Signal enhancement in ligand-receptor interactions using dynamic polymers at quartz crystal microbalance sensors.

The signal enhancement properties of QCM sensors based on dynamic, biotinylated poly(acrylic acid) brushes has been studied in interaction studies with an anti-biotin Fab fragment. The poly(acrylic acid) sensors showed a dramatic increase in signal response with more than ten times higher signal than the carboxyl-terminated self-assembled monolayer surface.

Using a quartz crystal microbalance biosensor for the study of metastasis markers on intact cells.

The use of biosensors has become a standard method to characterize biomolecular interactions. Data obtained from biosensor studies are widely used to evaluate drug candidates, particularly in relation to their binding properties towards a selected target. The importance of measuring such interactions in a biologically relevant environment has become the new challenge for the biosensor technologies. In this chapter we describe a method for studying interactions between proteins and targets at the surface of intact cells using a quartz crystal microbalance (QCM) biosensor.

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach.

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity.

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK(a), antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK(a) is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab(2) antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Esterification of self-assembled carboxylic-acid-terminated thiol monolayers in acid environment: a time-dependent study.

This contribution reports on the influence of acids on the quality of carboxylic-acid-terminated self-assembled monolayers (SAMs) on gold prepared from ethanolic solution of HS-(CH(2))(15)-COOH and HS-(CH(2))(11)CONH-(EG)(6)CH(2)-COOH. Null ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy are used to monitor the physical and chemical changes occurring within the SAMs upon acid post treatment; after incubation with acids present in the solution; and after incubation in aged acid containing solutions. The presence of acid has a positive effect on the crystallinity, packing, and orientation of the supporting alkyl and ethylene glycol subunits of the SAM. Our studies also confirm previous findings stating that the carboxylic groups are rapidly converted into ethyl ester groups in the presence of hydrochloric acid in the incubation solution. It is also evident that the conversion occurs in the presence of the weaker acid, acetic acid, although at a much slower rate than that for hydrochloric acid. This is a new observation that has not been reported on before. The physical and chemical characterization is also complemented with a functional bioaffinity study. The functional evaluation revealed that the present model system was surprisingly insensitive to the degree of esterification of the carboxylic acid groups, but that 4 weeks of storage of the two investigated thiols in hydrochloric acid containing ethanol resulted in SAMs that were completely inactive with respect to immobilization and subsequent binding of the antigen. It was encouraging to note that the nonspecific binding of both antigen and antibody was extremely low on the two SAMs, regardless of the relative amount of ethyl esters on the surface.

Quartz crystal microbalance, a valuable tool for elucidation of interactions between apoB-100 peptides and extracellular matrix components.

Atherosclerosis has received wide attention as a primary cause of premature death in developed countries. The retention of low-density lipoprotein (LDL) particles in the intima, the inner layer of the capillaries, has been imputed as the main cause of the development of atherosclerotic plaques. The entrapment of LDL is mainly due to the specific interaction between the lysine-rich site on apolipoprotein B-100 (apoB-100), a major apolipoprotein of LDL, and extracellular matrix (ECM) components such as collagen, proteoglycans, and glycosaminoglycans (GAGs). Although valuable techniques already exist for studies on apoB-100 and ECM interactions, there is continued need for miniaturized tools that can complement the tools already available and even provide totally new data. This work explores the applicability of the quartz crystal microbalance (QCM) for interaction studies between apoB-100 peptide fragments and various components of the ECM. Two positive peptide fragments, PP and PP(2), and two components of the ECM, collagen I and a selected GAG, chondroitin 6-sulfate (C6S), were immobilized on polystyrene and carboxyl sensor chips. C6S was injected as analyte for PP- and PP(2)-coated surfaces, while PP was the analyte for collagen I and C(6)S surfaces. The estimated dissociation constant (K(D)) indicates that the interactions occur via the positive residues, lysine and arginine, of apoB-100. The continuous-flow QCM system employed in this study is shown to be an excellent tool for the elucidation of interactions between these types of biomolecules.

Evidence for selective advantage of pathogenic FGFR2 mutations in the male germ line.

Observed mutation rates in humans appear higher in male than female gametes and often increase with paternal age. This bias, usually attributed to the accumulation of replication errors or inefficient repair processes, has been difficult to study directly. Here, we describe a sensitive method to quantify substitutions at nucleotide 755 of the fibroblast growth factor receptor 2 (FGFR2) gene in sperm. Although substitution levels increase with age, we show that even high levels originate from infrequent mutational events. We propose that these FGFR2 mutations, although harmful to embryonic development, are paradoxically enriched because they confer a selective advantage to the spermatogonial cells in which they arise.