those that have been fluorescently labeled to achieve the immunological test (tracers). This is especially critical for long-distance transport, field testing, or planetary exploration. A number of different environmental stresses may affect the antibody integrity, such as dryness, sudden temperature shift cycles, or, as in the case of space science, exposure to large quantities of the highly penetrating gamma radiation. Here, we report on the effect of certain stabilizing solutions for long-term storage of printed antibody microarrays under different conditions. We tested the effect of gamma radiation on printed and freeze- or vacuum-dried fluorescent antibodies at working concentrations (tracer antibodies), as well as the effect of multiple cycles of sudden and prolonged temperature shifts on the stability of fluorescently labeled tracer antibody cocktails. Our results show that (i) antibody microarrays are stable at room temperature when printed on stabilizing spotting solutions for at least 6 months, (ii) lyophilized and vacuum-dried fluorescently labeled tracer antibodies are stable for more than 9 months of sudden temperature shift cycles (-20°C to 25°C and 50°C), and (iii) both printed and freeze- or vacuum-dried fluorescent tracer antibodies are stable after several-fold excess of the dose of gamma radiation expected during a mission to Mars. Although different antibodies may exhibit different susceptibilities, we conclude that, in general, antibodies are suitable for use in planetary exploration purposes if they are properly treated and stored with the use of stabilizing substances.

AFM imaging of ALYGNSA polymer-protein surfaces: evidence of antibody orientation.

Previous investigations found the combination of recombinant bacterial protein G (rProG) and poly(methyl methacrylate) (PMMA) to produce a greater proportion of oriented antibodies. PMMA-rProG yielded a sixfold greater availability of antibody Fab regions compared with other bacterial affinity linker protein and polymer pairings, including commercially available polystyrene (PS) high-binding 96-well microplates. Given the name ALYGNSA, the PMMA-rProG combination was developed into a fluorescence assay and evaluated in conjunction with commercially available cancer biomarker enzyme-linked immunosorbent assays (ELISAs). In each study, a lower limit of detection was seen with the ALYGNSA assay. The purpose of this investigation was to examine the ALYGNSA substrate in contrast with a commonly used ELISA substrate and analyze the affinity-immobilized antibodies for additional evidence of orientation. Non-contact atomic force microscopy is a logical method as it operates in ambient conditions, can be used directly on biological samples without modification, and offers the resolution necessary to identify the position of the antibody on the surface. Dynamic contact angle studies were employed to examine untreated PMMA and PS samples and revealed important differences in their surface characters. Comparative height threshold grain analysis of the prepared ALYGNSA surface, a similarly treated mica surface, and a gold colloid sizing standard evaluated and confirmed the antibody orientation of the ALYGNSA system.

A photoelectrochemical immunosensor based on Au-doped TiO2 nanotube arrays for the detection of α-synuclein.

α-Synuclein (α-SYN) is a very important neuronal protein that is associated with Parkinson's disease. In this paper, we utilized Au-doped TiO(2) nanotube arrays to design a photoelectrochemical immunosensor for the detection of α-SYN. The highly ordered TiO(2) nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au-doped TiO(2) nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab(1)) with high stability and bioactivity to bind target α-SYN. The enhanced sensitivity was obtained by using {Ab(2)-Au-GOx} bioconjugates, which featured secondary antibody (Ab(2)) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H(2)O(2), which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO(2) nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α-SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL(-1) to 100 ng mL(-1) with a detection limit of 34 pg mL(-1). The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.

Solid-phase preparation of protein complexes.

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.