We report here on a new potentiometric biosensing principle for the detection of antibody-antigen interactions at the sensing membrane surface without the need to add a label or a reporter ion to the sample solution. This is accomplished by establishing a steady-state outward flux of a marker ion from the membrane into the contacting solution. The immunobinding event at the sensing surface retards the marker ion, which results in its accumulation at the membrane surface and hence in a potential response. The ion selective membranes were surface-modified with an antibody against respiratory syncytial virus using click chemistry between biotin molecules functionalized with a triple bond and an azide group on the modified poly (vinyl chloride) group of the membrane. The bioassay sensor was then built up with streptavidin and subsequent biotinylated antibody. A quaternary ammonium ion served as the marker ion. The observed potential was found to be modulated by the presence of respiratory syncytial virus bound on the membrane surface. The sensing architecture was confirmed with quartz crystal microbalance studies, and stir effects confirmed the kinetic nature of the marker release from the membrane. The sensitivity of the model sensor was compared to that of a commercially available point-of-care test, with promising results.

The unique localized surface plasmon resonance (LSPR) property of gold nanoparticles has been used to design a label-free biosensor in a chip format. In this research, a sensitive and low-cost microfluidic integrated LSPR-based biosensor is developed. The gold nanoparticles were synthesized in solution and immobilized on quartz substrates by a silane layer as molecular glue. The gold nanoparticle-coated substrate was further integrated with a microfluidic chip. An automated sample introduction system was developed to perform a variety of processes including sample loading, chip washing and sample change. A refractive index resolution of 1×10−4 RIU (refractive index unit) was demonstrated by using the on-chip biosensor combined with the automated sampling system. This developed microfluidic integrated system is capable of transporting a specific amount of bio-samples into the sensing chambers to achieve sensitive and specific biosensing with decreased reaction time and less reagent consuming. Proof-of-concept detection of antigen/antibody (biotin/anti-biotin) binding was performed and was quantitatively detected.

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10(-4) in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.

The increasing miniaturization of biochips based on microelectronics and the demand for higher biosensor sensitivities and specificities put severe demands on the process and methodology of coupling biomolecules to surfaces. More specifically, controlled thin film structures have to be created which allow the bio-affinity elements to be arranged and addressed in a reproducible and controlled manner. In this chapter, we describe how to create functional oxide and gold surfaces using Self-Assembled Monolayers (SAMs) and how they can be applied to construct immunosensor interfaces. Furthermore, we report on the direct self-assembly of low-molecular weight compounds and single stranded DNA onto gold surfaces. We conclude that immobilization approaches inspired by self-assembly are extremely versatile and are of the utmost importance for the development of highly sensitive and specific biosensors.

The secret of a successful affinity biosensor partially hides in the chemical interface layer between the transducer system and the biological receptor molecules. Over the past decade, several methodologies for the construction of such interface layers have been developed on the basis of the deposition of self-assembled monolayers (SAMs) of alkanethiols on gold. Moreover, mixed SAMs of polyethylene oxide (PEO) containing thiols have been applied for the immobilization of biological receptors. Despite the intense research in the field of thiol SAMs, relatively little is known about their biosensing properties in correlation with their long-term stability. Especially the impact of the storage conditions on their biosensing characteristics has not been reported before to our knowledge. To address these issues, we prepared mixed PEO SAMs and tested their stability and biosensing performance in several storage conditions, i.e., air, N2, ethanol, phosphate buffer, and H2O. The quality of the SAMs was monitored as a function of time using various characterization techniques such as cyclic voltammetry, contact angle, grazing angle Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In addition, the impact of the different storage conditions on the biosensor properties was investigated using surface plasmon resonance. Via the latter technique, the receptor immobilization, the analyte recognition, and the nonspecific binding were extensively studied using the prostate specific antigen as a model system. Our experiments showed that very small structural differences in the SAM can have a great impact in their final biosensing properties. In addition it was shown that the mixed SAMs stored in air or N2 are very stable and retain their biosensor properties for at least 30 days, while ethanol appeared to be the worst storage medium due to partial oxidation of the thiol headgroup. In conclusion, care must be taken to avoid SAM degradation during storage to retain typical SAM characteristics, which is very important for their general use in many proposed applications.

Three-dimensional (3D) self-assemblies of metal nanoparticles (NPs) are attracting intense attention for sensing applications since they display unique optical properties which are distinct from their respective bulk and individual NP characteristics. In this paper, 3D self-assembled gold (Au) NPs multilayer structures have been fabricated by the alternate deposition of a bifunctional cross-linker and Au NPs, to improve the refractive index sensitivity of the localized surface plasmon resonance sensor. UV–vis spectroscopy, atomic force microscopy (AFM) and cross-section transmission electron microscopy (TEM) have been used to characterize the obtained multilayer structures. The investigations show that the number of Au NP deposition cycles has a strong effect on the sensitivity of the sensor and the multilayer structure fabricated from four NP deposition cycles exhibits maximum sensitivity to the change of the environmental refractive index.

Today’s proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

Magnetic particles (MPs) have been attracting much interest as a labeling material for advanced biological and medical applications, such as biomagnetic separation, drug delivery, magnetic resonance imaging, and hyperthermia. In most of these applications, the MPs have been designed to specifically interact with a target, such as cells or proteins, moving freely in a solution. However, for surface-based applications, such as magnetic biosensing, these MPs must bind specifically with a target that is immobilized onto a planar substrate. Consequently, new interaction phenomena, which influence the binding of the MPs to the substrate, have to be taken into account. To achieve adequate binding characteristics and to optimize the MPs toward substrate labeling, these physicochemical interactions should be properly identified. In this paper, the interactions between 16 commercially available streptavidin MPs and a biotinylated gold substrate were monitored in real time by surface plasmon resonance technology and the particle surface coverage was calculated by optical microscopy. On the basis of the type of interactions, the MPs studied in this paper could be classified into three different cases: (I) MPs that bind to the biotinylated substrate via the specific streptavidin-biotin interactions, without showing any nonspecific interactions; (II) MPs that do not bind to the substrate; and (III) MPs that bind to the biotinylated substrate via nonspecific interactions rather than via specific streptavidin-biotin interactions. The three cases were understood by determining the surface charges of both the particle and the substrate in zeta potential measurements. It was found that binding of MPs to the substrate was strongly dependent on the amount and the sign of the charges on both surfaces. The strong influence of electrostatic interactions was validated by simulating the total interaction force between a streptavidin MP and a biotinylated substrate by use of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, while the gravitational force and the streptavidin-biotin force were accounted for. Finally, we conclude that apart from a well-controlled streptavidin coating, the surface charge of the particle and the substrate plays a pivotal role in the construction of MP assays on surfaces.

We present an approach for the controlled positioning of single gold colloids onto dot and line nanoarrays which have the potential to serve as label-free biosensor platforms. The separation between the high-resolution nanolithography step, extreme ultraviolet interference lithography, and the subsequent functionalization has many advantages, among them the independence of the linkage chemistry. The activation of the pre-patterned substrates is performed by transforming them into a surface of biotinylated nanopatches in a protein-resistant background. Complexes of streptavidin and single-stranded DNAs can then be selectively immobilized onto the biotinylated patches, which are embedded in an inert background. This approach enables the creation of single gold colloid dot and line arrays by directed self-assembly using the specificity of DNA hybridization.

Ferrite magnetic nanoparticles (MNPs) were functionalized with a variety of silanes bearing different functional endgroups to render them stable with respect to aggregation and keep them well-dispersed in aqueous media. The MNPs were prepared by the thermal decomposition method, widely used for the synthesis of monodisperse nanoparticles with controllable size. This method makes use of a hydrophobic surfactant to passivate the surface, which results in nanoparticles that are solely dispersible in nonpolar solvents. For use in biological applications, these nanoparticles need to be made water-dispersible. Therefore, a new procedure was developed on the basis of the exchange of the hydrophobic surface ligands with silanes bearing different endgroups to decorate ferrite magnetic nanoparticles with diverse functionalities . By this means, we could easily determine the influence of the endgroup on the nanoparticle stability and water-dispersibility. Amino-, carboxylic acid- and poly(ethylene glycol)-terminated silanes were found to render the MNPs highly stable and water-dispersible because of electrostatic and/or steric repulsion. The silane molecules were also found to form a protective layer against mild acid and alkaline environments. The ligand exchange on the nanoparticle surface was thoroughly characterized using SQUID, TEM, XPS, DLS, TGA, FTIR, UV-vis, and zeta potential measurements. The presented approach provides a generic strategy to functionalize magnetic ferrite nanoparticles and to form stable dispersions in aqueous media, which facilitates the use of these magnetic nanoparticles in biological applications.

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3 @ SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be similar to 8 x 10(14) molecules/cm(2). It was shown that the magnetic,'crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of alclehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle W surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 mu g antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 rim in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (similar to 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins W could be significantly suppressed down to similar to 3 mu g/mg. We conclude that self-assembly of silane cc films creates a generic platform for the controlled interactions of MNPs with biomolecules.

The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab′)2 and Fab′, with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab′ fragments. First, an optimisation of cleavage protocol to generate these F(ab′)2 and Fab′ fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab′ fragments on mixed SAMs leads to a >2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies.