Label-free genetic and proteomic marker detection within a single flowcell assay.

No one technique for multiplexing 100+ label-free measurements in a single well or flowcell has yet gained wide acceptance, probably because the added complexity introduced by the multiplexing element is seen to outweigh any possible cost advantage, or the multiplexing scheme itself is not flexible enough to accommodate the desired combinations of immobilization conditions and target/analyte molecules. Here, we demonstrate a simple yet highly versatile technology which uses microparticles, each bearing both an identifier code and an optical grating, to permit the inclusion of diverse molecules such as protein A, IgG and DNA, on chemically diverse -OH, -NH(2) and -COOH-terminated surfaces, within a single flowcell assay. Binding of avidin is used to reveal the presence of the immobilized biotinylated species, and to compare directly the binding of similar molecules on dissimilar surfaces, and vice-versa. Though we report the results of a 26-plex assay here, the technique itself has scope for increased throughput up to the level of hundreds of molecular species.

Encoded and multiplexed surface plasmon resonance sensor platform.

We present a flexible new sensor system that combines the joint advantages of (i) discretely functionalized, code-bearing, microparticles and (ii) label-free detection using grating-coupled surface plasmon resonance. This system offers the possibility of simultaneously investigating the real-time binding kinetics of a variety of molecular interactions. One single multiplexed assay could employ a wide range of immobilization chemistries, surface preparation methods, and formats. Thus, the new system offers a very high level of assay conformability to the end user, particularly when compared to fixed microarrays.

Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes.

The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed.

Partially reversible adsorption of annexin A1 on POPC/POPS bilayers investigated by QCM measurements, SFM, and DMC simulations.

The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall area occupied by the POPS-enriched phase is controlled by the CaCl2 concentration. Monte Carlo simulations suggest that the area of the POPS-enriched phase increases by a factor of 7 when the Ca2+ concentration is changed from 0.01 to 1 mM.

Scrutiny of annexin A1 mediated membrane-membrane interaction by means of a thickness shear mode resonator and computer simulations.

The dissipational quartz crystal microbalance (D-QCM) technology was applied to monitor the adsorption of vesicles to membrane-bound annexin A1 by simultaneously reading out the shifts in resonance frequency and dissipation. Solid-supported membranes (SSMs) composed of a chemisorbed octanethiol monolayer and a physisorbed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine monolayer were immobilized on the gold electrode of a 5 MHz quartz plate. Adsorption and desorption of annexin A1 to the SSM was followed by means of the QCM technique. After nonbound annexin A1 was removed from solution, the second membrane binding was monitored by the D-QCM technique, which allowed distinguishing between adsorbed and ruptured vesicles. The results show that vesicles stay always intact independent of the amount of bound annexin and the vesicle and buffer composition. It was shown that the vesicle adsorption process to membrane-bound annexin A1 is fully irreversible and is mediated by two-dimensional annexin clusters. For N-terminally truncated annexin A1, a decrease in the amount of bound vesicles was observed, which might be the result of fewer binding sites presented by the annexin A1 core. Supported by computer simulations, the results demonstrate that the vesicle adsorption process is electrostatically driven, but compared to those of sole electrostatic binding, the rate constants of adsorption are 1-2 orders of magnitude smaller, indicating the presence of a potential barrier.

Kinetics and thermodynamics of annexin A1 binding to solid-supported membranes: a QCM study.

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin A1 with lipid membranes was quantified using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of a first octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer obtained from vesicle fusion. This experimental setup enabled us to determine for the first time rate constants and affinity constants of annexin A1 binding to phosphatidylserine-containing layers as a function of the calcium ion concentration in solution and the cholesterol content within the outer leaflet of the solid-supported bilayer. The results reveal that a decrease in Ca(2+) concentration from 1 mM to 100 microM significantly increases the rate of annexin A1 binding to the membrane independent of the cholesterol content. However, the presence of cholesterol in the membrane altered the affinity constants considerably. While the association constant decreases with decreasing Ca(2+) concentration in the case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) membranes lacking cholesterol, it remains high in the presence of cholesterol.