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Surface plasmon resonance is one of the surface-oriented biosensing techniques that can be used to monitor biomolecular interactions. It is utilized in instrumentation for real-time biospecific interaction analysis capable of determining not only the concentrations of biomolecules but also kinetic constants, binding specificity, etc. In this contribution biosensing with surface plasmon resonance is reviewed. Special attention is given to an extended interaction matrix on the sensing surface, which enables the covalent binding of, e.g., antigens or antibodies. The surface plasmon resonance angle shifts are calculated as a function of the amount of organic material in the matrix. The influence of physical parameters, such as matrix thickness and wavelength of the light, on the expected performance is considered. Finally, a few illustrative experimental results obtained with a recently introduced commercial instrument are given.

It has been brought to our attention very recently that we had an omission error in our methods section of the paper [1]. This is Section 3.4, which should have read as follows.

This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization.Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance.Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.

A strategy for the synthesis of a series of closely related oligo(ethylene glycol)-terminated alkanethiol amides (principally HS(CH2)mCONH(CH2CH2O)nH; m = 2, 5, 11, 15, n = 1, 2, 4, 6, 8, 10, 12) and analogous esters has been developed. These compounds were made to study the structure and stability of self-assembled monolayers (SAMs) on gold in the prospect of designing new biosensing interfaces. For this purpose, monodisperse heterofunctional oligo(ethylene glycols) with up to 12 units were prepared. Selective monoacylation of the symmetrical tetra- and hexa(ethylene glycol) diols as their mesylates with the use of silver(I) oxide was performed. The synthetic approach was based on carbodiimide couplings of various oligo(ethylene glycol) derivatives to ω-(acetylthio) carboxylic acids via a terminal amino or hydroxyl function. SAM structures on gold were studied with respect to thickness, wettability (water contact angles ∼30°), and conformation. A good fit was obtained for the relation between monolayer t...

Optical characterization of ultrathin films and interfaces using surface plasmon polariton or surface plasmon resonance (SPR) excitation in “free-electron-like” metals of gold and silver was pioneered by two research groups from Germany in the late 1960s.1,2 They showed that the SPR phenomenon could be excited optically by the method of attenuated total reflection (ATR) either via a very thin air gap-“the Otto configuration”-or by a route where the thin metal film was deposited directly on the base of a glass prism-“the Kretschmann configuration.” Both methods were extensively used to study fundamental optical properties of thin metal films and inorganic/organic coatings deposited on top of the metal film surface. In the following years the SPR-ATR approach became very popular for studies of monomolecular assemblies of Langmuir-Blodgett films on metals3 and later for the determination of molecular orientation4 in such assemblies as well as for measurement of refractive index changes occurring during phase transitions in liquid crystals.5

The formation of highly ordered self-assembled monolayers (SAMs) on gold from an unusually long and linear compound HS(CH(2))(15)CONH(CH(2)CH(2)O)(6)CH(2)CONH(CH(2))(15)CH(3) is investigated by contact angle goniometry, ex situ null ellipsometry, cyclic voltammetry and infrared reflection-absorption spectroscopy. The molecules are found to assemble in an upright position as a complete monolayer within 60 min. The overall structure of the SAM reaches equilibrium within 24 h as evidenced by infrared spectroscopy, although a slight improvement in water contact angles is observed over a period of a few weeks. The resulting SAM is 60 A thick and it displays an advancing water contact angle of 112 degrees and excellent electrochemical blocking characteristics with typical current densities about 20 times lower as compared to those observed for HS(CH(2))(15)CH(3) SAMs. The dominating crystalline phases of the supporting HS(CH(2))(15) and terminal (CH(2))(15)CH(3) alkyl portions, as well as the sealed oligo(ethylene glycol) (OEG) "core," appear as unusually sharp features in the infrared spectra at room temperature. For example, the splitting seen for the CH(3) stretching and CH(2) scissoring peaks is normally only observed for conformationally trapped alkylthiolate SAMs at low temperatures and for highly crystalline polymethylenes. Temperature-programmed infrared spectroscopy in ultrahigh vacuum reveals a significantly improved thermal stability of the SAM under investigation, as compared to two analogous OEG derivatives without the extended alkyl chain. Our study points out the advantages of adopting a "modular approach" in designing novel SAM-forming compounds with precisely positioned in plane stabilizing groups. We demonstrate also the potential of using the above set of compounds in the fabrication of "hydrogel-like" arrays with controlled wetting properties for application in the ever-growing fields of protein and cell analysis, as well as for bioanalytical applications.

The surface interactions of exploring cyprids of the barnacle Semibalanus balanoides were studied in situ using imaging surface plasmon resonance. It was demonstrated how the deposition of a proteinaceous adhesive could be followed in real time as the cyprids explored and temporarily attached to a surface. Furthermore, the amount of protein left on the surface when the cyprids moved on could be quantified. Clear differences were demonstrated between an oligo(ethyleneglycol) coated surface and a bare gold substrate. It is anticipated that this technique will be a valuable tool in the development of novel surface chemistries that can prevent biofouling.

We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.

l -Cysteine adsorbates and multilayer films on gold and copper surfaces have been investigated by X-ray photoelectron spectroscopy. Both adsorbates, prepared by vapor deposition in UHV and prepared from solution, strongly indicate a dissociative chemisorption through the thiol group (−SH) on the metal surface. We suggest that an organized double layer is formed on gold, for the UHV-prepared layer. In the case of copper, evidence is found for the coordination of both amino and carboxyl groups to the surface, in addition to chemisorption through the thiol group. When l -cysteine is adsorbed from solution on copper, all of the thiol groups interact with copper ions, even in a 25-A-thick layer. This indicates copper ion diffusion and copper complex formation through the entire layer.

The cover picture illustrates a novel concept for colorimetric protein sensing based on the controllable assembly of polypeptide-functionalized gold nanoparticles. Recognition of the analyte is acc ...

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.

In this work, we have evaluated the haemocompatibility of different surface modifications, intended for biomaterials and bioanalytical applications. Polystyrene slides were coated with thin hydrogel films by self-initiated photografting and photopolymerization (SIPGP) of four different monomers. The hydrogel surface modifications were thoroughly characterized and tested for their protein resistance and ability to resist platelet adhesion and activation of the coagulation system. There was very little protein adsorption from human plasma on the hydrogels prepared from poly(ethylene glycol) methacrylate and 2-hydroxyethyl methacrylate. Platelet adhesion tests performed under both static and flow conditions showed that these coatings also demonstrated very high resistance towards platelet adhesion. A small amount of platelets were found to adhere to hydrogels formed from ethylene glycol methyl ether methacrylate and 2-carboxyethyl methacrylate. The polystyrene substrates themselves facilitated large amounts of platelet adhesion under both static and flow conditions. Utilizing a novel setup for imaging of coagulation, it was confirmed that none of the hydrogel surfaces activated the coagulation system to any great extent. We suggest that this simple fabrication method can be used to produce hydrogel coatings with unusually high blood compatibility, suitable for demanding biomaterials applications.

We recently reported on the strong interactions of zoospores of the green alga, Ulva linza with an arginine-rich oligopeptide self-assembled monolayer (SAM) [Biofouling 2008, 24, 303-312], where the arginine-rich peptide induced not only high spore settlement, but also a form of abnormal settlement, or "pseudo-settlement", whereby a proportion of spores do not go through the normal process of surface exploration, adhesive exocytosis, and loss of flagella. Further, it was demonstrated that both the total number of settled spores and the fraction of pseudosettled spores were related to the surface density of the arginine-rich peptide. Here we present a further investigation of the interactions of zoospores of Ulva with a set of oligomeric, de novo designed, arginine-rich peptides, specifically aimed to test the effect of peptide primary structure on the interaction. Via variations in the peptide length and by permutations in the amino acid sequences, we gain further insight into the spore-surface interactions. The interpretation of the biological assays is supported by physicochemical characterization of the SAMs using infrared spectroscopy, ellipsometry, and contact angle measurements. Results confirm the importance of arginine residues for the anomalous pseudosettlement, and we found that settlement is modulated by variations in both the total length and peptide primary structure. To elucidate the causes of the anomalous settlement and the possible relation to peptide-membrane interactions, we also compared the settlement of the "naked" zoospores of Ulva (which present a lipoprotein membrane to the exterior without a discrete polysaccharide cell wall), with the settlement of diatoms (unicellular algae that are surrounded by a silica cell wall), onto the peptide SAMs. Cationic SAMs do not notably affect settlement (attachment), adhesion strength, or viability of diatom cells, suggesting that the effect of the peptides on zoospores of Ulva is mediated via specific peptide-membrane interactions.

In recent years, biosensors and sensor arrays have developed into very important analytical tools, which found applications in many fields such as pharmaceutical (high-throughput) screening, medical diagnosis, or industrial process control. One of the major challenges for material research is the preparation of appropriate sensor surfaces, providing an interface with a high sensitivity and selectivity toward a given analyte. This chapter discusses some straightforward and flexible approaches to study structure and/or composition-function relationships and response characteristics of polymeric and molecular sensor materials. The controlled continuous deposition of self-assembled monolayers (SAMs), e.g. of substituted thiols or silanes, paves the way for the generation of molecular gradients on solid surfaces. These are useful for the preparation of interfaces with spatially controlled chemical composition and/ or physical properties. These tools can help to improve the selectivity and specificity of surfaces for biosensors and biochips. They can also be utilized for the study of fundamental protein adsorption and exchange phenomena.

3-Aminopropyl triethoxy silane (APTES) was deposited onto silicon oxide surfaces under various conditions of solvent, heat, and time, and then exposed to different curing environments, including air, heat, and ethanol. The macro- and micromolecular structure of APTES was probed on different levels using different techniques. The thickness of APTES layers was measured by ellipsometry, macromolecular structures were identified using microscopic methods (scanning electron microscopy and atomic force microscopy), and chemical form was investigated using angle-resolved X-ray photoelectron spectroscopy and also reflection infrared spectroscopy of APTES on gold and aluminum oxide surfaces. Coverage equivalent to one monolayer was achieved using very mild reaction and curing conditions (reaction in dry toluene for 15 min at room temperature, curing in air, or 15 min in 200°C oven), whereas thick layers were made by increasing reaction and curing times. APTES initially adsorbed to the surface, and curing was necessary to complete covalent binding between APTES and the surface. Deposition of APTES from water gave thin layers, probably electrostatically bound to silicon.

Infrared reflection-absorption spectroscopy (IRAS) has been used to study the chemisorption of l -cysteine ( l -Cys) and 3-mercaptopropionic acid (MPA) on evaporated gold and copper surfaces. The infrared data show that monomolecular films are formed for both l -Cys and MPA on gold, and that the bonding to the surface occurs primarily via the SH group. More complex reactions occur on the copper surface. MPA forms a multilayer structure on copper when adsorbed from solution at pH 3.5. In the very first layer the SH group is coordinated to the copper surface and the CO2− group to dissolved copper ions. The subsequent layers consist of MPA molecules in the dimeric form. Dissolution of copper is not very pronounced at high pH values as evidenced by the formation of a sodium salt instead of a cuprous (cupric) complex when MPA is adsorbed from a NaOH solution at pH 11.5. l -Cys also forms a complex ionic structure on copper where the SH, NH2 and CO2− groups appear to be involved in the bonding to the copper surface and/or dissolved copper ions.

Surface modification to provide, e.g., a biocompatible surface is an important molecular engineering method. As an example the FTIR-Attenuated Total Reflection (ATR) method has been applied to follow the different steps in the immobilization process of heparin on polyethylene (PE). This chemical multicomponent modification process is based on van der Waals and electrostatic interaction between alternating layers of cross-linked polyethylene imine and dextran sulfate, and finally covalent attachment of partly nitrous acid degraded heparin. Modified PE sheets were withdrawn and analyzed after each reaction step in the process. The overall spectral agreement between the ATR spectra and the spectra obtained from the pure substances used is good, except for slight changes in position and relative intensities of the sulfate and amino peaks. This observation indicates that the amino and sulfate groups are important (electrostatic) binding sites between the alternating polyethylene imine and dextran sulfate layers. The amount of covalently attached heparin was determined according to the “thin film model” by Harrick. The surface concentration, Γ, falls typically in the range of 2.4–2.7 μg/cm2, which is in good agreement with the result from a chemical analysis. The choice of peak areas used in the calculation of Γ is also discussed.

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (Ulva linza), diatoms (Navicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens). The biological results showed that the hydrogel coating exhibited excellent antifouling properties with respect to settlement and removal. © 2008 American Chemical Society.

Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These 'pseudo-settled' spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.

Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.

Heterodimerization between designed helix-loop-helix polypeptides was utilized in order to assemble gold nanoparticles on planar substrates. The peptides were designed to fold into four-helix bundl ...

Under the constraint that sulfur atoms form a hexagonal (square root 3 x square root 3)R30 degrees overlayer on the (111) gold surface, the optimized geometry of periodic arrays of HS(CH2)3CONH-(CH2CH2O)3H molecules has been found ab initio, by exploiting the BP86 exchange-correlation functional with 6-31G and "general" basis sets. The obtained data suggests that several prominent features of in-SAM molecular geometry and orientation stand out from conclusions based on single-molecule modeling. In particular, changing of amide-related dihedrals is shown to dominate in adjustment of molecular constituents to the assembly environment and to result in a substantial shortening of the hydrogen bond distance between nearest-neighbor amides. First demonstrated here, the full account to the intermolecular interaction within periodic arrays of amide-bridged, oligo(ethylene glycol)-terminated alkanethiolates forms a new platform for arguable modeling of SAM apparent properties.

Extensive ab initio modeling has been performed to explain quantitatively the apparent shapes of characteristic bands, which are systematically observed in the fingerprint region of infrared (IR) reflection-absorption (RA) spectra of oligo(ethylene glycol) (OEG)-terminated SAMs. The presence of defects was thoroughly examined by modeling the RA spectra using the DFT method BP86/6-31G* for all-helical and all-trans conformers of HS(CH2CH2O)nR (n = 5, 6, R = H, CH3) and HS(CH2)15CONH(CH2CH2O)6H molecules and for several defect-containing conformers. These data were then used to simulate RA spectra of SAMs with different content of defects and to compare them with experiments. It is shown that for SAMs of HS(CH2CH2O)nCH3 (n = 5, 6) the pronounced asymmetry of the dominating band can be attributed to the multimode nature of COC stretching vibrations of helical conformers combined with the contribution from few percent of all-trans conformers. Arguments are presented which disprove appreciable amounts of helical conformers with single trans and/or gauche defects. Much more complex combination of factors, which can come into play in the formation of the high-frequency shoulder of COC band, is exemplified by self-assemblies of OEG-terminated amide-bridged alkanethiolates. In particular, spectral signatures of defects with inverted OH terminus are compared with other contributions to the apparent shape of COC band formation. For this family of SAMs, the presence of about 10% of all-trans conformers gives a satisfactory quantitative agreement between the calculated RA spectra and experimental observations.

Infrared reflection-absorption spectroscopy and X-ray photoelectron spectroscopy (XPS) are used to characterize adsorbed layers of amino acids on evaporated copper surfaces. Thin layers of glycine, l -alanine, and β-alanine are formed by adsorption from 5 m M aqueous solutions at pH values near their isoelectric points. Glycine is most thoroughly studied, and much attention is paid to a comparison with synthesized complexes of cis -Cu(II)(Gly) 2 ·H 2 O and trans -Cu(II)(Gly) 2 ·2H 2 O. A very good agreement is obtained between calculated reflection—absorption spectra based on these model substances and observed spectra of adsorbed glycine on copper. This observation suggests that both the carboxylate oxygens and the amino nitrogens are involved in the bonding to copper and that an ionic lattice (≈10 A) consisting of Cu ions and glycine is formed on the surface. Further support for an ionic structure is obtained from a preliminary XPS study where differently prepared glycine layers on copper are compared with the cis -Cu(II)(Gly) 2 ·H 2 O complex. The composition of the ionic lattice is found to vary with the microstructure of the copper surface and with film orientation in particular. Our infrared data indicate that the cis form is more pronounced on copper films with preferred (111) orientation, whereas the trans form appears to dominate on “polycrystalline” copper. l -Alanine and β-alanine also react with copper via the carboxylate-oxygen and amino-nitrogen atoms. However, the layer thicknesses for l - and β-alanine appear to be smaller than those obtained for glycine.

Glycine adsorbed on polycrystalline copper has been studied by angle-dependent X-ray photoelectron spectroscopy, XPS(θ), and infrared reflection absorption spectroscopy, IRAS. At −50°C the molecule adsorbs with its molecular identity intact. Upon heating to 30°C the glycine molecule slowly decomposes on the copper surface. The adsorbate held at −50°C is compared with a thin film of glycine, which is known to be zwitterionic. The binding energy shifts, the intensity ratios in the N(1s), C(1s), and O(1s)XPS(θ) spectra, and the infrared spectra of the adsorbate show that the glycine molecule coordinates with both the nitrogen and the two oxygen atoms to the copper surface. The amino group exists as an −NH2 group in the adsorbate rather than as an −NH3+, which is the case in the zwitterionic form of glycine. This result is in contrast with the molecular orientation of glycine on hydrophilic gold, silicon, and highly oriented pyrolytic graphite (HOPG).

Pure aqueous Ti-peroxy gels with a pH of 4, made from metallic titanium and hydrogen peroxide, were heat dried at 60°C or freeze-dried at a final pressure of 1 × 10−2 Torr and then aged. Prior to measurements some samples were evacuated to 1 × 10−7 Torr during 4–24 h. Studies were made also on powders prepared from a solution of phosphate-buffered saline, metallic titanium, and hydrogen peroxide with a pH between 4.25 and 7.85 during the process period and on titania precipitates made from Ti(III) sulfate and KO2. The powders so obtained were examined with FT-IR and ESR methods. During aging, hydrogen peroxide is released from the dried Ti-peroxy gel powders. In a KBr matrix, gel powders first aged and then evacuated to 1 × 10−7 Torr show a vibration at 1385 cm−1. This vibration is shown to emanate from the KBrH2O2 interaction. Titanium superoxide (Ti(IV)O2−) radicals appear, as shown by ESR methods, both in dried pure Ti-peroxy gels and in powders made from buffered Ti-peroxo solutions under weakly acidic or neutral pH conditions.

Infrared reflection absorption spectroscopy (IRAS) and x-ray photoelectron spectroscopy (XPS) have been used to study the adsorption of potassium p-methyl benzyl xanthate and potassium p-trifluoromethyl benzyl xanthate on gold surfaces. Monolayer structures were formed by adsorption from 1 to 10 μM aqueous solutions of the xanthates for different adsorption times. The reflection-adsorption (R-A) spectra are compared with calculated R-A spectra, which are based on the optical constants n(v) and k(v) derived from KBr transmission spectra of the corresponding Au(I) salts. The experimentally obtained peak positions are in good agreement with the calculated ones, indicating that the chemical structure of the surface complexes are closely related to those of the metal salts. However, the relative intensities vary considerably between experimental and calculated R-A spectra. We discuss these intensity changes in terms of molecular orientation and packing. A detailed comparison of the relative ratios for the peaks belonging to the OCS2 and CF3 groups reveals that ordered structures are formed on the gold surface, and that a reorientation from a flat to an almost vertical orientation occurs with increasing immersion time and concentration of the xanthate solution. These observations are confirmed with angle-dependent XPS(Θ).

The physical adsorption of glycine molecules on highly oriented pyrolytic graphite substrates is studied using angle-dependent X-ray photoelectron spectroscopy, XPS(θ). A partially oriented double-layer is found to be stable on the surface. The structure of the double-layer is consistent with (but not identical to) the crystal structure of β-glycine, which is a layered crystal.

A protein array chip for label-free optical detection of low molecular weight compounds has been developed. As a proof of principle, the chip is proven capable of rapidly (approximately 1 min) determining hits from aqueous cocktails composed of four common narcotics, cocaine, ecstasy, heroin, and amphetamine, using imaging surface plasmon resonance (SPR) as the detection principle. The chip is produced by injecting a mixture of antibodies and letting them self-sort and bind to narcotic analog coupled proteins already present in a predefined pattern on the supporting substrate. An indirect detection method, where antibodies are displaced from the surface upon recognition of their corresponding narcotics, is used to obtain the optical contrast and thus a detectable SPR and/or ellipsometric signal. Two types of readouts are possible from the present setup: intensity SPR images and SPR/ellipsometric sensorgrams. Positive hits were routinely obtained for analyte concentrations of 50 pg/microL and the limit of detection, without any parameter optimizations, seems to fall in the range 0.5 pg/microL (1.4 nM) for heroin, 2.5 pg/microL (8.2 nM) for cocaine, and 5 pg/microL for the other two narcotics (26 nM for ecstasy and 37 nM for amphetamine). With improved readout possibilities (sampling frequency), signal evaluation algorithms, and antibody-antigen design strategies, we believe this limit can be further improved. The chip is shown to work for many measurement cycles with excellent reproducibility. Moreover, with a more advanced fluidic system, excess injected antibodies could be collected and reused for many cycles, which could make the running costs of the system very low. The chip is in no way limited to detection of narcotics. Other low molecular weight compounds could easily be detected on the same chip. For example, trinitrotoluene detection has already been demonstrated using our chip. Possible areas of application for the system are therefore envisaged in airport and underground transport security, customs, drug interdiction, forensics, and as warning alerts on military equipment and personnel.

A novel method of producing a poly(ethylene glycol) (PEG)-based gradient matrix that varies gradually in thickness from 0 to 500 A over a distance of 5-20 mm is presented. The gradient matrix is graft copolymerized from a mixture of PEG methacrylates onto organic thin films providing free radical polymerization sites initiated by UV irradiation at 254 nm. The films used as grafting platforms consist of either a spin-coated cycloolefin polymer or a self-assembled monolayer on planar gold. The thickness/irradiation gradient is realized by means of a moving shutter that slowly uncovers the modified gold substrate. The structural and functional characteristics of the gradient matrix are investigated with respect to thickness profile, degree of carboxylation, and subsequent immobilization of two model proteins of different sizes and shapes. These characteristics are studied with ellipsometry and infrared reflection-absorption microscopy using a grazing angle objective. It is revealed that the relatively small carboxylation agent used offers homogeneous activation throughout the gradient, even in the thick areas, whereas the diffusion/interpenetration and subsequent immobilization of large proteins is partially hindered. This is crucial information in biosensor design that can be easily obtained from a gradient experiment on a single sample. Moreover, the partially hindered protein interpenetration, the marginal swelling upon hydration, and the unspecific nature of the graft polymerization suggest a matrix growth mechanism that favors the formation of a bushlike polymer structure with a certain degree of cross linking.

This contribution describes how de novo designed synthetic helix–loop–helix polypeptides are utilized to control the assembly of gold nanoparticles and as scaffolds for biosensing. The synthetic polypeptides are designed to fold into a four-helix bundle upon dimerization. When immobilized on gold nanoparticles, dimerization and folding occur between peptides on neighbouring particles as an effect of particle aggregation and the folded polypeptides are rigid enough to keep the particles separated at a distance corresponding to the size of the four-helix bundle. Moreover, peptide dimerization offers a convenient route to assemble nanoparticles into hybrid multilayers on planar substrates. The drastic change in the resonance conditions of the localized nanoparticle surface plasmon upon particle aggregation is shown to be useful for optical detection of biomolecular interactions.

Dip-pen nanolithography (DPN) is employed to develop a generic array platform for the selective recruitment of membrane protein complexes. An atomic force microscope tip inked with HS(CH2)16NH2 is used to generate amino-terminated domains on gold. These domains can be arranged into microscopic and submicroscopic patterns, and the untreated gold substrate is subsequently blocked with HS(CH2)2CONH(CH2CH2O)15CH3, a compound known to resist the unspecific binding of proteins and cells. The patterned gold substrate is exposed to an enriched membrane fraction from mutant Rhodobacter sphaeroides, which contains photosynthetic core complexes consisting of the reaction center and the light-harvesting complex LH1. The selective recruitment to the patterned domains, governed primarily by electrostatic interactions, is confirmed by contact mode atomic force microscopy.

The nucleation and phase behavior of ultrathin D2O-ice overlayers have been studied on oligo(ethylene glycol) (OEG)-terminated and hydroxyl self-assembled monolayers (SAMs) at low temperatures in ultrahigh vacuum. Infrared reflection-absorption spectroscopy (IRAS) is used to characterize the ice overlayers, the SAMs, and the interactions occurring between the ice and the SAM surfaces. Spectral simulations, based on optical models in conjunction with Maxwell Garnett effective medium theory, point out the importance of including voids in the modeling of the ice structures, with void fractions reaching 60% in some overlayers. The kinetics of the phase transition from amorphous-like to crystalline-like ice upon isothermal annealing at 140 K is found to depend on the conformational state of the supporting OEG SAM surface. The rate is fast on the helical OEG SAMs and slow on the corresponding all-trans SAMs. This difference in kinetics is most likely due to a pronounced D2O interpenetration and binding to the all-trans segments of the ethylene glycol portion of the SAM. No such penetration and binding was observed on the helical OEG SAM.

Background The development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions without the need for chemical-, biological- or radiological-labelled reagents. Method We utilised SPR to detect hepatocyte growth factor (HGF) in reconstituted faecal samples and studied samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10). Mouse anti-human HGF monoclonal antibodies and recombinant human HGF receptor (c-Met)/Fc chimera were immobilised in flow cells of a CM5 biosensor chip. Results We found that infectious gastroenteritis produced a higher signal response compared to controls, due to binding of HGF to monoclonal anti-HGF antibody as well as binding of HGF to c-Met receptor (p < 0.01). The SPR signal response correlated with results from ELISA (r = 72%, p > 0.001). The signal response decreased significantly (p < 0.05) when samples were diluted with dextran, because of reduction in both specific as well as unspecific binding of HGF to dextran. The decrease in the specific response might imply that the dextran- binding site for HGF overlaps with the antibody binding epitope, or that dextran binding induces a conformational change of the HGF molecule. Bands corresponding to HGF were found by gel electrophoresis of purified faeces in an affinity chromatography column immobilised by HGF ligands. Conclusion Determination of HGF by SPR might be beneficial in diagnosis of acute situations that present with symptoms of gastroenteritis and may, possibly, guide appropriate medical treatments. This is to our knowledge the first report on the use of SPR for detection of HGF in faeces samples.

Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption-reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

We report orientation angles for the alkyl chain, amide group, and oligo(ethylene glycol) (OEG) portion within self‐assembled monolayers (SAMs) of OEG‐terminated and amide containing alkanethiolates which, depending on the OEG length and substrate temperature, display unique conformations — all‐trans or helical. Optimized geometries of the molecular constituents, characteristic vibration frequencies and transition dipole moments are obtained by using DFT methods with gradient corrections. These ab initio data are subsequently used to simulate infrared reflection‐absorption (RA) spectra associated with different conformations and orientations. The obtained results have generated a deeper knowledge of the internal SAM structure, which is crucial for understanding phase and folding characteristics, interaction with water and ultimately the protein repellent properties of OEG‐containing SAMs.

This paper reports on the structural characteristics of microcontact printed oligo(ethylene glycol)-terminated alkanethiol layers, HS(CH2)15CONH-(CH2CH2O)6-H (hereafter EG6), on gold. Microwetting, contact angle goniometry, imaging null ellipsometry, and infrared reflection-absorption spectroscopy (IRAS) are used to characterize the printed EG6 layers, and the quality of these layers in terms of layer thickness and the crystallinity of the alkyl and ethylene glycol portions is compared with data obtained from analogous layers prepared by solution self-assembly. The outcome of the printing process is critically dependent on the experimental parameters used to prepare the patterns. It is found that high quality layers, consisting of densely packed all-trans alkyl chains terminated with relatively helical hexa(ethylene glycol) tails, are formed by inking the poly(dimethylsiloxane) (PDMS) stamp with a 1 mM EG6 solution and contacting it with gold for 15 min. The homogeneity of printed layers is not as good as the homogeneity of those prepared from solution under similar conditions, most likely because of simultaneous transfer of low molecular weight residues from the PDMS stamp. These residues, however, can be easily removed upon ultrasonication in ethanol without affecting the quality of the printed layer. Further on, the microscopic square-shaped bare gold patterns formed after microcontact printing (muCP) are subsequently filled with 16-hexadecanoic acid (hereafter THA) or HS(CH2)15CONH-(CH2CH2O)6-COOH (hereafter EG6COOH) to provide a microarray platform for further covalent attachment of biomolecules. Well-defined structures in terms of wettability contrast, sharpness, and height differences between the printed and back-filled areas are confirmed by imaging null ellipsometry and microscopic wetting.

Tetraoctadecylammonium bromide (TOAB, (CH3(CH2)17)4N+Br−) has been used to print temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels to create a generic platform for protein microarray applications. The primary reason for printing temporary hydrophobic barriers is to prevent cross-contamination and overflow during microdrop dispensing. Equally important is to eliminate the risk for non-specific binding to the barriers during analyte exposure. This has been accomplished by introducing a regeneration step that removes the barriers after ligand immobilization. The overall fabrication process was characterized by microscopic wetting, atomic force microscopy, imaging ellipsometry, fluorescence microscopy, surface plasmon microscopy and biospecific interaction analysis. A series of model proteins including transferrin, Protein A, anti-myoglobin and bovine serum albumin was spotted into the TOAB-defined areas under different experimental conditions, e.g. at increased humidity and reduced substrate temperature or with glycerol as an additive in the protein solution. Much emphasis was devoted to studies aiming at exploring the homogeneity and activity of the immobilized proteins. The printed barriers were removed after protein immobilization using tert-n-butyl alcohol (TBA). TBA was found to be a very efficient agent as compared to previously used salt regeneration solutions, and the regeneration time could be reduced from ∼30 to 10 minutes. Finally, the potential of using the well established CMD hydrogel chemistry as a platform for protein microarrays was exploited using surface plasmon microscopy.

Hydrogen Bond Interaction between Ester Terminated Self-Assembled Monolayers and Adsorbed Water Molecules and Its Implications for Cluster Formation