Willem M. Albers

In situ quartz crystal microbalance monitoring of Fab'-fragment binding to linker lipids in a phosphatidylcholine monolayer matrix. Application to immunosensors

Abstract Linker lipids were embedded in a phosphatidylcholine monolayer matrix prepared at the air–water interface. The covalent coupling of antibody fragments, non-specific adsorption of bovine serum albumin and specific binding of antibodies was monitored in situ with a 10-MHz quartz crystal microbalance. The attachment of antibody fragments and the activity of the layers was also showed with standardized radioimmunoassay. The results demonstrate that the coupling of Fab′-fragments to linker lipids in a monolayer matrix is a promising approach to achieve a highly oriented layer of antibody fragments with a high density of binding sites on the sensor surface for immunological measurements.

Structural and functional characteristics of chimeric avidins physically adsorbed onto functionalized polythiophene thin films.

Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional avidins (wild-type avidin and streptavidin). In the present study, the applicability of chimeric avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric avidins showed remarkably increased binding characteristics compared with other avidins, such as wild-type avidin, streptavidin, and bacterial avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the avidins. Polythiophene layers functionalized with chimeric avidins thus offered a promising generic platform for biosensor applications.

Improved functionality of antibody-colloidal gold conjugates with the aid of lipoamide-grafted N-[tris(hydroxymethyl)methyl]acrylamide polymers

Colloidal gold has been used as a label in sandwich assays for human IgG, in which intercalating N-[tris(hydroxymethyl)methyl]acrylamide (pTHMMAA) polymers have been employed to stabilise the particles coated with antibody fragments. A direct absorbance reading of the particles could be obtained from sandwich assays on polystyrene, and a strongly amplified response was observed in similar assays based on Surface Plasmon Resonance (SPR): for h-IgG, detection limits below 100 pg/mL could be achieved. Three different polymer lengths and two different particles sizes were compared in sandwich assays performed on polystyrene and gold. The resulting binding curves fitted well to the Langmuir-Freundlich isotherm and the binding constants were in good agreement with the values found in earlier studies. The amplification afforded by the nanoparticles was strongly dependent on the antigen concentration, on the type of polymer and on the particle size. Compared to the direct response of the antigen, amplification factors larger than 100 could be achieved. The study proves that the polymers give stabilised particles, which can be used in highly sensitive sandwich assays.

Chapter 1 – INTERFACIAL AND MATERIALS ASPECTS OF THE IMMOBILIZATION OF BIOMOLECULES ONTO SOLID SURFACES

This chapter discusses the interfacial and material aspects of the immobilization of biomolecules onto solid surfaces. The controlled deposition of proteins to solid supports is a tedious task. Presently there is an explosive growth in information about adsorption of proteins to surfaces and about their interaction with other proteins. The further development of scanning probe microscopy gives additional opportunities for an increased understanding of adsorbed proteins. The challenge for the near future is first to reach lower detection limits. It has been proposed that the detection sensitivity of SPR and QCM methods can be increased by oriented immobilization of antibodies, and it has been confirmed that 50-100 ppb detection levels are feasible. Another challenge is to design LB-film deposition methods that meet the demands of mass production. Setting up LB-film production to an industrial scale has to date been quite difficult because of the strict conditions needed in the deposition process and the slowness of vertical deposition. Horizontal deposition methods are however much faster and allow mass production schemes. The chapter concludes that there is presently a great demand for immobilization methods that allow large libraries of DNA or biomolecules of various specificities to be attached to a small surface area in high-density arrays. For the production of high density arrays, spatial activation methods for attachment of biological molecules have to be used, and this can be done by photoactivation.

Surface Plasmon Resonance on Nanoscale Organic Films

Over the past 20 years, surface plasmon resonance (SPR) has evolved into a very versatile detection method, particularly in bioscience applications. Not only the scientific literature has greatly expanded, but also the various commercial vendors of instrumentation, detection chips, and reagents have emerged. In the scientific sphere, the accent lies more and more on fabrication of nanostructures with interesting optical behavior (plasmonics), while in the R&D area, there are many new miniaturization efforts and combination with other detection methods, such as electrochemistry and quartz crystal microbalance (QCM). The present chapter will focus on the latest developments in making functional biochemical coatings for SPR detection as well as will review the basic theory behind the detection techniques.

Structural aspects of self-assembly of thienoviologen molecular conductors on gold substrates

Abstract The adsorption of various monomethylated π -extended viologen compounds onto self-assembled layers of octadecylmercaptan (ODM) on gold was investigated with secondary ion mass spectroscopy (SIMS) and atomic force microscopy (AFM). SIMS results indicated that the monomethylated viologen 5-(4-pyridyl)-5′-[4-( N -methyl)pyridinio]-2,2′-bithiophene iodide (PT2) adsorbed much more strongly onto the ODM self-assembled layers at 50 μM adsorption concentration in ethanol, while very similar compounds were much less effective in their adsorption even at much higher adsorption concentrations. The formation of multilayers of PT2 could be observed by AFM. Some structural features of the viologens, (dipole moment and polarizability), were calculated with semi-empirical quantum chemical calculations (MOPAC/AM1). One compound, PF2, having a slightly larger molecular length, dipole moment and polarizability than PT2, did not assemble noticeably onto the surface due to its larger solubility. The large difference in efficiency of adsorption can be related to a combination of length and absence of hydrophilic groups at the free pyridine side of the monomethylated viologens.

Self-Assembly of Pyridine-Modified Lipoic Acid Derivatives on Gold and Their Interaction with Thyroxine (T4)

Pyridyl derivatives of lipoic acid were prepared as ligands for the study of the interaction with thyroxine (T4). Thin self-assembled films of the ligands were prepared in 70% ethanol on gold and their interaction with T4 was studied by titration experiments in an aqueous buffer solution using Surface Plasmon Resonance (SPR). The thickness and refractive index of the ligand layers were calculated from SPR spectra recorded in two media, also allowing for surface coverage and the density of the layers to be estimated. Two ligands, a 4-pyridyl and a bis(2-hydroxyethyl) derivative of lipoic acid, were selected to investigate the feasibility for producing molecularly imprinted self-assembled layers on gold for T4. The methodology was to co-assemble T4 and the ligand onto the gold surface, elute the T4 from the layer under alkaline conditions, and study the rebinding of T4 to the layer. Multiple elution/rebinding cycles were conducted in different buffer solutions, and rebinding of T4 could be observed, with a moderate binding affinity that depended greatly on the solvent used. More optimal binding was observed in HBS buffer, and the affinity of the interaction could be slightly increased when the 4-pyridyl and bis(2-hydroxy-ethyl) derivatives of lipoic acid were combined in the imprinted layer.