Monolayers of derivatized poly(L-lysine)-grafted poly(ethylene glycol) on metal oxides as a class of biomolecular interfaces.

We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers adsorbed on negatively charged surfaces. As a model system, we synthesized biotin-derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-[(PEGm)((1-x)) (PEG-biotin)(x)], where x varies from 0 to 1. Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy. The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures. The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins. This streptavidin layer can subsequently capture biotinylated proteins. Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning.

Atomic force microscope: a tool for studying ionophores

The aim of the investigations was to show the analytical use of an atomic force microscopy (AFM) tip coated with an ion-selective membrane and to show that the ion-selective boundary potential is detectable as a force induced by ion-selective electrostatic interactions, which are more pronounced than double-layer forces. This new technique allows the area-specific ion exchange over boundaries to be displayed with a destruction-free technique by AFM in an aqueous buffer. From experiments with ISEs (ion-selective electrodes), a boundary potential for valinomycin was assumed to be established in close vicinity to a K+-releasing surface. To trace this boundary potential, an AFM tip was coated with a potassium-selective polymer film containing valinomycin as used in preparing ISEs. The K+-releasing substrate was prepared by incorporating a lipophilic potassium salt into a plasticized PVC layer. In contact with an electrolyte such as sodium chloride solution, an ion exchange takes place. Analogue experiments were performed using a sodium-selective ionophore, DD16C5, incorporated into the coating of the AFM tip, with a Na+-releasing substrate. The boundary potential was traced and investigated with the help of force vs distance curves. The resulting adhesion forces for a valinomycin-coated tip in a 150 mM NaCl solution were 9.8+/-3.275 nN using a blank PVC substrate and 330.15+/-113.0 nN using a substrate containing 10 wt % potassium tetrakis(4-chlorophenyl) borate. The selectivity of the ion-selective AFM tips was measured on sodium relative to potassium-releasing substrates and studied in different salt solutions with concentrations between 10 mmol L(-1) and 1 mol L(-1). For valinomycin, a force selectivity coefficient log Kf(K,Na) of -2.5+/-0.5 for K+ against Na+ and a selectivity coefficient log Kf(Na,K) of -4 +/- -0.5 for DD16C5 were measured. In addition, the surface of the polymer substrate was imaged by AFM in contact mode with and without lipophilic potassium salt. The modulation of the force-distance curves induced by the ion exchange was compared to the experimental change in elasticity of the blank and ion-exchanging substrate. The Young's moduli measured with strain force analysis on a potassium-containing substrate were 5 times smaller than the ones registered with nanoindentation and did not explain the modulation of the force vs distance curves.

Microstructured bioreactive surfaces: covalent immobilization of proteins on Au(1 1 1)/silicon via aminoreactive alkanethiolate self-assembled monolayers.

Micrometer-scale patterns of a defined surface chemistry and structure were produced on both ultraflat Au(1 1 1) and on gold-coated monocrystalline silicon surfaces by a method combining microcontact printing, wet chemical etching and the replacement of etch-resist self-assembled monolayers (SAMs) by functionalized or reactive SAMs. Key steps in this methodology were characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry and contact angle measurements. The covalent immobilization of (functional) biological systems on these surfaces was tested using an N-hydroxysuccinimide ester omega-functionalized disulphide (DSU), which covalently binds primary amines without the need for further activation steps. Atomic force microscope images of native collagen V single molecules immobilized on these patterned surfaces revealed both high spatial resolution and strong attachment to the monolayer/gold surface. Microcontact printing of DSU is shown to be feasible on specially prepared, ultraflat Au(1 1 1) surfaces providing a valuable tool for scanning probe experiments with biomolecules. The retention of enzymatic activity upon immobilization of protein was demonstrated for the case of horseradish peroxidase. The described approach can thus be used to confine biological activity to predetermined sites on microstructured gold/silicon devices - an important capability in biomedical and biomolecular research.

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.