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Alkanethiolates have been widely used as chemisorbates to modify gold surfaces, in spite of their relatively poor oxidative stability. We introduce gold-chemisorbing block copolymers bearing an anchoring block of poly(propylene sulphide) (PPS), selected in the expectation of greater stability. These materials offer a more robust approach to surface modification of gold. As an example, a triblock copolymer with poly(ethylene glycol) (PEG) was selected, with the goal of minimizing biological adsorption and adhesion. The copolymer PEG17-bl-PPS25-bl-PEG9 chemisorbed to form a dense monolayer of 226 +/- 26 ng cm(-2), approximately 2.2 nm thick. The copolymeric adlayer was much more stable to oxidation than commonly used alkanethiolates. Its presence greatly reduced protein adsorption (>95%), even after exposure to whole blood serum (>55 mg x ml(-1)), as well as cell adhesion over long culture durations (>97%). PPS-containing copolymers are an attractive alternative to alkanethiolates, and PEG-bl-PPS-bl-PEG presents a powerful example for use in biodiagnostic and bioanalytical devices.

A method is presented to form gold-electrode-tethered lipid bilayers with exceptionally high electrical resistances. Electrical impedance spectroscopy is used to monitor the bilayer incorporation of a ligand-gated ion channel protein and the modulation of its channel activity by the selective binding of an antibody. Due to the low defect density of the tethered membrane, the effect of a few channels can be resolved thus opening the way to single-channel experiments on this highly stable and versatile platform. In turn, a minute quantity of analyte, here antibodies, can be measured, which is of great interest for bioanalytics.

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The outer membrane protein OmpF from Escherichia coli is a member of a large family of ␤-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with ␤-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its ␤-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these ␤-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.

Abstract. Relatively simple electrochemical probes of redox active proteins can yield a wealth of diverse information concerning their kinetic and thermodynamic reactivity, charge state, and transport rates. In this report, the electrochemical properties of three heme-containing proteins are probed using Au electrodes derivatized with self-assembled monolayers of co-hydroxyalkanethiols. Cytochrome c and b s (wild type from horse and rat, respectively) display low reorganization energies (0.58 eV and 0.44 eV, respectively) and electronic coupling terms which are quite comparable to small redox molecule models. In contrast, metmyoglobin reduction is characterized by a somewhat higher reorganization energy (0.76 eV) and an order of magnitude reduction in its electronic coupling to the electrode. Upon binding oxygen, the reduced oxymyoglobin becomes electroinactive. This radical decrease in the oxymyoglobin redox reactivity is associated with structural and electronic differences caused by the spin-state change induced by oxygen binding. The reactivity differences between these heme-containing proteins are discussed in light of their biochemical function, evolutionary pressures, and structural differences.

The structure of w-hydroxyalkanethiol monolayers containing an intemal ether group is probed by traditional surface infrared reflection-absorption spectroscopy and a novel electrochemical method based on the measured shifts in the potential of zero charge (pzc) upon introduction of the ether group. The analyses of the infrared and electrochemical data give complementary structural information. Vibrational bandwidth measurements indicate that these monolayers exhibit crystalline packing. From a comparison of the integrated infrared absorption intensities between monolayer and bulk samples of the thiols, an average tilt angle of 29" and an average twist angle of 40" are determined for the ether modified w-hydroxyalkanethiol monolayers. The pzc of the ether modified monolayer coated Au electrodes is observed to shift by 130 i 40 mV from that characteristic of an unmodified w-hydroxyalkanethiol monolayer. A simple dipole analysis of the magnitude of these pzc shifts, assuming a twist angle of 40", gives an average tilt angle of 23 & 8", in reasonable agreement with the infrared determination. In addition, the monolayers modified by an oxygen located at an even methylene site (from the S atom) are observed to give a positive pzc shift, while those located at odd sites give strictly negative pzc shifts. Such an evedodd effect on the sign of the pzc shift indicates that the S-C bond is directed away from the surface normal. This electrochemical approach to structural analysis is generally applicable to crystalline monolayers which contain a net static internal dipole.